AChEi Brief: Reference Abstracts - U.S. Department of ... · Web view61. Mosca, L., et al., Modulation of apoptosis and improved redox metabolism with the use of a new antioxidant

Research Advisory Committee on Gulf War Veterans’ Illnesses (RAC-GWVI)AChEi Brief: Reference Abstracts

Acetylcholinesterase Inhibitor Brief: Reference Abstracts

1. Fricker, R.D., et al., A Review of the Scientific Literature as It Pertains to Gulf War Illnesses. Volume 12: Pesticide Use During the Gulf War: A Survey of Gulf War Veterans. 2000, Santa Monica, CA: RAND, MR-1018/12-OSD.

2. Golomb, B.A., Illness in Persian Gulf War veterans: Evidence for a causal role for acetylcholinesterase inhibitors (pyridostigmine bromide, pesticides, and nerve agent). Submitted.

3. Nisenbaum, R., et al., Deployment stressors and a chronic multisymptom illness among Gulf War veterans. J Nerv Ment Dis, 2000. 188(5): p. 259-66. Unusual health problems have been reported by Gulf War (GW) veterans, but no single etiology has been linked to these illnesses. This study was conducted to determine the association between self-reported GW deployment stressors and an illness defined by a combination of fatigue, mood-cognition, and musculoskeletal symptoms. A total of 1002 GW veterans from this cross-sectional survey of four Air Force units completed a self-administered questionnaire that asked about symptoms, demographic and military characteristics, and stressors during deployment. Severe and mild-moderate illness was positively associated with self-reports of pyridostigmine bromide use, insect repellent use and belief in a threat from biological or chemical weapons. Injuries requiring medical attention were only associated with severe illness. These results suggest a link between self-reported chemical, emotional, and physical exposures, and GW veterans' illness. Further research is needed to determine physiological and psychological mechanisms through which such stressors could have contributed to this symptom complex.

4. Unwin, C., et al., Health of UK servicemen who served in the Persian Gulf war. Lancet, 1999. 353: p. 169-178. BACKGROUND: Various symptoms in military personnel in the Persian Gulf War 1990-91 have caused international speculation and concern. We investigated UK servicemen. METHODS: We did a cross-sectional postal survey on a random sample of Gulf War veterans (Gulf War cohort, n=4248) and, stratified for age and rank, servicemen deployed to the Bosnia conflict (Bosnia cohort, n=4250) and those serving during the Gulf War but not deployed there (Era cohort, n=4246). We asked about deployment, exposures, symptoms, and illnesses. We analysed men only. Our outcome measures were physical health, functional capacity (SF-36), the general health questionnaire, the Centers for Disease Control and Prevention (CDC) multisymptom criteria for Gulf War illness, and post-traumatic stress reactions. FINDINGS: There were 8195 (65.1%) valid responses. The Gulf War cohort reported symptoms and disorders significantly more frequently than those in the Bosnia and Era cohorts, which were similar. Perception of physical health and ability were significantly worse in the Gulf War cohort than in the other cohorts, even after adjustment for confounders. Gulf War veterans were more likely than the Bosnia cohort to have substantial fatigue (odds ratio 2.2 [95% CI 1.9-2.6]), symptoms of post-traumatic stress (2.6 [1.9-3.4]), and psychological distress (1.6 [1.4-1.8]), and were nearly twice as likely to reach the CDC case definition (2.5 [2.2-2.8]). In the Gulf War, Bosnia, and Era cohorts, respectively, 61.9%, 36.8%, and 36.4% met the CDC criteria, which fell to

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25.3%, 11.8%, and 12.2% for severe symptoms. Potentially harmful exposures were reported most frequently by the Gulf War cohort. All exposures showed associations with all of the outcome measures in the three cohorts. Exposures specific to the Gulf were associated with all outcomes. Vaccination against biological warfare and multiple routine vaccinations were associated with the CDC multisymptom syndrome in the Gulf War cohort. INTERPRETATION: Service in the Gulf War was associated with various health problems over and above those associated with deployment to an unfamiliar hostile environment. Since associations of ill health with adverse events and exposures were found in all cohorts, however, they may not be unique and causally implicated in Gulf-War-related illness. A specific mechanism may link vaccination against biological warfare agents and later ill health, but the risks of illness must be considered against the protection of servicemen.

5. Golomb, B.A., A Review of the Scientific Literature as it Pertains to Gulf War Illnesses, Vol 2: Pyridostigmine Bromide. 1999, Santa Monica, CA: RAND. 385.

6. Cecchine, G., et al., A Review of the Scientific Literature as it Pertains to Gulf War Illnesses, Vol 8: Pesticides. 2000, Santa Monica, CA: RAND. 182.

7. Li, L., et al., Reactive oxygen species mediate pyridostigmine-induced neuronal apoptosis: involvement of muscarinic and NMDA receptors. Toxicol Appl Pharmacol, 2001. 177(1): p. 17-25. Pyridostigmine bromide (PB) is a reversible cholinesterase inhibitor used for treatment of myasthenia gravis and for prophylactic protection against organophosphate nerve agent. We previously showed PB can induce apoptotic death in rat brain following systemic treatment. To study mechanisms by which PB induces brain cell death, cultured rat cerebellar granule cells were used. Cytotoxicity was determined after exposure to PB (10-1000 microM) for 24 h; a high concentration of PB (>500 microM) significantly increased lactate dehydrogenase release, which was reduced by pretreatment with the antioxidant, N-t-butyl-alpha-phenyl-nitrone (PBN). Apoptosis, as determined by TUNEL staining, was concentration dependent (10-250 microM) after a 24-h exposure and cytotoxicity was confirmed by gel electrophoresis of DNA, release of cytochrome c from mitochondria, elevation of caspase activity, and electron microscopy. The oxidant-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate was used to detect reactive oxidative species (ROS) generation. Pretreatment with PBN, superoxide dismutase, catalase, or the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) blocked PB-induced ROS generation and apoptotic cell death. Pretreatment with atropine or MK-801 blocked ROS generation and the subsequent neurotoxicity, showing that both muscarinic and NMDA receptors mediate the response. DNA extracted from PB-treated cells revealed oligonucleosomal fragmentation on gel electrophoresis and antioxidants attenuated the DNA fragmentation, providing further evidence for a link of ROS generation and apoptosis. These results indicate that muscarinic receptor-mediated ROS generation is an initiating factor in PB-induced apoptotic cell death and activation of the NMDA glutamate receptor is directly linked to the response.

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8. Li, L., et al., Muscarinic receptor-mediated pyridostigmine-induced neuronal apoptosis. Neurotoxicology, 2000. 21(4): p. 541-552. Abstract: Pyridostigmine is a reversible cholinesterase (ChE) inhibitor that is associate with neurologic dysfunction involving both central and peripheral nervous systems. To determine the neurotoxic potential of pyridostigmine, rats were sacrificed at intervals after drug administration (0.5-1.85 mg/kg, i.p., twice daily for 4 days) and brains examined histologically. ChE inhibition was used as a biomarker of pyridostigmine activity. Using the in situ terminal deoxynucleotidyl transferase nick-end labeling of DNA fragments (TUNEL) method and electron microscopy, apoptotic brain cell death was noted in cerebral cortex over a dose range of 0.5-1.85 mg/kg and at the higher dose (1.85 mg/kg), apoptosis was also noted in striatum and hippocampus. These responses were blocked by pretreatment with atropine. Rat cortical cells in culture also underwent apoptosis when exposed to pyridostigmine (250 microM for 24 hr), indicating that the pyridostigmine can initiate apoptosis, independent of peripheral mechanisms. Pretreatment of cells with atropine (10 microM) inhibited pyridostigmine-induced apoptosis, confirming the response was mediated by muscarinic receptors. Short term treatment of rats with pyridostigmine (1.85 mg/kg twice daily for 4 days) induced a prolonged apoptotic response, which was evident in rat cortex up to 30 days after the last dose. Active apoptosis persisted, despite recovery of serum ChE activity. These in vivo and in vitro observations indicate that pyridostigmine can initiate a prolonged neurodegeneration.

9. Pascuzzo, G.J., et al., The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex I. Agonist, desensitizing, and binding properties. Mol Pharmacol, 1984. 25: p. 92-101.

10. Tandon, P., et al., Fenthion produces a persistent decrease in muscarinic receptor function in the adult rat retina. Toxicol Appl Pharmacol, 1994. 125: p. 271-280.

11. Tandon, P., et al., Dose-response study of the tissue-specific effects of fenthion on receptor function in rat CNS. Toxicologist, 1994. 14: p. 257.

12. Sherby, S.M., et al., Comparison of actions of carbamate anticholinesterses on the nicotinic acetylcholine receptor. Molecular Pharmacology, 1985. 27: p. 343-348.

13. Gupta, R.C., G.T. Patterson, and W.-D. Dettbarn, Mechanisms of toxicity and tolerance to diisopropylphosphorofluoridate at the neuromuscular junction of the rat. Toxicology and Applied Pharmacology, 1986. 84: p. 541-550.

14. Eskenazi, B., A. Bradman, and R. Castorina, Exposures of children to organophosphate pesticides and their potential adverse health effects. Environ Health Perspect, 1999. 107 Suppl 3: p. 409-19. Recent studies show that young children can be exposed to pesticides during normal oral exploration of their environment and their level of dermal contact with floors and other surfaces. Children living in agricultural areas may be exposed to higher pesticide levels than other children because of pesticides tracked into their homes by household members, by pesticide drift, by breast milk from their farmworker mother, or by playing in nearby fields. Nevertheless, few studies have

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assessed the extent of children's pesticide exposure, and no studies have examined whether there are adverse health effects of chronic exposure. There is substantial toxicologic evidence that repeated low-level exposure to organophosphate (OP) pesticides may affect neurodevelopment and growth in developing animals. For example, animal studies have reported neurobehavorial effects such as impairment on maze performance, locomotion, and balance in neonates exposed (italic)in utero(/italic) and during early postnatal life. Possible mechanisms for these effects include inhibition of brain acetylcholinesterase, downregulation of muscarinic receptors, decreased brain DNA synthesis, and reduced brain weight in offspring. Research findings also suggest that it is biologically plausible that OP exposure may be related to respiratory disease in children through dysregulation of the autonomic nervous system. The University of California Berkeley Center for Children's Environmental Health Research is working to build a community-university partnership to study the environmental health of rural children. This Center for the Health Assessment of Mothers and Children of Salinas, or CHAMACOS in Monterey County, California, will assess (italic)in utero(/italic) and postnatal OP pesticide exposure and the relationship of exposure to neurodevelopment, growth, and symptoms of respiratory illness in children. The ultimate goal of the center is to translate research findings into a reduction of children's exposure to pesticides and other environmental agents, and thereby reduce the incidence of environmentally related disease.

15. Costa, L.G., B.W. Schwab, and S.D. Murphy, Tolerance to anticholinesterase compounds in mammals. Toxicology, 1982. 25: p. 79-97.

16. Buccafusco, J.J., Chronic organophosphorus exposure and cognition. 1999 Annual Report to Congress on Gulf War Veterans' Illnesses, 2000: p. 101.

17. Lev-Lehman, E., et al., Synaptogenesis and myopathy under acetylcholinesterase overexpression. Journal of Molecular Neuroscience, 2000. 14(1-2): p. 93-105. Abstract: Environmental, congenital, and acquired immunological insults perturbing neuromuscular junction (NMJ) activity may induce a variety of debilitating neuromuscular pathologies. However, the molecular elements linking NMJ dysfunction to long-term myopathies are unknown. Here, we report dramatically elevated levels of mRNA encoding c-Fos and the "readthrough" (R) variant of acetylcholinesterase (AChE) in muscles of transgenic mice overexpressing synaptic (S) AChE in motoneurons and in control mice treated with the irreversible cholinesterase inhibitor diisopropylfluorophosphonate (DFP). Tongue muscles from DFP-treated and AChE-S transgenic mice displayed exaggerated neurite branching and disorganized, wasting fibers. Moreover, diaphragm muscles from both transgenic and DFP-treated mice exhibited NMJ proliferation. 2'-O-methyl-protected antisense oligonucleotides targeted to AChE mRNA suppressed feedback upregulation of AChE and ameliorated DFP-induced NMJ proliferation. Our findings demonstrate common transcriptional responses to cholinergic NMJ stress of diverse origin, and implicate deregulated AChE expression in excessive neurite outgrowth, uncontrolled synaptogenesis, and myopathology.

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18. Robinson, D. and R. McGee, Agonist-induced regulation of neuronal nicotinic acetylcholine receptor of PC12 cells. Molecular Pharmacology, 1985. 27: p. 409-417.

19. Sherby, S.M., et al., Comparison of actions of carbamate anticholinesterses on the nicotinic acetylcholine receptor. Molecular Pharmacology, 1985. 27: p. 343-348.

20. Shaw, K.-P., et al., The reversible cholinesterase inhibitor physostgmine has channelblocking and agonist efects on the acetylcholine receptor-ion channel complex. Molecular Pharmacology, 1985. 28: p. 527-238.

21. Anderson, R.J., et al., Decreased tetanic contracture of rat skeletal muscle induced by pyridostigmine. J Toxicol Environ Health, 1986. 18: p. 221-230.

22. Haley, R.W., S. Billecke, and B.N. La Du, Association of low PON1 type Q (type A) arylesterase activity with neurologic symptom complexes in Gulf War veterans. Toxicol Appl Pharmacol, 1999. 157(3): p. 227-33. Previously Haley et al. described six possible syndromes identified by factor analysis of symptoms in Gulf War veterans and demonstrated that veterans with these symptom complexes were more neurologically impaired than age-sex-education-matched well controls. They also uncovered strong associations (relative risks 4-8) suggesting that these symptom complexes were related to wartime exposure to combinations of organophosphate pesticides, chemical nerve agents, high concentration DEET insect repellant, and symptoms of advanced acute toxicity after taking pyridostigmine. Here we have shown that compared to controls, ill veterans with the neurologic symptom complexes were more likely to have the R allele (heterozygous QR or homozygous R) than to be homozygous Q for the paraoxonase/arylesterase 1 (PON1) gene. Moreover, low activity of the PON1 type Q (Gln192, formerly designated type A) arylesterase allozyme distinguished ill veterans from controls better than just the PON1 genotype or the activity levels of the type R (Arg192, formerly designated type B) arylesterase allozyme, total arylesterase, total paraoxonase, or butyrylcholinesterase. A history of advanced acute toxicity after taking pyridostigmine was also correlated with low PON1 type Q arylesterase activity. Type Q is the allozyme of paraoxonase/arylesterase that most efficiently hydrolyzes several organophosphates including sarin, soman, and diazinon. These findings further support the proposal that neurologic symptoms in some Gulf War veterans were caused by environmental chemical exposures.

23. La Du, B.N., et al., Serum paraoxonase (PON1) isozymes: the quantitative analysis of isozymes affecting individual sensitivity to environmental chemicals. Drug Metab Dispos, 2001. 29(4 Pt 2): p. 566-9. In a recent study on Gulf War veterans who developed delayed neurotoxicity symptoms, we found their levels of serum paraoxonase (PON1) isozyme type Q to be significantly lower than in the control, unaffected veteran group. These results were obtained in 25 ill veterans and 20 well control subjects, of which 10 were deployed and 10 were nondeployed battalion members who remained in the United States during the Gulf War. The blood samples were also assayed for serum butyrylcholinesterase in our laboratory, and more recently in Dr. C. Broomfield's laboratory for somanase and sarinase activities. The cholinesterase activities showed no

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significant correlation with the PON1 isozyme levels or the severity of the clinical symptoms, but the somanase and sarinase levels ran parallel to the PON1 type Q isozyme concentrations. Although there is no direct evidence that these Gulf War veterans were directly exposed to or encountered either of these nerve gases, they may have been exposed to some environmental or chemical toxin with a similar preference for hydrolysis by the PON1 type Q isozyme. The number of subjects is relatively small, but the results should encourage other investigators to examine both the individual phenotypes and the levels of PON1 isozymes in other groups exhibiting neurological symptoms.

24. Mackness, B., P.N. Durrington, and M.I. Mackness, Low paraoxonase in Persian Gulf War Veterans self-reporting Gulf War Syndrome. Biochem Biophys Res Commun, 2000. 276(2): p. 729-33. Exposure to organophosphate (OP's) insecticides and nerve gases during the Persian Gulf War has been implicated in the development of Gulf War Syndrome. Paraoxonase (PON1) present in human serum detoxifies OP's. We determined the levels of PON1 in the serum of Gulf War Veterans and compared these to those found in a control population. One hundred fifty-two Gulf War Veterans from the UK who self-reported the presence of Gulf War Syndrome via a questionnaire and 152 age and gender matched controls were studied. PON1 activity, concentration, and genotype were determined. In the Gulf War Veterans, paraoxon hydrolysis was less than 50% of that found in the controls (100.3 (14.8-233.8) vs 214.6 (50.3-516.2) nmol/min/ml, P < 0.001). This low activity was independent of the effect of PON1 genotype. The serum PON1 concentration was also lower in the Gulf War Veterans (75.7 (18.1-351.3) vs 88.2 (34.5-527.4) microg/ml, P < 0.00025), which was again independent of PON1 genotype. There was no difference in the rate of diazoxon hydrolysis between the groups (10. 2 +/- 4.1 micromol/min/ml vs 9.86 +/- 4.4, P = NS). A decreased capacity to detoxify OP insecticides resulting from low serum PON1 activity may have contributed to the development of Gulf War Syndrome.

25. Cherry, N., et al., Paraoxonase (PON1) polymorphisms in farmers attributing ill health to sheep dip. Lancet, 2002. 359(9308): p. 763-4. Human serum paraoxonase (PON1) hydrolyses diazinonoxon, the active metabolite of diazinon, which is an organophosphate used in sheep dip. In a case-referent study, 175 farmers with ill health that they attributed to sheep dip nominated 234 referent farmers who also dipped sheep and whom they believed to be in good health. We calculated odds ratios for polymorphisms in PON1 at positions 192 and 55, and for PON1 activity with diazinonoxon as substrate. Cases were more likely than referents to have at least one R allele at position 192 (glutamine to arginine aminoacid substitution; odds ratio 1center dot93, 95% CI 1center dot24--3center dot01), both alleles of type LL (1center dot70, 1center dot07--2center dot68) at position 55, and to have diazoxonase activity below normal median (1center dot77, 1center dot18--2center dot67). Our results support the hypothesis that organophosphates contribute to the reported ill health of people who dip sheep.

26. Cecchine, G., et al., A Review of the Scientific Literature as it Pertains to Gulf War Illnesses, Vol 8: Pesticides. 2000, Washington, DC: RAND. 182.

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27. Cherry, N., et al., Health and exposures of United Kingdom Gulf war veterans. Part II: The relation of health to exposure. Occup Environ Med, 2001. 58(5): p. 299-306. OBJECTIVES: To investigate whether, in personnel who served with the United Kingdom forces in the Gulf war, self reported exposures were related to symptoms in a way that was consistent, specific, and credible. METHODS: Responses to symptom and exposure questionnaires, completed 7 or more years after the war, were collected from 7971 subjects deployed in the Gulf, from two exposed cohorts, in a study with an overall response rate of 85.5%. Exposures were considered in three groups, those outside the control of the subjects, the use of prophylaxis, and indicators of susceptibility. Health indices derived from symptom questionnaires were related to reports of 14 exposures in these three groups in a series of multiple regression analyses to allow for confounding. The relation of exposure to complaints of widespread pain and to symptoms suggesting peripheral neuropathy were examined by logistic regression. RESULTS: Consistent but weak correlations between exposures and with health effects were found in independent analyses of the two (main and validation) cohorts. Three exposures outside the control of the subject, the number of inoculations, the number of days handling pesticides, and the days exposed to smoke from oil fires, were consistently and independently related to severity. The number of inoculations was also associated with higher scores on a factor weighted on symptoms associated with skin and musculoskeletal complaints. The number of days handling pesticides related particularly to scores on a neurological factor and to symptoms consistent with toxic neuropathy. CONCLUSION: The relations between exposures and ill health were generally weak. Consistent, specific, and credible relations, warranting further investigation, were found between health indices and two exposures, the reported number of inoculations and days handling pesticides.

28. Gray, G.C., et al., Self-reported symptoms and medical conditions among 11,868 Gulf War-era veterans: the Seabee Health Study. Am J Epidemiol, 2002. 155(11): p. 1033-44. US Navy Seabees have been among the most symptomatic Gulf War veterans. Beginning in May 1997, the authors mailed Gulf War-era Seabees a health survey in serial mailings. As of July 1, 1999, 68.6% of 17,559 Seabees contacted had returned the questionnaire. Compared with other Seabees, Gulf War Seabees reported poorer general health, a higher prevalence of all 33 medical problems assessed, more cognition difficulties, and a higher prevalence of four physician-diagnosed multisymptom conditions: chronic fatigue syndrome, posttraumatic stress disorder, multiple chemical sensitivity, and irritable bowel syndrome. Because the four multisymptom conditions were highly associated with one another, the authors aggregated them into a working case definition of Gulf War illness. Among the 3,831 (22% cases) Gulf War Seabee participants, multivariable modeling revealed that female, Reserve, and enlisted personnel and participants belonging to either of two particular Seabee units were most likely to meet the case definition. Twelve of 34 self-reported Gulf War exposures were mildly associated with meeting the definition of Gulf War illness, with exposure to fumes from munitions having the highest odds ratio (odds ratio = 1.9, 95% confidence interval: 1.5, 2.4). While these data do not implicate a specific etiologic exposure, they demonstrate a strong association and a high prevalence of self-reported multisymptom conditions in a large group of symptomatic Gulf War veterans.

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29. Haley, R.W. and T.L. Kurt, Self-reported exposure to neurotoxic chemical combinations in the Gulf War. A cross-sectional epidemiologic study. Jama, 1997. 277(3): p. 231-7. OBJECTIVE: To identify risk factors of factor analysis-derived Gulf War-related syndromes. DESIGN: A cross-sectional survey. PARTICIPANTS: A total of 249 Gulf War veterans from the Twenty-fourth Reserve Naval Mobile Construction Battalion. DATA COLLECTION: Participants completed standardized booklets measuring self-reported wartime exposures and present symptoms. MAIN OUTCOME MEASURES: Associations of factor analysis-derived syndromes with risk factors for chemical interactions that inhibit butyrylcholinesterase and neuropathy target esterase. RESULTS: Risk of syndrome 1 ("impaired cognition") was greater in veterans who reported wearing flea collars during the war (5 of 20, 25%) than in those who never wore them (7 of 229, 3%; relative risk [RR], 8.7; 95% confidence interval [CI], 3.0-24.7; P<.001). Risk of syndrome 2 ("confusion-ataxia") increased with a scale of advanced adverse effects from pyridostigmine bromide (chi2 for trend, P<.001), was greater among veterans who believed they had been involved in chemical weapons exposure (18 of 108, 17%) than in those who did not (3 of 141, 2%; RR, 7.8; 95% CI, 2.3-25.9; P<.001), and was increased in veterans who had been in a sector of far northeastern Saudi Arabia on the fourth day of the air war (6 of 21, 29%) than in those who had not been (15 of 228, 7%; RR, 4.3; 95% CI, 1.9-10.0; P=.004). Effects of perceived chemical weapons exposure and advanced adverse effects from pyridostigmine were synergistic (Rothman S, 5.3; 95% CI, 1.04-26.7). Risk of syndrome 3 ("arthro-myo-neuropathy") increased with an index of frequency and amount of government-issued insect repellent containing 75% DEET (N,N-diethyl-m-toluamide) in ethanol applied during the war (chi2 for trend, P<.001) and with advanced adverse effects from pyridostigmine (chi2 for trend, P<.001). CONCLUSION: Some Gulf War veterans may have delayed, chronic neurotoxic syndromes from wartime exposure to combinations of chemicals that inhibit butyrylcholinesterase and neuropathy target esterase.

30. Kang, H.K., et al., Illnesses among United States veterans of the Gulf War: a population-based survey of 30,000 veterans. J Occup Environ Med, 2000. 42(5): p. 491-501. Despite numerous studies on veterans of the 1990 to 1991 Gulf War, the fundamental questions of how healthy they are and how their health compares with that of their military peers who were not deployed to the Gulf have not been fully answered. We conducted a health survey in which the health outcomes of a population-based sample of 15,000 Gulf veterans representing various military branches and unit components (regular, reserve, National Guard) were compared with those of 15,000 non-Gulf veterans who were randomly sampled to mirror the number in the same military strata in the Gulf veteran group. In comparison with their peers, Gulf veterans had a higher prevalence of functional impairment, health care utilization, symptoms, and medical conditions and a higher rate of low general health perceptions. A longitudinal follow-up of the health of these veterans will be needed to detect changes in health status and to detect diseases with a long latency period.

31. Kang, H., et al., Evidence for a deployment-related Gulf War syndrome by factor analysis, . 2002, Arch Environ Health. p. 58.

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32. Kroenke, K., P. Koslowe, and M. Roy, Symptoms in 18,495 Persian Gulf War veterans. Latency of onset and lack of association with self-reported exposures. Journal of Occupational and Environmental Medicine, 1998. 40(6): p. 520-8. Toxic or environmental exposures have been suggested as a possible cause of symptoms reported by Gulf War veterans. To further explore this hypothesis, we analyzed findings in 18,495 military personnel evaluated in the Department of Defense Comprehensive Clinical Evaluation Program. The program was established in 1994 to evaluate Persian Gulf veterans eligible for Department of Defense medical care who had health concerns after service in the Persian Gulf during Operation Desert Shield/Desert Storm. The evaluation included a structured clinical assessment, a physician-administered symptom checklist, and a patient questionnaire addressing self-reported exposures, combat experiences, and work loss. Among 18,495 patients examined, the most common symptoms were joint pain, fatigue, headache, memory or concentration difficulties, sleep disturbances, and rash. Symptom onset was often delayed, with two-thirds of symptoms not developing until after individuals returned from the Gulf War and 40% of symptoms having a latency period exceeding one year. There was no association between individual symptoms and patient demographics, specific self-reported exposures, or types of combat experience. Increased symptom counts were associated with work loss, the number of self-reported exposures, the number of types of combat experience, and certain ICD-9 diagnostic categories, particularly psychological disorders. Prolonged latency of symptom onset and the lack of association with any self-reported exposures makes illness related to toxic exposure less likely.

33. Proctor, S.P., et al., Health-related quality of life in Persian Gulf War Veterans. Mil Med, 2001. 166(6): p. 510-9. OBJECTIVE: The objective of this investigation is to describe the health-related quality of life of Persian Gulf War (GW) veterans and to examine the effects of current chronic medical conditions and psychiatric status on physical functioning. METHODS: To measure health-related quality of life, the Medical Outcomes Short Form Survey (SF36) was administered approximately 4 years after the GW to a stratified, random sample of New England-area GW-deployed veterans and a group of military personnel deployed to Germany during the GW. The SF36 scores for the GW-deployed study population (N = 141) were compared with those for the Germany-deployed group (N = 46) and with published U.S. population norms. Multiple linear regression analyses were performed to identify risk factors associated with lower physical health functioning in the GW-deployed study group. RESULTS: Functional health status was significantly lower in the GW-deployed group compared with the Germany-deployed group for each of the SF36 subscales and the two summary scores (Physical Component Summary [PCS] and Mental Component Summary). Compared with the general U.S. population, the GW-deployed group median was between the 25th and 50th percentile for the Physical Functioning subscale and the PCS score. Within the GW-deployed group, lower education, psychological symptomatology, and a higher number of chronic self-reported medical conditions were significant predictors of the PCS score. CONCLUSION: GW-deployed veterans report lower functional health status compared with a group of Germany-deployed veterans and published general U.S. population norms. Within the group of GW-deployed veterans, several current medical

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and psychological conditions predictive of lower physical functioning levels were identified.

34. Reid, S., et al., Multiple chemical sensitivity and chronic fatigue syndrome in British Gulf War veterans. Am J Epidemiol, 2001. 153(6): p. 604-9. The objective of this study was to measure the prevalence of multiple chemical sensitivity (MCS) and chronic fatigue syndrome (CFS) in British Gulf War veterans and to investigate their association with reported exposures and psychologic morbidity. In 1997--1998, the authors undertook a cross-sectional survey of three cohorts of British military personnel comprising Gulf veterans (n = 3,531), those who had served in Bosnia (n = 2,050), and those serving during the Gulf War but not deployed there (Era cohort, n = 2,614). MCS and CFS were defined according to operational criteria. The prevalence of MCS in the Gulf, Bosnia, and Era cohorts was 1.3%, 0.3%, and 0.2%, respectively. For CFS, the prevalence was 2.1% (Gulf cohort), 0.7% (Bosnia cohort), and 1.8% (Era cohort). In Gulf veterans, MCS was strongly associated with exposure to pesticides (adjusted odds ratio = 12.3, 95% confidence interval: 5.1, 30.0). Both syndromes were associated with high levels of psychologic morbidity. These findings suggest that CFS and MCS account for some of the medically unexplained illnesses reported by veterans after deployment to the Gulf. MCS was particularly associated with Gulf deployment and self-reported exposure to pesticides, findings that merit further exploration given the controversial status of this diagnosis and the potential for recall bias in a questionnaire survey.

35. Schumm, W.R., et al., Pyridostigmine bromide and the long-term subjective health status of a sample of female reserve component Gulf War veterans: a brief report. Psychol Rep, 2001. 88(1): p. 306-8. The role of pyridostigmine bromide (PB) pills in explaining the long-term subjective health status of a sample of over 100 female Reserve Component Gulf War veterans was examined through regression analysis. Results fell just short of significance (p < .06) for the prediction of subjective health approximately six years after the war and were clearly not significant for the prediction of subjective health at previous times. Results parallel Golomb's 1999 RAND report, which found suggestive but not conclusive evidence for the possible adverse effects of Gulf War veterans' consumption of pyridostigmine bromide pills. Our data suggest that use of more than 10 pills may have been especially risky with respect to long-term subjective health.

36. Schumm, W.R., et al., Pyridostigmine bromide and the long-term subjective health status of a sample of over 700 male Reserve Component Gulf War era veterans. Psychol Rep, 2002. 90(3 Pt 1): p. 707-21. Data from a 1996-1997 survey of approximately 700 Reserve Component male veterans indicate that the consumption of pyridostigmine bromide pills, used as a pretreatment for potential exposure to the nerve agent Soman, was a significant predictor of declines in reported subjective health status after the war, even after controlling for a number of other possible factors. Reported reactions to vaccines and other medications also predicted declines in subjective health. While higher military rank generally predicted better health during and after the war, educational attainment, minority status, number of days in theater, and age generally did not predict changes in subjective health. Although servicemembers were directed to take three pills a day, veterans reported a range of compliance--less than a fourth (24%)

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followed the medical instructions compared to 61% who took fewer than three pills daily and 6% who took six or more pills a day. Implications for use of pyridostigmine bromide are discussed.

37. White, R.F., et al., Neuropsychological function in Gulf War veterans: relationships to self-reported toxicant exposures. Am J Ind Med, 2001. 40(1): p. 42-54. BACKGROUND: The present study was aimed at (1) exploring evidence of central nervous system (CNS) dysfunction among Gulf War (GW) veterans on neuropsychological tests and (2) examining whether performance on neuropsychological tests was related to specific neurotoxicant exposures experienced in the Gulf. METHODS: The GW-deployed groups were selected using stratified random sampling methods from two distinct cohorts of GW veterans. A comparison group that had been called up for GW service but deployed to Germany rather than the Gulf also was examined. Neuropsychological function was assessed using a pre-determined battery chosen to include tests known to be highly sensitive to the behavioral effects of the neurotoxicants thought to have been present in the Gulf. RESULTS: Self-reported exposures were related to neuropsychological test performance controlling for post-traumatic stress disorder, major depression, and other known covariates of neuropsychological test performance. Results showed that GW-deployed veterans performed more poorly than the Germany-deployed veterans on several specific neuropsychological tests, but after adjustment for multiple comparisons, only the differences in mood complaints remained significant. Within the GW-deployed group, self-reported exposure to chemical warfare agents was associated with poorer performance on cognitive tests involving specific functional domains. CONCLUSIONS: Results provide evidence that there are subtle differences in CNS function among GW-deployed veterans who report chemical warfare agent exposure while in the GW theater.

38. McCauley, L.A., et al., Symptoms of Gulf War veterans possibly exposed to organophosphate chemical warfare agents at Khamisiyah, Iraq. Int J Occup Environ Health, 2001. 7(2): p. 79-89. During the 1991 Gulf War, some Allied troops were potentially exposed to sarin/cyclosarin as the result of the destruction of Iraqi munitions at Khamisiyah. To evaluate the prevalence of past and current symptoms known to be associated with exposure to these chemical warfare agents, the authors conducted a computer-assisted telephone survey of 2,918 U.S. Gulf War veterans. Veterans who had participated in or witnessed the demolition in 1991 were more likely to report historical or extant symptoms than were veterans from other military units. These results should be viewed cautiously because they are based on symptoms recalled nine years after the event without precise characterization of exposure. Nonetheless, the findings suggest that symptoms consistent with low-level sarin exposure may have initially occurred, and health effects may have persisted in the veterans who were nearest to the demolition activity. Further research is warranted.

39. McCauley, L., et al., Illness experience of Gulf War veterans possibly exposed to chemical warfare agents. Am J Prev Med, 2002. 23(3): p. 200. BACKGROUND: During the 1991 Gulf War, some Allied troops were potentially exposed to chemical warfare agents as the result of the detonation of Iraqi munitions at Khamisiyah.METHODS: In

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1999, we conducted a computer-assisted telephone survey of 2918 Gulf War veterans from Oregon, Washington, California, North Carolina, and Georgia to evaluate the prevalence of self-reported medical diagnoses and hospitalizations among this potentially exposed population and among comparison groups of veterans deployed and nondeployed to the Southwest Asia theater of operations.RESULTS: Troops reported to be within 50 kilometers of the Khamisiyah site did not differ from other deployed troops on reports of any medical conditions or hospitalizations in the 9 years following the Gulf War. Hospitalization rates among deployed and nondeployed troops did not differ. Deployed troops were significantly more likely to report diagnoses of high blood pressure (odds ratio [OR]=1.7); heart disease (OR=2.5); slipped disk or pinched nerve (OR=1.5); post-traumatic stress disorder (OR=14.9); hospitalization for depression (OR=5.1); and periodontal disease (OR=1.8) when compared to nondeployed troops. There was a trend for deployed veterans to report more diagnoses of any cancer (OR=3.0).CONCLUSIONS: These findings do not provide evidence of any long-term health effect associated with exposure to the detonation of chemical warfare agents, but support findings from other investigations of increased morbidity among deployed troops. The prevalence of cancer among this population of deployed troops merits ongoing attention.

40. Gray, G., et al., The postwar hospitalization experience of US veterans of the Persian Gulf War. New Engl J Med, 1996. 335(20): p. 1505-1513.

41. Gray, G.C., et al., Are Gulf War veterans suffering war-related illnesses? Federal and civilian hospitalizations examined, June 1991 to December 1994. Am J Epidemiol, 2000. 151(1): p. 63-71. A previous epidemiologic study demonstrated no unexplained increase in risk for postwar hospitalization among Gulf War veterans who had remained on active duty. The authors sought to expand this study to include Reserve and separated military personnel. They examined hospitalization data from the Department of Defense, the Department of Veterans Affairs (VA), and the California Office of Statewide Health Planning and Development hospital systems for the years 1991-1994. Since denominator data were not available, the authors compared the proportional morbidity ratios (PMRs) of hospitalization discharge diagnoses (both large categories and specific diagnoses) between Gulf War veterans and other veterans of the same era. There were no indications that Gulf War veterans were suffering increased PMRs for infectious diseases; neoplasms; endocrine diseases; blood diseases; skin conditions; or diseases of the nervous system, circulatory system, or musculoskeletal system. However, these veterans did experience proportionally more hospitalizations for various specific diagnoses, namely, fractures and bone and soft-tissue injuries (Department of Defense and California Office of Statewide Health Planning and Development), various diseases of the respiratory (including asthma) and digestive systems (VA), and diverse symptom diagnoses (VA). While these findings may be influenced by chance or by a number of potential confounders, including health registry participation, they merit further examination using other study designs.

42. Kaiser, K.S., Pyridostigmine bromide intake during the Persian Gulf War is not associated with postwar handgrip strength. Mil Med, 2000. 165(3): p. 165-8. Many

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Persian Gulf War veterans took pyridostigmine bromide (PB) during the Persian Gulf War. Previous research suggests that PB intake and insecticide exposure may reduce muscular strength. During 1994 and 1995, we examined the relationships between self-reported PB intake, self-reported exposures, and handgrip strength among 527 Gulf War veterans (GWVs) and 969 nondeployed veterans of that era (NDVs). We found that 25.4% and 6.7% of the GWVs and NDVs, respectively, reported generalized musucle weakness (for 1 month or longer) since the Gulf War (July 1990). Many veterans also reported exposure to insecticide during the war. Dominant handgrip strength was measured three times with a hand-held dynamometer in subjects standing with the elbow bent at a right angle. Multiple linear regression revealed that handgrip strength was negatively associated with age (p = 0.001) and female gender (p < 0.001). Handgrip strength was also found to be positively associated with height (p < 0.001), but it was not associated with PB intake (p = 0.558). Exposure to insecticides had no major effect on handgrip strength. These data suggest no association between PB intake and postwar handgrip strength.

43. Golomb, B.A., Chapter 14. Chronic Effect. In: A Review of the Scientific Literature as it Pertains to Gulf War Illnesses, Vol 2: Pyridostigmine Bromide. 1999, Santa Monica, CA: RAND. 385.

44. Lotti, M., Low-level exposures to organophosphorus esters and peripheral nerve function. Muscle Nerve, 2002. 25(4): p. 492-504. This review evaluates the epidemiological and clinical evidence linking low-level and prolonged exposures to organophosphorus esters, used as insecticides or nerve agents, to peripheral nerve dysfunction. The clinical effects of large doses of these chemicals-including the cholinergic syndrome, the intermediate syndrome, and the delayed polyneuropathy-are all well established and are summarized. Based on these clinical observations and experimental studies, dose-effect relationships indicate that peripheral neuropathy always develops after cholinergic toxicity. However, several studies have suggested that this relationship may be different after low-level prolonged exposures, as, for instance, those experienced by Gulf War veterans and British sheep farmers, thereby leading to the development of peripheral neuropathy without preceding cholinergic toxicity. A critical assessment of these studies, involving subjects with either current or past exposures, indicates that changes in peripheral nerve function were mild, inconsistent, and unexplained and that most studies lack exposure data. Suggestions made about individual hypersusceptibility to delayed polyneuropathy lack support. It is concluded that there is no evidence of peripheral nerve dysfunction caused by low-level prolonged exposures to organophosphate insecticides or nerve agents.

45. Ray, D.E., Chronic effects of low level exposure to anticholinesterases. Toxicology Letters, 1998. 95(1001): p. 26.

46. Glass-Marmor, L. and R. Beitner, Effects of carbamylcholine and pyridostigmine on mitochondrial-bound hexokinase in skeletal muscle and heart. Biochem Mol Med, 1996. 57(1): p. 67-70. We show here that carbamylcholine (acetylcholine agonist) or pyridostigmine (acetylcholinesterase inhibitor), drugs which are widely used in medical

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treatments, exerted a rapid reduction in mitochondrial-bound hexokinase. This reduction was inversely proportional to the changes in glucose-6-phosphate levels in skeletal and heart muscle. Increased concentration of acetylcholine, occurring in various diseases or induced by acetylcholinesterase inhibitors, was reported to cause deterioration of mitochondrial function, resulting in heart and muscle damage. The present experiments suggest that the reduction in mitochondrial-bound hexokinase, which is closely linked to intramitochondrial oxidative metabolism, may play an important role in the mechanism which leads to tissue damage.

47. Francesconi, R., R. Hubbard, and M. Mager, Effects of pyridostigmine on ability of rats to work in the heat. J Appl Physiol, 1984. 56(4): p. 891-5. Adult, male rats (300-325 g) were treated with pyridostigmine bromide (n = 22) or saline (n = 22) to quantitate the effects of cholinesterase inhibition (64%) on the ability to work (9.14 m/min, level treadmill) in the heat (35 degrees C). Pyridostigmine-treated rats had a mean endurance of 23 min, whereas saline-treated animals ran for nearly 35 min (P less than 0.001). Rates of rectal and skin temperature increments were significantly higher (P less than 0.001) in pyridostigmine-treated rats as were water losses (P less than 0.001). Exercise in the heat to hyperthermic exhaustion effected anticipated increments in circulating urea nitrogen, creatinine, lactate dehydrogenase, and potassium levels, whereas pyridostigmine pretreatment had additive effects on lactate and creatine kinase concentrations. Additionally, pyridostigmine elicited a significant (P less than 0.01) hyperglycemia before exercise, an effect noted also with other organophosphate simulants. We concluded that pyridostigmine-induced cholinesterase inhibition had a variety of debilitating effects during work in the heat.

48. Babu, S.R., et al., Effect of physostigmine on plasma lactate and pyruvate in untrained/trained rats. Pharmacol Biochem Behav, 1992. 42(1): p. 67-73. Physostigmine (Phy) is metabolized to eseroline, a phenolic compound that appears to alter mitochondrial functions. The effect of Phy on recovery from exercise and on time course of plasma lactate and pyruvate levels following an acute bout of exercise (AE) was examined in untrained and trained (ET) rats. Phy alone elicited significantly higher plasma lactate and pyruvate levels than sedentary control. AE + Phy had a significantly higher plasma lactate and pyruvate levels compared to AE 2 min postadministration. From 5-30 min postexercise, lactate and pyruvate levels did not differ between these two acutely exercised groups. ET + Phy exhibited significantly lower levels of plasma lactate and pyruvate from 5-60 min postexercise compared to ET. The data show that the "additive" effect of Phy on postexercise plasma lactate and pyruvate levels can be attenuated by an enhanced fitness level in these rats.

49. Buckenmeyer, P.J., et al., Effect of concurrent exercise and physostigmine on lactate and pyruvate in plasma, muscle, and brain tissue of rats. Pharmacol Biochem Behav, 1994. 47(4): p. 779-88. The purpose of this investigation was to determine the effect of physostigmine (Phy) and/or concurrent exercise on lactate, pyruvate, and L/P ratio in plasma, skeletal muscle, and brain tissue in male Sprague-Dawley rats. The Phy-dosed (Phy-D) and Phy-dosed + concurrent acute exercise (Phy-D + CAE) groups elicited significantly higher L/P ratios in plasma compared to the acutely exercised (AE)

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group at 30 min postexercise. Physostigmine dosing, with or without exercise, resulted in significantly lower muscle pyruvate levels, from 30 to 50 min postdrug administration, in Phy-D and Phy-D + CAE groups compared to the AE group. In the brain, lactate values were significantly elevated in the acutely exercised groups at 5 min postexercise with or without Phy dosing. However, at 15 to 30 min postexercise, lactate values were significantly elevated in the Phy-D + CAE compared to the AE group. These data suggest that when Phy is administered prior to a 20-min moderately intensive exercise bout, there is an accumulation of lactate for a prolonged period of time in recovery.

50. De Pinieux, G., et al., Lipid-lowering drugs and mitochondrial function: effects of HMG-CoA reductase inhibitors on serum ubiquinone and blood lactate/pyruvate ratio. British Journal of Clinical Pharmacology, 1996. 42(3): p. 333-7. 1. Statins inhibit synthesis of mevalonate, a precursor of ubiquinone that is a central compound of the mitochondrial respiratory chain. The main adverse effect of statins is a toxic myopathy possibly related to mitochondrial dysfunction. 2. This study was designed to evaluate the effect of lipid-lowering drugs on ubiquinone (coenzyme Q10) serum level and on mitochondrial function assessed by blood lactate/pyruvate ratio. 3. Eighty hypercholesterolaemic patients (40 treated by statins, 20 treated by fibrates, and 20 untreated patients, all 80 having total cholesterol levels > 6.0 mmol l-1) and 20 healthy controls were included. Ubiquinone serum level and blood lactate/pyruvate ratio used as a test for mitochondrial dysfunction were evaluated in all subjects. 4. Lactate/pyruvate ratios were significantly higher in patients treated by statins than in untreated hypercholesterolaemic patients or in healthy controls (P < 0.05 and P < 0.001). The difference was not significant between fibratetreated patients and untreated patients. 5. Ubiquinone serum levels were lower in statin-treated patients (0.75 mg l-1 +/- 0.04) than in untreated hypercholesterolaemic patients (0.95 mg l-1 +/- 0.09; P < 0.05). 6. We conclude that statin therapy can be associated with high blood lactate/ pyruvate ratio suggestive of mitochondrial dysfunction. It is uncertain to what extent low serum levels of ubiquinone could explain the mitochondrial dysfunction.

51. Chariot, P., et al., Determination of the blood lactate:pyruvate ratio as a noninvasive test for the diagnosis of zidovudine myopathy. Arthritis Rheum, 1994. 37(4): p. 583-6. OBJECTIVE. To evaluate the use of the lactate: pyruvate ratio as a test for the detection of zidovudine myopathy. METHODS. Twenty consecutive human immunodeficiency virus-infected patients with muscle involvement and 20 without muscle involvement were studied prospectively. Blood lactate and pyruvate levels and serum creatine kinase levels were tested, muscle involvement was assessed both clinically and electrophysiologically, and muscle biopsy was performed in patients with myopathy. RESULTS. Nine patients had biopsy-proven zidovudine myopathy. All 9 had a high lactate:pyruvate ratio, with elevations on 2 of 2 determinations in 6 patients and on 1 of 2 in 3 patients. Two of 11 patients with other myopathies and 2 of 20 patients without myopathy had a high lactate:pyruvate ratio on 1 of 2 determinations. CONCLUSION. The lactate:pyruvate ratio, when determined repeatedly, is a sensitive test for detecting mitochondrial muscular toxicity of zidovudine.

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52. Barbiroli, B., et al., Coenzyme Q10 improves mitochondrial respiration in patients with mitochondrial cytopathies. An in vivo study on brain and skeletal muscle by phosphorous magnetic resonance spectroscopy. Cellular and Molecular Biology, 1997. 43(5): p. 741-9.

53. Boitier, E., et al., A case of mitochondrial encephalomyopathy associated with a muscle coenzyme Q10 deficiency. Journal of the Neurological Sciences, 1998. 156(1): p. 41-6.

54. Chen, R.S., C.C. Huang, and N.S. Chu, Coenzyme Q10 treatment in mitochondrial encephalomyopathies. Short-term double-blind, crossover study. European Neurology, 1997. 37(4): p. 212-8.

55. Sobreira, C., et al., Mitochondrial encephalomyopathy with coenzyme Q10 deficiency. Neurology, 1997. 48(5): p. 1238-43.

56. Fosslien, E., Mitochondrial medicine--molecular pathology of defective oxidative phosphorylation. Ann Clin Lab Sci, 2001. 31(1): p. 25-67. Different tissues display distinct sensitivities to defective mitochondrial oxidative phosphorylation (OXPHOS). Tissues highly dependent on oxygen such as the cardiac muscle, skeletal and smooth muscle, the central and peripheral nervous system, the kidney, and the insulin-producing pancreatic beta-cell are especially susceptible to defective OXPHOS. There is evidence that defective OXPHOS plays an important role in atherogenesis, in the pathogenesis of Alzheimer's disease, Parkinson's disease, diabetes, and aging. Defective OXPHOS may be caused by abnormal mitochondrial biosynthesis due to inherited or acquired mutations in the nuclear (n) or mitochondrial (mt) deoxyribonucleic acid (DNA). For instance, the presence of a mutation of the mtDNA in the pancreatic beta-cell impairs adenosine triphosphate (ATP) generation and insulin synthesis. The nuclear genome controls mitochondrial biosynthesis, but mtDNA has a much higher mutation rate than nDNA because it lacks histones and is exposed to the radical oxygen species (ROS) generated by the electron transport chain, and the mtDNA repair system is limited. Defective OXPHOS may be caused by insufficient fuel supply, by defective electron transport chain enzymes (Complexes I - IV), lack of the electron carrier coenzyme Q10, lack of oxygen due to ischemia or anemia, or excessive membrane leakage, resulting in insufficient mitochondrial inner membrane potential for ATP synthesis by the F0F1-ATPase. Human tissues can counteract OXPHOS defects by stimulating mitochondrial biosynthesis; however, above a certain threshold the lack of ATP causes cell death. Many agents affect OXPHOS. Several nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit or uncouple OXPHOS and induce the 'topical' phase of gastrointestinal ulcer formation. Uncoupled mitochondria reduce cell viability. The Helicobacter pylori induces uncoupling. The uncoupling that opens the membrane pores can activate apoptosis. Cholic acid in experimental atherogenic diets inhibits Complex IV, cocaine inhibits Complex I, the poliovirus inhibits Complex II, ceramide inhibits Complex III, azide, cyanide, chloroform, and methamphetamine inhibit Complex IV. Ethanol abuse and antiviral nucleoside analogue therapy inhibit mtDNA replication. By contrast, melatonin stimulates Complexes I and IV and Gingko biloba stimulates Complexes I and III. Oral

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Q10 supplementation is effective in treating cardiomyopathies and in restoring plasma levels reduced by the statin type of cholesterol-lowering drugs.

57. Alleva, R., et al., Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection. FEBS Lett, 2001. 503(1): p. 46-50. Generation of free radicals is often associated with the induction and progression of apoptosis. Therefore, antioxidants can prove anti-apoptotic, and can help to elucidate specific apoptotic pathways. Here we studied whether coenzyme Q, present in membranes in reduced (ubiquinol) or oxidised (ubiquinone) forms, can affect apoptosis induced by various stimuli. Exposure of Jurkat cells to alpha-tocopheryl succinate (alpha-TOS), hydrogen peroxide, anti-Fas IgM or TRAIL led to induction of apoptosis. Cell death due to the chemical agents was suppressed in cells enriched with the reduced form of coenzyme Q. However, coenzyme Q did not block cell death induced by the immunological agents. Ubiquinol-10 inhibited reactive oxygen species (ROS) generation in cells exposed to alpha-TOS, and a mitochondrially targeted coenzyme Q analogue also blocked apoptosis triggered by alpha-TOS or hydrogen peroxide. Therefore, it is plausible that ubiquinol-10 protects cells from chemically-induced apoptosis by acting as an antioxidant in mitochondria. Our results also indicate that generation of free radicals may not be a critical step in induction of apoptosis by immunological agents.

58. Di Giovanni, S., et al., Coenzyme Q10 reverses pathological phenotype and reduces apoptosis in familial CoQ10 deficiency. Neurology, 2001. 57(3): p. 515-8. Two brothers with myopathic coenzyme Q10 (CoQ10) deficiency responded dramatically to CoQ10 supplementation. Muscle biopsies before therapy showed ragged-red fibers, lipid storage, and complex I + III and II + III deficiency. Approximately 30% of myofibers had multiple features of apoptosis. After 8 months of treatment, excessive lipid storage resolved, CoQ10 level normalized, mitochondrial enzymes increased, and proportion of fibers with TUNEL-positive nuclei decreased to 10%. The authors conclude that muscle CoQ10 deficiency can be corrected by supplementation of CoQ10, which appears to stimulate mitochondrial proliferation and to prevent apoptosis.

59. Fernandez-Ayala, D.J., et al., Coenzyme Q protects cells against serum withdrawal-induced apoptosis by inhibition of ceramide release and caspase-3 activation. Antioxid Redox Signal, 2000. 2(2): p. 263-75. Coenzyme Q10 (CoQ10) is a component of the antioxidant machinery that protects cell membranes from oxidative damage and decreases apoptosis in leukemic cells cultured in serum-depleted media. Serum deprivation induced apoptosis in CEM-C7H2 (CEM) and to a lesser extent in CEM-9F3, a subline overexpressing Bcl-2. Addition of CoQ10 to serum-free media decreased apoptosis in both cell lines. Serum withdrawal induced an early increase of neutral-sphingomyelinase activity, release of ceramide, and activation of caspase-3 in both cell lines, but this effect was more pronounced in CEM cells. CoQ10 prevented activation of this cascade of events. Lipids extracted from serum-depleted cultures activated caspase-3 independently of the presence of mitochondria in cell-free in vitro assays. Activation of caspase-3 by lipid extracts or ceramide was prevented by okadaic acid, indicating the implication of a phosphatase in this process. Our results support the hypothesis that plasma membrane CoQ10 regulate the initiation phase of serum

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withdrawal-induced apoptosis by preventing oxidative damage and thus avoiding activation of downstream effectors as neutral-sphingomyelinase and subsequent ceramide release and caspase activation pathways.

60. Kagan, T., et al., Coenzyme Q10 can in some circumstances block apoptosis, and this effect is mediated through mitochondria. Ann N Y Acad Sci, 1999. 887: p. 31-47. The mitochondrial component coenzyme Q10 (CoQ10) has been used for many years as a dietary supplement intended to promote good health by trapping free radicals, thus preventing lipid peroxidation and DNA damage. We have tested its use as a generic anti-apoptotic compound and have found that its ability to protect against apoptosis varies depending on both cell type and mode of cell death induction. We have further established that this protection may be mediated by its effect on mitochondrial function and viability. We provide additional evidence that CoQ10's protective effect on mitochondrial membrane potential does not always result in altered mitochondrial enzyme activity and neither does it guarantee survival. These observations open the way for further investigations into the mechanisms involved in mitochondrial control of apoptosis.

61. Mosca, L., et al., Modulation of apoptosis and improved redox metabolism with the use of a new antioxidant formula. Biochem Pharmacol, 2002. 63(7): p. 1305-14. Oxidative stress is involved in the pathogenesis of a wide spectrum of diseases, implicating that strategies directed at counterbalancing oxidative processes could have a role in clinical medicine. There is also an evidence that oxidative stress acts as a major determinant of apoptotic cell death. Many studies have reported favourable effects of antioxidant formulas on several parameters of the oxidant-antioxidant balance, but none of them has focused whether antioxidant formulas could modulate apoptosis. We investigated in 20 healthy individuals the effect of supplementation with a formula containing alpha-tocopherol, alpha-lipoic acid, coenzyme Q(10), carnitines, and selenomethionine, on plasma oxidant status and peroxide levels, erythrocyte antioxidant enzymes, lymphocyte apoptosis, and generation of ROS at the mitochondrial level. Control subjects received only carnitines or an incomplete formula with alpha-tocopherol, alpha-lipoic acid, coenzyme Q(10), and selenomethionine. Supplementation with the complete formula resulted in a significant increase in the plasma antioxidant status that was mirrored by a decrease in blood peroxide levels and a reduced generation of ROS at the mitochondrial level. This was associated with a significant decrease in the frequency of peripheral blood lymphocytes, with either CD4 or CD8 phenotype, undergoing apoptosis. Less consistent results were found when either incomplete formula was used. Our study suggests that supplementation with antioxidant formulas can modulate the process of apoptosis under in vivo conditions. The clinical potential of this strategy in the treatment of diseases with an elevated commitment to apoptosis should be explored.

62. Ogasahara, S., et al., Muscle coenzyme Q deficiency in familial mitochondrial encephalomyopathy. Proc Natl Acad Sci U S A, 1989. 86(7): p. 2379-82. The electron transport system of muscle mitochondria was examined in a familial syndrome of lactacidemia, mitochondrial myopathy, and encephalopathy. The propositus, a 14-year-

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old female, and her 12-year-old sister had suffered from progressive muscle weakness, abnormal fatigability, and central nervous system dysfunction since early childhood. In the propositus, the state 3 respiratory rate of muscle mitochondria with NADH-linked substrates and with succinate was markedly reduced. The levels of cytochromes a + a3, b, and c + c1 were normal. The activities of complexes I, II, III, and IV of the electron transport chain were normal or increased. By contrast, the activities of complex I-III and of complex II-III, both of which need coenzyme Q10 (CoQ10), were abnormally low. On direct measurement, the mitochondrial CoQ10 content was 3.7% of the mean value observed in 10 controls. Serum and cultured fibroblasts of the propositus had normal CoQ10 contents. In the younger sister, the respiratory activities and CoQ10 level of muscle mitochondria were similar to those observed in the propositus. The findings establish CoQ10 deficiency as a cause of a familial mitochondrial cytopathy and suggest that the disease results from a tissue-specific defect of CoQ10 biosynthesis.

63. Tedeschi, D., et al., Potential involvement of ubiquinone in myotonic dystrophy pathophysiology: new diagnostic approaches for new rationale therapeutics. Neurol Sci, 2000. 21(5 Suppl): p. S979-80. An impairment of mitochondrial function may contribute to the pathophysiology of myotonic dystrophy (MyD). Coenzyme Q10 (CoQ10) deficiency has been previously observed, even if in a restricted sample of patients. The aim of this investigation was to obtain more information about coenzyme Q10 and its relationships to the aerobic metabolism in a group of MyD patients. Serum CoQ10 appeared significantly reduced with respect to normal controls: 0.93 +/- 0.22 vs. 1.58 +/- 0.28 micrograms/ml (p < 0.05). Moreover, the results demonstrated an inverse tendency between CoQ10 levels and the CTG expansion degree. Basal blood lactate levels were significantly higher than controls (p < 0.05). A borderline inverse correlation between CoQ10 and lactate, corresponding to lactate threshold, was found. These data suggest a possible role of CoQ10 in the pathogenesis of MyD, which may be mediated by mechanisms of cellular damage common to the oxidative pathway. Therapeutic strategies may be devised by virtue of this rationale.

64. Takusa, Y. and S. Yamaguchi, [Myopathies with miscellaneous disorders related to mitochondrial fatty acid oxidation: defective synthesis of ketone body, long-chain fatty acid transport defect, and muscular coenzyme Q10 deficiency]. Ryoikibetsu Shokogun Shirizu, 2001(36): p. 90-4.

65. Folkers, K. and R. Simonsen, Two successful double-blind trials with coenzyme Q10 (vitamin Q10) on muscular dystrophies and neurogenic atrophies. Biochimica et Biophysica Acta, 1995. 1271(1): p. 281-6.

66. Shimomura, Y., et al., Protective effect of coenzyme Q10 on exercise-induced muscular injury. Biochem Biophys Res Commun, 1991. 176(1): p. 349-55. The effect of coenzyme Q10 administration on exercise-induced muscular injury was examined in rats. Coenzyme Q10-treated and control rats were exercised by 90 min of downhill treadmill running. A part of the animals in both groups were killed immediately after exercise and the others were 40 h postexercise. After the exercise bout, serum creatine kinase and lactate dehydrogenase activities were elevated in the control rats, but not in the coenzyme

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Q10-treated rats. These enzyme activities in the latter increased to the similar level of the former 40 h postexercise. The muscle coenzyme Q10 content increased by the coenzyme Q10 treatment. These results suggest that the coenzyme Q10 treatment protected skeletal muscles against injury caused during exercise, but not against damage related with inflammatory processes after exercise.

67. Elble, R., E. Giacobini, and C. Higgins, Choline levels are increased in cerebrospinal fluid of Alzheimer patients. Neurobiol Aging, 1989. 10(1): p. 45-50. We measured choline (Ch), acetylcholinesterase activity (AChE) and total protein in the cerebrospinal fluid (CSF) of 66 Alzheimer patients (ages 54-89 years) and 22 age-matched controls (ages 52-80 years), looking for markers of the well-established cholinergic deficit and neuronal degeneration in Alzheimer disease (AD). Three or more lumbar punctures were performed in 21 patients over a span of 24 months in order to study the changes in these CSF components with disease progression. We found a statistically significant reduction in AChE and an increase in Ch with advancing dementia. These changes were not related to patient age. We suggest that the rise in CSF choline is related to neuronal membrane breakdown and reduced Ch uptake by cholinergic neurons. The reduction in CSF AChE is consistent with the depletion of cholinergic neurons in AD.

68. Berrettini, W.H., et al., Intravenous physostigmine increases cerebrospinal fluid neuropeptide-Y. Biol Psychiatry, 1989. 26(6): p. 623-30. The ability to measure directly central nervous system (CNS) neurotransmitter changes after an acute pharmacological challenge would be a useful clinical tool in psychiatric research. As one approach to this possibility, we attempted to measure cerebrospinal fluid (CSF) neuropeptide changes produced by an intravenous infusion of the indirect cholinergic agonist physostigmine. Six rhesus monkeys, with indwelling CSF catheters, had serial CSF samples removed before and after a 15 micrograms/kg physostigmine infusion. Five of six monkeys studied showed at least a 50% increase in CSF neuropeptide-Y (NPY) levels. Normal human subjects (n = 27) had CSF sampled before and 15, 30, and 45 min after an acute intravenous infusion of physostigmine (either 0, 5, or 15 micrograms/kg). An Analysis of Variance revealed a significant (p = 0.04) dose-time interaction, suggesting that physostigmine increased CSF NPY at the 15 micrograms/kg dose. CSF levels of seven other neuropeptides remained unchanged. These results suggest that the pharmacological challenge paradigm can be adapted to CSF neuropeptides, providing new measures of CNS stimulus-induced response beyond the peripheral plasma determinations usually employed.

69. Davis, K.L., et al., Alterations in cerebrospinal fluid dopamine metabolites following physostigmine infusion. Psychopharmacology (Berl), 1981. 72(2): p. 155-60. The effect of IV physostigmine administration on the cerebrospinal fluid (CSF) levels of homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC) in normal subjects was determined. After an adjustment for differing CSF concentrations of probenecid, physostigmine was found to elevate CSF HVA and DOPAC concentrations. The authors discuss these changes in CSF HVA and DOPAC and their possible relationship to the

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ability of physostigmine to decrease the symptoms of some patients with tardive dyskinesia.

70. Kaye, J.A., et al., Cerebrospinal fluid neurochemistry in the myoclonic subtype of Alzheimer's disease. Ann Neurol, 1988. 24(5): p. 647-50. Monoamine metabolites, biopterin, acetylcholinesterase (AChE) activity, and somatostatin-like immunoreactivity (SLI) were determined in the lumbar cerebrospinal fluid (CSF) of 24 patients with dementia of the Alzheimer type (DAT) without myoclonus or extrapyramidal signs, in 8 patients with DAT and myoclonus, and in 14 age-matched healthy control subjects. In patients with DAT with myoclonus as compared with both DAT patients without myoclonus and control subjects, the concentrations of homovanillic acid and biopterin were significantly decreased. 5-Hydroxyindoleacetic acid was significantly lower in patients with myoclonic DAT as compared to patients with nonmyoclonic DAT, but not significantly lower than in control subjects. CSF AChE and SLI were significantly reduced in patients with DAT with or without myoclonus, as compared with control subjects, but AChE and SLI were not significantly different between dementia groups. These results suggest that DAT patients with myoclonus represent a distinct clinical and neurochemical DAT subtype.

71. Jolkkonen, J., et al., Somatostatin-like immunoreactivity in the cerebrospinal fluid of patients with Parkinson's disease and its relation to dementia. J Neurol Neurosurg Psychiatry, 1986. 49(12): p. 1374-7. Acetylcholinesterase, somatostatin-like immunoreactivity, and homovanillic acid levels were measured in the cerebrospinal fluid of 36 patients with early stages of Parkinson's disease and in 19 control patients. In patients with Parkinson's disease the levels of somatostatin-like immunoreactivity were lower than in the controls (p less than 0.01); these values were lowest in the demented Parkinsonian patients. Concentrations of homovanillic acid were also significantly lower in Parkinsonian patients (p less than 0.05). In contrast, no changes were observed in the acetylcholinesterase activity of patients with Parkinson's disease. The reduced somatostatin-like immunoreactivity in CSF agrees with previous post-mortem studies and indicates that Parkinson's disease and Alzheimer's disease may have some neurochemical features in common.

72. Gomez, S., et al., Somatostatin-like immunoreactivity and acetylcholinesterase activities in cerebrospinal fluid of patients with Alzheimer disease and senile dementia of the Alzheimer type. Psychoneuroendocrinology, 1986. 11(1): p. 69-73. Somatostatin-like immunoreactivity (SLI) and acetylcholinesterase (AchE) activities were measured in the cerebrospinal fluid of 25 patients with senile dementia of the Alzheimer type (SDAT). Both SLI levels and AchE activities were reduced in the CSF of SDAT patients. The SLI levels and AchE activities were not correlated with the duration and the dementia score. However, in two patients the CSF SLI concentration was in agreement with the SLI levels in the frontal cortex obtained by biopsy. Our findings suggest that CSF SLI may be a good index of cortical SLI activities.

73. Hartikainen, P., et al., Neurochemical markers in the cerebrospinal fluid of patients with Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis

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and normal controls. J Neural Transm Park Dis Dement Sect, 1992. 4(1): p. 53-68. Several neurotransmitter markers were investigated in the cerebrospinal fluid (CSF) from patients with Alzheimer's disease (AD) (n = 27), Parkinson's disease (PD) (n = 35) and ALS (n = 26) and from control subjects (n = 34) to compare the possible alterations in the biochemical profiles of these different neurodegenerative diseases. The main proportion of the patients represented an early phase of the illness at the time of the diagnosis. Correlations of the degree of dementia and the stage of the disease with CSF measures were evaluated. The CSF levels of somatostatin like-immunoreactivity (SLI) were significantly reduced in AD patients when compared with those of normals and ALS patients. The CSF concentrations of homovanillic acid (HVA) were significantly decreased for PD patients and the decrease focused on the non-demented patients. A trend of decreasing HVA values towards the most advanced stage of Parkinson's disease assessed by Webster's scale was also displayed. The content of 3-methoxy-4-hydroxyphenylglycol (MHPG) in the CSF was higher for ALS patients than for other groups. The lowest 5-hydroxy-indoleacetic acid (5HIAA) levels were observed in the PD group and the lowest acetylcholinesterase (AChE) activities were found in the PD patients with the most severe disease. Changes in CSF measures were too subtle to be beneficial for diagnostic purposes, but adequate for reflecting the different neurochemical profiles of these three degenerative neurological disorders.

74. Davis, K.L., et al., Neurotransmitter metabolites in the cerebrospinal fluid of man following physostigmine. Life Sci, 1977. 21(7): p. 933-6.

75. Popovic, M., et al., Effect of physostigmine and verapamil on active avoidance in an experimental model of Alzheimer's disease. Int J Neurosci, 1997. 90(1-2): p. 87-97. The present study was performed to investigate and compare the effect of acetylcholinesterase inhibitor, physostigmine (0.045, 0.060 and 0.075 mg/kg sc, 30 min before the tests) and Ca-antagonist, verapamil (1.0, 2.5, 5.0 and 10.0 mg/kg sc, 30 min before the tests), on two-way active avoidance (AA) learning (acquisition and performance) in nucleus basalis magnocellularis (NBM)-lesioned rats. Bilateral electrolytic lesions of NBM induced significant decrease of acquisition and performance of AA responses in rats. Physostigmine (0.060 mg/kg) significantly improved only acquisition of AA, while verapamil (2.5 and 5.0 mg/kg) significantly improved both type of AA behavior in NBM-lesioned rats. These results suggest that altered calcium homeostasis might play significant role in pathogenesis of experimental induced Alzheimer's disease (AD) and that administration of calcium antagonist such as verapamil might successfully ameliorate disturbances of learning and memory appeared after lesions of NBM.

76. Peskind, E.R., et al., Oral physostigmine in Alzheimer's disease: effects on norepinephrine and vasopressin in cerebrospinal fluid and plasma. Biol Psychiatry, 1995. 38(8): p. 532-8. Physostigmine is a cholinesterase inhibitor which enhances central and peripheral cholinergic activity. In this study, we explored in persons with Alzheimer's disease (AD) the effects of an acute dose of physostigmine in patients receiving chronic physostigmine treatment on the activity of the cholinergically regulated noradrenergic and arginine vasopressin (AVP) systems. Specifically, we estimated the

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effects of sustained release oral physostigmine on central and peripheral noradrenergic and AVP systems by measuring norepinephrine (NE) and AVP in cerebrospinal fluid (CSF) and plasma. Lumbar punctures were performed in both physostigmine and no drug treatment conditions. In some subjects the effects of physostigmine on the plasma AVP response to the osmolar stimulus of a hypertonic saline infusion also were measured. NE concentrations in both CSF and plasma were significantly lower in the physostigmine than in the no drug condition. AVP concentrations did not differ between conditions in either compartment, nor did physostigmine affect the AVP response to hypertonic saline. Physostigmine appears to decrease both central and peripheral noradrenergic activity in AD.

77. degli Uberti, E.C., et al., Acute administration of human galanin in normal subjects reduces the potentiating effect of pyridostigmine-induced cholinergic enhancement on release of norepinephrine and pancreatic polypeptide. Neuroendocrinology, 1996. 64(5): p. 398-404. The neuropeptide galanin (GAL) is widely distributed in the central and peripheral nervous systems where it often coexists with catecholamines and acetylcholine. Recently we have reported that human GAL (hGAL) in man depresses the release of norepinephrine (NE) and the responses to both assumption of upright posture and insulin-induced hypoglycemia. To gain an insight into the action of hGAL on sympathetic nervous system activity in man, we investigated the effects of a 60-min infusion (80 pmol/kg/min) of hGAL or saline on the release of NE, epinephrine (E) and pancreatic polypeptide (PP) induced by an acetylcholinesterase inhibitor, pyridostigmine bromide (PD), in nine healthy volunteers. PD (120 mg orally) induced a significant rise in plasma concentrations of NE (1.6 +/- 0.04 vs. 1.08 +/- 0.06 nmol/l), E (0.34 +/ 0.05 vs. 0.12 +/- 0.04 nmol/l) and PP (178.06 +/- 33 vs. 37.57 +/- 7.35 pmol/l), whilst it significantly reduced heart rate (HR; 61 +/- 2 vs. 71 +/- 4 beats/min). Changes in plasma levels of PP were determined as an indirect measure of amplification of endogenous cholinergic activity produced by PD. Administration of hGAL blunted the release of NE and PP evoked by PD. The mean (+/- SEM) area under the curve produced by PD of NE (50.05 +/- 3.97 nmol/l.90 min) and PP (8,692.87 +/- 1,724 pmol/l.90 min) was significantly (p < 0.001) reduced by hGAL infusion (2.65 +/- 1.57 nmol/l.90 min and 248.1 +/- 148 pmol/l.90 min, for NE and PP, respectively). hGAL failed to affect significantly the E release evoked by PD. hGAL was able to enhance HR significantly (104 +/- 5 vs. 69 +/- 3 beats/min), and completely prevented the PD-induced slowing of HR. Both PD and hGAL did not alter supine systolic and diastolic blood pressure. We conclude that hGAL significantly reduces the release of NE and PP stimulated by PD-induced enhancement of cholinergic activity. These findings are consistent with a functional interrelationship between GAL and the cholinergic system in man, and may suggest the participation of a cholinergic pathway in the galaninergic modulation of the autonomic nervous system.

78. Araki, S. and H. Shibasaki, [Variations in serum amylase responding to the neostigmine test in amyotrophic lateral sclerosis]. Igaku To Seibutsugaku, 1966. 73(5): p. 233-9.

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79. Gowen, G.F., A second look at the neostigmine morphine test. Am Surg, 1989. 55(10): p. 640-4. In a prospective five-year study of 65 consecutive patients with upper abdominal pain the Neostigmine Morphine Test (NMT) was applied as a screen for biliary, ampullary, and pancreatic disease. Three facts emerged from this study: 1) the amylase and lipase were overly sensitive, but not specific and had only a 10 per cent predictive value for ampullary obstruction; 2) the bilirubin, alkaline phosphatase, and serum glutamic oxaloacetic transaminase (SGOT) were positive in patients with ampullary obstruction if they were postcholecystectomy but not so in patients with an intact gallbladder, which may be explained by the third observation; 3) the gallbladder can compensate for partial ampullary obstruction by dilatation five to ten times and by its ability to absorb 90 per cent of the water content of the bile. The Neostigmine Morphine Test is not a reliable screen for ampullary obstruction and positive findings must be confirmed by other studies.

80. Bell, I.R., et al., Serum neopterin and somatization in women with chemical intolerance, depressives, and normals. Neuropsychobiology, 1998. 38(1): p. 13-8. The symptom of intolerance to low levels of environmental chemicals (CI, chemical intolerance) is a feature of several controversial polysymptomatic conditions that overlap symptomatically with depression and somatization, i.e., chronic fatigue syndrome, fibromyalgia, multiple chemical sensitivity, and Persian Gulf syndrome. These syndromes can involve many somatic symptoms consistent with possible inflammation. Immunological or neurogenic triggering might account for such inflammation. Serum neopterin, which has an inverse relationship with l-tryptophan availability, may offer a marker of inflammation and macrophage/monocyte activation. This study compared middle-aged women with CI (who had high levels of affective distress; n = 14), depressives without CI (n = 10), and normals (n = 11). Groups did not differ in 4 p.m. resting levels of serum neopterin. However, the CI alone had strong positive correlations between neopterin and all of the scales measuring somatization. These preliminary findings suggest the need for additional research on biological correlates of 'unexplained' multiple somatic symptoms in subtypes of apparent somatizing disorders.

81. Amici, S., et al., Cerebrospinal fluid acetylcholinesterase activity after long-term treatment with donepezil and rivastigmina. Mech Ageing Dev, 2001. 122(16): p. 2057-62. At present acetylcholinesterase (AChE) inhibitors (AChEIs) represent the only reliable therapeutic resource for symptomatic treatment of Alzheimer Disease (AD). This study was designed to assess the effects of 6-12 month treatment with AChEIs donepezil and rivastigmine on cerebrospinal fluid (CSF) AChE and butyrylcholinesterase (BuChE) activity in AD patients. The pattern of AChE isoforms (G4, G1, G2) before and after treatment was investigated as well. In AD patients treated with donepezil a significant increase of CFS AChE activity was observed, whereas treatment with rivastigmine induced a significant decrease of AChE activity. Both drugs did not change BuChE activity and tended to restore the physiological pattern of AChE isoform. The possible significance of the influence of AChEIs on CSF AChE activity and isoforms is discussed.

82. Appleyard, M.E., et al., Cholinesterase activities in cerebrospinal fluid of patients with senile dementia of Alzheimer type. Brain, 1987. 110 ( Pt 5): p. 1309-22.

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Acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) activities of lumbar ceresbrospinal fluid (CSF) from patients with a clinical or histological diagnosis of Alzheimer's disease have been compared with those of normal age-matched control patients and patients with dementia of non-Alzheimer aetiology. No significant differences in the AChE activity of lumbar CSF from histologically and clinically diagnosed Alzheimer's disease patients and normal age-matched controls were found, although they could be distinguished from controls and other dements by their lower lumbar CSF levels of nsChE activity and by their elevated ratio of AChE/nsChE. A lower level of AChE activity was observed in the lumbar CSF of patients with dementia of non-Alzheimer aetiology. The AChE and nsChE activities of ventricular CSF obtained at postmortem have also been examined. The AChE activity of the ventricular CSF of patients with histologically confirmed Alzheimer's disease was 66% lower than that of age-matched controls; these patients could also be distinguished from normals by their lower levels of nsChE and by the elevated ratio of AChE/nsChE activities. A molecular defect in the AChE in the ventricular CSF of Alzheimer patients is indicated by the finding that the enzyme failed to show inhibition by high concentrations of substrate. The lower level of AChE in ventricular CSF may reflect the changes in this enzyme in forebrain regions of Alzheimer patients. Although it is at present not possible to correlate the lower levels of nsChE found in CSF with any known brain pathology, the significantly altered ratio of AChE/nsChE activities in lumbar CSF may possibly form the basis for a diagnostic test of Alzheimer type dementia.

83. Appleyard, M.E. and B. McDonald, Acetylcholinesterase and butyrylcholinesterase activities in cerebrospinal fluid from different levels of the neuraxis of patients with dementia of the Alzheimer type. J Neurol Neurosurg Psychiatry, 1992. 55(11): p. 1074-8. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities of cerebrospinal fluid (CSF) collected post mortem from the lateral ventricles, cisterna magna, and lumbar regions of the spinal cord of patients with a histologically confirmed diagnosis of Alzheimer's disease were compared with those of normal, age matched control patients, patients with dementia of non-Alzheimer aetiology, and patients with non-dementing neurological disorders. The AChE activity of the ventricular CSF of patients with Alzheimer's disease was 48% lower (p < 0.005) than that of age matched controls or patients with other types of dementia, and the AChE activity of CSF sampled from the basal cistern was 40% lower (p < 0.005) in patients with Alzheimer's disease. There were no significant differences between the AChE activity in Alzheimer's disease and control patients in CSF collected from the lumbar cistern. AChE activity was lower in CSF sampled from the basal and lumbar cistern of patients with dementia of non-Alzheimer aetiology, while ventricular activity was in the normal range. BuChE activity in ventricular CSF of Alzheimer's disease patients was 41% lower than normal (p < 0.05) and in the normal range in all other samples. The secretion of AChE from forebrain and hindbrain regions is reduced in Alzheimer's disease patients, leading to decreased ventricular and cisternal levels of the enzyme. Secretion from more caudal regions of the central nervous system seems to be unaffected by the disease, resulting in AChE in the lumbar CSF of patients with Alzheimer's disease being in the control range.(ABSTRACT TRUNCATED AT 250 WORDS).

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84. Abeyta, B.J., R.M. Albrecht, and C.R. Schermer, Retrospective study of neostigmine for the treatment of acute colonic pseudo-obstruction. Am Surg, 2001. 67(3): p. 265-8; discussion 268-9. Acute colonic pseudo-obstruction (ACPO) typically develops postoperatively or after severe illness. Studies suggest that pharmacologic manipulation with intravenous (i.v.) neostigmine (NSM) may be an effective and less invasive treatment modality for ACPO with minimal side effects. The purpose of this study was to retrospectively assess the efficacy and incidence of complications of an i.v. NSM bolus in patients with ACPO. Eight patients with ten episodes of ACPO were treated with a bolus dose of NSM. Rapid and effective decompression of the colon was achieved in six episodes after a single dose of NSM at a mean of 22.8 +/- 13.5 minutes. In three episodes decompression occurred after a second dose of NSM at a mean of 44.7 +/- 37.7 minutes. One patient failed NSM treatment but responded to a Cystografin enema. One patient experienced significant bradycardia. NSM is a simple, safe, and effective treatment for ACPO and based on result comparison of this study and previous studies both bolus and slow infusion dosing practices of NSM are effective. The NSM bolus dosing side effect profile has been shown to include significant bradycardia, whereas when NSM was infused over one hour significant bradycardic episodes requiring treatment have not been encountered. We propose that a prospective study evaluating NSM dosing as an i.v. bolus versus an i.v. infusion would be useful in determining whether NSM infusion can be proven safer than bolus dosing for the treatment of ACPO.

85. Adams, S.B. and M.A. MacHarg, Neostigmine methylsulfate delays gastric emptying of particulate markers in horses. Am J Vet Res, 1985. 46(12): p. 2498-9. Eight horses were allotted to 2 groups, each of 4 horses. All horses were given 100 plastic markers intragastrically via a nasogastric tube. One group of animals (control group) was not given medication after marker administration. The other group (test group) was given neostigmine methylsulfate (0.022 mg/kg of body weight) in the subcutaneous tissue at the time of marker administration and 30, 60, and 90 minutes later. All horses were killed 135 minutes after marker administration to locate the beads in the gastrointestinal tract. Gastric emptying of the markers was significantly delayed (P less than 0.05) in horses given neostigmine methylsulfate.

86. Amaro, R. and A.I. Rogers, Neostigmine infusion: new standard of care for acute colonic pseudo-obstruction? Am J Gastroenterol, 2000. 95(1): p. 304-5. This study was designed to assess the efficacy of i.v. infusion of neostigmine in patients with acute colonic pseudo-obstruction, which was defined as colonic distention with a cecal diameter of at least 10 cm on plain radiographs and no radiographic evidence of mechanical obstruction. Patients who failed to respond to conventional management (nothing by mouth, nasogastric suction, postural changes, i.v. fluids, electrolyte replacement, and discontinuation of any drugs that affect colonic motility) for 24 h were included in the study. Those with bradycardia (heart rate <60/min), hypotension (systolic blood pressure <90 mm Hg), active bronchospasm, clinical or radiographic evidence of perforation, history of partial colonic resection, active gastrointestinal bleeding, pregnancy, or serum creatinine >3 mg/dL were excluded. Twenty patients were included in this prospective, randomized, double-blind, placebo-controlled study. Eleven patients received neostigmine 2.0 mg i.v. over 3-5 min with electrocardiographic monitoring, and

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10 received placebo. Patients were evaluated for immediate clinical response (passage of flatus or stools associated with decreased abdominal distention within 30 min) and sustained response with decreased abdominal girth and reduced colonic dilation on radiographs 3 h after infusion. Ten patients in the neostigmine group had an immediate clinical response (median time, 4 min) compared to none in the placebo group (p<0.001). Three patients in the neostigmine group (27%) and eight in the placebo group (80%) failed to show sustained improvement 3 h after infusion (p = 0.04). Eight patients (one-neostigmine; seven-placebo) who failed to respond received open-label treatment with neostigmine. Seven patients responded; one patient from the placebo group failed and eventually required colonic resection. In conclusion, from a total of 18 patients treated with neostigmine, 17 (94%) had immediate clinical response, and 16 (89%) did not have recurrent colonic dilation. The most common side effect was crampy abdominal pain reported in 13 patients, although usually mild (nine). Symptomatic bradycardia requiring atropine occurred in two patients. Two patients in the neostigmine group died, but death was felt not to be related to acute colonic pseudo-obstruction or its treatment.

87. Alitalo, I. and A. Hietala, Effects of atropine, isoprenaline, neostigmine and practocol on cardiac arrhythmias induced by potassium in anesthetized dogs. Nord Vet Med, 1977. 29(11): p. 498-501. Either atropine, isoprenaline, neostigmine or practolol was given intravenously in therapeutic doses to dogs anesthetized with pentobarbitone sodium. Cardiac arrhythmias were induced by potassium chloride intravenously as a bolus injection in a dose of 0.3 mmol/kg. The standard electrocardiographic lead II was recorded continuously. pH, Po2 and Pco2 were checked from arterial blood samples before and after the injection. The isoprenaline treated dogs showed the smallest amount of arrhythmias and all the dogs survived. More severe arrhythmias were seen in the neostigmine and practolol treated dogs and the most severe occurrences were encountered in the atropine treated dogs.

88. Anzueto, A., et al., Acute inhalation toxicity of soman and sarin in baboons. Fundam Appl Toxicol, 1990. 14(4): p. 676-87. Adult baboons (Papio sp.; 8-12 kg) were anesthesized with sodium pentobarbital (20 mg/kg iv). The animals were instrumented for measurement of mean blood pressure (MBP), pulmonary artery pressure (PAP), ECG, arterial and mixed venous blood gases, lung volumes, lung pressures, and efferent phrenic nerve activity. Bronchoalveolar lavage (BAL) was performed. Studies were done prior to exposure, at intervals during the first 4 hr postexposure, and at 4 and 28 days after exposure. Control animals received a sham exposure to 2-propanol (N = 5). Soman (pinacolyl methylphosphonofluoridate) at 13.14 micrograms/kg (2 X LD50) was vaporized into the upper airway in a second group of animals (N = 5), and sarin (isopropyl methylphosphonofluoride) 30 micrograms/kg (2 X LD50) was vaporized into a third group of animals (N = 4). Controls showed no change in any parameter either immediately after diluent exposure or during the monitoring period. Soman and sarin produced a decline in MBP and bradyarrhythmias that were reversed with atropine. Apnea occurred in all soman- and sarin-exposed animals within 5 min postexposure, and was associated with absence of phrenic nerve signal. Ventilation was mechanically supported until the animal could maintain normal arterial blood gases during spontaneous breathing. BAL studies revealed an increase in total white cell population and neutrophils

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at 4 hr in all three groups. There were signs of impaired hemodynamics and persistent lung injury for 4 days that resolved by 28 days after exposure. In conclusion, inhalation of soman and sarin in the baboon is associated with cardiac arrhythmias, development of apnea, and a significant decrease in MBP. Inhalation exposure also resulted in a persistent influx of neutrophils and hypoxemia.

89. Haley, R.W., et al., Abnormal control of heart rate variability in veterans with Gulf War Syndrome. Submitted for publication.

90. Ally, A., et al., Cardiovascular effects elicited by central administration of physostigmine via M2 muscarinic receptors in conscious cats. Brain Res, 1995. 677(2): p. 268-76. The cardiovascular effects of an intracerebroventricular (i.c.v.) injection of physostigmine were studied using conscious cats. Physostigmine (5-25 micrograms: 5 microliters) caused a dose-dependent increase in mean arterial pressure (MAP) and heart rate (HR). The highest dose (25 micrograms) increased MAP and HR by 32 +/- 3 mmHg and 45 +/- 5 beats/min, respectively (n = 5). Pre-administration of the muscarinic receptor antagonist, atropine (25 micrograms; i.c.v.) blocked the effects of physostigmine (25 micrograms; i.c.v.). Also, the pre-administration of the M2 muscarinic antagonist, methoctramine (25 micrograms; i.c.v.), antagonized the cardiovascular effects of physostigmine without altering the baseline variables. However, the M1 muscarinic antagonist, pirenzepine (100 micrograms; i.c.v.) did not alter baseline MAP or HR, and also failed to inhibit the cardiovascular responses to physostigmine. Similarly, the M3 muscarinic blocker, 4-diphenyl-acetoxy-N-methylpiperidine methiodide (50 micrograms; i.c.v.), neither changed baseline cardiovascular variables nor blocked the effects of physostigmine. When the same cats were anesthetized with intravenous injection of sodium pentobarbital (25-30 mg/kg), physostigmine (25 micrograms; i.c.v.) evoked a decrease in MAP and HR of 13 +/- 6 mmHg and 15 +/- 6 bpm, respectively (n = 5). These results demonstrate that the increases in MAP and HR to the i.c.v. administration of physostigmine in conscious cats are possibly mediated through stimulation of central M2 muscarinic receptors. In addition, anesthesia reverses the effects elicited by the central administration of physostigmine to a decrease in MAP and HR.

91. Ally, A., G.A. Hand, and J.H. Mitchell, Central injection of physostigmine attenuates exercise-induced pressor response in conscious cats. Am J Physiol, 1997. 273(1 Pt 2): p. R393-9. The effects of intracerebroventricular administration of physostigmine, a cholinesterase inhibitor, on the cardiovascular responses evoked by static voluntary exercise were investigated using conscious cats. Four cats were trained to press a bar (200-650 g) with one forelimb for at least 20 s. The changes in mean arterial pressure (MAP), heart rate (HR), and developed force during the first five trials in 30 min by each individual cat were averaged, and a mean of the four values was then calculated. After the cats exercised for 30 min, either artificial cerebrospinal fluid (CSF) or physostigmine (5 micrograms) was administered intracerebroventricularly. Before physostigmine, exercise trials by the cats increased MAP and HR by 17 +/- 3 mmHg and 42 +/- 4 beats/min, respectively. Administration of physostigmine did not alter the resting MAP and HR but attenuated the MAP and HR responses to exercise (5-30 min postphysostigmine: MAP = 8 +/- 3 mmHg, HR = 25 +/- 7 beats/min; 30-60 min

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postphysostigmine: MAP = 4 +/- 3 mmHg, HR = 19 +/- 8 beats/min). Intracerebroventricular administration of CSF had no effect on the cardiovascular responses to static exercise. Pretreatment with the muscarinic antagonist, atropine (25 micrograms icv), blocked the attenuating effects of subsequent intracerebroventricular administration of physostigmine. These results demonstrate that stimulation of central muscarinic receptors attenuates the cardiovascular responses to static exercise by conscious cats. In addition, the present study suggests that there is no tonic effect of central muscarinic receptors on the cardiovascular responses to voluntary exercise.

92. Jancin, B., Pyridostigmine reduced orthostatic intolerance. Internal Medicine News, 2002. June 15: p. 32. Cites Dr. Wolfgang Singer at annnual meeting of Amer Acad of neurology.

18 pts with orthostatic intolerance with HR increase of =30 w standing, w sx cerebral hypoperfusion or symp overactivity. All underwent tilt-table testing before and at 1h after taking 60mg of oral PB. Mean HR increas of 45 dropped to 38 with rx (significant). Also supine HR fell 79 to 73; upright fell 124 to 111. Sx severity score 0-10 scale improved 5.6 on baseline tilt table to 4.2 after PB (on 0-10 scale)

Plasma NE increased predrug 214 and 491pg/ml supine and upright to 263 and 5552pg/ml after drug. "No side effects were noted in assoc with PB therapy".

Current most widely used drugs are beta blockers, vasoconstrictors, and volume expansion agents, but resp's are highly variable.

93. Park, K.H., J.P. Long, and J.G. Cannon, Reversal of antihypertensive agent-induced postural hypotension with physostigmine. Arch Int Pharmacodyn Ther, 1991. 311: p. 155-65. Physostigmine, administered intravenously, reversed postural hypotension induced by hypotensive agents, guanethidine, clonidine and dopamine2-receptor agonists. Postural hypotension, induced by pentobarbital sodium, was also reversed by physostigmine. Neostigmine reversed postural hypotension induced by clonidine following intracerebroventricular, but not intravenous administration. It is proposed that centrally acting cholinomimetic agents may be used to manage postural hypotension resulting from suppression of sympathetic nervous system activity.

94. Hata, S., H. Kunida, and Y. Oyama, [Antihypertensive effects of coenzyme Q10 in essential hypertension--in relation to the renin-aldosterone system]. Horumon To Rinsho, 1977. 25(9): p. 1019-23.

95. Aoyagi, M., et al., Central cholinergic control of cerebral blood flow in the baboon. Effect of cholinesterase inhibition with neostigmine on autoregulation and CO2 responsiveness. J Neurosurg, 1975. 43(6): p. 689-705. Cerebral autoregulation and vastomotor responsiveness to carbon dioxide (CO2) were measured quantitatively by the use of the autoregulation index and chemical index, respectively, in normal baboons before and after intravertebral and intracarotid infusion of the anticholinesterase agent, neostigmine methylsufate (Prostigmin). Continuous measurements were made of cerebral

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blood flow (measured as bilateral internal jugular venous outflow), arterial and cerebral venous pO2 and pCO2, cerebral arteriovenous oxygen differences, and endotracheal CO2. The effect of intravertebral infusion of neostigmine (12.5 mug/kg body weight) was compared to intravertebral infusion of neostigmine (25 mug/kg body weight) for assessment of any specific action of the drug on a hypothetical cholinergic vasomotor center, presumed to be located in the territory of the vertebrobasilar supply. No significant or persistent changes in cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) followed either intravertebral or intracarotid infusion of neostigmine. Cerebral vascular resistance (CVR) and cerebral perfusion pressure (CPP), however, decreased significantly after intravertebral infusion. Cerebral autoregulatory vasoconstriction during increases of CCP was significantly reduced following both intravertebral and intracarotid infusion. Cerebral autoregulatory vasodilatation was not altered as CPP was lowered. Cerebral vasodilatory reactivity to CO2 inhalation was significantly enhanced following intravertebral neostigime but not following intracarotid neostigmine. Cerebral vasoconstrictive response to hyperventilation was not influenced by neostigmine. These results support the view that central cholinergic cerebrovascular influences exist, and are vasodilatory in nature.

96. Eneroth-Grimfors, E., et al., Noninvasive test of microvascular endothelial function in normal and hypertensive pregnancies. Br J Obstet Gynaecol, 1993. 100(5): p. 469-71. OBJECTIVE: To examine microvascular endothelial cell function in vivo in pre-eclampsia. DESIGN: Iontophoresis of acetylcholine (ACh), which gives rise to endothelial cell dependent vasodilatation, and of sodium nitroprusside (SNP), which elicits vasodilatation independently of functioning vascular endothelium. SETTING: Sodersjukhuset, Stockholm, Sweden. SUBJECTS: Ten pre-eclamptic patients, ten healthy pregnant women and ten healthy nonpregnant women were examined. MAIN OUTCOME MEASURES: The degree of vasodilatation following iontophoretic administration of ACh compared with SNP was recorded with a laser Doppler technique, the data being analysed on a personal computer. RESULTS: Both ACh and SNP administration resulted in marked vasodilatation; the magnitude of the vasodilatation was similar in the three groups of women. CONCLUSION: Following iontophoretic administration of endothelial cell dependent or independent vasodilatators, laser Doppler measurement of blood flow demonstrated no microvascular endothelial cell dysfunction in pre-eclamptic women.

97. Arora, S., et al., Estrogen improves endothelial function. J Vasc Surg, 1998. 27(6): p. 1141-6; discussion 1147. PURPOSE: To determine the effect of estrogen on endothelium-dependent relaxation in the cutaneous microcirculation of women. METHODS: Three groups of women participated in the study. Group 1 (n = 20) was premenopausal and had a mean age of 39 years (range 24-50 years). Group 2 (n = 9) was postmenopausal and had a mean age of 58 years (range 53-65 years). Group 3 (n = 11) was postmenopausal and taking estrogen replacement therapy; the mean age was 53 years (range 43-58 years). Eleven women in group 1 underwent testing twice, once during menstruation (mean serum estradiol level 73 +/- 30 pg/ml) and once during midcycle (mean serum estradiol level 268 +/- 193 pg/ml; p = 0.003). Single-point laser Doppler ultrasound and laser Doppler imaging with a scanner were used to measure vasodilatation

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in the forearm skin in response to iontophoresis of 1% acetylcholine (endothelium dependent) and 1% sodium nitroprusside (endothelium-independent smooth muscle relaxant). RESULTS: All three groups were matched for body mass index and fasting glucose, total, high-density lipoprotein, and low-density lipoprotein cholesterol and triglyceride levels. All women had normal blood pressure, and none smoked. Mean serum estradiol levels were 196 +/- 170 pg/ml (group 1), 35 +/- 12 pg/ml (group 2), and 107 +/- 78 pg/ml (group 3) (p = 0.004). Maximum microvascular vasodilatation (percentage increase over baseline) in response to acetylcholine was reduced in group 2 (93% +/- 43%) compared with group 1 (187% +/- 63%) and group 3 (142% +/- 56%) (p = 0.001). The response to sodium nitroprusside also was diminished in group 2 (73% +/- 27%) compared with group 1 (126% +/- 45%) and group 3 (100% +/- 32%) (p = 0.02). Within group 1 the acetylcholine response was higher during the midcycle phase (186% +/- 31%) compared with the menstrual phase (147% +/- 57%) (p < 0.05). The sodium nitroprusside response also was higher during the midcycle phase (144% +/- 31%) compared with the menstrual phase (94% +/- 41%) (p < 0.05) CONCLUSION: The results indicate that estrogens might enhance endothelium-dependent and endothelium-independent vasodilatation in the microcirculation of women.

98. Kubli, S., et al., Reproducibility of laser Doppler imaging of skin blood flow as a tool to assess endothelial function. J Cardiovasc Pharmacol, 2000. 36(5): p. 640-8. Endothelial dysfunction might be an important and early event in the pathogenesis of major cardiovascular diseases. Therefore, the evaluation of endothelial function in humans may be of great clinical relevance. Usual methods for that purpose are either invasive and/or technically demanding. In the dermal microcirculation, endothelial function may be assessed noninvasively from the laser Doppler measurement of increases in blood flow after either the transdermal application of acetylcholine by iontophoresis, or the release of transient arterial occlusion (reactive hyperemia). An endothelium-independent response may be provided by the iontophoresis of sodium nitroprusside. This approach is notable for technical simplicity, but of uncertain reproducibility. Sixteen young, healthy, nonsmoking males were examined in the fasting state. Changes in skin blood flow were measured with a laser Doppler imager during the iontophoresis of acetylcholine and sodium nitroprusside, as well as during reactive hyperemia, on two different days, at each of two different sites on the volar face of the forearm. Nonspecific effects related to the stimulation of terminal nerve fibers by the iontophoretic current were suppressed by prior surface anesthesia. The iontophoresis of acetylcholine and sodium nitroprusside induced a seven- to eightfold increase in dermal blood flow. The corresponding figure for peak reactive hyperemia was approximately fourfold. The mean coefficients of variation of responses recorded on different days, on the same site, in the same individual were <10% for iontophoresis of acetylcholine and for peak reactive hyperemia, and between 10 and 20% for iontophoresis of sodium nitroprusside. This day-to-day variation was significantly smaller than the site-to-site variation (p < 0.01 for all three responses). Endothelium-dependent and -independent responses of dermal blood flow evaluated with laser Doppler imaging are highly reproducible from day to day, at least in healthy nonsmoking young male subjects, and provided some simple precautions are observed, foremost among which is the strict standardization of the recording site.

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These observations may have implications for the testing of endothelial function in clinical studies.

99. Williams, M.R., et al., Variations in endothelial function and arterial compliance during the menstrual cycle. J Clin Endocrinol Metab, 2001. 86(11): p. 5389-95. Female sex hormones have been implicated in the cardioprotection of premenopausal women. However, the cardiovascular actions of these hormones and the effects of their natural fluctuations during the menstrual cycle are not fully understood. We studied changes in vascular function during the menstrual cycle in 15 healthy premenopausal women. Four noninvasive procedures were performed during the early follicular (EF), late follicular (LF), early luteal (EL), and late luteal (LL) phases: flow-mediated dilatation (FMD) of the brachial artery during reactive hyperemia, laser Doppler velocimetry (LDV) with direct current iontophoresis of acetylcholine (ACh) and nitroprusside, whole body arterial compliance (WBAC), and pulse wave velocity. Hormone levels were consistent with predicted cycle phase and showed that all subjects ovulated during the cycle studied. FMD, LDV with ACh, and WBAC varied cyclically, with significant increases from the F to LF phase, sharp falls in the EL phase, and significant recoveries in the LL phase. These changes were most marked for FMD [EF, 8.8 +/- 0.6% (mean +/- SEM); LF, 10.0 +/- 0.7; EL, 4.2 +/- 0.6; LL, 8.6 +/- 0.9] and the LDV response to ACh (EF, 2.7 +/- 0.2 V/min; LF, 3.3 +/- 0.4; EL, 1.8 +/- 0.3; LL, 2.7 +/- 0.4). WBAC changed similarly (EF, 0.58 +/- 0.08 arbitrary units; LF, 0.84 +/- 0.06; EL, 0.65 +/- 0.05; LL, 0.68 +/- 0.06). Sodium nitroprusside-induced vasodilatation decreased significantly from EF to EL, with no other significant difference, and pulse wave velocity did not vary significantly over the four time points. Conductance and resistance artery endothelial reactivity and smooth muscle sensitivity to nitric oxide and arterial compliance are modulated significantly in response to the changing hormonal patterns of the menstrual cycle. These findings emphasize the importance of menstrual phase in the interpretation of data on endothelial function and may provide insights into the mechanisms underlying sex differences in cardiovascular risk and other disease processes in premenopausal women.

100. Newton, D.J., F. Khan, and J.J. Belch, Assessment of microvascular endothelial function in human skin. Clin Sci (Lond), 2001. 101(6): p. 567-72. Endothelial dysfunction is an important factor in many cardiovascular diseases, and is commonly associated with impaired endothelium-mediated vasodilatation. Information about the mechanisms behind this dysfunction has come largely from animal studies or, in humans, through invasive techniques that are not specific to one vascular bed. We have developed protocols to assess endothelial function non-invasively in the cutaneous microcirculation by measuring blood flow responses to four receptor-specific vasoactive compounds. Cumulative doses of acetylcholine, methacholine, bradykinin and substance P were administered iontophoretically to the forearm skin of healthy volunteers on two to three occasions. Dose-dependent increases in skin microvascular blood flow in response to these drugs were measured with laser Doppler imaging. Vascular responses to acetylcholine and methacholine were reasonably consistent, with coefficients of variation of approx. 17%. The coefficients of variation for bradykinin and substance P were much poorer, as high as 70% for some doses. This might partly be a consequence of the more unpredictable effects of histamine release in the vasoactive behaviour of these two

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agonists. Although it might be advantageous to find other agonists with which to test the function of different receptor pathways, we have shown that just acetylcholine and methacholine can currently be used with iontophoresis to allow sensitive and reproducible assessment of endothelial function.

101. Elherik, K., et al., Circadian variation in vascular tone and endothelial cell function in normal males. Clin Sci (Lond), 2002. 102(5): p. 547-52. The existence of circadian rhythms in the time of onset of acute cardiovascular events has been described previously. This report describes the circadian variation in endothelial cell products, such as nitric oxide (NO) and endothelin-1 (ET-1) levels, and endothelium-dependent and -independent vasodilation in normal males. Plasma ET-1 and NO were measured every 4 h in nine subjects (20-41 years old) over a 24 h period. Endothelium-dependent and -independent vascular responses were measured in the forearm skin every 4 h using laser Doppler imaging after iontophoresis of increasing doses of acetylcholine (ACh) and sodium nitroprusside respectively. A statistically significant circadian variation was demonstrated for the mean ACh response (P=0.0001, ANOVA). The peak response [in arbitrary perfusion units (AU)] occurred at 16.00 hours (8.90+/-1.91 AU) and the lowest response at 08.00 hours (4.57+/-0.66 AU). A significant circadian variation was also seen for the highest dose of sodium nitroprusside (P=0.036, ANOVA), the peak occurred at 16.00 hours (3.97+/-1.80 AU), and the lowest at 04.00 hours (2.62+/-0.58 AU) and 08.00 hours (2.58+/-1.16 AU). There was a significant circadian variation in the ET-1 levels (P=0.04) with two peaks, one at 20.00 hours (0.80+/-0.28 pg/ml) and the other at 08.00 hours (0.84+/-0.15 pg/ml). The lowest value occurred at 16.00 hours (0.61+/-0.24 pg/ml). There was also a borderline trend for a circadian variation in NO levels (P=0.06), with higher levels at 20.00 hours (15.53+/-8.42 micromol/l), and low levels at 04.00 hours (10.87+/-4.70 micromol/l) and 08.00 hours (9.82+/-3.15 micromol/l). ACh responses were significantly correlated with ET-1 (r=-0.3, P=0.02) and NO (r=0.30, P=0.02) levels. Our findings suggest that endothelial activity has a circadian variation with attenuation in the morning. These circadian variations in endothelial activity might play an important role in the occurrence of acute cardiovascular events at this time, which are precipitated through the interplay between ET-1, NO and vascular function.

102. Lim, S.C., et al., The effect of hormonal replacement therapy on the vascular reactivity and endothelial function of healthy individuals and individuals with type 2 diabetes. J Clin Endocrinol Metab, 1999. 84(11): p. 4159-64. Estrogens protect healthy women from cardiovascular disease. However, epidemiological data suggest that women with diabetes are denied the cardioprotection associated with estrogens. Whether or not hormonal replacement therapy (HRT) confers cardiovascular benefits in postmenopausal women with diabetes is not known. The aim of this study was to examine the effects of HRT on the microvascular reactivity and endothelial function of individuals with and without diabetes. We studied the following groups of individuals: premenopausal healthy women [n = 28, age 41 +/- 8 yr (mean +/- SD)], premenopausal women with type 2 diabetes (n = 16, age 43 +/- 6 yr); postmenopausal healthy women (n = 12, age 57 +/- 4 yr), postmenopausal women with diabetes (n = 17, age 62 +/- 5 yr); postmenopausal healthy women on HRT (n = 13, age 51 +/- 5 yr), postmenopausal women with diabetes on HRT (n = 11, age 57 +/- 7 yr). We used laser Doppler flowmetry to measure forearm

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cutaneous vasodilatation in response to iontophoresis of 1% acetylcholine (endothelium dependent) and 1% sodium nitroprusside (endothelium independent). The endothelium-dependent vasodilation was significantly higher in premenopausal healthy women (180 +/- 67%; increase over baseline) compared to premenopausal diabetic women (87 +/- 41%; P < 0.001). endothelium-dependent vasodilation was also higher in postmenopausal healthy women on HRT (143 +/- 52) compared with postmenopausal diabetic women on HRT (86 +/- 61), postmenopausal healthy women without HRT (104 +/- 43), and postmenopausal diabetic women without HRT (74 +/- 28; P < 0.001). A similar pattern of responses was observed in the endothelium-independent vasodilation (premenopausal healthy women, 126 +/- 56; premenopausal diabetic women, 88 +/- 26; postmenopausal healthy women on HRT, 121 +/- 37; postmenopausal diabetic women on HRT, 88 +/- 41; postmenopausal healthy women without HRT, 84 +/- 36; and postmenopausal diabetic women without HRT, 73 +/- 36; P < 0.001). Soluble intercellular adhesion molecule (sICAM) was also measured among all the women with diabetes. Premenopausal women with diabetes (248.9 +/- 56 ng/ml) and postmenopausal women with diabetes on HRT (257.7 +/- 49 ng/ml) had lower sICAM levels compared with the postmenopausal diabetic women without HRT (346.4 +/- 149 ng/ml; P < 0.05). We conclude that menopausal status and type 2 diabetes are associated with impaired microvascular reactivity. HRT substantially improves microvascular reactivity in postmenopausal healthy women. In contrast, the effect of HRT on the microvascular reactivity of postmenopausal diabetic women is less apparent. However, the use of HRT among women with diabetes is associated with lower sICAM levels, suggesting an attenuation in endothelial activation.

103. Wirtschafter, J.D., C.R. Volk, and R.J. Sawchuk, Transaqueous diffusion of acetylcholine to denervated iris sphincter muscle: a mechanism for the tonic pupil syndrome (Adie syndrome). Ann Neurol, 1978. 4(1): p. 1-5. The accepted hypothesis for the pathophysiology of tonic pupil syndrome (Adie syndrome) was reexamined in light of recent developments concerning denervation supersensitivity of cholinergically innervated smooth muscle. Kinetic analysis suggests that enzymatic hydrolysis is unimportant relative to convective diffusion in the turnover of acetylcholine in the aqueous humor. We postulate that the greater response to near stimuli than to light stimuli and the delay in iris sphincter contraction and relaxation can be explained by release of acetylcholine from the neuromuscular junction of the ciliary muscle followed by transaqueous diffusion to receptor sites on denervated, supersensitive iris sphincter muscle.

104. Fountoulakis, K., et al., Changes in pupil reaction to light in melancholic patients. Int J Psychophysiol, 1999. 31(2): p. 121-8. The aim of the current study was to compare the pupil reaction to light in depressed patients and normal control subjects. Seven depressed patients with melancholic features according to DSM-IV criteria and 14 age- and gender-matched control subjects took part in the study. All were free of any medication for at least 2 weeks. All were aged between 25 and 50 years. An optical method was used to assess the pupil reaction to a single flash. Depressed patients manifested shorter latency for constriction than control subjects, and a marginal difference in the total work produced by acetylcholine. The results of the current study

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support the theory that there is a norepinephrine hypoactivity in melancholic depression, with less affected acetylcholine activity.

105. Elliott, A. and C. Carter, Pupil size after extracapsular cataract extraction and posterior chamber lens implantation: a prospective randomized trial of epinephrine and acetylcholine. Ophthalmic Surg, 1989. 20(8): p. 591-4. The effects of using epinephrine in the irrigating fluid and intracameral acetylcholine were studied by measuring changes in pupil size in the 48 hours following extracapsular cataract extraction and intraocular lens implantation in 39 eyes. Epinephrine reduced peroperative pupil constriction, but its effect was insignificant thereafter. The pupil constriction following acetylcholine was maximal at 2 hours and was still significant at 4 hours, but pupils redilated by 6 hours. Neither drug had any effect after this time. The edge of most lens implants was visible at 6 hours, after which pupils steadily constricted.

106. Suzuki, R., S. Kobayashi, and H. Yoshino, [The inhibitory cholinergic innervation of the dilator on the movement of the pupil]. Nippon Ganka Gakkai Zasshi, 1984. 88(12): p. 1555-7.

107. Naito, M., [The autonomic nervous system and eyes: the role of the sympathetic nerve on extra-ocular muscle and pupil]. Nippon Ganka Gakkai Zasshi, 1971. 75: p. 988-95.

108. Pohjanpelto, P., [Carbachol in cataract surgery used for constriction of the pupil]. Duodecim, 1969. 85(24): p. 1504-7.

109. Cook, J.E., M.A. Kolka, and C.B. Wenger, Chronic pyridostigmine bromide administration: side effects among soldiers working in a desert environment. Mil Med, 1992. 157(5): p. 250-4. The side effects of chronic pyridostigmine bromide administration were studied in seven male soldiers performing moderate-intensity exercise in a desert environment. A 2-week, double-blind, placebo-controlled crossover design was employed in which pyridostigmine was administered for 7 consecutive days (30 mg orally, t.i.d.). Four hours each day were spent in the heat (42 degrees C, 20% relative humidity); 2 hours rest followed by 2 hours moderate exercise (40% maximal aerobic power). Each day, subjects completed four symptom questionnaires and received three focused physical examinations. Symptoms reported did not differ between treatment groups except for fewer headaches during pyridostigmine treatment. Soldiers were unable to distinguish the effects of pyridostigmine from placebo. Pyridostigmine was associated with lower resting diastolic blood pressure (approximately 4 mmHg, p less than 0.05), smaller pupil diameter (approximately 0.5 mm, p less than 0.01), decreased handgrip strength (approximately 3%, p less than 0.05), and higher final rectal temperature (approximately 0.1 degree C, p less than 0.01). Effects of this magnitude are not likely to appreciably limit performance. We conclude that chronic pyridostigmine administration does not negatively impact on soldiers' ability to perform physical work over repeated days in a desert environment.

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110. Borland, R.G., et al., Studies on the possible central and peripheral effects in man of a cholinesterase inhibitor (pyridostigmine). Hum Toxicol, 1985. 4(3): p. 293-300. The effect of a reversible cholinesterase inhibitor (pyridostigmine: 30 mg, 8-hourly for 3 days) on psychomotor performance and visual function and on the electrical activity of the brain was studied in healthy man. It was not possible to detect any change during the six individual experimental sessions over the 3 days, but with pooling of the data there was a drug effect. The threshold for detection of a flickering light was increased and less responses were missed on a dynamic visual-acuity task. Visuo-motor coordination was impaired. The observations were consistent with the known activity of the drug and suggest an increase in central arousal and minimal alteration in motor coordination.

111. Goel, A., S. Joseph, and T.K. Dutta, Organophosphate poisoning: predicting the need for ventilatory support. J Assoc Physicians India, 1998. 46(9): p. 786-90. The present study evaluated 103 consecutive patients of organophosphate poisoning with special reference to the need for ventilatory support. Of 103 patients, 36(34.95%) required assisted ventilation. The need for ventilatory support was significantly more with greater time duration for institution of specific treatment, low level of sensorium at admission, pin-point pupils and generalized fasciculations, presence of convulsions, presence of respiratory insufficiency at admission, and higher initial atropine requirement for atropinization. The study also proposes a system of grading of severity, modified from the existing systems proposed.

112. Kay, C.D. and J.D. Morrison, The effects of ingestion of 60 mg pyridostigmine bromide on contrast sensitivity in man. Hum Toxicol, 1988. 7(4): p. 347-52. The effects on vision of ingestion of the anticholinesterase pyridostigmine bromide (60 mg), assessed from pharmacokinetic data to provide at least 20% inhibition of blood cholinesterase over the 1 1/2-4 1/2 h experimental period, was compared with 60 mg lactose in a double-blind crossover protocol. Contrast sensitivity to stationary oscilloscope-generated gratings of 3-40 c/deg showed a small but significant increase of 7% which was consistent with a small reduction in pupil diameter, surmised to cause a small improvement in optical quality. This reduction in pupil diameter was, however, overshadowed by a larger though still non-significant reduction on the second visit to the laboratory compared with the first. Contrast sensitivities to laser interference fringes observed in the Maxwellian view, by which the effects of the optical media are essentially bypassed, thus providing an entirely neural assessment, were unchanged after pyridostigmine. It is concluded that pyridostigmine may be given as a prophylactic in anticipation of exposure to an organophosphorus anticholinesterase without a deleterious effect on stationary visual function.

113. Loupe, D.N., et al., Pupillary response to tropicamide in patients with Alzheimer disease. Ophthalmology, 1996. 103(3): p. 495-503. PURPOSE: To determine whether pupillary responses to dilute tropicamide could be used as a diagnostic test for Alzheimer disease (AD). The authors also investigated whether concurrent use of an oral acetylcholinesterase inhibitor (tacrine) alters the pupillary response to dilute tropicamide in patients with AD, and whether pupillary responses to dilute tropicamide differ in young versus older control subjects. METHODS: Pupillary diameter and area of both

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eyes were measured in light and darkness, at 10-minute intervals for 40 minutes after random instillation of 0.01% tropicamide to one eye. Four groups of subjects were studied: 9 patients with AD, 10 who were treated with tacrine, 11 older control subjects, and 10 young control subjects. RESULTS: Mean change in anisocoria was not significantly different among groups at any of the measurement time points. Mean percent change in diameter of the treated eyes showed a trend toward faster maximum dilatation in the AD groups, but change in pupillary measurements did not identify individuals with AD. CONCLUSION: Pupillary response to dilute tropicamide did not effectively distinguish individual patients with AD from young or older control subjects.

114. Macic, I., et al., [Antagonism of miosis induced by sarin and VX in rabbits]. Vojnosanit Pregl, 1986. 43(2): p. 103-8.

115. Nozaki, H., et al., Secondary exposure of medical staff to sarin vapor in the emergency room. Intensive Care Med, 1995. 21(12): p. 1032-5. OBJECTIVE: To clarify the risk of secondary exposure of medical staff to sarin vapor in the emergency room, and to warn emergency room staffs of the hazard. DESIGN: Retrospective observational survey. SETTING: Emergency department of a university hospital in a metropolitan area of Japan. PARTICIPANTS: Fifteen doctors treating victims of a terrorist attack with sarin in the Tokyo subways on the day of the attack. MEASUREMENTS AND RESULTS: Of the 15 doctors who worked in the emergency room treating the victims, 13 became simultaneously aware of symptoms during the resuscitation of two victims who were exposed to sarin. Among 11 doctors (73%) who complained of dim vision, the pupils were severely miotic (<2 mm) in 8 (73%). Other symptoms included rhinorrhea in eight (53%), dyspnea or tightness of the chest in four (27%), and cough in two (13%). Atropine sulfate was given to six, and pralidoxime was given to one of these six doctors. To decontaminate the emergency room of sarin vapor, ventilation was facilitated and all belongings of the patients were sealed up. None of the doctors noticed worsening of their symptoms thereafter. CONCLUSIONS: Careful attention to the risks of secondary exposure to toxic gas in the emergency room and prompt decontamination if such exposure should occur are necessary in the case of large-scale disasters caused by sarin.

116. Nozaki, H., et al., Relationship between pupil size and acetylcholinesterase activity in patients exposed to sarin vapor. Intensive Care Med, 1997. 23(9): p. 1005-7. OBJECTIVE: To elucidate the effect of sarin vapor on pupil size and erythrocyte acetylcholinesterase activity (AchE). DESIGN: Retrospective observational survey. SETTING: Emergency department of an urban teaching hospital. PATIENTS: 80 patients who were exposed to sarin in a terrorist attack in Tokyo subways. MEASUREMENTS AND RESULTS: Pupil size and AchE activity on the day of exposure were measured. Among the 80 patients, the pupils were miotic (< 3 mm) in 50 patients (62.5%), while AchE activity was below the normal range (< 1.2 U) in 34 patients (42.5%). AchE was significantly lower in the miotic group than in the group with normal pupils (1.0 +/- 0.5 U vs 1.5 +/- 0.3 U, p < 0.01). In the miotic group, AchE activity was lower than normal in 32 patients (64.0%) but was decreased in only 2 patients in the normal pupil group (6.7%) (p < 0.01). CONCLUSIONS: Miosis is a more sensitive index of exposure to

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sarin vapor than erythrocyte AchE. Systemic poisoning is apparently less likely to develop if the patient's pupil size is normal on arrival at the hospital.

117. Rengstorff, R.H., Accidental exposure to sarin: vision effects. Arch Toxicol, 1985. 56(3): p. 201-3. Two men were accidentally exposed to vapors of sarin, a cholinesterase inhibitor and extremely toxic nerve gas. Diagnosis was confirmed by depressed cholinesterase activity, and fixed extremely miotic pupils. No other signs or symptoms developed and neither man required treatment. Recovery to normal cholinesterase activity was gradual over a 90-day period. Pupillary reflexes were not detectable until 11 days after exposure; the miotic pupils dilated slowly over a 30-45 day-period. Eye pain and blurred vision did not occur; visual acuity and amplitude of accommodation were improved for several weeks. Other functions not affected significantly were intraocular pressure, visual fields, color vision, heterophorias, and vergences.

118. Sidell, F.R. and W.A. Groff, The reactivatibility of cholinesterase inhibited by VX and sarin in man. Toxicol Appl Pharmacol, 1974. 27(2): p. 241-52.

119. Wiley, R.W., J.C. Kotulak, and I. Behar, The effects of pyridostigmine bromide on visual performance. Aviat Space Environ Med, 1992. 63(12): p. 1054-9. The effects of pyridostigmine bromide (PB) on selected visual functions were measured on four healthy aviator candidates. Following a pretreatment day during which baseline measurements were completed, subjects were administered currently recommended doses (30 mg, t.i.d.) of PB for 3 d during which their visual functions were assessed using a repeated measures design. Spatial resolution ability was evaluated with high and low contrast visual acuity charts and contrast sensitivity charts at three luminance levels. Dark adaptation was evaluated by measuring visual thresholds for 40 min after a standardized retinal photopigment bleach. Also, refractive error and several oculomotor functions (lateral phoria, fusional vergence, accommodative amplitude, and pupil size) were measured. On days that the subjects ingested PB, only refractive error and pupil diameter were significantly different, and these only minimally. We conclude that the use of PB at doctrinal doses will not significantly compromise an aviator's visual ability.

120. Schneider, R., A test for the assessment of the duration of the action of neostigmine and of the relative potencies of various anticholinesterases using the pupil of the mouse. J Pharm Pharmacol, 1970. 22(4): p. 298-301.

121. Beaver, K.M. and T.J. Gavin, Treatment of acute anticholinergic poisoning with physostigmine. Am J Emerg Med, 1998. 16(5): p. 505-7. Five cases of acute anticholinergic poisoning presenting to an inner-city emergency department (ED) are discussed. All five patients presented with classic signs and symptoms of anticholinergic toxicity, which included tachycardia, hot, dry and flushed skin, markedly dilated and fixed pupils, and pronounced delirium. The patients were violently agitated, and physical restraint was required. Initial treatment with benzodiazepines did not diminish their combative behavior. Treatment with intravenous physostigmine salicylate resulted in a

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decrease in agitation within 15 to 20 minutes of therapy. No untoward effects occurred as a result of treatment with physostigmine.

122. Crowell, E.B., Jr. and J.S. Ketchum, The treatment of scopolamine-induced delirium with physostigmine. Clin Pharmacol Ther, 1967. 8(3): p. 409-14.

123. Wilhelm, B., et al., [Pupillography as an objective attention test]. Wien Med Wochenschr, 1996. 146(13-14): p. 387-9. Infrared video pupillography (IVP) allows continuous recording of spontaneous pupillary oscillations in darkness which change characteristically with fatigue. Pupil size in darkness is age-related, has its maximum in the second decade and subsequently decreases during life time. Normally, the pupil oscillates in darkness with a frequency of about 1 Hz and amplitudes of less than 0.3 mm. Excessive daytime sleepiness causes instability of this pupillary behaviour which is constant in alert normals, and so called fatigue waves appear with an amplitude reaching several millimeters. The frequency profile is dominated by slow frequencies below 0.5 Hz, while average pupil size decreases continuously with time. As IVP is an objective and time-saving method it could become an important supplement to test procedures used in sleep medicine and sleep research to measure daytime sleepiness.

124. Wilhelm, B., et al., [Pupillography for objective vigilance assessment. Methodological problems and possible solutions]. Ophthalmologe, 1996. 93(4): p. 446-50. To measure vigilance disorders in healthy normals or in patients (narcolepsy, sleep apnea syndrome) is difficult, time-consuming and hardly objective with present methods. Recording and analysis of spontaneous pupillary behaviour in darkness by infrared video pupillography is an objective and time-saving method to measure daytime sleepiness. However, certain external conditions must be satisfied (avoid light, noise, stress) to get reliable results. Spontaneous pupillary oscillations are recorded in darkness over 10 min and data are analyzed by fast Fourier transformation, with additional calculation of the mean pupillary diameter for each time segment (approx. 1 min). While in the alert normal, pupil remains dilated during the measurement in darkness and oscillates with an amplitude below 0.3 mm and a frequency about 1 Hz, there are characteristic changes in fatigue: (1) low-frequency components dominate the spontaneous pupillary oscillations, with an amplitude reaching several millimeters, and (2) pupil diameter decreases with time. Infrared video pupillography could play a role as a screening method and therapy control for hypersonic patients (most frequent: sleep apnea syndrome) with excessive daytime sleepiness. An objective, time-saving method like infrared video pupillography would be useful in sleep medicine and psychiatry when testing the level of vigilance, and in psychology or industrial medicine as well, providing informations about acute vigilance problems in healthy normals.

125. Morad, Y., et al., Pupillography as an objective indicator of fatigue. Curr Eye Res, 2000. 21(1): p. 535-42. PURPOSE: To determine whether parameters calculated from pupillary activity can identify subjects with sleep deprivation, and whether the objective values correlate with a subjective feeling of fatigue. METHODS: pupil size in the dark was recorded continuously for 10 minutes in 12 healthy volunteers using an infrared video camera. Two recordings were made for each subject: after a full night's

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sleep, and after 24 hours of sleep deprivation. Several parameters calculated from pupil size and activity were analyzed and compared with a subjective rating of the state of alertness provided by the participants in each test. RESULTS: All pupillary parameters differed significantly between alertness and fatigue (p = 0.0076-0. 0186). Changes in one of the parameters - average pupillary diameter - correlated with changes in the subjective level of sleepiness (r = -0.51, p = 0.028). Although the values of most parameters differed among subjects, an absolute value of more than 25 in one parameter, cumulative pupillary variability ratio, was always associated with sleep deprivation. CONCLUSION: On-line analysis of the pupillogram using the suggested parameters can be performed easily to produce a real-time assessment of an individual's state of alertness or fatigue that correlates with his/her subjective assessment of this state.

126. Danker-Hopfe, H., et al., Time-of-day variations in different measures of sleepiness (MSLT, pupillography, and SSS) and their interrelations. Psychophysiology, 2001. 38(5): p. 828-35. The aim of the present study is to analyze how well physiological measures of sleepiness derived from pupillography and the Multiple Sleep Latency Test correlate with a subjective measure, the Stanford Sleepiness Scale (SSS) score. The results are based on data from 12 healthy participants, who underwent these tests every 2 hr from 7:00 a.m. until 11:00 p.m. Sleep latencies were correlated with four different variables derived from pupillography and the SSS score. The results indicate that the physiologically based variables correspond very well. This is reflected by similar patterns of time-of-day variations, a good agreement at the group level, and correlations at the individual level, whereas the SSS shows a quite different pattern of variation. The two physiological measures of sleepiness seem to reflect the same aspect of the level of tonic central nervous activation, which is not correlated with the subjective feeling of sleepiness.

127. Coelho, F. and J. Birks, Physostigmine for Alzheimer's disease. Cochrane Database Syst Rev, 2001(2): p. CD001499. BACKGROUND: The main pharmacological approach for the treatment of Alzheimer's disease (AD) has been based on the use of agents potentiating cholinergic transmission, particularly by inhibiting acetylcholinesterase (AChE), the enzyme that destroys acetylcholine after it has been secreted into the synaptic clefts. Physostigmine is an AChE inhibitor originally extracted from calabar beans. It is licensed in many countries as an agent for reversing the effect of drugs and poisons causing the anticholinergic syndrome. Studies conducted more than 20 years ago suggested that physostigmine could improve memory in people with or without dementia. Investigation of this property has been limited by the very short half-life of physostigmine. Various forms of administering the drug have been tried to overcome this problem, most recently a controlled-release (CR) oral formulation, and a skin patch. It has been proposed as a potential drug for the symptomatic treatment of AD. OBJECTIVES: To determine whether there is evidence of beneficial effects for the use of physostigmine in Alzheimer's disease. To assess the incidence and severity of adverse effects. SEARCH STRATEGY: The Cochrane Controlled Trials Register was searched using the following terms: 'physostigmine', 'physostigmine salicylate', 'Synapton' and 'Antilirium' in accordance with the Cochrane Dementia and Cognitive Improvement Group's search strategy. The pharmaceutical company was contacted. SELECTION

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CRITERIA: All relevant unconfounded, double-blind, randomized, placebo-controlled trials in which physostigmine was administered for more than one day to patients with dementia of Alzheimer type. Trials in which the allocation to the treatment was not randomized, or in which the allocation to the treatment was not concealed were excluded. DATA COLLECTION AND ANALYSIS: Data were extracted independently by two reviewers (JMC & JB), pooled where appropriate and possible, and the weighted or standardized mean differences or Peto odds ratios (95% CI) were estimated. Where possible, intention-to-treat analysis was used. MAIN RESULTS: Fifteen studies were included using four different methods of administration of physostigmine. Four studies, involving 29 people in total, used intravenous infusion; seven, involving 131 people, used a conventional oral form; four, involving 1456 participants, used a controlled-release oral form, and one study of 181 people used a verum skin patch. There are no usable results from the intravenous infusion trials, and the few results from the conventional oral form showed no benefit of physostigmine compared with placebo. The results from two of the four studies of the controlled-release physostigmine apply only to a group of patients identified as responders in a pre-randomization titration period. The best dose physostigmine (mean 25mg/day) was associated with a 1.75 point improvement on ADAS-Cog score (mean difference -1.75, 95% confidence interval -2.90, -0.60 on an intention-to-treat basis) and a 0.26 point improvement on the CGIC score (treated as a continuous scale) (mean difference -0.26, 95% confidence interval 0.06, 0.46 on an intention-to-treat basis) compared with placebo at 6 weeks. There were statistically significantly higher numbers of patients from the physostigmine group withdrawing from the trial (22/183 vs 2/183)(OR 5.92, 95% confidence limits 2.59, 13.54) and suffering at least one event of nausea, vomiting, diarhoea, anorexia, dizziness, stomach pain, flatulence or sweating compared with placebo at 6 weeks. The best dose physostigmine (mean 27mg/day) was associated with a 2.0 point improvement on ADAS-Cog score (mean difference -2.02, 95% confidence interval -3.59, -0.45 on an intention to treat basis) compared with placebo at 12 weeks. There were statistically significantly higher numbers of patients from the physostigmine group withdrawing from the trial due to adverse events (13/83 vs 5/93)(OR 3.05, 95% confidence limits 1.15, 8.07) and suffering at least one event of nausea, vomiting, diarhoea, anorexia, dizziness, stomach pain, tremor, asthenia or sweating compared with placebo at 12 weeks.

When no attempt was made to identify responders and all relevant patients with Alzheimer's disease were randomized, fixed dose physostigmine (mean 33 mg/day) was associated with a statistically significantly higher number withdrawing (234/358 vs 31/117)(OR 4.82, 95% confidence limits 3.17, 7.33), withdrawing due to adverse events (196/358 vs 10/117) (OR 6.54, 95%confidence limits 4.29, 9.95) and suffering at least one event of nausea, vomiting, diarhoea, anorexia, dizziness, stomach pain, dyspepsia, sweating, asthenia, dyspnoea or abnormal dreaming compared with placebo at 24 weeks. The results from the study of the verum patch physostigmine show that the double dose (delivering mean dose 12mg/day) was associated with statistically significantly higher numbers suffering at least one adverse event of vomiting, nausea or abdominal cramps compared with placebo at 24 weeks, but placebo was associated with statistically significantly greater numbers of gastrointestinal complaints at 24 weeks compared with single-dose physostigmine. [BG: Does not specify what the placebo was] REVIEWERS'

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CONCLUSIONS: The evidence of effectiveness of physostigmine for the symptomatic treatment of Alzheimer's disease is limited. Even in a controlled release formulation designed to overcome the short half-life, physostigmine showed no convincing benefit and adverse effects remained common leading to a high rate of withdrawal.

128. Thal, L.J., et al., A 24-week randomized trial of controlled-release physostigmine in patients with Alzheimer's disease. Neurology, 1999. 52(6): p. 1146-52. OBJECTIVE: To evaluate the safety and efficacy of controlled-release physostigmine, an acetylcholinesterase inhibitor, in patients with probable AD of mild to moderate severity. METHODS: A prospective, 24-week, randomized, multicenter, double-blind, parallel group study of patients was conducted. The study enrolled 475 patients at 24 sites. Patients met criteria for probable AD and were randomized to one of three arms: placebo, controlled-release (CR) physostigmine 30 mg daily, or CR physostigmine 36 mg daily. Dosage was escalated by a forced upward titration during the first 6 to 9 weeks of the trial, then maintained at a constant dose to 24 weeks. Primary outcome measures were the Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) and the Clinician's Interview-Based Impression of Change-Plus with caregiver input (CIBIC+). Secondary outcome measures included the Clinical Global Impression of Change (CGIC), the Geriatric Evaluation by Relatives Rating Instrument, and an Instrumental Activities of Daily Living Scale. RESULTS: In an intent-to-treat population, the last observation carried forward analysis revealed a 2.9-point ADAS-Cog (p = 0.002) difference between physostigmine and placebo-treated patients for both dosages, and a 0.26 to 0.31-point difference on the CIBIC+ (p = 0.048). There were no significant differences on the secondary outcome measures except for a difference on the CGIC when analyzed by use of the Cochran-Mantel-Haenszel statistic (p = 0.014). There were significant increases in gastrointestinal side effects including nausea, vomiting, diarrhea, anorexia, dyspepsia, and abdominal pain for patients on either dose of physostigmine, resulting in a high dropout rate. Agitation was decreased significantly. There was no evidence of cardiac rhythm disturbance or liver function abnormalities. CONCLUSION: CR physostigmine enhanced cognitive and global function. It is relatively safe for the treatment of cognitive dysfunction secondary to AD. However, in light of the gastrointestinal side effects, a lower starting dose and a flexible titration schedule might lead to a more favorable adverse event profile in the clinical arena.

129. van Dyck, C.H., et al., Extended-release physostigmine in Alzheimer disease: a multicenter, double-blind, 12-week study with dose enrichment. Physostigmine Study Group. Arch Gen Psychiatry, 2000. 57(2): p. 157-64. BACKGROUND: The efficacy of extended-release physostigmine salicylate, an acetylcholinesterase inhibitor, was evaluated in 850 subjects with mild-to-moderate Alzheimer disease (AD) in a multicenter trial. METHODS: Subjects initially entered a dose-enrichment phase in which they received 1 week each of physostigmine salicylate, 24 mg/d and 30 mg/d, and daily placebo. Among the subjects who completed this phase, 35.9% responded to physostigmine treatment, whereas 62.4% were considered nonresponders, and 1.6% could not be evaluated because of missing data. After a 4-week placebo-washout phase, 176 responder subjects were randomized to receive their best dose of physostigmine or placebo in a 12-week double-blind phase. Primary efficacy measures included the

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cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-Cog), the Clinician's Interview-Based Impression of Change With Caregiver Input (CIBIC+), and the Clinical Global Impression of Change (CGIC). RESULTS: In the intent-to-treat analysis of the double-blind phase, physostigmine-treated subjects scored -2.02 points better than placebo-treated subjects on the ADAS-Cog (F1,167 = 6.42 [P = .01]) and 0.33 points higher on the CIBIC+ (F1,150 = 5.68 [P = .02]). No significant improvement was observed on the CGIC or the secondary outcome measures. Nausea and vomiting were experienced by 47.0% of all physostigmine-treated subjects during the double-blind phase. CONCLUSIONS: Physostigmine demonstrated a statistically significant benefit compared with placebo on a clinical global rating of change and an objective test of cognitive function. Given the frequency of gastrointestinal side effects, the role of this agent in clinical use remains to be determined.

130. Memory enhancement with oral physostigmine in Alzheimer's disease. N Engl J Med, 1983. 308(12): p. 720-1.

131. Aigner, T.G. and M. Mishkin, The effects of physostigmine and scopolamine on recognition memory in monkeys. Behav Neural Biol, 1986. 45(1): p. 81-7. The visual recognition of rhesus monkeys was evaluated by means of a delayed nonmatching-to-sample task with trial-unique objects. Each daily session consisted of two lists of 20 objects each, which untreated animals were able to recognize at approximately 75% accuracy. When they were performing at this level reliably, doses of physostigmine (0.32, 1.0, 3.2, 10.0, 32.0, 56.0 micrograms/kg), scopolamine (1.0, 3.2, 5.6, 10.0, 17.8, 32.0 micrograms/kg), or saline were administered 20 min prior to the session. Physostigmine and scopolamine produced dose-related increases and decreases, respectively, in the number of objects correctly remembered. The systematic changes in performance support the view that cholinergic mechanisms contribute to recognition memory and suggest that tasks with trial-unique objects may be particularly useful for studying the mnemonic effects of cholinergic drugs.

132. Aigner, T.G., et al., Effects of scopolamine and physostigmine on recognition memory in monkeys with ibotenic-acid lesions of the nucleus basalis of Meynert. Psychopharmacology (Berl), 1987. 92(3): p. 292-300. Monkeys with bilateral ibotenic-acid lesions of the nucleus basalis of Meynert, an area rich in cholinergic neurons that innervate the cerebral cortex, were compared with unoperated control monkeys on a recognition memory task. Although animals with large lesions had substantial reductions of cortical choline acetyltransferase activity, none showed impairment in the task. Lesion effects were observed, however, when performance was assessed following administration of a muscarinic receptor blocker (scopolamine) or a cholinesterase inhibitor (physostigmine). Although scopolamine produced dose-related impairments in both groups, this effect was greater in the experimental animals. Conversely, whereas physostigmine produced modest improvement in performance in the control group, no such improvement was observed in the experimental animals. The altered sensitivity to the mnemonic effects of cholinergic agents in the experimental group suggests that the cholinergic neurons of the nucleus basalis of Meynert contribute to recognition memory.

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133. Alagiakrishnan, K., W. Wong, and P.L. Blanchette, Use of donepezil in elderly patients with Alzheimer's disease--a Hawaii based study. Hawaii Med J, 2000. 59(2): p. 57-9. Donepezil (Aricept) is a reversible acetylcholinesterase inhibitor which is indicated for the treatment of mild to moderate dementia of the Alzheimer's type. We did a retrospective analysis of 41 elderly Alzheimer's subjects of different ethnic groups including a large number of Asian and Hawaiian patients. Donepezil appears to be clinically effective in patients of different ethnicities with mild to moderate Alzheimer's disease, even at advanced age.

134. Almkvist, O., et al., Responder characteristics to a single oral dose of cholinesterase inhibitor: a double-blind placebo-controlled study with tacrine in Alzheimer patients. Dement Geriatr Cogn Disord, 2001. 12(1): p. 22-32. A proportion of Alzheimer's disease (AD) patients treated for several months with cholinesterase (ChE) inhibitors have shown some favorable response on cognition, but the characteristics of the responders are still unclear. This study attempts to identify the characteristics of individuals with a positive behavioral response after a double-blind randomized administration of a single oral dose of tacrine (40 mg) and placebo to AD patients. Furthermore, the relationship between single-dose and long-term responders are examined. Twenty-four mildly to very mildly demented AD patients participated in the study. They all fulfilled the diagnosis of probable AD according to NINCDS-ADRDA criteria. Active treatment (tacrine 40 mg) and placebo was administered in random order on 2 consecutive days, and the effects were evaluated within 2 h using neuropsychological tests (assessing visuospatial ability, episodic memory and attention), registration of EEG activity and measurement of red blood cells (RBC) acetylcholinesterase (AChE), ChE activity and concentrations of tacrine and its metabolites in plasma. Results demonstrated significant improvement, tacrine compared to placebo, in measures of attention, but not in episodic memory or visuospatial ability. A single-dose response was therefore defined in terms of improvement in attention. The tacrine plasma concentration (pcTHA) showed a positively skewed distribution (mean +/- SD: 10.5 +/- 11.8, range: 1.0-51.8 ng/ml). There were no significant differences between single-dose responders compared to nonresponders in pcTHA, metabolites of tacrine, inhibition of AChE in RBC, tau levels in CSF, AChE activity in CSF or plasma and demographic variables. However, single-dose responders showed a higher right frontal alpha/theta ratio on EEG and had lower glucose metabolism in the parietal-temporal association cortex at baseline. In addition, the frequency of apolipoprotein E (APOE) epsilon 4 alleles was higher in responders. Interestingly, the single-dose response was related to the long-term response, although not significantly, which probably was due to lack of power. To conclude, the present study identified single-dose responders in terms of improved attentional performance associated with a relatively higher alpha/theta activity in the right frontal regions of the brain measured on EEG and predominance of APOE epsilon 4 allele.

135. Arsland, D. and K. Laake, [Should Alzheimer disease be treated with tacrine? Review of the literature]. Tidsskr Nor Laegeforen, 1996. 116(23): p. 2791-4. The authors describe the rationale for use of the acetylcholinesterase inhibitor, tacrine, in Alzheimer's disease and critically review the controlled clinical trials using this drug. The data shows

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that tacrine improves cognition and function, and that 20-40% of the patients may benefit from such treatment. A large proportion of the patients do not tolerate the highest dose, mainly because of an increase in serum liver-enzymes, which is reversible and asymptomatic. The authors recommend that tacrine should be considered for patients with Alzheimer's disease of mild and moderate severity, and present guidelines for clinical use.

136. Arendt, T., M.M. Schugens, and V. Bigl, The cholinergic system and memory: amelioration of ethanol-induced memory deficiency by physostigmine in rat. Acta Neurobiol Exp (Warsz), 1990. 50(4-5): p. 251-61. Male Sprague-Dawley rats were treated with a solution of ethanol (20% v/v) as the only source of fluid for 28 weeks while controls received tap water. Profound reductions of acetylcholine content, the activities of choline acetyltransferase and acetylcholinesterase and choline uptake were observed in the cortex, hippocampus, substantia innominata and striatum four weeks after withdrawal from ethanol. These changes in cholinergic markers were accompanied by an impaired performance in a spontaneous alternation task tested at long acquisition-retention intervals. This ethanol induced behavioural deficiency, which most likely represents a memory impairment, could be reversed by application of physostigmine (0.45 g/kg; i.p) whereas neostigmine did not show any behavioural effect. The results suggest that ethanol-induced memory impairment can be ameliorated by pharmacological manipulation of central cholinergic function.

137. Ashford, J.W., et al., Physostigmine and its effect on six patients with dementia. Am J Psychiatry, 1981. 138(6): p. 829-30.

138. Asthana, S., et al., Clinical pharmacokinetics of physostigmine in patients with Alzheimer's disease. Clin Pharmacol Ther, 1995. 58(3): p. 299-309. OBJECTIVE: To study the pharmacokinetic and pharmacodynamic properties of physostigmine in subjects with Alzheimer's disease. METHODS: Plasma physostigmine concentration and butyrylcholinesterase inhibition were measured in blood samples collected during and after a single high-dose (1 to 1.5 mg for 45 to 60 minutes) and a sustained low-dose steady-state intravenous infusion in nine subjects with Alzheimer's disease. Escalating doses (0.5 to 25 mg/day) were administered during a 2-week period. A dose (2 to 12 mg/day) that optimized cognition in each subject was identified and then administered in a randomized, double-blind, placebo-controlled crossover design for 1 week. RESULTS: The elimination half-life of physostigmine was 16.4 +/- 3.2 (SE) minutes. Clearance and volume of distribution were 7.7 +/- 0.9 (SE) L/min and 2.4 +/- 0.6 (SE) L/kg, respectively. Butyrylcholinesterase inhibition half-life was 83.7 +/- 5.2 (SE) minutes. During sustained steady-state infusion, plasma physostigmine concentration (r = 0.95) and butyrylcholinesterase inhibition (r = 0.99) were linearly correlated with the dose. In five cognitive responders, the memory enhancement was significantly correlated (r = 0.86; p < 0.05) with butyrylcholinesterase inhibition. CONCLUSIONS: These results showed that, in cognitive responders, memory enhancement by physostigmine in Alzheimer's disease is correlated directly to the magnitude of plasma cholinesterase inhibition. Furthermore, during single-dose conditions, the dynamic half-life is five-fold longer than the kinetic half-life.

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139. Lacey, S.C., et al., Evaluation of working memory in Persian Gulf War veterans with chronic fatigue [abstract]. Proceedings of the Conference on Federally Sponsored Gulf War Veteransí Illnesses Research, Pentagon City, Jun 1999, 133.134, 1999. thus, for instance, in a set of PGW veterans with chronic fatigue, slower reaction times were found only during a dual-task condition (p < 0.007), suggesting to the authors that there may be ìinefficient allocation of attentional resources necessary to process simultaneous tasks adequately.

140. Leclercq, M., et al., Dual task performance after severe diffuse traumatic brain injury or vascular prefrontal damage. J Clin Exp Neuropsychol, 2000. 22(3): p. 339-50. The ability to perform two tasks simultaneously is a key function of the central executive of working memory (Baddeley, 1986). This study addressed dual-task performance after diffuse very severe traumatic brain injury (TBI) (mean coma duration = 21 days, mean post-traumatic amnesia = 70 days) or prefrontal damage due to a ruptured aneurysm of the anterior communicating artery (AACA). Mean time since injury was 8 and 16 months in the TBI and the AACA group respectively. A simple visual reaction time and random number generation were used as single and dual tasks. Randomization was self-paced, to control for individual differences in speed. Both patient groups had greater reaction time decrements than controls under the dual-task condition, suggesting a divided attention deficit. In addition, patients with AACA performed significantly poorer in random generation. These results suggest that patients with AACA and with severe TBI suffer from an impairment of the central executive system.

141. Overstreet, D.H., et al., Potential animal model of multiple chemical sensitivity with cholinergic supersensitivity. Toxicology, 1996. 111(1-3): p. 119-34. Multiple Chemical Sensitivity (MCS) is a clinical phenomenon in which individuals, after acute or intermittent exposure to one or more chemicals, commonly organophosphate pesticides (OPs), become overly sensitive to a wide variety of chemically-unrelated compounds, which can include ethanol, caffeine and other psychotropic drugs. The Flinders Sensitive Line (FSL) rats were selectively bred to be more sensitive to the OP diisopropylfluorophosphate (DFP) compared to their control counterparts, the Flinders Resistant Line (FRL) rats. The present paper will summarize evidence which indicates that the FSL rats exhibit certain similarities to individuals with MCS. In addition to their greater sensitivity to DFP, the FSL rats are more sensitive to nicotine and the muscarinic agonists arecoline and oxotremorine, suggesting that the number of cholinergic receptors may be increased, a conclusion now supported by biochemical evidence. The FSL rats have also been found to exhibit enhanced responses to a variety of other drugs, including the serotonin agonists m-chlorophenylpiperazine and 8-OH-DPAT, the dopamine antagonist raclopride, the benzodiazepine diazepam, and ethanol. MCS patients report enhanced responses to many of these drugs, indicating some parallels between FSL rats and MCS patients. The FSL rats also exhibit reduced activity and appetite and increased REM sleep relative to their FRL controls. Because these behavioral features and the enhanced cholinergic responses are also observed in human depressives, the FSL rats have been proposed as a genetic animal model of depression. It has also been reported that MCS patients have a greater incidence of depression, both before and after onset of

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their chemical sensitivities, so cholinergic supersensitivity may be a state predisposing individuals to depressive disorders and/or MCS. Further exploration of the commonalities and differences between MCS patients, human depressives, and FSL rats will help to elucidate the mechanisms underlying MCS and could lead to diagnostic approaches and treatments beneficial to MCS patients.

142. Servatius, R.J., et al., Persistently exaggerated startle responses in rats treated with pyridostigmine bromide. J Pharmacol Exp Ther, 1998. 287(3): p. 1020-8. Troops in the Persian Gulf War have registered complaints consistent with CNS dysfunction that emerged after returning from the Gulf. A common experience among Persian Gulf War veterans was exposure to pyridostigmine bromide (PB) for prophylaxis against nerve gas exposure. To determine whether PB causes emergent CNS dysfunction, Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats were given PB for 7 consecutive days in their drinking water. The WKY, but not the SD, rats exhibited a delayed-onset, persistently exaggerated startle response. The WKY rats exhibited exaggerated startle responses that appeared 15 days after the end of PB treatment and were still evident 22 days after the end of treatment. Both the duration and the magnitude of the exaggerated startle responses were related to the dosage of PB. The PB-treated rats exhibited normal short-term and long-term habituation. However, exaggerated startle responses were related to the development of enhanced short-term sensitization. Treating the rats for a second time, 7 weeks after the end of the first PB treatment, induced an exaggerated startle response that appeared sooner and dissipated faster than was evident after the first PB treatment. Inasmuch as the WKY rat has inherently low butyrylcholinesterase activity, a scavenger for PB, these results suggest that prophylactic PB may influence CNS function in individuals with low butyrylcholinesterase activity. Elaboration of the factors that mediate enhanced sensitization in the WKY rat may provide insight into some of the complaints registered by veterans of the Persian Gulf War.

143. Janowsky, D.S., et al., Parasympathetic suppression of manic symptoms by physostigmine. Arch Gen Psychiatry, 1973. 28(4): p. 542-7.

144. Janowsky, D., et al., Increased vulnerability to cholinergic stimulation in affective disorder patients. Psychopharmacol Bull, 1980. 16: p. 29-31.

145. Janowsky, D.S., et al., Cholinergic supersensitivity in affect disorder patients: Behavioral and neuroendocrine observations. Psychopharmacol Bull, 1981. 17: p. 129-132.

146. Janowsky, D., C. Risch, and J. Gillin, Adrenergic-cholinergic balance and the treatment of affective disorders. Prog Neuropsychopharmacol Biol Psychiatry, 1983. 7: p. 297-307.

147. Janowsky, D. and C. Risch, Cholinomimetic and anti cholinergic drugs used to investigate an acetylcholine hypothesis of affective disorders adn stress. Drug Dev Res, 1984.

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148. Bannon, A.W., K.L. Gunther, and M.W. Decker, Is epibatidine really analgesic? Dissociation of the activity, temperature, and analgesic effects of (+/-)-epibatidine. Pharmacol Biochem Behav, 1995. 51(4): p. 693-698. The experiments in the present study were designed to determine if

the activity, temperature, and analgesic effects of (+/-)-epibatidine

treatment could be dissociated. Initially (i.e., 15 min) (+/-)-epibatidine

treatment (0.1 mumol/kg = 28 micrograms/kg, IP) impaired rotorod

performance, decreased activity, decreased temperature, and increased jump

latency (e.g., analgesic effect). For the remaining time points measured

(i.e., 30, 60, and 120 min), activity and temperature remained

significantly reduced. In contrast, by 120 min (+/-)-epibatidine's effects

on rotorod performance and analgesia (jump latency) were not observed. When

administered after (+/-)-epibatidine (0.05 mumol/kg, IP), mecamylamine

treatment (5 mumol/kg = 1 mg/kg, IP) produced a potentiation of analgesia.

This potentiation effect was not observed on activity and temperature

measures. The effect of (+/-)-epibatidine treatment (0.1 mumol/kg, IP) was

also determined in mice with central nicotinic receptor blockade induced by

treatment with chlorisondamine (23 mumol/kg = 10 mg/kg, IP). An

(+/-)-epibatidine-induced reduction in activity was not attenuated in

chlorisondamine-treated mice and only a minimal effect was observed on

(+/-)-epibatidine-induced hypothermia in chlorisondamine-treated mice. In

contrast, in chlorisondamine-treated mice (+/-)-epibatidine's analgesic

effect was attenuated. Taken together, these data suggest that various

centrally mediated effects of (+/-)-epibatidine can be dissociated.

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149. Avlonitou, E. and R. Elizondo, Effects of atropine and pyridostigmine in heat-stressed patas monkeys. Aviat Space Environ Med, 1988. 59(6): p. 544-8. The effects of a single intramuscular atropine injection (0.03 mg.kg-1) and a chronic oral pyridostigmine treatment (0.4 mg.kg-1, 3 times/day over a period of 7 d) on the thermoregulatory effector responses of unanesthetized patas monkeys were investigated using indirect calorimetry. The effects of atropine treatment on the thermoregulatory effector responses of patas monkeys exposed to 25 degrees and 35 degrees C were qualitatively similar but quantitatively greater at 35 degrees C. At 35 degrees C atropine decreased sweating (Esw) 52%, increased rectal temperature (Tre), mean skin temperature (Tsk), metabolic rate (MR), and whole body conductance (K), and elicited a consistent 11% increase in heart rate (HR). Daily oral pyridostigmine treatment to patas monkeys produced a significant 25-30% drop in serum cholinesterase activity with no chronic effects on thermoregulatory or cardiovascular functions. The acute effects of oral pyridostigmine treatment in this species included transient 12% and 15% decreases in MR and HR, respectively, and a transient 25% increase in Esw. The latter was associated with significant acute reductions in Tre and Tsk which lasted at least 120 min following pyridostigmine administration. It is concluded that the patas monkey is an excellent animal model for studies to evaluate the effects of neuroactive agents on thermoregulatory and other physiological functions which are difficult, if not impossible, to perform on humans.

150. Barar, F.S. and B.R. Madan, Tremorine-oxotremorine-induced tremor, hypothermia and analgesia, and physostigmine toxicity, in mice after pretreatment with beta-adrenoceptor antagonists. J Pharm Pharmacol, 1976. 28(4): p. 286-9. The beta-adrenoceptor blockers propranolol, PhQA33 and LB-46 exhibited appreciable activity against tremorine-(TMN) and oxotremorine-(OTMN) induced tremor, whereas pronethalol, (+)-H56/28, (-)-H56/28, Ko-592 and L(+)-INPEA possessed weak action. The two beta-blockers, namely D,L(+/-)-INPEA and D(-)-INPEA acted as weak tremorgens. None of the above compounds suppressed the induced peripheral cholinergic phenomena; or possessed any central anticholinergic activity, as they were unable to afford protection against physostigmine-induced death. Propranolol, PhQA33 and LB-46 antagonized TMN-induced hypothermia and analgesia, but were inactive against OTMN-induced changes. A correlation of the beta-blocking and anti-tremor activity of these agents is unlikely.

151. Clement, J.G., Variability of sarin-induced hypothermia in mice: investigation into incidence and mechanism. Biochem Pharmacol, 1991. 42(6): p. 1316-8.

152. Francesconi, R., et al., Oral pyridostigmine administration in rats: effects on thermoregulation, clinical chemistry, and performance in the heat. Pharmacol Biochem Behav, 1986. 25(5): p. 1071-5. We have recently reported that acute intraperitoneal administration of pyridostigmine bromide to rats resulted in significant decrements in physical performance in the heat, adverse thermoregulatory effects, and exacerbated elevations in several indices of heat/exercise injury. Since it will be consumed orally as a prophylaxis for organophosphate poisoning, pyridostigmine was dissolved in the drinking water of rats. Consumption of pyridostigmine for 7 days (n = 34, 6.6 mg/day) resulted in

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a 23% (p less than 0.001) reduction of circulating cholinesterase when compared with a control group (n = 31) while ingestion for 14 days (n = 35, 8.9 mg/day) elicited a 39% (p less than 0.001) inhibition of circulating cholinesterase when compared to a second control group (n = 33). Water and food consumption, rate of weight gain, and overt behavior were unaffected by pyridostigmine consumption. When approximately half the animals in each group were exercised (9.14 m/min) in the heat (35 degrees C) to hyperthermic exhaustion (Tre = 42.5-43 degrees C, rats unable to right themselves), pyridostigmine consumption for 14 days effected a significantly (p less than 0.05) increased rate of weight loss, but no further effects on thermoregulation or performance were noted. Several minor increments were observed in clinical indices of heat/exercise injury in rats consuming pyridostigmine for 14 days. These data indicate that oral dosages of pyridostigmine can probably be titrated to levels of cholinesterase inhibition which are efficacious in prophylaxis against organophosphate toxicity without significant effects on selected physiologic and metabolic processes.

153. Gordon, C.J., 24-hour control of body temperature in the rat: II. Diisopropyl fluorophosphate-induced hypothermia and hyperthermia. Pharmacol Biochem Behav, 1994. 49: p. 747-754.

154. Epstein, Y., et al., Heat-exercise performance of pyridostigmine-treated subjects wearing chemical protective clothing. Aviat Space Environ Med, 1990. 61(4): p. 310-3. Pyridostigmine bromide is currently the pretreatment of choice for operation in a chemical warfare (CW) environment. Under CW conditions, subjects are exposed to thermal stress caused by CW protective clothing. This investigation was conducted to determine if pyridostigmine affects various physiological and biophysical parameters of human temperature regulation in subjects wearing CW protective clothing. Pyridostigmine was administered orally in a randomized double-blind cross-over study in four doses of 30 mg every 8 h. An average of 33% whole blood cholinesterase inhibition was induced in the pyridostigmine treated group 4 h after ingestion of last tablet. The subjects were exposed to 170 min exercise-heat stress (Tdb = 33 degrees C; rh = 60%) consisting of 60 min in a sitting position and two 50-min walks (1.39 m.s-1, 5% grade) separated by 10 min of rest. Non-evaporative heat exchange was significantly higher, -14.0 and -10.6 W.m-2 (p less than 0.03), for the pyridostigmine-treated subjects. No additional differences were found between treatments in the physiological responses and heat balance parameters at the end of exposure: heart rate (HR) was (mean +/- S.D.) 154 +/- 16 and 151 +/- 24 bpm, rectal temperature (Tre) was 39.0 +/- 0.4 and 38.9 +/- 0.2 degrees C, heat storage over the 2 h of exercise was 62 +/- 15 and 70 +/- 15 W.m-2, and sweat rate was 832 +/- 185 and 748 +/- 52 g.h-1, in the pyridostigmine and placebo treatments, respectively.(ABSTRACT TRUNCATED AT 250 WORDS).

155. Epstein, Y., et al., Effect of pyridostigmine on the exercise-heat response of man. Eur J Appl Physiol Occup Physiol, 1990. 61(1-2): p. 128-32. The effect of pyridostigmine on thermoregulatory responses was evaluated during exercise and heat stress. Eight heat acclimated, young adult male subjects received four doses of pyridostigmine (30 mg) or identical placebo tablets every 8 h, in a double blind, randomized, cross-over trial. A 30.3%, SD 4.6% inhibition of the circulating

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cholinesterase (ChE) activity was induced in the pyridostigmine-treated group. The subjects were exposed to 170-min exercise and heat-stress (dry bulb temperature, 33 degrees C; relative humidity 60%) consisting of 60 min in a sitting position and two bouts of 50-min walking (1.39 m.s-1, 5% gradient) which were separated by 10-min rest periods. No differences were found between treatments in the physiological responses and heat balance parameters at the end of exposure: heart rate (fc) was 141 beats.min-1, SD 16 and 150 beats.min-1, SD 12, rectal temperature (Tre) was 38.5 degrees C, SD 0.4 degrees and 38.6 degrees C, SD 0.3 degrees, heat storage was 60 W.m-2, SD 16 and 59 W.m-2, SD 15 and sweat rate was 678 g.h-1, SD 184 and 661 g.h-1, SD 133, in the pyridostigmine and placebo treatments, respectively. The changes in Tre and fc over the heat-exercise period were parallel in both study and control groups. Pyridostigmine caused a slight slowing of fc (5 beats.min-1) which was consistent throughout the entire exposure (P less than 0.001) but was of no clinical significance. The overall change in fc was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS).

156. Kassa, J., et al., The long-term influence of low-level sarin exposure on behavioral and neurophysiological functions in rats. Acta Medica (Hradec Kralove), 2001. 44(1): p. 21-7. 1. Long term effects of low doses of highly toxic organophosphorus agent sarin on behavioral and neurophysiological functions were studied in rats exposed to sarin by inhalation. The toxic effects of sarin were monitored using a functional observational battery (FOB), an automatic measurement of motor activity and a test of excitability of central nervous system at 3, 6 and 12 months following sarin exposure. 2. The results indicate that sarin at symptomatic as well as asymptomatic doses (level 2 and 3) is able to induce some neurotoxic effects (a decrease in activity and mobility, an alteration of gait, an increase in stereotyped behavior) including an increase in the excitability of central nervous system (an increase in convulsive activity following the administration of pentamethylenetetrazole) in rats at 3 months following inhalation exposure. Some sings of increased excitability were also observed in sarin-exposed rats following 6 or 12 months (an increase in exploratory activity, body temperature and a hindlimb grip strength at 6 months following exposure to sarin at asymptomatic doses, an increase in tail-pinch response at 12 months following exposure to sarin at symptomatic doses). 3. Therefore, nerve agents such as sarin seem to be harmful not only at high, clinically symptomatic doses but also at low, clinically asymptomatic doses because of long term manifestation of alteration of neurophysiological functions in sarin-exposed rats without disruption of cholinergic nervous system.

157. Kolka, M.A. and L.A. Stephenson, Human temperature regulation during exercise after oral pyridostigmine administration. Aviat Space Environ Med, 1990. 61(3): p. 220-4. Four healthy males exercised in two experiments at ambient temperatures of 22, 29, and 36 degrees C with the relative humidity at 30% in all environments (Tdp = 3.9, 9.9, and 15.8 degrees C). One experiment in each environment was done 150 min after 30 mg oral pyridostigmine bromide (PYR) administration, and the second experiment was done on a separate day with no medication (CON). Red blood cell cholinesterase was 39 +/- 7% lower after PYR (11.8 vs 7.2 micromol.ml-1.min-1). Esophageal (Tes) and mean skin temperature (Tsk), forearm blood flow (FBF), forearm sweating, and skin blood flow (SkBF) were measured twice each minute during a 15-min

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rest period and during 30-min of seated cycle exercise at approximately 58% Vo2peak. Whole body sweating was determined from weight changes before and after exercise. PYR decreased heart rate at rest and during exercise at 29 degrees C and 36 degrees C (8bpm, p less than 0.05). Resting SkBF was 40% lower at 29 degrees C and 30% lower at 36 degrees C after PYR compared to CON (p less than 0.05). There was no effect of PYR on heat production at rest or during exercise. Tsk was different in the three conditions by design, but was unchanged by PYR. Tes was not different at rest in any condition, but was elevated during exercise at 36 degrees C (0.1 degree C, p less than 0.05) in PYR compared to CON. These data suggest that pyridostigmine ingestion decreased skin blood flow, which may limit exercise thermoregulation in more severe environments.

158. Lin, M.T., H.C. Wang, and A. Chandra, The effects on thermoregulation of intracerebroventricular injections of acetylcholine, pilocarpine, physostigmine, atropine and hemicholinium in the rat. Neuropharmacology, 1980. 19(6): p. 561-5.

159. Matthew, C.B., R.W. Hubbard, and R.P. Francesconi, Atropine, diazepam, and physostigmine: thermoregulatory effects in the heat-stressed rat. Life Sci, 1989. 44(25): p. 1921-7. We have previously reported that administration of atropine (A) to unrestrained, sedentary, heat-stressed rats resulted in a dose-dependent increase in heating rate (rate of rise of core temperature, degree C/min). Additionally, we have demonstrated that the decrements in treadmill endurance and increments in heating rate of physostigmine (PH)-treated running rats can both be restored to control levels by pretreating the animals with A and diazepam (D). Our objective in the present work was to determine if the administration of D + PH to A-treated unrestrained, sedentary, heat-stressed rats (N = 16/group, 510-530 g) could improve their thermal tolerance. The following drugs were administered singly (at 10 min intervals) via lateral tail vein: vehicle-control (C), A (200 micrograms/kg), D (500 micrograms/kg), and PH (200 micrograms/kg). After drug administration, the rats were heat-stressed (Tamb = 41.5 degrees C) until a core temperature of 42.6 degrees C was attained when they were removed to a 26 degrees C chamber. The heating rates (degrees C/min) and tolerance times (min) of the respective groups were: C- 0.02, 235; A- 0.08, 58; A D- 0.06, 94; and A + D + PH- 0.04, 143. Administration of D with A significantly decreased heating rate, and D + PH more than doubled the thermal tolerance of A-treated rats. Thus, the combination of A + D + PH not only restores PH-induced performance and thermoregulatory decrements of rats exercised in a moderate environment, but also reduces A-induced heat intolerance.

160. Matthew, C.B., R.P. Francesconi, and R.W. Hubbard, Physostigmine: dose-response effects on endurance and thermoregulation during exercise. Life Sci, 1992. 50(1): p. 39-44. We previously reported that the administration of 200 micrograms/kg of physostigmine (PH) to rats exercising on a treadmill resulted in decrements in both endurance (decreased running time to exhaustion) and thermoregulation. However, it was necessary to determine the dose-response effects of PH administration before PH-treated exercising rats could be used as a model with which to examine the relative anticholinergic potency of drugs. In the present work saline, 50, 100, or 200 micrograms/kg of physostigmine salicylate (0%, 40%, 50%, and 60% whole blood

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cholinesterase inhibition) was administered to rats (N = 12/group) prior to treadmill exercise (26 degrees C, 50% rh, 11 m/min, 6 degrees incline). The saline control group ran for 67 +/- 6 min (mean +/- SE) with a rate of rise of core temperature of 0.051 +/- 0.007 degrees C/min. The run times declined (80%, 64% and 48% of control) as rate of rise of core temperature increased (116%, 180%, and 214% of control) in a dose-dependent manner (50, 100, 200 micrograms/kg PH). Cholinergic symptoms such as salivation, tremors, and defecation were also affected in a dose-dependent manner by PH administration. Since cholinergic symptoms, thermoregulatory effects, and endurance decrements all vary in a dose-dependent manner with physostigmine administration, the exercising rat represents a useful model for examining the relative potency of cholinergic therapies.

161. Maickel, R.P., et al., Antagonism of physostigmine induced hypothermia and neuroendocrine changes following exposure to different environmental temperatures. Prog Neuropsychopharmacol Biol Psychiatry, 1991. 15(6): p. 873-84. 1. The magnitude of physostigmine-induced hypothermia increased with decreasing environmental temperature. 2. The hypothermic response was accompanied by significant changes in plasma levels of corticosterone, glucose and fatty acids. 3. Central cholinergic mediation appears to be a significant component of physostigmine-induced hypothermia and neuroendocrine changes at moderate temperature. 4. At lower ambient temperatures cholinergic blockers produced less antagonism of physostigmine-induced effects. 5. The decreased effectiveness of cholinergic blockers at low environmental temperatures and the increased plasma fatty acid levels under almost all conditions studied may be of importance in considering long term therapy with cholinergic agonists.

162. Monda, M., A. Viggiano, and V. De Luca, Administration of muscimol into the posterior hypothalamus reduces hyperthermia induced by hippocampal neostigmine injection. Brain Res, 2000. 887(2): p. 344-9. The firing rate of the sympathetic nerves innervating interscapular brown adipose tissue (IBAT), IBAT and colonic temperatures (T(IBAT) and T(C)) and oxygen (O(2)) consumption were monitored in urethane-anesthetized male Sprague-Dawley rats. These variables were measured for 40 min before (baseline values) and 40 min after an injection of neostigmine (5 x 10(-7) mol in 1 microl of saline) into the hippocampus and a bilateral administration of a GABA(a)-agonist, muscimol (28 ng in 0.5 microl of saline, per side) into the posterior hypothalamus. The same variables were recorded in other rats, but the muscimol was replaced by saline. Control animals were used with muscimol or saline alone. The results show an increase of sympathetic firing rate, T(IBAT), T(C) and O(2) consumption after neostigmine injection. Muscimol significantly reduces this enhancement. The findings suggest that hippocampus controls the sympathetic and thermogenic activation induced by neostigmine through an influence on GABAergic tone of the posterior hypothalamus.

163. Prusaczyk, W.K. and M.N. Sawka, Effects of pyridostigmine bromide on human thermoregulation during cold water immersion. J Appl Physiol, 1991. 71(2): p. 432-7. This study examined the effects of an oral 30-mg dose of pyridostigmine bromide (PYR) on thermoregulatory and physiological responses of men undergoing cold stress. Six men were immersed in cold water (20 degrees C) for up to 180 min on two occasions, once

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each 2 h after ingestion of PYR and 2 h after ingestion of a placebo. With PRY, erythrocyte cholinesterase inhibition was 33 +/- 12% (SD) 110 min postingestion (10 min preimmersion) and 30 +/- 7% at termination of exposure (mean 117 min). Percent cholinesterase inhibition was significantly related to lean body mass (r = -0.91, P less than 0.01). Abdominal discomfort caused termination in three of six PYR experiments but in none of the control experiments (mean exposure time 142 min). During immersion, metabolic rate, ventilatory volume, and respiratory rate increased significantly (P less than 0.05) over preimmersion levels and metabolic rate increased with duration of immersion (P less than 0.01) in both treatment but did not differ between conditions. PYR had no significant effect on rectal temperature, mean body temperature, thermal sensations, heart rate, plasma cortisol, or change in plasma volume. It was concluded that a 30-mg dose of PYR does not increase an individual's susceptibility to hypothermia during cold water immersion; however, in combination with cold stress, PYR may result in marked abdominal cramping and limit cold tolerance.

164. Roberts, D.E., et al., Pyridostigmine bromide does not alter thermoregulation during exercise in cold air. Can J Physiol Pharmacol, 1994. 72(7): p. 788-93. This study examined the effects of acute and chronic pyridostigmine bromide (PB) administration on thermoregulatory and metabolic responses to exercise in cold air (5 degrees C). Seven healthy men completed two 7-day trials in a double-blind, crossover experimental design: during one trial they received PB (30 mg three times daily) and during the other trial they received placebo. For each trial, subjects attempted four (3 h) exercise tests: low-intensity exercise (approximately 25% VO2max) and moderate-intensity exercise (approximately 50% VO2max), on days 2 and 3 and again on days 6 and 7. Metabolic rate, body temperatures, and venous blood samples were obtained before and during exercise. Red blood cell acetylcholinesterase inhibition induced by PB increased (p < 0.05) from 34% on day 1 to 43% on days 3-7. Metabolic rate, body temperatures, and regional heat conductance responses were not different between trials. Plasma glucose, glycerol, free fatty acid, lactate, sodium, and potassium concentrations were not different between trials. In addition, differences were not found between acute and chronic experiments for any thermoregulatory or metabolic responses. These findings demonstrate that the PB dosage used by military personnel, as a pharmacological defense against nerve-agent poisoning, should not cause any adverse thermoregulatory or metabolic effects during moderate activity in cold climates.

165. Sharkawi, M., Morphine hyperthermia in the rat: its attenuation by physostigmine. Br J Pharmacol, 1972. 44(3): p. 544-8.

166. Stephenson, L.A. and M.A. Kolka, Acetylcholinesterase inhibitor, pyridostigmine bromide, reduces skin blood flow in humans. Am J Physiol, 1990. 258(4 Pt 2): p. R951-7. Five subjects exercised on a cycle ergometer for 30 min at 55% peak oxygen consumption on two occasions in an environmental test chamber (ambient temperature = 29 degrees C; dew point temperature = 10 degrees C). Pyridostigmine bromide (PYR), an acetylcholinesterase (AChE) inhibitor, was ingested (30 mg) approximately 150 min before one experiment, and no drug was administered during the other experiment (control). Red blood cell AChE inhibition averaged 40 (+/- 7)% during PYR treatment.

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Esophageal temperature (Tes), an eight site-derived mean skin temperature, forearm blood flow (FBF; venous occlusion plethysmography), skin blood flow (SkBF; laser-Doppler velocimetry), and metabolic rate (indirect calorimetry) were measured. SkBF decreased 37% after PYR treatment compared with control (P less than or equal to 0.05). The Tes threshold for initiation of cutaneous vasodilation was 36.8 (+/- 0.3) degrees C for the control treatment and 37.0 (+/- 0.3) degrees C for the PYR treatment (P less than or equal to 0.01). FBF was not significantly different between treatments, whereas heart rate was reduced by 7 and 9 beats/min during rest and exercise, respectively (P less than or equal to 0.01). The increased threshold for initiation of cutaneous vasodilation with AChE inhibition by PYR is compatible with nonthermal modulation of the control of thermoregulation through increased acetylcholine (ACh) accumulation. This could potentiate preganglionic transmission to enhance adrenergic vasoconstrictor tone. One suggested mechanism possible at the neuroeffector junction of the sweat gland may be that accumulated ACh diffusion across the adventitia of adjacent arterioles to muscarinic receptors initiates contraction of the smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS).

167. Wenger, C.B. and W.A. Latzka, Effects of pyridostigmine bromide on physiological responses to heat, exercise, and hypohydration. Aviat Space Environ Med, 1992. 63(1): p. 37-45. Five men underwent eight heat stress tests (HSTs) at 35 degrees C, each consisting of four 25-min treadmill walks (35% Vo2max), separated by 5-min rests, in four conditions: 1) 20% relative humidity (rh), subjects euhydrated and drinking ad libitum; 2) 20% rh, euhydrated; 3) 75% rh, euhydrated; and 4) 20% rh, hypohydrated 3% of body weight. In Conditions 2-4 subjects drank during the walks to maintain their pre-HST weight. In each condition we tested subjects once after 30 mg pyridostigmine bromide (PB) by mouth and once after placebo. PB lowered heart rate a mean of 3 beats/min overall, most with hypohydration. PB did not significantly affect rectal temperature (Tre), but reduced the rise in Tre during hypohydrated exercise. In Condition 2, chest skin temperature decreased more during exercise with PB. PB had no significant effect on other skin temperatures, sweating, hematocrit, hemoglobin, total plasma protein, osmolality, ad libitum drinking, rate of O2 uptake, or subject ratings of temperature, discomfort, or exertion. PB thus had little effect on physiological responses to moderate exercise-heat stress.

168. Wenger, B., M.D. Quigley, and M.A. Kolka, Seven-day pyridostigmine administration and thermoregulation during rest and exercise in dry heat. Aviat Space Environ Med, 1993. 64(10): p. 905-11. Seven men participated in a double-blind study of effects of multiple-dose oral pyridostigmine bromide (PB) on physiological responses to 4-h heat stress tests (HST's) in a hot dry environment, 42 degrees C, 20% relative humidity. Subjects underwent 2 7-d series of tests, separated by 72 h, taking 30 mg PB every 8 h in one series, and placebo in the other. Each HST began right after the 0800 dose of PB or placebo. Subjects drank ad libitum during each HST, and performed two 55-min treadmill walks at about 40% VO2max during the last 2 h. Inhibition of red cell cholinesterase at the start of exercise averaged 30.0% in subjects taking PB, and did not differ significantly among HST's with PB. PB increased sweating and evaporative water loss by about 4%, and lowered chest skin temperature during exercise by 0.7 degrees C;

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but it had no significant effect on rectal temperature, other skin temperatures, O2 uptake, or fluid balance. PB alone had no significant effect on heart rate (HR), but had a significant interaction with day: although PB had essentially no effect on HR in the 1st HST, its effect increased progressively so that HR during exercise in the 4th HST was 8 beats.min-1 lower with PB. Multiple-dose PB had only slight effects on responses to moderate exercise-heat stress beyond those described after single-dose PB, and we found no adverse effects of multiple-dose PB administration.

169. Billet, S., N.B. Cant, and W.C. Hall, Cholinergic projections to the visual thalamus and superior colliculus. Brain Res, 1999. 847(1): p. 121-3. The parabrachial region of the brainstem reticular formation projects to the dorsal lateral geniculate nucleus of the thalamus and to the intermediate gray layer of the superior colliculus. We used the retrograde axonal transport of two fluorescent labels to demonstrate that individual parabrachial cells project to both structures. The results suggest that cholinergic cells of the parabrachial region may coordinate the relay of visuosensory information to the cortex with the onset of orienting movements.

170. Lee, P.H., M. Schmidt, and W.C. Hall, Excitatory and inhibitory circuitry in the superficial gray layer of the superior colliculus. J Neurosci, 2001. 21(20): p. 8145-53. Stratum griseum superficiale (SGS) of the superior colliculus receives a dense cholinergic input from the parabigeminal nucleus. In this study, we examined in vitro the modulatory influence of acetylcholine (ACh) on the responses of SGS neurons that project to the visual thalamus in the rat. We used whole-cell patch-clamp recording to measure the responses of these projection neurons to electrical stimulation of their afferents in the stratum opticum (SO) before and during local pressure injections of ACh. These colliculothalamic projection neurons (CTNs) were identified during the in vitro experiments by prelabeling them from the thalamus with the retrograde axonal tracer wheat germ agglutinin-apo-HRP-gold. In a group of cells that included the prelabeled neurons, EPSCs evoked by SO stimulation were significantly reduced by the application of ACh, whereas IPSC amplitudes were significantly enhanced. Similar effects were observed when the nicotinic ACh receptor agonist lobeline was used. Application of the selective GABA(B) receptor antagonist 3-[[(3,4-dichlorophenyl)-methyl]amino]propyl](diethoxymethyl)phosphinic acid blocked ACh-induced reduction in the evoked response. In contrast, the ACh-induced reduction was insensitive to application of the GABA(A) receptor antagonist bicuculline. The ACh-induced reduction was also diminished by bath application of muscimol at the low concentrations that selectively activate GABA(C) receptors. Because GABA(C) receptors may be specifically expressed by GABAergic SGS interneurons (Schmidt et al., 2001), our results support the hypothesis that ACh reduces CTN activity by nicotinic receptor-mediated excitation of local GABAergic interneurons. These interneurons in turn use GABA(B) receptors to inhibit the CTNs.

171. Aizawa, H., et al., Injection of nicotine into the superior colliculus facilitates occurrence of express saccades in monkeys. J Neurophysiol, 1999. 82(3): p. 1642-6. To clarify the role of cholinergic inputs to the intermediate layer of the superior colliculus (SC), we examined the effect of microinjection of nicotine into the SC on visually guided saccades in macaque monkeys. After injection of 0.4-2 microl of 1-100 mM nicotine into

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the SC, frequency of extremely short latency saccades (express saccades; reaction time = 70-120 ms) dramatically increased, for the saccades the direction and amplitude of which were represented at the location of the injection site on the collicular map. However, no marked change was observed for the relationship between the peak velocities and the amplitudes of saccades. These results suggested that activation of nicotinic acetylcholine receptors in the SC can facilitate initiation but causes no major change in dynamics of visually guided saccades.

172. Binns, K.E., The synaptic pharmacology underlying sensory processing in the superior colliculus. Prog Neurobiol, 1999. 59(2): p. 129-59. The superior colliculus (SC) is one of the most ancient regions of the vertebrate central sensory system. In this hub afferents from several sensory pathways converge, and an extensive range of neural circuits enable primary sensory processing, multi-sensory integration and the generation of motor commands for orientation behaviours. The SC has a laminar structure and is usually considered in two parts; the superficial visual layers and the deep multi-modal/motor layers. Neurones in the superficial layers integrate visual information from the retina, cortex and other sources, while the deep layers draw together data from many cortical and sub-cortical sensory areas, including the superficial layers, to generate motor commands. Functional studies in anaesthetized subjects and in slice preparations have used pharmacological tools to probe some of the SC's interacting circuits. The studies reviewed here reveal important roles for ionotropic glutamate receptors in the mediation of sensory inputs to the SC and in transmission between the superficial and deep layers. N-methyl-D-aspartate receptors appear to have special responsibility for the temporal matching of retinal and cortical activity in the superficial layers and for the integration of multiple sensory data-streams in the deep layers. Sensory responses are shaped by intrinsic inhibitory mechanisms mediated by GABA(A) and GABA(B) receptors and influenced by nicotinic acetylcholine receptors. These sensory and motor-command activities of SC neurones are modulated by levels of arousal through extrinsic connections containing GABA, serotonin and other transmitters. It is possible to naturally stimulate many of the SC's sensory and non-sensory inputs either independently or simultaneously and this brain area is an ideal location in which to study: (a) interactions between inputs from the same sensory system; (b) the integration of inputs from several sensory systems; and (c) the influence of non-sensory systems on sensory processing.

173. Wichmann, T., R.B. Illing, and K. Starke, Evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus. Neuroscience, 1987. 23(3): p. 991-1000. Acetylcholinesterase staining and studies on the uptake of [3H]choline into the subsequent efflux of tritium from collicular slices were carried out in order to provide evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus. Acetylcholinesterase staining was dense and homogeneous in superficial layers whereas the staining was arranged in patches with slightly higher density caudally than rostrally in the intermediate layers. The accumulation of tritium in slices incubated with [3H]choline depended on time, temperature and concentration, and was inhibited by hemicholinium-3. Accumulation was slightly higher in caudal than in rostral slices. Electrical stimulation enhanced tritium outflow from slices preincubated with [3H]choline. Tetrodotoxin and a low calcium medium inhibited the evoked overflow whereas hemicholinium-3 caused an

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enhancement. Oxotremorine decreased the evoked overflow; atropine prevented this effect. The opioids [D-Ala2, MePhe4, Glycol5]enkephalin, [D-Ala2, D-Leu5]enkephalin and ethylketocyclazocine caused an inhibition. The effects of the latter two agonists were antagonized by naloxone. The GABAB-receptor-agonist (-)-baclofen decreased the evoked overflow at lower concentrations than GABA, whereas the GABAA-receptor-agonist muscimol was ineffective. Serotonin produced an inhibition which was prevented by metitepin, alpha- and beta-adrenoceptor as well as dopamine-receptor ligands caused no change. It is concluded that in the rabbit superior colliculus the pattern of acetylcholinesterase staining is comparable, but not identical to the distribution in other species. The accumulation of [3H]choline, as well as the tetrodotoxin-sensitive and calcium-dependent overflow of tritium upon electrical stimulation (reflecting presumably release of [3H]acetylcholine) indicate that acetylcholine has a neurotransmitter function in this tissue. The release of [3H]acetylcholine was modulated by various transmitter substances and related compounds. The pattern of modulation of release differed from the pattern in other cholinergically innervated tissues.

174. Beninato, M. and R.F. Spencer, A cholinergic projection to the rat superior colliculus demonstrated by retrograde transport of horseradish peroxidase and choline acetyltransferase immunohistochemistry. J Comp Neurol, 1986. 253(4): p. 525-38. Acetylcholinesterase (AChE) has been localized by histochemistry in the superior colliculus and in the tegmentum of the caudal midbrain and rostral pons of the rat. The pattern of AChE localization in the superior colliculus was characterized by homogeneous staining in the superficial layers and patchlike staining in the intermediate gray layer. In the tegmentum, AChE was localized in the pedunculopontine nucleus (PPN), beginning rostrally at the caudal pole of the substantia nigra and extending caudally to the level of the parabrachial nuclei, and in the lateral dorsal tegmental nucleus (LDTN) of the central gray. The localization of AChE in these nuclei overlapped the distribution of neurons stained by immunohistochemistry using an antibody to choline acetyltransferase (ChAT), the synthesizing enzyme of the neurotransmitter acetylcholine. Other neighboring areas that were stained with AChE, but that did not contain ChAT-immunoreactive neurons, included the microcellular tegmental nucleus and the ventral tegmental nucleus. Neurons in the PPN and LDTN were determined to be potential sources of the cholinergic projection to the intermediate gray layer of the rat superior colliculus by double labelling with retrograde transport of horseradish peroxidase (HRP) combined with the immunohistochemical localization of ChAT. Three populations of neurons were identified. A predominantly ipsilateral ChAT-immunoreactive population was located in the pars compacta subdivision of PPN (PPNpc). Retrograde HRP-labelled neurons in the pars dissipata subdivision of the PPN (PPNpd), located ventral to the superior cerebellar peduncle (SCP) at the level of the inferior colliculus, composed a second population that was predominantly contralateral but was not ChAT immunoreactive. A third population of retrogradely labelled neurons was predominantly ipsilateral and ChAT immunoreactive and was located in the LDTN. These findings compared favorably with the full extent of the projection from this tegmental region revealed by retrograde transport of HRP from the superior colliculus when more compatible fixation and chromogen procedures were used. The results suggest that the PPN and the LDTN are two sources of the cholinergic input to the superior colliculus.

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Since the PPN also has extensive efferent, and afferent, connections with basal-ganglia-related structures, this cholinergic excitatory input to the superior colliculus, like the GABA-ergic inhibitory input from the substantia nigra pars reticulata, may provide the basis for an additional influence of the basal ganglia on visuomotor behavior.

175. Weldon, D.A., L.C. Calabrese, and K.J. Nicklaus, Rotational behavior following cholinergic stimulation of the superior colliculus in rats. Pharmacol Biochem Behav, 1983. 19(5): p. 813-20. Rate which received microinjections of carbachol into the superior colliculus exhibited pronounced dose-dependent rotational behavior contralateral to the site of injection (Experiment 1). Wet dog shakes were also observed in some animals. Similar injections in the midbrain reticular formation produced immobility with slight contralateral flexion of the neck. Convulsions were observed in some rats after injections into either anatomical location. In Experiment 2, circling induced by carbachol in the superior colliculus was blocked by prior injection of either the muscarinic receptor antagonist scopolamine or the nicotinic receptor antagonist mecamylamine, suggesting that both nicotinic and muscarinic receptors are involved in the effect. In Experiment 3 contralateral rotational behavior was induced by intracollicular microinjections of the combination of acetylcholine chloride and physostigmine. The results suggest that collicular mediation of contralateral rotational behavior, and perhaps orientation, might involve cholinergic receptors.

176. Pazdernik, T.L., et al., Superior colliculus activation by retinal nicotinic ganglion cells: a 2-deoxyglucose study. Brain Res, 1982. 243(1): p. 197-200. Systemic injection of the acetylcholinesterase inhibitor, di-isopropylfluorophosphate, in rats causes a marked increase in glucose use in the superficial layers of the superior colliculus. This activation of the superior colliculus is largely a retinal effect. Furthermore, since this response can be blocked by intraocular as well as systemic injections of mecamylamine, it is postulated that retinal nicotinic receptors are involved.

177. Kasser, R.J. and P.D. Cheney, DFP action on rat superior colliculus: localization and role of cholinergic receptors. Neurotoxicology, 1987. 8(4): p. 607-22. DFP, an irreversible acetylcholinesterase inhibitor, markedly increases spontaneous unit activity and reduces light-evoked responses in the superficial layers of the rat superior colliculus (Cheney et al., 1987). The purpose of this study was to investigate: (1) the sites of DFP action within the retino-tectal pathway (retinal, central or both), and (2) the types of cholinergic receptors (muscarinic, nicotinic, or both) involved. DFP increased SGS unit activity when injected intraocularly, confining its action to the retina, or when given systemically in bilateral enucleate rats. Thus, DFP acts at both retinal and central sites to increase unit activity in the SGS. Pretreating with muscarinic receptor antagonists such as atropine or scopolamine blocked DFP's effects at both sites whereas the nicotinic receptor antagonist mecamylamine was ineffective. Moreover, DFP's actions were mimicked by injections of the muscarinic receptor agonist, oxotremorine. The oxotremorine effects were also blocked or reversed by treatment with atropine or scopolamine. We conclude that DFP acts at both retinal and central sites to influence SGS unit activity and, at both sites, muscarinic receptors mediate DFP's effects.

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178. Kang, H.K. and T.A. Bullman, Mortality among US veterans of the Persian Gulf War: 7-year follow-up. Am J Epidemiol, 2001. 154(5): p. 399-405. To assess the long-term health consequences of the 1991 Persian Gulf War, the authors compared cause-specific mortality rates of 621,902 Gulf War veterans with those of 746,248 non-Gulf veterans, by gender, with adjustment for age, race, marital status, branch of service, and type of unit. Vital status follow-up began with the date of exit from the Persian Gulf theater (Gulf veterans) or May 1, 1991 (control veterans). Follow-up for both groups ended on the date of death or December 31, 1997, whichever came first. Cox proportional hazards models were used for the multivariate analysis. For Gulf veterans, mortality risk was also assessed relative to the likelihood of exposure to nerve gas at Khamisiyah, Iraq. Among Gulf veterans, the significant excess of deaths due to motor vehicle accidents that was observed during the earlier postwar years had decreased steadily to levels found in non-Gulf veterans. The risk of death from natural causes remained lower among Gulf veterans compared with non-Gulf veterans. This was mainly accounted for by the relatively higher number of deaths related to human immunodeficiency virus infection among non-Gulf veterans. There was no statistically significant difference in cause-specific mortality among Gulf veterans relative to potential nerve gas exposure. The risk of death for both Gulf veterans and non-Gulf veterans stayed less than half of that expected in their civilian counterparts. The authors conclude that the excess risk of mortality from motor vehicle accidents that was associated with Gulf War service has dissipated after 7 years of follow-up.

179. Kang, H.K. and T.A. Bullman, Mortality among U.S. veterans of the Persian Gulf War [see comments]. N Engl J Med, 1996. 335(20): p. 1498-504. BACKGROUND: Since the 1990-1991 Persian Gulf War, there has been persistent concern that U.S. war veterans may have had adverse health consequences, including higher-than-normal mortality. METHODS: We conducted a retrospective cohort study of postwar mortality according to cause among 695,516 Gulf War veterans and 746,291 other veterans. The follow-up continued through September 1993. A stratified, multivariate analysis (with Cox proportional-hazards models) controlled for branch of service, type of unit, age, sex, and race in comparing the two groups. We used standardized mortality ratios to compare the groups of veterans with the general population of the United States. RESULTS: Among the Gulf War veterans, there was a small but significant excess of deaths as compared with the veterans who did not serve in the Persian Gulf (adjusted rate ratio, 1.09; 95 percent confidence interval, 1.01 to 1.16). The excess deaths were mainly caused by accidents (1.25; 1.13 to 1.39) rather than disease (0.88; 0.77 to 1.02). The corresponding rate ratios among 49,919 female veterans of the Gulf War were 1.32 (0.95 to 1.83) for death from all causes, 1.83 (1.02 to 3.28) for accidental death, and 0.89 (0.45 to 1.78) for death from disease. In both groups of veterans the mortality rates were significantly lower overall than those in the general population. The adjusted standardized mortality ratios were 0.44 (95 percent confidence interval, 0.42 to 0.47) for Gulf War veterans and 0.38 (0.36 to 0.40) for other veterans. CONCLUSIONS: Among veterans of the Persian Gulf War, there was a significantly higher mortality rate than among veterans deployed elsewhere, but most of the increase was due to accidents rather than disease, a finding consistent with patterns of postwar mortality among veterans of previous wars.

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180. Bell, N.S., et al., Proposed explanations for excess injury among veterans of the Persian Gulf War and a call for greater attention from policymakers and researchers. Inj Prev, 2001. 7(1): p. 4-9. INTRODUCTION: Death rates among US veterans of the Persian Gulf War were lower than rates among non-deployed veterans and the US population at large, with the exception of injury deaths; returning veterans were at significantly greater risk of injury mortality. Similar patterns of excess injury mortality were documented among US and Australian veterans returning from Vietnam. In spite of these consistent findings little has been done to explain these associations and in particular to determine whether or not, and how, war related exposures influence injury risk among veterans returning home after deployments. HYPOTHESIZED PATHWAYS: Several potential pathways are proposed through which injury might be related to deployment. First, increases in injury mortality may be a consequence of depression, post-traumatic stress disorder, and symptoms of other psychiatric conditions developed after the war. Second, physical and psychological traumas experienced during the war may result in the postwar adoption of "coping" behaviors that also increase injury risk (for example, heavy drinking). Third, greater injury risk may be the indirect consequence of increased experiences of ill defined diseases and symptoms reported by many returning veterans. Fourth, veterans may experience poorer survivability for a given injury event resulting in greater mortality but not morbidity. Finally, the process that selects certain individuals for deployment may lead to a spurious association between deployment status and injury mortality by preferentially selecting individuals who are risk takers and/or exposed to greater hazards. CONCLUSIONS: More research and attention from policymakers is needed to clarify the link between deployment and postwar increased risk of injury.

181. Haley, R.W., et al., Brain abnormalities in Gulf War syndrome: Evaluation with 1H MR spectroscopy. Radiology, 2000. 215: p. 807-817. PURPOSE: To test for neuronal brain damage in the basal ganglia and brainstem in Gulf War veterans by using magnetic resonance (MR) spectroscopy. MATERIALS AND METHODS: Twenty-two Gulf War veterans with one of three factor analysis-derived syndromes (case patients); 18 well veterans matched for age, sex, and education level (control subjects); and six Gulf War veterans with syndrome 2 from a different population (replication sample) underwent long echo time (272 msec) proton (hydrogen 1) MR spectroscopy on a 4 x 2 x 2-cm voxel in the basal ganglia bilaterally and a 2 x 2 x 2-cm voxel in the pons. Syndromes 1-3 are described as "impaired cognition," "confusion-ataxia," and "central pain," respectively. RESULTS: The N-acetylaspartate-to-creatine (NAA/Cr) ratio, which reflects functional neuronal mass, was significantly lower in the basal ganglia and brainstem of Gulf War veterans with the three syndromes than in those structures of the control subjects (P =.007). The finding was corroborated in the replication sample (P =.002). Veterans with syndrome 2 (the most severe clinically) had evidence of decreased NAA/Cr in both the basal ganglia and the brainstem; those with syndrome 1, in the basal ganglia only; and those with syndrome 3, in the brainstem only. CONCLUSION: Veterans with different Gulf War syndromes have biochemical evidence of neuronal damage in different distributions in the basal ganglia and brainstem.

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182. Mayberg, H.S., Clinical correlates of PET- and SPECT-identified defects in dementia. Journal of Clinical Psychiatry, 1994. 55(11 SUPPL): p. 12-21. Functional imaging studies in patients with dementia have focused primarily on the reliability of scan patterns to correctly diagnose specific diseases, particularly Alzheimer's disease. Results from these studies have important implications as disease-specific treatments become available. A complementary approach is to examine the relationships between patterns of altered brain function and specific behaviors. This tactic has potential impact on understanding normal brain organization as well as targeting symptom-specific treatments. Regional abnormalities have been identified in dementia patients that correlate with specific behavioral deficits: disturbances of language and visuospatial function, impaired verbal fluency and selective attention, and the presence of delusions and depression. These patterns are seen in patients with Alzheimer's disease as well as with dementias of other etiologies. The specificity of these patterns for disease-specific and disease-independent symptoms is unknown.

183. Eustache, F., et al., Entorhinal cortex disruption causes memory deficit in early Alzheimer's disease as shown by PET. Neuroreport, 2001. 12(4): p. 683-685. Voxel-based mapping of the correlations between cognitive scores and resting-state brain glucose utilization measured by PET has recently emerged as a novel way to reveal in living patients with Alzheimer's disease (AD) the neural systems whose disruption underlies particular neuropsychological, especially mnemonic, deficits. We have now applied this approach using a novel cognitive paradigm designed to selectively assess verbal episodic memory, and show that in early AD disruption of the left entorhinal cortex underlies this memory deficit, consistent with post mortern data showing that this brain area is affected earliest and most severely by tau pathology in AD.

184. Tani, Y., et al., 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (R-THBP, SUN 0588) acts on the brain muscarinic and nicotinic cholinergic receptors as evaluated by positron emission tomography (PET) in rhesus monkey. Adv Exp Med Biol, 1993. 338: p. 331-4.

185. Nordberg, A., et al., Long-term tacrine treatment in three mild Alzheimer patients: effects on nicotinic receptors, cerebral blood flow, glucose metabolism, EEG, and cognitive abilities. Alzheimer Dis Assoc Disord, 1998. 12(3): p. 228-37. The effect of long-term treatment with tacrine (tetrahydroaminoacridine) was studied in three Alzheimer patients (aged 57, 64, and 68 years) with mild dementia. All three patients had a Mini-Mental State Examination score of 24/30 and carried at least one apolipoprotein E (ApoE) epsilon4 allele. Tacrine was given in doses between 80 and to 160 mg daily for 13-31 months. A lower tacrine concentration was observed generally in cerebrospinal fluid (CSF) compared with plasma. The acetylcholinesterase activity in CSF tended to be increased following longer periods of tacrine treatment, whereas the butyrylcholinesterase activity was decreased. The three patients repeatedly underwent positron emission tomography investigation of cerebral blood flow, nicotinic receptors, cerebral glucose metabolism, and electroencephalogram (EEG) and cognitive tests. Positive influences on these parameters were observed following both short-term and long-term treatment with tacrine. Improvement of nicotinic receptors (measured as 11C-nicotine binding), cerebral blood flow, EEG, and some cognitive tests (trail making test,

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block design test) occurred earlier after initiation of tacrine treatment compared with the glucose metabolism, which was increased after several months of tacrine treatment. An improvement in attention (trail making test) was observed following tacrine as sign for frontal lobe activation (EEG). The functional effects of tacrine in Alzheimer patients appeared to be related to both dose and length of cholinesterase inhibitor treatment.

186. Ding, Y.S., et al., Mapping nicotinic acetylcholine receptors with PET. Synapse, 1996. 24(4): p. 403-7.

187. Chefer, S.I., et al., 2-[18F]F-A-85380: a PET radioligand for alpha4beta2 nicotinic acetylcholine receptors. Neuroreport, 1999. 10(13): p. 2715-21. 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]F-A-85380), a ligand for nicotinic acetylcholine receptors (nAChRs) was evaluated in an in vitro binding assay with membranes of rat brain and in vivo by PET in Rhesus monkey brain. The ligand has high affinity for alpha4beta2 nAChRs (K(D)=50 pM), crosses the blood-brain barrier, and distributes in the monkey brain in a pattern consistent with that of alpha4beta2 nAChRs. The specific/non-specific binding ratio increased steadily, reaching a value of 3.3 in the thalamus at 4 h. The specific binding of 2-[18F]F-A-85380 was reversed by cytisine. These results, in combination with the data demonstrating low toxicity of 2-[18F]F-A-85380, indicate that this ligand shows promise for use with PET in human subjects.

188. Dolci, L., et al., Synthesis of a fluorine-18 labeled derivative of epibatidine for in vivo nicotinic acetylcholine receptor PET imaging. Bioorg Med Chem, 1999. 7(3): p. 467-79. Epibatidine (exo-2-(2'-chloro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a natural compound isolated from the skin of the Ecuadorian poison frog Epipedobates tricolor, is the most potent nicotinic acetylcholine receptor (nAChR) agonist reported to date. In order to visualize and quantify in vivo these receptors in human brain using Positron Emission Tomography (PET), [18F]norchlorofluoroepibatidine (exo-2-(2'-[18F]fluoro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a fluorine-18 (t(1/2): 110 min) radiolabeled derivative of epibatidine has been designed. The corresponding 2'-bromo-, 2'-iodo- and 2'-nitro exo-2-(5'-pyridyl)-7-azabicyclo[2.2.1]heptane analogues as labeling precursors, as well as norchlorofluoroepibatidine as a reference compound have been synthesized by reductive, stereoselective, palladium-catalyzed Heck-type coupling between an N-Boc protected azanorbornene and the corresponding halopyridine. [18F]Norchlorofluoroepibatidine has been radiolabeled with fluorine-18 by nucleophilic aromatic substitution from the corresponding Boc-protected halo- and nitro precursors using [18F]FK-K222 complex in DMSO by conventional heating (at 150-180 degrees C for 10 min) or microwave activations (at 100 Watt, for 1 to 2.5 min), followed by TFA-removal of the protective group. Typically, using the microwave activation procedure, 60-80 mCi (2.22-2.96 GBq) of pure [18F]norchlorofluoroepibatidine could be obtained in less than 2 h (110-115 min) from the bromo labeling precursor, with specific radioactivities of 1.5-2.5 Ci/micromol (55.5-92.5 GBq/micromol) calculated for End of Bombardment. The preliminary PET experiments in baboon (Papio papio) with [18F]norchlorofluoroepibatidine show a high uptake and a rapid accumulation of the radiotracer into the brain within 30 min. In the thalamus, a nAChR rich area, uptake of radioactivity reached a maximum at 40 min (10% I.D./100 mL tissue). The ratio of

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radioactivity thalamus/cerebellum (the latter being a nAChR poor area) was 2 at 40 min and increased with time, up to 4.3 at 160 min. Its specific regiodistribution and its high ratio of specific-to-nonspecific binding confirm the ideal profile of [18F]norchlorofluoroepibatidine as a suitable radioligand for PET imaging of nAChRs in the brain.

189. Horti, A., et al., Fluorine-18-FPH for PET imaging of nicotinic acetylcholine receptors. J Nucl Med, 1997. 38(8): p. 1260-5. Visualization of central nicotinic acetylcholine receptors (nAChRs) with modern PET or SPECT imaging techniques has been hampered by the lack of a radioligand with suitable in vivo binding characteristics (i.e., high target-to-nontarget ratios and kinetics appropriate for the half-life of the tracer and imaging modality used). This paper describes in vivo binding, kinetics and pharmacology of a highly potent 18F-labeled analog of epibatidine, (+/-)-exo-2-(2-[18F]fluoro-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]FPH), in the mouse brain with the view towards application of this tracer for PET imaging of nAChR in human brain. METHODS: Fluorine-18-FPH was administered intravenously to mice, and time-activity curves were determined for several regions in the brain and other organs. Saturation and pharmacology of [18F]FPH binding was demonstrated in vivo by preinjecting unlabeled FPH or other drugs with known pharmacological action before [18F]FPH was injected. The effect of the drugs on [18F]FPH accumulation was evaluated. RESULTS: [18F]FPH was rapidly incorporated into the mouse brain; peak activity (2.4% of the injected dose) was measured at 5 min after intravenous administration, followed by washout to 1.1% injected dose (ID) at 60 min. Highest concentrations of 18F occurred at 15 min in areas known to contain high densities of nAChR inverted question marke.g., thalamus [9.7% of injected dose per gram tissue (ID/g inverted question mark] and superior colliculus (8.3% ID/g)]. Accumulation of the 18F tracer in hippocampus, striatum, hypothalamus and cortical areas was intermediate (5.0, 5.6, 4.2 and 5.6% ID/g, respectively) and low in the cerebellum (2.8% ID/g). The distribution of [18F]FPH in the mouse brain matched that of other in vivo nAChR probes such as 3H-labeled epibatidine or norchloroepibatidine, [3H](-)-nicotine and [3H]cytisine and that of nAChR densities determined in postmortem autoradiographic studies in rodents. Preinjection of blocking doses of unlabeled epibatidine, (-)-nicotine, lobeline and cytisine significantly inhibited [18F]FPH binding in thalamus and superior colliculus, but not in cerebellum, whereas drugs that interact with binding sites other than acetylcholine recognition sites of nAChR (e.g., mecamylamine, scopolamine, N-methylspiperone and ketanserin) had no effect on [18F]FPH accumulation in any of the brain regions examined. CONCLUSION: Fluorine-18-FPH labels nAChR in vivo in the mouse brain. Because of its high uptake into the brain and high ratios of specific-to-nonspecific binding, this radioligand appears to be ideally suited for PET imaging of nAChR in the mammalian brain.

190. Ding, Y., et al., Synthesis and evaluation of 6-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine as a PET tracer for nicotinic acetylcholine receptors. Nucl Med Biol, 2000. 27(4): p. 381-9. Both ABT-594 ((R)-2-chloro-5-(2-azetidinylmethoxy)pyridine) and A-85380 (3-[2(S)-2-azetidinylmethoxy]pyridine), novel nicotinic agonists that possess potent non-opioid analgesic properties, have high affinity for neuronal nicotinic acetylcholine receptors (nAChR) but do not elicit the pronounced

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toxicity of epibatidine. 6-[(18)F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[(18)F]fluoro-A-85380), a F-18 labeled analogue of these two compounds, is therefore a promising radioligand for positron emission tomography (PET) studies in humans. The use of trimethylammonium as a leaving group in nucleophilic aromatic substitution reactions has proven to be a versatile and efficient strategy, and offers several advantages over other leaving groups. Here, we report the synthetic strategy for the preparation of a precursor, as a trimethylammonium iodide salt, and its use in the radiosynthesis to 6-[(18)F]fluoro-A-85380. Preliminary compartative PET studies of 6-[(18)F]fluoro-A-85380 and 2-[(18)F]fluoro-A-85380 were carried out in baboon to examine their suitability as tracers for studying nAChR system.

191. Asahina, M., et al., Brain muscarinic receptors in progressive supranuclear palsy and Parkinson's disease: a positron emission tomographic study. J Neurol Neurosurg Psychiatry, 1998. 65(2): p. 155-63. OBJECTIVES: To assess muscarinic acetylcholine receptors (mAChRs) in the brains of patients with progressive supranuclear palsy and Parkinson's disease, and to correlate the cholinergic system with cognitive function in progressive supranuclear palsy and Parkinson's disease. METHODS: Positron emission tomography (PET) and [11C]N-methyl-4-piperidyl benzilate ([11C]NMPB) was used to measure mAChRs in the brain of seven patients with progressive supranuclear palsy, 12 patients with Parkinson's disease, and eight healthy controls. All of the patients with progressive supranuclear palsy were demented. The Parkinson's disease group consisted of 11 non-demented patients and one demented patient. The mini mental state examination (MMSE) was used to assess the severity of cognitive dysfunction in all of the subjects. The modified Wisconsin card sorting test (WCST) was used to evaluate frontal cognitive function in the non-demented patients with Parkinson's disease and controls. RESULTS: The mean K3 value, an index of mAChR binding, was significantly higher for the frontal cortex in the patients with Parkinson's disease than in the controls (p<0.01). By contrast, the patients with progressive supranuclear palsy had no significant changes in the K3 values of any cerebral cortical regions. The mean score of the MMSE in the progressive supranuclear palsy group was significantly lower than that in the control group. Although there was no difference between the Parkinson's disease and control groups in the MMSE, the non-demented patients with Parkinson's disease showed significant frontal lobe dysfunction in the WCST. CONCLUSIONS: The increased mAChR binding in the frontal cortex of the patients with Parkinson's disease may reflect denervation hypersensitivity caused by loss of the ascending cholinergic input to that region from the basal forebrain and may be related to frontal lobe dysfunction in Parkinson's disease. The cerebral cortical cholinergic system may not have a major role in cognitive dysfunction in progressive supranuclear palsy.

192. Mentis, M.J., et al., Muscarinic versus nicotinic modulation of a visual task. a pet study using drug probes. Neuropsychopharmacology, 2001. 25(4): p. 555-64. Little is known about acetylcholine (ACh) modulation of central visual processing in humans. Receptor densities in visual brain regions are differentially distributed suggesting that receptor subtypes have different functions. Using PET, we have previously described the brain regions activated by a simple pattern-flash stimulus in healthy elderly subjects. To evaluate muscarinic and nicotinic contributions to ACh modulation of visual processing,

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we scanned elderly subjects watching the pattern-flash stimulus during no drug, during physostigmine augmentation, and during scopolamine antagonism of physostigmine's action. These manipulations of ACh significantly altered regional cerebral blood flow (rCBF) in brain regions activated by the task. The pattern of rCBF values across drug conditions suggested that muscarinic and nicotinic effects were dissociated. Muscarinic action predominated in striate cortex (Brodmann Area, BA 17) and lateral visual association areas (BA 18, 19), while nicotinic action predominated in the thalamus and inferior parietal regions (BA 39/40). Both muscarinic and nicotinic actions increased rCBF in some regions while decreasing it in others. A parsimonious reconciliation of these results with functional anatomy suggests that muscarinic action modulates visual attribute processing, while nicotinic action modulates arousal and selective attention to the visual task.

193. Furey, M.L., et al., Cholinergic stimulation alters performance and task-specific regional cerebral blood flow during working memory. Proc Natl Acad Sci U S A, 1997. 94(12): p. 6512-6. Modulation of the cholinergic neurotransmitter system results in changes in memory performance, including working memory (WM), in animals and in patients with Alzheimer disease. To identify associated changes in the functional brain response, we studied performance measures and regional cerebral blood flow (rCBF) using positron emission tomography (PET) in healthy subjects during performance of a WM task. Eight control subjects received an infusion of saline throughout the study and 13 experimental subjects received a saline infusion for the first 2 scans followed by a continuous infusion of physostigmine, an acetylcholinesterase inhibitor, for the subsequent 8 scans. rCBF was measured using H215O and PET in a sequence of 10 PET scans that alternated between rest and task scans. During task scans, subjects performed the WM task for faces. Physostigmine both improved WM efficiency, as indicated by faster reaction times, and reduced WM task-related activity in anterior and posterior regions of right midfrontal gyrus, a region shown previously to be associated with WM. Furthermore, the magnitudes of physostigmine-induced change in reaction time and right midfrontal rCBF correlated. These results suggest that enhancement of cholinergic function can improve processing efficiency and thus reduce the effort required to perform a WM task, and that activation of right prefrontal cortex is associated with task effort.

194. Kuhl, D.E., et al., In vivo mapping of cerebral acetylcholinesterase activity in aging and Alzheimer's disease. Neurology, 1999. 52(4): p. 691-9. OBJECTIVE: To validate an in vivo method for mapping acetylcholinesterase (AChE) activity in human brain, preparatory to monitoring inhibitor therapy in AD. BACKGROUND: AChE activity is decreased in postmortem AD brain. Lacking a reliable in vivo measure, little is known about central activity in early AD, when the disease is commonly targeted by AChE inhibitor drug therapy. METHODS: Intravenous N-[11C]methylpiperidin-4-yl propionate ([11C]PMP) served as an in vivo AChE substrate. AChE activity was defined using cerebral PET for tracer kinetic estimates of the local rate of [11C]PMP hydrolysis in 26 normal controls and 14 patients with AD. Eleven AD patients also had concomitant in vivo cerebral measures of vesicular acetylcholine transporter (cholinergic terminal) density and glucose metabolism. RESULTS: Cerebral AChE activity measures 1) were independent of changes in tracer delivery to cerebral cortex; 2) agreed with reported

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postmortem data concerning normal relative cerebral distributions, absence of large age-effect in normal aging, and deficits in AD; 3) correlated in AD cerebral cortex with concomitant in vivo measures of cholinergic terminal deficits, but not with metabolic deficits; and 4) agreed quantitatively with predicted level of cerebral AChE inhibition induced by physostimine. CONCLUSIONS: This in vivo PET method provided valid measures of central AChE activity in normal subjects and AD patients. Applied in early AD, it should facilitate inhibitor treatment by confirming central inhibition, optimizing drug dosage, identifying likely responders, and testing surrogate markers of therapeutic response.

195. Shinotoh, H., et al., Progressive loss of cortical acetylcholinesterase activity in association with cognitive decline in Alzheimer's disease: a positron emission tomography study. Ann Neurol, 2000. 48(2): p. 194-200. We measured brain acetylcholinesterase activity in 30 patients with Alzheimer's disease (AD) and 14 age-matched controls by positron emission tomography (PET) and using a carbon 11-labeled acetylcholine analogue. Seven AD patients had repeat PET scans. The k3 values were calculated as an index of acetylcholinesterase activity in a three-compartment analysis using the metabolite corrected arterial input function. Twenty-eight of the 30 AD patients (14 each in the early and late onset subgroups) were retained in the study so as to equalize the range and average severity of cognitive impairment within the early and late onset subgroups. The k3 values were significantly reduced in the neocortex, hippocampus, and amygdala in the early onset AD patients, although the k3 values were significantly reduced only in the temporoparietal cortex and amygdala in the late onset AD patients. In the longitudinal study, all 7 repeat AD patients showed further reduction of cortical k3 values in the second PET scans, with a mean interval of 2 years, suggesting a progressive loss of the ascending cholinergic system from the nucleus basalis of Meynert in AD. In 37 AD patients, there was a highly significant correlation between the cortical k3 values and Mini-Mental State Examination scores, supporting the cholinergic hypothesis in AD.

196. Meltzer, C.C., et al., Serotonin in aging, late-life depression, and Alzheimer's disease: The emerging role of functional imaging. Neuropsychopharmacology, 1998. 18(6): p. 407-430. Serotonin (5-HT) neuron and neurotransmitter loss in normal aging and neuropsychiatric diseases of late life may contribute to behavioral changes commonly observed in the elderly population. Extensive evidence implicates a deficit in serotonergic neurotransmission in the development of major depression. It has been further suggested that the age-related changes in 5-HT neurons may predispose the elderly to develop depression. There is also increasing evidence that a combination of disturbances in cholinergic and serotonergic function may play a role in cognitive impairment in Alzheimer's disease (AD), with serotonergic dysfunction potentially responsible for a significant portion of the behavioral aspects of the disease. This implication of the 5-HT system in aging and age-related cognitive and mood disorders rests in large part on post mortem studies and animal models, which are limited in their capacity to predict dynamic human biochemical-behavior relationships or to accurately model the living human brain. Initial applications of functional brian imaging with positron emission tomography (PET) in the in vivo study of the brain in aging,

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depression, and dementia focused on characterizing alterations in physiological measurements of cerebral metabolism and perfusion. However, recent advances in PET radiochemistry, instrumentation, and image processing have paved the way for noninvasive means to test specific hypotheses regarding the direct involvement of 5-HT neurons in the behavioral features of aging and to define and monitor therapeutic regimens for neuropsychiatric conditions of late life. Coupling of clinical trials in well-characterized subject populations with PET imaging using ligands specific for 5-HT receptor subtypes and transporter proteins promises to increase our understanding of the role of the 5-HT system in affective and cognitive aspects of treatment response. Longitudinal studies in aging, late-life depression, and AD are also needed to evaluate the complex interplay between neurodegenerative processes and serotonergic neurotransmission.

197. Price, J.C., et al., PET imaging of serotonin-1A receptors in type 2 diabetes: Evidence of increased binding. Journal of Nuclear Medicine, 2001. 42(5 Supplement): p. 18P.

198. Backman, L., et al., Cognitive deficits in Huntington's disease are predicted by dopaminergic PET markers and brain volumes. Brain, 1997. 120(12): p. 2207-2217. The main aim of this study was to investigate the relationship between dopaminergic markers and brain volumes for striatal and cortical structures, and cognitive performance in patients with Huntington's disease and control subjects. We used PET and MRI data as predictors of performance in tasks assessing executive function, visuospatial ability, episodic memory, verbal fluency, perceptual speed and reasoning. The dopamine neurotransmission parameters (D1 and D2 receptor density and dopamine transporter density) and the volumetric measurements for caudate and putamen accounted for substantial portions of the variance across the majority of cognitive tasks. In addition, frontal volume showed a strong relationship with all cognitive tasks. D1 binding and volume, measurements for the temporal cortex and thalamic volume showed associations with a select number of cognitive tasks. The overall data pattern is consistent with the view that Huntington's disease may be characterized as a frontostriatal dementia, in which cognitive deficits may result from pathological changes at multiple sites in the frontostriatal circuitry.

199. Schroeder, U., et al., Subthalamic nucleus stimulation affects striato-anterior cingulate cortex circuit in a response conflict task: A PET study. Brain, 2002. 125(9): p. 1995-2004. The subthalamic nucleus (STN) has generally been considered as a relay station within frontal-subcortical motor control circuitry. Little is known about the influence of the STN on cognitive networks. Clinical observations and studies in animals suggest that the STN participates in non-motor functions which can now be probed in Parkinson's disease patients with deep brain stimulation of the STN, allowing selective and reversible modulation of this nucleus. Using PET, we studied changes in regional cerebral blood flow (rCBF) associated with a response conflict task (Stroop task) in Parkinson's disease patients ON and OFF bilateral STN stimulation. The Stroop task requires subjects to name the font colour of colour words (e.g. 'blue') printed in an incongruent colour ink (e.g. yellow). During STN stimulation, impaired task performance

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(prolonged reaction times) was associated with decreased activation in both right anterior cingulate cortex (ACC) and right ventral striatum. Concomitant increased activation in left angular gyrus indicative of ongoing word processing during stimulation is consistent with an impairment to inhibit habitual responses. ACC and ventral striatum are part of the ACC circuit associated with response conflict tasks. The decreased activation during STN stimulation in the ACC circuit, while response conflict processing worsened, provides direct evidence of STN modulating non-motor basal ganglia-thalamocortical circuitry. Impairment in ACC circuit function could account for the subtle negative effects on cognition induced by STN stimulation.

200. de Leon, M.J., et al., Prediction of cognitive decline in normal elderly subjects with 2-(18F)fluoro-2-deoxy-D-glucose/positron-emission tomography (FDG/PET). Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(19): p. 10966-10971. Neuropathology studies show that patients with mild cognitive impairment (MCI) and Alzheimer's disease typically have lesions of the entorhinal cortex (EC), hippocampus (Hip), and temporal neocortex. Related observations with in vivo imaging have enabled the prediction of dementia from MCI. Although individuals with normal cognition may have focal EC lesions, this anatomy has not been studied as a predictor of cognitive decline and brain change. The objective of this MRI-guided 2-(18F)fluoro-2-deoxy-D-glucose/positron-emission tomography (FDG/PET) study was to examine the hypothesis that among normal elderly subjects, EC METglu reductions predict decline and the involvement of the Hip and neocortex. In a 3-year longitudinal study of 48 healthy normal elderly, 12 individuals (mean age 72) demonstrated cognitive decline (11 to MCI and 1 to Alzheimer's disease). Nondeclining controls were matched on apolipoprotein E genotype, age, education, and gender. At baseline, metabolic reductions in the EC accurately predicted the conversion from normal to MCI. Among those who declined, the baseline EC predicted longitudinal memory and temporal neocortex metabolic reductions. At follow-up, those who declined showed memory impairment and hypometabolism in temporal lobe neocortex and Hip. Among those subjects who declined, apolipoprotein E E4 carriers showed marked longitudinal temporal neocortex reductions. In summary, these data suggest that an EC stage of brain involvement can be detected in normal elderly that predicts future cognitive and brain metabolism reductions. Progressive E4-related hypometabolism may underlie the known increased susceptibility for dementia. Further study is required to estimate individual risks and to determine the physiologic basis for METglu changes detected while cognition is normal.

201. Cools, R., et al., Dopaminergic modulation of high-level cognition in Parkinson's disease: The role of the prefrontal cortex revealed by PET. Brain, 2002. 125(3): p. 584-594. This study examined the effects of L-dopa medication in patients with Parkinson's disease on cortical and subcortical blood flow changes during two tasks known to involve frontostriatal circuitry. Eleven patients with Parkinson's disease were scanned on two occasions, one ON L-dopa medication and one OFF L-dopa medication, during performance of the Tower of London planning task and a related test that emphasized aspects of spatial working memory. L-dopa-induced decreases were observed in the right dorsolateral prefrontal cortex during performance of both the planning and the spatial

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working memory tasks compared with the visuomotor control task. Conversely, L-dopa-induced blood flow increases were observed in the right occipital lobe during the memory task relative to the control task. Data from age-matched healthy volunteers demonstrated that L-dopa effectively normalized blood flow in these regions in the patient group. Moreover, a significant correlation was found between L-dopa-induced, planning related blood flow decreases in the right dorsolateral prefrontal cortex and L-dopa-induced changes in performance on the planning task. These data suggest that L-dopa ameliorates high-level cognitive deficits in Parkinson's disease by inducing relative blood flow changes in the right dorsolateral prefrontal cortex.

202. Arakawa, K., et al., Component-specific effects of physostigmine on the cat visual evoked potential. Exp Brain Res, 1993. 95(2): p. 271-6. Pattern visual evoked potentials (VEPs) were recorded from the pial surface of the cat primary visual cortex prior to and following the intravenous administration of physostigmine, an agent which blocks the enzyme responsible for the breakdown of synaptically released acetylcholine. The control VEP was composed of a small initial positive deflection (P1), a subsequent large negative wave (N1) and a second large positive wave (P2). Following physostigmine, the amplitude of P1-N1 was diminished whereas that of N1-P2 increased. These effects were long lasting and were blocked by prior treatment with scopolamine, a result consistent with mediation by a muscarinic cholinergic pathway. Waveform subtraction revealed that the physostigmine-sensitive component had a slow, negative polarity waveform while the physostigmine-insensitive component was also slow, but positive in polarity. The fundamental nature of these components remains to be assessed. Nevertheless, the results indicate that waveforms of different polarity combine algebraically to yield the conventional VEP.

203. Thierry, G., B. Doyon, and J.-F. Demonet, ERP mapping in phonological and lexical semantic monitoring tasks: A study complementing previous PET results. NeuroImage, 1998. 8(4): p. 391-408. Previous PET results identified distinct neural systems involved with phonology (vicinity of the left sylvian fissure) and lexical semantics (left inferior temporal, left superior frontal, bilateral inferior parietal regions). In the time domain, the phonological task was thought to involve serial parsing of pseudo-words, whereas the lexical semantic task would correspond to probabilistic automated access to meaning. Auditory event-related potentials (ERPs) elicited by the same tasks were explored on 32 channels in 12 male volunteers. Subjects had to categorize as target a second element of a pair of pseudo-words or words if a preceding target was detected in the first element. Depending on the absence/presence of target in the first element, a RELEASE condition and a HOLD condition were distinguished. RELEASE and HOLD ERPs split earlier in the semantic task than in the phonological task (300 versus 412 ms after SOT, respectively), although words lasted longer than pseudo-words. Corresponding paired t test maps showed a predominance of differences over left perisylvian regions for the phonological task and posterior bilateral regions for the semantic task. Underlying generators were investigated using BESA (Scherg, 1990) with global task ERPs. Six dipoles-constrained according to PET clusters-brought residual variance down to 0.36%, from 364 to 565 ms after SOT, in both tasks. Relative dipole amplitudes suggested a left-sided functional asymmetry for phonology. These

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results support the hypothesis of left perisylvian serial processing for phonology contrasting with bihemispheric parallel access for semantics and substantiates BESA for temporally tackling cognitive processes.

204. Abdullaev, Y.G. and M.I. Posner, Event-related brain potential imaging of semantic encoding during processing single words. NeuroImage, 1998. 7(1): p. 1-13. Functional brain imaging studies with positron emission tomography (PET) have identified blood flow changes in widely separated areas of brain during the performance of word processing tasks. In the present study we have utilized event-related brain potentials (ERPs) to investigate the temporal relationships among cortical areas previously identified by PET to be differentially activated when performing semantic tasks with visual words. ERPs revealed task-related differences over the central and left inferior frontal regions around 170 and 220 ms, respectively, over a left occipital region around 200 ms, over a large left parietotemporal region around 600 ms, and finally over the right temporal lobe around 800 ms after the word presentation. Analysis of topographic maps and dipole sources as well as PET data allowed relating frontal midline positivity around 170 ms to the anterior cingulate activation, and left inferior frontal positivity around 220 ms to the PET activation of the left inferior prefrontal cortex. The left parieto-temporal positivity around 600 ms seems to reflect the activity of Wernicke's area. The right anterior temporal negativity beginning around 800 ms and peaking around 1100 ms may reflect the activity of the right insula. The left occipital negativity around 200 ms is likely to reflect activation of a visual word-form area in the left occipital lobe. These results provide the time course for parts of the circuitry involved in semantic processing of words and also demonstrate how combining the spatial localization of PET with the temporal resolution of ERPs helps to understand the brain mechanisms involved in human cognition.

205. Gazzard, M.F. and D.P. Thomas, A comparative study of central visual field changes induced by sarin vapour and physostigmine eye drops. Exp Eye Res, 1975. 20(1): p. 15-21.

206. Alderdice, M.T., Physostigmine, but not neostigmine, inhibits acetylcholine release. Brain Res, 1979. 178(2-3): p. 596-9.

207. Alderdice, M.T., Further comparison of the effects of physostigmine and neostigmine on frog neuromuscular transmission. Clin Exp Pharmacol Physiol, 1982. 9(1): p. 35-43. 1. The effects of physostigmine and neostigmine were compared at frog neuromuscular junctions using intracellular microelectrodes. 2. Both drugs increased the amplitudes of miniature endplate potentials (MEPP) in a dose-dependent manner because of their ability to competitively inhibit cholinesterase activity. 3. In addition, physostigmine (1.0-20.0 mumol/l) decreased by approximately 20% the quantal content (determined by two methods), indicating a decrease in the amount of transmitter released upon nerve stimulation, whereas neostigmine did not alter quantal content. 4. Because the endplate potential (EPP) amplitude is a composite of pre- and post-junctional effects, the dose-response curve for neostigmine on EPP amplitude approximately paralleled that obtained on MEPP amplitude, whereas the effect of physostigmine on EPP amplitude

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was depressed because of its action to decrease transmitter release. 5. Upon washout of neostigmine, the increase in EPP amplitude reversed more rapidly than effects on MEPP amplitude, a difference dependent on the magnitude of quantal content and explained by an action on the postjunctional membrane. Similar results were obtained with physostigmine. 6. Neither drug significantly affected MEPP frequency although small decreases were sometimes observed. 7. These results suggest that at the frog neuromuscular junction the effects of neostigmine are explained primarily by its inhibition of cholinesterase, whereas physostigmine are explained primarily by its inhibition of cholinesterase, whereas physostigmine has an additional action on nerve terminals of decreasing nerve-stimulated release of acetylcholine.

208. Abdel-Rahman, A., A. Shetty, and M. Abou-Donia, Disruption of the blood-brain barrier and neuronal cell death in cingulate cortex, dentate gyrus, thalamus, and hypothalamus in a rat model of gulf-war syndrome. Neurobiol Dis, 2002. 10(3): p. 306. We investigated the effects of a combined exposure to restraint stress and low doses of chemicals pyridostigmine bromide (PB), N, N-diethyl-m-toluamide (DEET), and permethrin in adult male rats, a model of Gulf-War syndrome. Animals were exposed daily to one of the following for 28 days: (i) a combination of stress and chemicals (PB, 1.3 mg/kg/day; DEET, 40 mg/kg/day; and permethrin, 0.13 mg/kg/day); (ii) stress and vehicle; (iii) chemicals alone; and (iv) vehicle alone. All animals were evaluated for: (i) the disruption of the blood-brain barrier (BBB) using intravenous horseradish peroxidase (HRP) injections and endothelial barrier antigen (EBA) immunostaining; (ii) neuronal cell death using H&E staining, silver staining, and glial fibrillary acidic protein (GFAP) immunostaining; and (iii) acetylcholinesterase (AChE) activity and m2-muscarinic acetylcholine receptors (m2-AChR). Animals subjected to stress and chemicals exhibited both disruption of the BBB and neuronal cell death in the cingulate cortex, the dentate gyrus, the thalamus, and the hypothalamus. Other regions of the brain, although they demonstrated some neuronal cell death, did not exhibit disruption of the BBB. The neuropathological changes in the above four brain regions were highly conspicuous and revealed by a large number of HRP-positive neurons (21-40% of total neurons), a decreased EBA immunostaining (42-51% reduction), a decreased number of surviving neurons (27-40% reduction), the presence of dying neurons (4-10% of total neurons), and an increased GFAP immunostaining (45-51% increase). These changes were also associated with decreased forebrain AChE activity and m2-AchR (19-25% reduction). In contrast, in animals exposed to stress and vehicle or chemicals alone, the above indices were mostly comparable to that of animals exposed to vehicle alone. Thus, a combined exposure to stress and low doses of PB, DEET, and permethrin leads to significant brain injury. The various neurological symptoms reported by Gulf-War veterans could be linked to this kind of brain injury incurred during the war.

209. Abdel-Rahman, A., A.K. Shetty, and M.B. Abou-Donia, Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: dose-response relationships. Neuroscience, 2002. 113(3): p. 721-41. We hypothesize that a single exposure to an LD(50) dose of sarin induces widespread early neuropathological changes in the adult brain. In this study, we evaluated the early changes in the adult brain after a single exposure to different doses of sarin. Adult male

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rats were exposed to sarin by a single intramuscular injection at doses of 1, 0.5, 0.1 and 0.01xLD(50). Twenty-four hours after the treatment, both sarin-treated and vehicle-treated (controls) animals were analyzed for: (i) plasma butyrylcholinesterase (BChE) activity; (ii) brain acetylcholinesterase (AChE) activity, (iii) m2 muscarinic acetylcholine receptor (m2 mAChR) ligand binding; (iv) blood brain barrier (BBB) permeability using [H(3)]hexamethonium iodide uptake assay and immunostaining for endothelial barrier antigen (EBA); and (v) histopathological changes in the brain using H&E staining, and microtubule-associated protein (MAP-2) and glial fibrillary acidic protein immunostaining. In animals treated with 1xLD(50) sarin, the significant changes include a decreased plasma BChE, a decreased AChE in the cerebrum, brainstem, midbrain and the cerebellum, a decreased m2 mAChR ligand binding in the cerebrum, an increased BBB permeability in the cerebrum, brainstem, midbrain and the cerebellum associated with a decreased EBA expression, a diffuse neuronal cell death and a decreased MAP-2 expression in the cerebral cortex and the hippocampus, and degeneration of Purkinje neurons in the cerebellum. Animals treated with 0.5xLD(50) sarin however exhibited only a few alterations, which include decreased plasma BChE, an increased BBB permeability in the midbrain and the brain stem but without a decrease in EBA expression, and degeneration of Purkinje neurons in the cerebellum. In contrast, animals treated with 0.1 and 0.01xLD(50) did not exhibit any of the above changes. However, m2 mAChR ligand binding in the brainstem was increased after exposure to all doses of the sarin. Collectively, the above results indicate that, the early brain damage after acute exposure to sarin is clearly dose-dependent, and that exposure to 1xLD(50) sarin induces detrimental changes in many regions of the adult rat brain as early as 24 hours after the exposure. The early neuropathological changes observed after a single dose of 1xLD(50) sarin could lead to a profound long-term neurodegenerative changes in many regions of the brain, and resulting behavioral abnormalities.

210. Li, L., et al., Muscarinic receptor-mediated pyridostigmine-induced neuronal apoptosis. Neurotoxicology, 2000. 21(4): p. 541-52. Pyridostigmine is a reversible cholinesterase (ChE) inhibitor that is associated with neurologic dysfunction involving both central and peripheral nervous systems. To determine the neurotoxic potential of pyridostigmine, rats were sacrificed at intervals after drug administration (0.5-1.85 mg/kg, i.p., twice daily for 4 days) and brains examined histologically. ChE inhibition was used as a biomarker of pyridostigmine activity. Using the in situ terminal deoxynucleotidyl transferase nick-end labeling of DNA fragments (TUNEL) method and electron microscopy, apoptotic brain cell death was noted in cerebral cortex over a dose range of 0.5-1.85 mg/kg and at the higher dose (1.85 mg/kg), apoptosis was also noted in striatum and hippocampus. These responses were blocked by pretreatment with atropine. Rat cortical cells in culture also underwent apoptosis when exposed to pyridostigmine (250 microM for 24 hr), indicating that the pyridostigmine can initiate apoptosis, independent of peripheral mechanisms. Pretreatment of cells with atropine (10 microM) inhibited pyridostigmine-induced apoptosis, confirming the response was mediated by muscarinic receptors. Short term treatment of rats with pyridostigmine (1.85 mg/kg twice daily for 4 days) induced a prolonged apoptotic response, which was evident in rat cortex up to 30 days after the last dose. Active apoptosis persisted, despite recovery of serum

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ChE activity. These in vivo and in vitro observations indicate that pyridostigmine can initiate a prolonged neurodegeneration.

211. Alberts, P. and V.R. Ogren, Sarin and soman depress [3H]acetylcholine secretion in guinea-pig ileum myenteric plexus. Acta Physiol Scand, 1985. 125(2): p. 347-8.

212. Arroyo, G., et al., [Nicotinic Receptor, galantamine and Alzheimer disease]. Rev Neurol, 2002. 34(11): p. 1057-65. Population aging has increased and will drastically increase the prevalence of Alzheimer disease. The disease develops inexorably towards a syndrome of marked cognitive impairment, accompanied of emotional alterations and profound changes of personality. The patient loses its autonomy, and requires special attention of caregivers; this leads to a decrease of the quality of life, not only of the patient but also of its caregivers and family. The reduction of the number of functional nicotinic receptors in brain keeps pace with neurological symptoms and the severity of the disease (cholinergic theory of Alzheimer disease). There is a pleyade of data and observations reinforcing the idea that improving cholinergic neurotransmission is an investment in memory. Up to now, although with limited success, this improvement has been achieved only with the reversible inhibitors of acetylcholinesterase tacrine, rivastigmine and donepezil, available in the clinic since a few years. The last approved has been galantamine that in spite of being a modest inhibitor of acetylcholinesterase, improves memory (ADAS cog test) and slows down cognitive impairment of Alzheimer patients. To explain this therapeutic effect, a second mechanism of action for galantamine has been suggested, the positive allosteric modulation of presynaptic nicotinic receptors, that will favour the release of acetylcholine and other neurotransmitters involved in memory formation. Furthermore, galantamine possesses neuroprotectant antiapoptotic effects, according to recent data from our laboratory. These effects provide new ideas and therapeutic targets that might help to find novel and efficacious treatments for patients suffering Alzheimer disease.

213. Akaike, A., et al., The nature of the interactions of pyridostigmine with the nicotinic acetylcholine receptor-ionic channel complex. II. Patch clamp studies. Mol Pharmacol, 1984. 25(1): p. 102-12. Patch clamping of myoballs to record single channels was performed to examine the interaction of the anticholinesterase agent pyridostigmine (Pyr) with the acetylcholine (ACh) receptor-ion channel complex. Single ACh channel currents were recorded from tissue-cultured muscle cells of neonatal rats (myoballs). Pyr (50-100 microM) decreased the frequency of channel-opening events activated by ACh, and induced a modified form of the ACh channel currents. Channel conductance was lower in the presence of Pyr, and channel lifetime remained unaltered or only slightly prolonged. In addition, channel openings were frequently interrupted by fast flickers in the presence of Pyr. Higher concentrations (200 microM-1 mM) of the drug induced irregular waves of bursting activity during the initial phase of the application, and, subsequently, significantly reduced the frequency of channel openings. Infrequent channel openings with low conductance were observed in the patch when the micropipette was filled with Pyr alone. These results suggest that, in addition to its anticholinesterase activity, Pyr reacts with the ACh receptor, and both alone or in

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combination with ACh induces an altered, desensitized species of the nicotinic receptor-ion channel complex.

214. Arendt, T., et al., Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms. Neurochem Int, 1992. 21(3): p. 381-96. The pattern of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) separated by density gradient centrifugation was investigated in the brain and cerebrospinal fluid in Alzheimer's disease (AD), in human embryonic brain and in rat brain after experimental cholinergic deafferentation of the cerebral cortex. While a selective loss of the AChE G4 form was a rather constant finding in AD, a small but significant increase of G1 for both AChE and BChE was found in the most severely affected cases. Both in normal human brain and in AD a significant relationship could be established between the AChE G4/G1 ratio in different brain regions and the activity of choline acetyltransferase (ChAT). A similar decrease of the AChE G4 form as observed in AD can be induced in rat by experimental cholinergic deafferentation of the cerebral cortex. The increase in G1 of both AChE and BChE in different brain regions in AD is quantitatively related to the local density of neuritic plaques which are histochemically reactive for both enzymes. In human embryonic brain, a high abundance of G1 and a low G4/G1 ratio for both AChE and BChE was found resembling the pattern observed in AD. Furthermore, both in embryonic brain and in AD AChE shows no substrate inhibition which is a constant feature of the enzyme in the adult human brain. It is, therefore, concluded that the degeneration of the cholinergic cortical afferentation in AD as reflected by a decrease of AChE G4 is accompanied by the process of a neuritic sprouting response involved in plaque formation which is probably associated with the expression of a developmental form of the enzyme.

215. Antonelli, T., et al., Changes in synaptosomal high affinity choline uptake following electrical stimulation of guinea-pig cortical slices: effect of atropine and physostigmine. Br J Pharmacol, 1981. 74(3): p. 525-31. 1 Superfused guinea-pig cortical slices were electrically stimulated at different frequencies and the changes in acetylcholine (ACh) content measured. Synaptosomes were prepared at the end of the stimulation period and high affinity choline uptake (HACU) rate was measured. 2 The effect of increasing KC1 concentrations was compared on ACh content of the slices and on synaptosomal HACU. 3 Electrical stimulation (2, 5, 10, 20 Hz) elicited a frequency-dependent linear increase in synaptosomal HACU rate and a decrease in ACh content of the slices. 4 The addition of atropine (1.5 x 10(-8) M) to the slices enhanced and that of physostigmine (3 x 10(-5) M) reduced the frequency-dependent increase in HACU rate. Atropine (1.5 x 10(-6) M) not only antagonized the effect of physostigmine, but the HACU rate measured after treatment with both drugs was larger than that found after atropine alone. 5 These results indicate that in the cortical cholinergic nerve endings, depolarization caused by electrical stimulation is coupled with an increase in choline transport which can be modulated by the addition of atropine or physostigmine. Furthermore, within given experimental conditions a linear relationship exists between the reciprocal of ACh content in the slices and synaptosomal HACU.

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216. Abdel-Rahman, A., A.K. Shetty, and M.B. Abou-Donia, Subchronic dermal application of N,N-diethyl m-toluamide (DEET) and permethrin to adult rats, alone or in combination, causes diffuse neuronal cell death and cytoskeletal abnormalities in the cerebral cortex and the hippocampus, and Purkinje neuron loss in the cerebellum. Exp Neurol, 2001. 172(1): p. 153-71. N,N-Diethyl m-toluamide (DEET) and permethrin have been implicated as potential neurotoxic agents that may have played an important role in the development of illnesses in some veterans of the Persian Gulf War. To determine the effect of subchronic dermal application of these chemicals on the adult brain, we evaluated histopathological alterations in the brain of adult male rats following a daily dermal dose of DEET (40 mg/kg in 70% ethanol) or permethrin (0.13 mg/kg in 70% ethanol) or a combination of the two for 60 days. Control rats received a daily dermal dose of 70% ethanol for 60 days. Animals were perfused and brains were processed for morphological and histopathological analyses following the above regimen. Quantification of the density of healthy (or surviving) neurons in the motor cerebral cortex, the dentate gyrus, the CA1 and CA3 subfields of the hippocampus, and the cerebellum revealed significant reductions in all three treated groups compared with the control group. Further, animals receiving either DEET or permethrin exhibited a significant number of degenerating (eosinophilic) neurons in the above brain regions. However, degenerating neurons were infrequent in animals receiving both DEET and permethrin, suggesting that neuronal cell death occurs earlier in animals receiving combined DEET and permethrin than in animals receiving either DEET or permethrin alone. The extent of neuron loss in different brain regions was similar among the three treatment groups except the dentate gyrus, where neurodegeneration was significantly greater with exposure to DEET alone. The neuron loss in the motor cerebral cortex and the CA1 subfield of all treated groups was also corroborated by a significant decrease in microtubule associated protein 2-immunoreactive elements (15-52% reduction), with maximal reductions occurring in rats receiving DEET alone; further, the surviving neurons in animals receiving both DEET and permethrin exhibited wavy and beaded dendrites. Analysis of glial fibrillary acidic protein immunoreactivity revealed significant hypertrophy of astrocytes in the hippocampus and the cerebellum of all treated groups (24-106% increase). Thus, subchronic dermal application of DEET and permethrin to adult rats, alone or in combination, leads to a diffuse neuronal cell death in the cerebral cortex, the hippocampal formation, and the cerebellum. Collectively, the above alterations can lead to many physiological, pharmacological, and behavioral abnormalities, particularly motor deficits and learning and memory dysfunction.

217. Aizenman, E., G.G. Bierkamper, and E.F. Stanley, Botulinum toxin prevents stimulus-induced backfiring produced by neostigmine in the mouse phrenic nerve-diaphragm. J Physiol, 1986. 372: p. 395-404. The origin of motor nerve antidromic activity (backfiring) induced by anticholinesterase treatment was examined in the mouse phrenic nerve-hemidiaphragm preparation. Botulinum toxin was used to determine whether backfiring is due to (a) a direct effect of the cholinesterase inhibitor on the nerve terminal, or (b) an indirect effect via the prolongation of the action of acetylcholine. In previously untreated control preparations, neostigmine produced spontaneous and stimulus-induced antidromic activity in the phrenic nerve when rapidly introduced into the diaphragm via its vasculature. This activity could be reversibly blocked by d-

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tubocurarine and decamethonium, but not by atropine. Neostigmine-induced backfiring did not occur in preparations in which transmitter release was blocked with botulinum toxin. Infusion of a small bolus of a high concentration of acetylcholine following neostigmine treatment resulted in a short-term increase in the incidence of antidromic activity, followed by block, in both controls and botulinum toxin-treated preparations. It is concluded that transmitter release is necessary for the production of backfiring following cholinesterase inhibition since neostigmine alone does not elicit antidromic activity in botulinum toxin-treated preparations at concentrations which are effective in controls. Our results support the hypothesis that the effects of neostigmine on the motoneurone terminal are mediated by the prolonged action of acetylcholine that occurs with inhibition of acetylcholinesterase.

218. Amaki, Y., K. Kobayashi, and M. Kaneko, [The effect of neostigmine and edrophonium on the MEPP frequency]. Masui, 1989. 38(6): p. 760-6. Edrophonium may have stronger effect on presynaptic site than neostigmine. Comparative study was done by measuring the miniature endplate potential (MEPP) frequency which reflect the presynaptic activity using rat phrenic nerve hemidiaphragm preparation. Preparations were placed in an organ bath, filled with Krebs solution and bubbled with 95% O2 and 5% CO2 at room temperature. A glass electrode filled with 3M KCL was inserted as close to the endplate as possible and 20 spots of MEPP were recorded for each drug. For recorded MEEP with amplification, frequency was analyzed with the signal processor. The highest effective level was different, being 10(-6)M in neostigmine and 10(-5)M in edrophonium. At these levels, MEPP frequencies of both drugs showed increases 2-4 times as high as the level in the control group and the degrees of increase were about the same with the two drugs. Our study revealed that both drugs have a strong effect on presynaptic sites and that these stimulating effects of edrophonium and neostigmine were about the same degree if 10 times as high a dosage as that of the former is used.

219. Anderson, R.J., et al., Decreased tetanic contracture of rat skeletal muscle induced by pyridostigmine. J Toxicol Environ Health, 1986. 18(2): p. 221-30. The purpose of this study was to compare the effects of various regimens of pyridostigmine administration on the contractile strength of skeletal muscle. Rats were exposed to pyridostigmine according to 3 dosing schedules: 2 mg/kg ip daily, 5 mg/kg X d by sc infusion, and 25 mg/kg X d by sc infusion. After 1, 4, 10, and 20 d of exposure, measurements were made of muscle tension during tetanic stimulation, and of muscle mass, erythrocyte acetylcholinesterase activity, and body weight. Pyridostigmine produced a dose-dependent decrement in the contracture generated during tetanic stimulation. Peak effect was observed after 4 d of exposure but remained depressed after 20 d. The magnitude of the decrement correlated with the frequency of the tetanic stimulation (from 20 to 100 Hz). Muscle tension at the end of the tetanic stimulus was affected to a greater extent than the initial tension. The 25-mg/kg infusion of pyridostigmine significantly depressed erythrocyte acetylcholinesterase activity throughout the study and also decreased body weight on d 1-4. No change in muscle mass was observed in any treatment group. These results show that pyridostigmine exposure results in decrements in skeletal muscle contracture that are dose-dependent, frequency-dependent, and time-dependent. The effect is probably not the result of muscle wasting

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and does not correlate well with erythrocyte acetylcholinesterase inhibition. The results are consistent with effects of pyridostigmine, both presynaptically and postsynaptically, at the neuromuscular junction that affect neurotransmitter release and receptor responsiveness.

220. Anderson, R.J., Effect of pyridostigmine on tetanic contracture of innervated and denervated rat skeletal muscle. Neurotoxicology, 1987. 8(4): p. 553-60. Pyridostigmine has been reported to cause histological damage and decrements in tetanic contracture of skeletal muscle. Cholinesterase inhibition, decreased transmitter release and desensitization of postsynaptic receptors have all been proposed as mechanisms for these effects. The purpose of the present study was to determine whether the effect of pyridostigmine to decrease tetanic contracture was due to pre- or postsynaptic action. Rats with innervated or chronically denervated hindlimbs were given pyridostigmine (25 mg/kg) s.c. by infusion for four days. Tetanic contracture (20, 50, 100 Hz) was evoked by sciatic nerve stimulation in the innervated muscles and direct muscle stimulation in the denervated muscles. Pyridostigmine significantly decreased the contracture of innervated triceps surae muscle in a frequency dependent manner. Post-tetanic potentiation was reduced from control muscle responses. In denervated muscle pyridostigmine had no effect on contracture or post-tetanic potentiation. These results show that innervation is required for the detrimental effect of pyridostigmine and that the drug has no effect on skeletal muscle.

221. Anderson, R.J. and W.L. Chamberlain, Pyridostigmine-induced decrement in skeletal muscle contracture is not augmented by soman. Neurotoxicology, 1988. 9(1): p. 89-96. Previous studies have reported that pyridostigmine induces a decrement in contractile force generated during tetanic stimulation of skeletal muscle. Although our studies suggested that pyridostigmine affected release of transmitter from the motor nerve terminal, we could not exclude the possibility that the drug's action was due to depolarization blockade of the muscle brought on by excessive transmitter in the synaptic cleft. The purpose of this study was to determine whether the effect of combined treatment with pyridostigmine and an irreversible cholinesterase inhibitor (soman) would potentiate the decrement in muscle contracture observed with pyridostigmine alone. As reported previously, pyridostigmine (25 mg/kg) significantly reduced muscle contracture during tetanic stimulation (20-100 Hz), and soman (0.075 mg/kg) increased muscle contracture. Combined treatment with pyridostigmine and soman produced a decrease in muscle contracture equivalent to the effect of pyridostigmine alone. Since there was no evidence of depolarization blockade of the muscle despite aggressive treatment with two cholinesterase inhibitors, these results support the view that pyridostigmine has a significant presynaptic action to decrease neurotransmitter release. This action opposes the drug's inhibition of cholinesterase, and the net effect of combined treatment with pyridostigmine and soman is a muscle response which is largely unchanged from the effect of pyridostigmine alone.

222. Damodaran, T.V., et al., Sarin causes early differential alteration and persistent overexpression in mRNAs coding for glial fibrillary acidic protein (GFAP) and vimentin genes in the central nervous system of rats. Neurochem Res, 2002. 27(5): p. 407-15.

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Neurotoxic effects of single dose of 0.5 x LD50 sarin (O-isopropylmethylphosphonoflouridate) on central nervous system (CNS) of male Sprague-Dawley rats were studied. We investigated the mRNA expression of the astroglial marker genes glial fibrillary acidic protein (GFAP) and vimentin to evaluate the fate of astroglial and neuronal cells, because reactive gliosis is very often used to assess the extent of CNS damage. Rats were treated with 50 microg/kg/ml of sarin and terminated at the time-points 1 and 2 hours and 1, 3, and 7 days post-treatment. Control rats were treated with normal saline. Total RNA was extracted and Northern blots were hybridized with cDNA probes for GFAP and vimentin, as well as 28S RNA (control). The data obtained indicate that a single dose of sarin (0.5 x LD50) showed induction in the transcript levels of GFAP and vimentin in the cortex, cerebellum, brainstem and midbrain, and spinal cord. The induction showed distinct spatial-temporal differences for each tissue studied. Both GFAP and vimentin were induced at 1 hour in all the tissues studied except brainstem, where moderate and high levels of GFAP induction were noted at 1 and 3 days. Overexpressed transcript levels of GFAP and vimentin remained high in more responsive tissues such as the brainstem and midbrain. Other tissues, such as the cortex, spinal cord, and cerebellum showed a more downward trend for either GFAP or vimentin, or both, transcript levels at 7 days. It is noteworthy that both cortex (318 +/- 12%) and spinal cord (368 +/- 12%) showed relatively higher induction of GFAP, whereas cortex alone showed the highest level of overexpressed vimentin transcript levels (284 +/- 11%). Overall it is also clear that both GFAP and vimentin are needed for the effective recovery involving co-ordinated alternating up- and downregulation of these two key astrocyte genes, depending on tissue specificity. The changes seen in the transcript levels of GFAP and vimentin may be the result of astrocyte dysfunction and loss, accompanied by compensatory proliferation and dedifferentiation of the astroglia. These changes could affect the neuronal cell types, thus altering the neuron-glia homeostasis.

223. Glass-Marmor, L., M. Chen-Zion, and R. Beitner, Effects of carbamylcholine and pyridostigmine on cytoskeleton-bound and cytosolic phosphofructokinase and ATP levels in different rat tissues. Gen Pharmacol, 1996. 27(7): p. 1241-6. 1. The effects of carbamylcholine (CaCh) (acetylcholine agonist) and pyridostigmine (Pyr) (acetylcholinesterase inhibitor), on the activity of cytoskeleton-bound and cytosolic phosphofructokinase (PFK), the rate-limiting enzyme in glycolysis, and ATP levels, were studied in rat tibialis anterior (TA) muscle, heart, and brain. 2. In the TA muscle, a marked (about three-fold) increase in the allosteric activity of cytosolic (soluble) PFK was found, 3-5 min following the injection of CaCh or Pyr. The intracellular distribution of the enzyme was not affected by both drugs. Stimulation of glycolysis in this muscle was also expressed by a significant increase in the concentrations of glycolytic intermediates and lactate. Glucose 1,6-bisphosphate (Glc-1,6-P2) levels were unchanged, whereas fructose-2,6-bisphosphate (Fru-2,6-P2) was increased. Glycogenolysis was also stimulated, as deduced from the decrease in glycogen content. The stimulation of glycolysis, induced by both drugs, was accompanied by an increase in ATP level in the TA muscle. 3. In contrast to the stimulatory action of CaCh or Pyr on glycolysis in the TA muscle, both drugs had no effect on cytosolic and cytoskeletal PFK in heart and brain. However, ATP content in both heart and brain was markedly reduced by these drugs, most probably due to their reported harmful effects on mitochondrial function,

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leading to tissue damage. 4. Electron microscopic studies of TA muscle and heart from rats treated with CaCh or Pyr, revealed severe damage of heart but no harmful effects on TA muscle, which is a muscle with high glycolytic and low oxidative capacity. The present experiments suggest that the accelerated glycolysis in this muscle induced by both drugs, supplies ATP, thus preventing muscle damage.

224. Somani, S.M., R.K. Kutty, and G. Krishna, Eseroline, a metabolite of physostigmine, induces neuronal cell death. Toxicol Appl Pharmacol, 1990. 106(1): p. 28-37. The toxic effects of physostigmine, an anticholinesterase drug, and its metabolite eseroline were investigated in three neuronal cell culture systems, mouse neuroblastoma N1E-115, rat glioma C6, and neuroblastoma-glioma hybrid NG 108-15. Physostigmine and eseroline (0.5 nM) elicited a time-dependent leakage of lactic acid dehydrogenase (LDH) from all three cell types. An increased release of [14C]adenine nucleotides was also detected from cells when they were prelabeled with [14C]adenine. Eseroline was comparatively more toxic than the parent compound, physostigmine. Eseroline elicited a dose- and time-dependent leakage of LDH and release of adenine nucleotides from the neuronal cells. A nonneuronal cell line, rat liver ARL-15, was comparatively the most resistant cell type to eseroline toxicity. The concentrations of eseroline needed for 50% release of adenine nucleotides or 50% leakage of LDH from NG-108-15 and N1E-115 cells in 24 hr ranged from 40 to 75 microM. The concentrations of eseroline needed to obtain similar responses in C6 and ARL-15 cells were much higher and ranged from 80 to 120 microM. Phase contrast microscopy showed extensive damage to three neuronal cell lines at concentrations of eseroline as low as 75 microM. The loss of ATP from N1E-115 cells exceeded 50% when they were treated with 0.3 mM eseroline for 1 hr--at which time the leakage of LDH was not detectable. It seems that eseroline causes neuronal cell death by a mechanism involving loss of cell ATP. Thus, the formation of eseroline may contribute to the toxic effect of physostigmine.

225. Spencer, P.S., et al., Self-reported exposures and their association with unexplained illness in a population-based case-control study of Gulf War veterans. J Occup Environ Med, 2001. 43(12): p. 1041-56. Many factors have been considered as possible causes of the unexplained illness reported by veterans of the Gulf War (GW). In this study, we report an analysis of risk factors and unexplained illness in a population-based sample of GW veterans who underwent clinical evaluation. Multiple risk factors were compared in 241 veterans who met criteria for unexplained illness and 113 healthy controls. Results suggest that GW unexplained illness is most highly associated with combat conditions, heat stress, and having sought medical attention during the GW. When controlling for multiple simultaneous exposures during the GW, interactions around pyridostigmine bromide, insecticides and repellents, and stress were not significant. These results indicate that most unexplained illness in GW veterans cannot be explained by neurotoxic effects of exposures to chemicals that inhibit cholinesterase activity.

226. Abu-Qare, A.W. and M.B. Abou-Donia, Combined exposure to sarin and pyridostigmine bromide increased levels of rat urinary 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine, biomarkers of oxidative stress. Toxicol Lett, 2001. 123(1): p. 51-8. In

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this study concentrations of markers of oxidative stress 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OhdG) were determined in rat urine following a single oral dose of pyridostigmine bromide (PB) 13 mg/kg and a single intramuscular dose of sarin 80 microg/kg alone or in combination. Urine samples were collected 16, 24, 48, 72, and 96 h following dosing. Control urine samples of five rats treated with normal saline were also collected at the same time intervals. A combined dose of PB and sarin significantly increased levels of 3-nitrotyrosine and (8-OhdG) starting 48 h after dosing. An increase in the concentration of these markers was not detected following a single dose of PB or sarin alone. Maximal increase in 3-nitrotyrosine and 8-OhdG was detected 48 h after administration of a combination PB and sarin. The results indicate that concurrent exposure to PB and sarin could generate free radical species that may cause oxidative stress in rats. The results may have significant impact if veterans were expose to sarin following an oral dose of PB.

227. Abu-Qare, A.W., H.B. Suliman, and M.B. Abou-Donia, Induction of urinary excretion of 3-nitrotyrosine, a marker of oxidative stress, following administration of pyridostigmine bromide, DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination in rats. Toxicol Lett, 2001. 121(2): p. 127-34. In this study, we determined levels of 3-nitrotyrosine in rat urine following administration of a single oral dose of 13 mg/kg pyridostigmine bromide (PB) (3-dimethylaminocarbonyloxy-N-methylpyridinum bromide), a single dermal dose of 400 mg/kg N,N-diethyl-m-toluamide (DEET) and a single dermal dose of 1.3 mg/kg permethrin, alone and in combination. Urine samples were collected from five treated and five control rats at 4, 8, 16, 24, 48, and 72 h following dosing. Solid-phase extraction coupled with high-performance liquid chromatography with ultraviolet detection at 274 nm was used for the determination of tyrosine and 3-nitrotyrosine. A single oral dose of PB and a single dermal dose of DEET or their combination significantly (P<0.05) increased levels of 3-nitrotyrosine starting 24 h after dosing compared with control urine samples. The maximum increase of 3-nitroytyrosine was detected 48 h after combined administration of PB and DEET. The ratio of 3-nitrotyrosine to tyrosine in urine excreted 48 h after dosing was 0.19+/-0.04, 0.20+/-0.05, 0.28+/-0.03, 0.32+/-0.04, 0.19+/-0.05, 0.42+/-0.04, 0.27+/-0.03, 0.36+/-0.04, and 0.48+/-0.04 following administration of water, ethanol, PB, DEET, permethrin, PB+DEET, PB+permethrin, DEET+permethrin, and PB+DEET+permethrin, respectively. The results indicate that an oral dose of PB and a dermal administration of DEET, alone and in combination, could generate free radical species, and thus increase levels of 3-nitrotyrosine in rat urine. Induction of 3-nitrotyrosine, a marker of oxidative stress, following exposure to these compounds could be significant in understanding the proposed enhanced toxicity following combined exposure to these compounds.

228. Artru, A.A. and J.D. Michenfelder, A re-examination of physostigmine-induced cerebral protection in the hypoxic mouse. A critical assessment of the model. Stroke, 1980. 11(2): p. 197-9. Using the hypoxic (FiO2 = 0.05) mouse model as originally described, the survival time following pretreatment with physostigmine was examined. The maximum increase in survival time was 87% following a physostigmine dose of 0.4 mg/kg. This increase was considerably less than that previously reported for this drug in a hypoxic mouse study wherein the standard method for exposing mice to hypoxia was

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altered. We speculate that this alteration in methodology resulted in small variations in FiO2 sufficient to account for the differences between these studies.

229. Andjelkovic, D.S. and M.R. Terzic, The effects of physostigmine on the oxygen uptake in rat brain tissue. Experientia, 1979. 35(3): p. 372-3. Physostigmine in a dose of 0.1 mg/kg i.v. expressly stimulated the oxygen uptake in the rat cerebral cortex. This effect was blocked by propranolol and seems be mediated by catecholamines. Since atropine also antagonized the stimulant effect of physostigmine, it appears that the action of physostigmine is primarily cholinergic and that the adrenergic effect is a secondary phenomenon. The higher dose of physostigmine (0.4 mg/kg i.v.) caused a depression of rat brain oxygen uptake.

230. Rupreht, J., H.J. Schneck, and B. Dworacek, [Physostigmine--recent pharmacologic data and their significance for practical use]. Anaesthesiol Reanim, 1989. 14(4): p. 235-41. Physostigmine is widely used for treatment of the central anticholinergic syndrome during recovery from anaesthesia. The drug is also very useful in treatment of intoxicated patients, in differential-diagnostic procedures of coma of unknown origin, and in restoration of vigilance after prolonged sedation for mechanical ventilation. Besides the specific central cholinergic action of physostigmine, several new pharmacological actions have now been established. Analgesic action is dependent on the interaction with the 5-HT (serotoninergic) system and is independent of narcotic or cholinergic agonists. The antianalgetic stress hormone, ACTH, also does not interfere with this action. Physostigmine does not interfere with the anaesthetic state when given during general anaesthesia. It attenuates several withdrawal states, especially alcohol delirium, opiate and nitrous oxide withdrawal syndromes. The drug may offer a protective mechanism against hypoxic damage of the brain and may also be beneficial in amnestic syndromes and sleep disorders. Physostigmine produces central and peripheral cardiovascular stimulation. It has been shown that physostigmine can be useful in prevention and treatment of postanaesthetic behavioural disturbances following anaesthesia with propofol. Number of indications for use of physostigmine has increased considerably.

231. Bradley, R.J., R. Sterz, and K. Peper, Agonist and inhibitory effects of pyridostigmine at the neuromuscular junction. Brain Res, 1986. 376(1): p. 199-203. When all of the AChE at the endplate is irreversibly inhibited by phospholine iodide the ionophoretically induced ACh endplate currents are increased more than 10-fold in amplitude. The reversible AChE inhibitor pyridostigmine only increases the current to about half this value because its effects are obscured by receptor blocking. It was found that pyridostigmine can activate the receptor ion channels when released by ionophoresis at the endplate, thus suggesting that agonist-like desensitization could contribute to the blocking effects.

232. Zheng, J.Q., et al., Neostigmine competitively inhibited nicotinic acetylcholine receptors in sympathetic neurons. Life Sci, 1998. 62(13): p. 1171-8. The present experiment investigates the effect of neostigmine on nicotinic acetylcholine receptors (nAChRs) in the cultured neurons from neonatal rat superior cervical ganglia (SCG).

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Using whole-cell patch clamp techniques, we found that the amplitudes of the currents induced by 50 microM dimethylphenylpiperazinium (DMPP) were 21.5+/-10.7%, 52.9+/-9.2% and 86.9+/-4.9% depressed at the increased concentrations of neostigmine 100, 200 and 400 microM, respectively. The inhibition of neostigmine decreased gradually with the increased concentration of nicotine from 10 to 160 microM. Lineweaver-Burk's double-reversible plot illustrated that neostigmine blocked neuronal nAChRs in a competitive manner. Hyperpolarization of membrane potential from -40 mV to -100 mV did not significantly influence the blockade of neostigmine. Neostigmine could not accelerate the decay of the DMPP-induced currents, neither evoke any detectable currents in SCG neurons. The results indicate that neostigmine depress neuronal nAChRs in a competitive, concentration-dependent and voltage-independent manner, and can not facilitate desensitization of the receptors. The present data suggest that neostigmine blocks neuronal nAChRs by interacting with the ACh binding sites of the receptors.

233. Shaw, K.P., et al., The reversible cholinesterase inhibitor physostigmine has channel-blocking and agonist effects on the acetylcholine receptor-ion channel complex. Mol Pharmacol, 1985. 28(6): p. 527-38. The actions of the carbamate cholinesterase inhibitors, physostigmine (Phy) and physostigmine methiodide (MetPhy), were studied on the acetylcholine receptor-ion channel complex (AChR) of skeletal muscles. Low concentrations of these agents produced cholinesterase inhibition which resulted in potentiation of nerve-elicited muscle twitches and an increased peak amplitude and prolongation of the decay time constant (tau EPC) of endplate currents (EPCs) elicited in frog (Rana pipiens) sartorius muscles. However, increasing concentrations of Phy depressed the peak amplitude and shortened the decay phase of the EPC with an apparent loss in the voltage dependence of tau EPC. At higher concentrations and depolarized potentials, EPC decays were double exponential. The effects of both Phy and MetPhy on the postsynaptic AChR complex were also evident in preparations pretreated with diisopropylfluorophosphate. Under these conditions, a linear relationship between the reciprocal of tau EPC and the concentration of these agents was observed. Single channel studies revealed that Phy (20-600 microM) shortened channel lifetime and decreased channel conductance at very high concentrations. In addition, Phy (0.5 microM) induced the appearance of channel openings with conductance similar to that of acetylcholine. High concentrations (greater than 50 microM) of this agent activated channel openings with decreased conductance. Similar results were obtained with MetPhy. Thus, the reversible cholinesterase inhibitors Phy and MetPhy altered the properties of the AChR by interacting as agonists capable of inducing desensitization and blockade.

234. Zheng, J.Q., et al., Physostigmine blocked nicotinic acetylcholine receptors in rat sympathetic ganglion neurons. Zhongguo Yao Li Xue Bao, 1997. 18(6): p. 508-11. AIM: To study the blocking mechanism of physostigmine (Phy) on nicotinic acetylcholine receptors (NAChR) in sympathetic neurons. METHODS: The whole-cell patch-clamp technique was used to observe the effects of Phy on NAChR in the cultured sympathetic neurons from neonatal rat superior cervical ganglia (SCG). RESULTS: Phy 5 -20 mumol.L-1 inhibited neuronal NAChR in a concentration-dependent manner and accelerated the desensitization of NAChR. Changing the membrane potential from -50 to -90 mV did not affect the blocking effect of Phy. Phy 200 mumol.L-1 did not induce any

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noticeable response in SCG neurons. CONCLUSION: Phy blocked NAChR in the sympathetic ganglion neurons by interacting with the allosteric sites out of the binding sites and the open ionic channels of the receptors. Phy did not possess excitative effect on NAChR in SCG neurons.

235. Navratil, J., et al., The labilizing and stabilizing effect of physostigmine on CNS. Acta Univ Palacki Olomuc Fac Med, 1990. 126: p. 39-44. The authors observed biphasic changes of dopamine, norepinephrine and serotonin in the rat brain stem after a single application of physostigmine (1 mg/kg of body weight). The results confirm the hypothesis of "labilization and stabilization" of membranes.

236. Navratil, J., et al., Increase of nonesterified fatty acids in CNS after physostigmine application. Homeost Health Dis, 1991. 33(4): p. 172-3.

237. Tapia, R., Release and uptake of glutamate as related to excitotoxicity. Rev Bras Biol, 1996. 56 Su 1 Pt 1: p. 165-74. It has been established that neurons exposed to high concentrations of glutamate or other excitatory amino acids degenerate and die. Neuronal damage appears to be due to the activation of different types of glutamate receptors, among which the ionotropic N-methyl-D-aspartate (NMDA) type seems particularly involved, since its channel is permeable to Ca2+ and an increase in the cytoplasmic concentration of this cation promotes a chain of events leading to cell death. The mechanism of such glutamate receptor-mediated neurodegeneration has been defined as excitotoxicity, and several pieces of evidence suggest that this mechanism might contribute to the neuronal death associated with certain neurological disorders, such as ischemia, cerebral trauma and some chronic neurodegenerative diseases. A relevant question is whether the origin of endogenous extracellular glutamate is important for the induction of excitotoxicity. An excess of glutamate release, or a deficiency in its clearance from the synaptic cleft, which depends mainly on its transport by high affinity carriers, are potential sources for the accumulation of extracellular glutamate. In the present article some experimental results from our laboratory, aimed at obtaining information on this question, are reviewed. These experiments include the use of 4-aminopyridine, a convulsant drug that enhances the release of glutamate, and of some inhibitors of glutamate transport, in vivo and in neuronal cell cultures. The results obtained indicate that an increase of endogenous extracellular glutamate due to these procedures is not sufficient to induce neuronal death, at least under the experimental conditions used.

238. Pitt, D., P. Werner, and C.S. Raine, Glutamate excitotoxicity in a model of multiple sclerosis. Nat Med, 2000. 6(1): p. 67-70. Glutamate excitotoxicity mediated by the AMPA/kainate type of glutamate receptors damages not only neurons but also the myelin-producing cell of the central nervous system, the oligodendrocyte. In multiple sclerosis, myelin, oligodendrocytes and some axons are lost as a result of an inflammatory attack on the central nervous system. Because glutamate is released in large quantities by activated immune cells, we expected that during inflammation in MS, glutamate excitotoxicity might contribute to the lesion. We addressed this by using the AMPA/kainate antagonist NBQX to treat mice sensitized for experimental autoimmune

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encephalomyelitis, a demyelinating model that mimics many of the clinical and pathologic features of multiple sclerosis. Treatment resulted in substantial amelioration of disease, increased oligodendrocyte survival and reduced dephosphorylation of neurofilament H, an indicator of axonal damage. Despite the clinical differences, treatment with NBQX had no effect on lesion size and did not reduce the degree of central nervous system inflammation. In addition, NBQX did not alter the proliferative activity of antigen-primed T cells in vitro, further indicating a lack of effect on the immune system. Thus, glutamate excitotoxicity seems to be an important mechanism in autoimmune demyelination, and its prevention with AMPA/kainate antagonists may prove to be an effective therapy for multiple sclerosis.

239. Chen, Q., C.L. Veenman, and A. Reiner, Cellular expression of ionotropic glutamate receptor subunits on specific striatal neuron types and its implication for striatal vulnerability in glutamate receptor-mediated excitotoxicity. Neuroscience, 1996. 73(3): p. 715-31. Glutamate receptors are composed of subtype-specific subunits. Variation in the precise subunit composition of a receptor may result in significant functional differences. Thus, a precise knowledge of subunit composition on striatal neurons is a prerequisite for understanding the selective vulnerability of striatal neurons to excitatory amino acids. In the present study, we used an immunohistochemical double-labelling approach to localize ionotropic glutamate receptor subunits (NMDAR1, GluR1, GluR2/3, GluR4 and GluR5/6/7) on specific striatal neuron populations. Our results showed that striatal cholinergic and somatostatin interneurons were not labelled for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate, receptor subunits GluR1, GluR2/3 and GluR4. Most cholinergic and somatostatin interneurons (83.3% to 100%), however, were double-labelled for the N-methyl-D-aspartate receptor subunit NR1 and kainic acid receptor subunits GluR5/6/7. All parvalbumin interneurons were labelled for GluR1 and GluR4, and 96% GluR1 positive and 95% GluR4 positive neurons were also double-labelled as parvalbumin interneurons. About half of all parvalbumin interneurons co-localized with GluR2/3, and over 97% were labelled for NR1 and GluR5/6/7. Among striatal projection neurons, enkephalin-positive (mainly striatopallidal) neurons, striatonigral neurons (mainly substance P-positive) and calbindin-positive matrix neurons were not immunostained for GluR1 or GluR4. In contrast, 95% to 100% of each of these types of projection neurons were double-labelled for NR1, GluR2/3 and GluR5/6/7. Our results demonstrate that striatal neuron types differ in their expression of ionotropic glutamate receptor subunits and subtypes. The clear difference between striatal interneurons and projection neurons in ionotropic glutamate receptor subtypes/subunits supports the idea that differential glutamate receptor expression mechanism may account for the selective vulnerability of striatal projection neurons to excitotoxicity, and that glutamate receptor-mediated excitotoxicity may be involved in the striatal neurodegenerative diseases.

240. Paschen, W., Glutamate excitotoxicity in transient global cerebral ischemia. Acta Neurobiol Exp (Warsz), 1996. 56(1): p. 313-22. The glutamate excitotoxicity hypothesis of ischemic cell damage holds that cell damage caused by transient cerebral ischemia is triggered by glutamate, released during ischemia from the intracellular compartment into the synaptic cleft: high extracellular glutamate levels activate ionotropic glutamate

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receptors, thus inducing an overflow of calcium ions into the neurones and a calcium-induced activation of catabolic processes. However, several arguments (and much of the evidence) in favour of this hypothesis do not bear closer examination. On the other hand, evidence is accumulating that, after transient ischemia, calcium fluxes through ionotropic glutamate receptors of the non-NMDA type may play a major role in the manifestation of ischemic cell damage. Calcium fluxes through non-NMDA receptors are determined by mRNA editing of non-NMDA receptor subunits: calcium fluxes are blocked in the presence of an edited subunit. A possible role of mRNA editing in the development of ischemic cell damage is discussed.

241. Zhang, J., et al., Secondary excitotoxicity contributes to dopamine-induced apoptosis of dopaminergic neuronal cultures. Biochem Biophys Res Commun, 1998. 248(3): p. 812-6. Dopamine (DA) and related catechols may contribute to selective degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease. To investigate whether DA induces apoptosis of dopaminergic neurons, we characterized the effects of various concentrations of exogenous DA on a substantia nigra/neuroblastoma hybrid cell line (MES 23.5 or MES). The hybrid MES cells were maintained in the presence of 50 microM glutamate in logarithmic growth on poly-D-lysine-precoated T-75 flasks and plated either onto petri dishes with glass coverslips for morphological studies or onto 6-well plates for quantification of apoptosis by flow cytometry. The results showed that DA exposure (0.5-20 microM) induced time- and dose-dependent apoptotic cell death of MES cells. To further analyze the mechanism responsible for DA-mediated apoptosis, we repeated the experiments at 20 microM DA in the presence or absence of 40 microM nomifensine, a DA re-uptake inhibitor, and 25 microM 2-amino-5-phosphonopentanoic acid (AP5), an N-methyl-D-aspartate (NMDA) receptor antagonist. The data indicate that both compounds significantly prevented DA-induced apoptosis of MES cells and that combination of AP5 and nomifensine provided greater protection against DA toxicity than AP5 alone. These results suggest for the first time that DA-induced apoptosis in dopaminergic neurons is partially attributable to increased vulnerability of these cells to non-toxic levels of excitatory amino acids, i.e., secondary excitotoxicity.

242. Hugon, J., J.M. Vallat, and M. Dumas, [Role of glutamate and excitotoxicity in neurologic diseases]. Rev Neurol (Paris), 1996. 152(4): p. 239-48. Glutamate is one of the major excitatory neurotransmitter in the central nervous system. Glutamate acts on 4 different post synaptic receptors; NMDA (N-Methyl-D-aspartate) AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), Kainate and metabotropic receptors. The three former receptors are linked to membrane ion channels whereas metabotropic receptors are coupled with a G protein. Glutamate is involved in the physiologic processes of learning, memory and motricity. Glutamate is also a potent neurotoxin responsible for toxic neuronal death of post synaptic neurons. This action has been denominated excitotoxicity and occurs as a consequence of a prolonged or a strong activation of glutamate post-synaptic receptors. The rise in intracellular calcium seems to play a major role in the pathological events following excitotoxicity. The pathophysiology of several acute or chronic neurological disorders has been linked to excitotoxicity. This excitotoxic process could be present in acute neuronal death observed

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in stroke, hypoglycemia and traumatisms of the central nervous system and in chronic neuronal degeneration observed in Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease and neuro AIDS. A better knowledge of the cellular events induced by excitotoxicity will allow to consider new therapeutic approaches in various neurological disorders.

243. Shaw, P.J. and P.G. Ince, Glutamate, excitotoxicity and amyotrophic lateral sclerosis. J Neurol, 1997. 244 Suppl 2: p. S3-14. The "glutamate hypothesis" is one of three major pathophysiological mechanisms of motor neurone injury towards which current research effort into amyotrophic lateral sclerosis (ALS) is directed. There is great structural and functional diversity in the glutamate receptor family which results from combinations of 14 known gene products and their splice variants, with or without additional RNA editing. It is possible that motor neurones express a unique molecular profile of glutamate receptors. Abnormal activation of glutamate receptors is one of five main candidates as a final common pathway to neuronal death. In classical acute excitotoxicity, there is influx of Na+ and CI-, and destabilisation of intracellular Ca2+ homeostasis, which activates a cascade of harmful biochemical events. The concept of secondary excitotoxicity, where cellular injury by glutamate is triggered by disturbances in neuronal energy status, may be particularly relevant to a chronic neurodegenerative disease such as ALS. Data are now beginning to emerge on the fine molecular structure of the glutamate receptors present on human motor neurones, which have a distinct profile of AMPA receptors. Two important molecular features of motor neurones have been identified that may contribute to their vulnerability to neurodegeneration. The low expression of calcium binding proteins and the low expression of the GluR2 AMPA receptor subunit by vulnerable motor neurone groups may render them unduly susceptible to calcium-mediated toxic events following glutamate receptor activation. Eight lines of evidence that indicate a disturbance of glutamatergic neurotransmission in ALS patients are reviewed. The links between abnormal activation of glutamate receptors and other potential mechanisms of neuronal injury, including activation of calcium-mediated second messenger systems and free radical mechanisms, are emphasised. Riluzole, which modulates the glutamate neurotransmitter system, has been shown to prolong survival in patients with ALS. Further research may allow the development of subunit-specific therapeutic targeting of glutamate receptors and modulation of "downstream" events within motor neurones, aimed at protecting vulnerable molecular targets in specific populations of ALS patients.

244. US links motor neurone disease with Gulf War service. BMJ, 2002. 324: p. 65. ALS.

245. Nguimfack Mbodie, P.C., [Do the glutamate excitotoxicity theory and potential free radicals implication in schizophrenia aetiopathogenesis provide a new enlightenment to links between: genome, environment and biology in the determinism of that disorder?]. Encephale, 2002. 28(2): p. 147-53. The aetiopathogenesis of schizophrenia constitutes nowadays one of the major points of interest for researchers on this cosmopolitan disorder which involves about 1% of the world population and which significantly alters the social functioning of the individual. Numerous studies have

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focused on the role played by genome, environmental factors and biology in the development of symptoms. The neurodevelopmental theory is an illustration with the perinatal period considered as the main provider of environmental factors (hypertension, infections, bleedings during pregnancy, acute and chronic fetal distress.). Many authors found significant associations between such factors, the occurrence of brain lesions and finally schizophrenic symptoms. Although no convincing genetic model had been established to date for schizophrenia, nevertheless it appears that a predisposition not inheritable under the mendelian mode exists and authors showed that disease gets more and more severe over schizophrenic descendants. The risk to be schizophrenic being a first degree relative of the schizophrenic person is about ten time superior than in general population. Indeed, this risk is also about ten time superior in biological parents of schizophrenic adoptees than in biological parents of healthy adoptees. Studies done in monozygotic comparing to dizygotic twins are in favour of an important role played by genetic factors more than socioeducational or psychological factors. Concerning biology, the dopaminergic hypothesis remains shared by numerous authors although direct links with incriminated factors are not well established. Now is suspected the glutamate excitotoxicity with implication of free radicals in schizophrenia. These free radicals are products of various enzymatic activations led by overstimulation of post synaptic receptors (NMDA and AMPA) by the excess glutamate. Therefore, according to that concept, some amino acids as glutamate and derivatives could have through free radicals a noxious effect on neuronal synapses. This could be due to a failing of their recapture at the presynaptic level in addition to a dysfunctioning of the antioxidizing system (glutathion, carnosine, superoxide dismutase, aspartate) to which dopamine and other monoamines might participate. The question is whether or not this theory contributes to shed light on links between: genome, environmental factors and biology in schizophrenia. Through the review and discussion of genetical aspects of schizophrenia, environmental factors and the biological aspect, we intend to revive debate on that question. The articles and authors were selected with regard to the aptness of their publications on that subject, their evolving ideas and finally the interest of their works for neurosciences. This new approach perhaps is opening the way to new therapeutic perspectives in the treatment of schizophrenia based on the antioxidizing substances as shown for some neurological diseases (amyotrophic lateral sclerosis, Parkinson's disease and Huntington's chorea) for which experiments are going on.

246. Doble, A., The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacol Ther, 1999. 81(3): p. 163-221. Glutamic acid is the principal excitatory neurotransmitter in the mammalian central nervous system. Glutamic acid binds to a variety of excitatory amino acid receptors, which are ligand-gated ion channels. It is activation of these receptors that leads to depolarisation and neuronal excitation. In normal synaptic functioning, activation of excitatory amino acid receptors is transitory. However, if, for any reason, receptor activation becomes excessive or prolonged, the target neurones become damaged and eventually die. This process of neuronal death is called excitotoxicity and appears to involve sustained elevations of intracellular calcium levels. Impairment of neuronal energy metabolism may sensitise neurones to excitotoxic cell death. The principle of excitotoxicity has been well-established experimentally, both in in vitro systems and in vivo, following administration

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of excitatory amino acids into the nervous system. A role for excitotoxicity in the aetiology or progression of several human neurodegenerative diseases has been proposed, which has stimulated much research recently. This has led to the hope that compounds that interfere with glutamatergic neurotransmission may be of clinical benefit in treating such diseases. However, except in the case of a few very rare conditions, direct evidence for a pathogenic role for excitotoxicity in neurological disease is missing. Much attention has been directed at obtaining evidence for a role for excitotoxicity in the neurological sequelae of stroke, and there now seems to be little doubt that such a process is indeed a determining factor in the extent of the lesions observed. Several clinical trials have evaluated the potential of antiglutamate drugs to improve outcome following acute ischaemic stroke, but to date, the results of these have been disappointing. In amyotrophic lateral sclerosis, neurolathyrism, and human immunodeficiency virus dementia complex, several lines of circumstantial evidence suggest that excitotoxicity may contribute to the pathogenic process. An antiglutamate drug, riluzole, recently has been shown to provide some therapeutic benefit in the treatment of amyotrophic lateral sclerosis. Parkinson's disease and Huntington's disease are examples of neurodegenerative diseases where mitochondrial dysfunction may sensitise specific populations of neurones to excitotoxicity from synaptic glutamic acid. The first clinical trials aimed at providing neuroprotection with antiglutamate drugs are currently in progress for these two diseases.

247. Rodriguez, M.C., J.A. Obeso, and C.W. Olanow, Subthalamic nucleus-mediated excitotoxicity in Parkinson's disease: a target for neuroprotection. Ann Neurol, 1998. 44(3 Suppl 1): p. S175-88. Dopamine deficiency causes disinhibition and overactivity of the subthalamic nucleus (STN). Output neurons from the STN are excitatory and use glutamate as a neurotransmitter. They project to the external and internal segments of the globus pallidum (GPe and GPi), the substantia nigra pars reticulata (SNr), and the pedunculopontine nucleus (PPN). In addition, STN neurons provide excitatory innervation to dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) that contain glutamate receptors. Stimulation of the STN induces bursting activity in SNc dopaminergic neurons. This raises the possibility that the disinhibition of STN neurons that occurs as a result of a dopamine lesion might induce excitotoxic damage in target structures, including the SNc. In addition, the reduction in complex I activity found in the nigra in Parkinson's disease (PD) may cause mitochondrial dysfunction and make SNc dopaminergic neurons vulnerable to even physiologic concentrations of glutamate. We postulate that the dopamine loss that occurs in PD produces augmented STN activity which, in turn, causes further damage to vulnerable dopaminergic neurons, thereby creating a scenario for an increasing cycle of neuronal loss in the SNc. In addition, STN overactivity could, in theory, cause damage to the GPi, SNr, and PPN and thereby account for the development of parkinsonian features that do not respond to levodopa in patients with advanced disease. This hypothesis suggests that pharmacologic or surgical therapies that reduce STN neuronal overactivity or block glutamate receptors in the SNc and other target structures might be neuroprotective and might slow or halt the progression of neurodegeneration in PD.

248. Beal, M.F., Excitotoxicity and nitric oxide in Parkinson's disease pathogenesis. Ann Neurol, 1998. 44(3 Suppl 1): p. S110-4. A potential role for excitotoxic processes in

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Parkinson's disease (PD) has been strengthened by the recent observations that there appears to be a mitochondrially encoded defect in complex I activity of the electron transport chain. An impairment of oxidative phosphorylation will enhance vulnerability to excitotoxicity. Substantia nigra neurons possess N-methyl-D-aspartate receptors and there are glutamatergic inputs into the substantia nigra from both the cerebral cortex and the subthalamic nucleus. After activation of excitatory amino acid receptors, there is an influx of calcium followed by activation of neuronal nitric oxide (NO) synthase, which can then lead to the generation of peroxynitrite. Consistent with such a mechanism, studies of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity in both mice and primates have shown that inhibition of neuronal NO synthase exerts neuroprotective effects. Studies utilizing excitatory amino acid receptor antagonists have been inconsistent in mice but show significant neuroprotective effects in primates. These results raise the prospect that excitatory amino acid antagonists for neuronal NO synthase inhibitors might be useful in the treatment of PD.

249. Zhang, J., et al., Secondary excitotoxicity contributes to dopamine-induced apoptosis of dopaminergic neuronal cultures. Biochem Biophys Res Commun, 1998. 248(3): p. 812-6. Dopamine (DA) and related catechols may contribute to selective degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease. To investigate whether DA induces apoptosis of dopaminergic neurons, we characterized the effects of various concentrations of exogenous DA on a substantia nigra/neuroblastoma hybrid cell line (MES 23.5 or MES). The hybrid MES cells were maintained in the presence of 50 microM glutamate in logarithmic growth on poly-D-lysine-precoated T-75 flasks and plated either onto petri dishes with glass coverslips for morphological studies or onto 6-well plates for quantification of apoptosis by flow cytometry. The results showed that DA exposure (0.5-20 microM) induced time- and dose-dependent apoptotic cell death of MES cells. To further analyze the mechanism responsible for DA-mediated apoptosis, we repeated the experiments at 20 microM DA in the presence or absence of 40 microM nomifensine, a DA re-uptake inhibitor, and 25 microM 2-amino-5-phosphonopentanoic acid (AP5), an N-methyl-D-aspartate (NMDA) receptor antagonist. The data indicate that both compounds significantly prevented DA-induced apoptosis of MES cells and that combination of AP5 and nomifensine provided greater protection against DA toxicity than AP5 alone. These results suggest for the first time that DA-induced apoptosis in dopaminergic neurons is partially attributable to increased vulnerability of these cells to non-toxic levels of excitatory amino acids, i.e., secondary excitotoxicity.

250. Hugon, J., J.M. Vallat, and M. Dumas, [Role of glutamate and excitotoxicity in neurologic diseases]. Rev Neurol (Paris), 1996. 152(4): p. 239-48. Glutamate is one of the major excitatory neurotransmitter in the central nervous system. Glutamate acts on 4 different post synaptic receptors; NMDA (N-Methyl-D-aspartate) AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), Kainate and metabotropic receptors. The three former receptors are linked to membrane ion channels whereas metabotropic receptors are coupled with a G protein. Glutamate is involved in the physiologic processes of learning, memory and motricity. Glutamate is also a potent neurotoxin responsible for toxic neuronal death of post synaptic neurons. This action has been

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denominated excitotoxicity and occurs as a consequence of a prolonged or a strong activation of glutamate post-synaptic receptors. The rise in intracellular calcium seems to play a major role in the pathological events following excitotoxicity. The pathophysiology of several acute or chronic neurological disorders has been linked to excitotoxicity. This excitotoxic process could be present in acute neuronal death observed in stroke, hypoglycemia and traumatisms of the central nervous system and in chronic neuronal degeneration observed in Amyotrophic Lateral Sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease and neuro AIDS. A better knowledge of the cellular events induced by excitotoxicity will allow to consider new therapeutic approaches in various neurological disorders.

251. Olney, J.W., et al., Excitotoxicity of L-dopa and 6-OH-dopa: implications for Parkinson's and Huntington's diseases. Exp Neurol, 1990. 108(3): p. 269-72. Despite several decades of research aimed at elucidating the mechanisms underlying neuronal degeneration in Parkinson's and Huntington's diseases, these mysteries remain unfathomed. The brain contains high concentrations of the putative transmitters, glutamate and aspartate, which have neurotoxic (excitotoxic) potential and are thought to cause neuronal degeneration in certain acute neurological disorders. However, no mechanism has been identified by which these diffusely distributed agents might cause the regionally selective patterns of neuronal degeneration characterizing Parkinson's and Huntington's diseases. Here we report that L-DOPA, the natural precursor to dopamine, is a weak excitotoxin and its ortho-hydroxylated derivative, 6-OH-DOPA, is a powerful excitotoxin. We propose that an excitotoxic process mediated by L-DOPA or an acidic derivative such as 6-OH-DOPA might be responsible for degeneration of nigral neurons in Parkinson's disease or striatal neurons in Huntington's disease.

252. Greene, J.G. and J.T. Greenamyre, Bioenergetics and glutamate excitotoxicity. Prog Neurobiol, 1996. 48(6): p. 613-34. Bioenergetic defects and abnormalities in glutamate neurotransmission have both been proposed to play important roles in neurological diseases of varying chronology, etiology and pathology. Recent experimental evidence suggests an intimate relationship between these two systems. Metabolic inhibition predisposes neurons to glutamate-mediated "excitotoxic" damage. The exact mechanism of this increased susceptibility is yet to be defined, but may involve, singly or in combination, decreased voltage-dependent Mg2+ blockade of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, abnormalities in cellular Ca2+ homeostasis, or elevated production of reactive oxygen species. It is believed that enhancement of excitotoxicity by impaired metabolism may be a ubiquitous mechanism of neuronal death in neurological disease. Further elucidation of the exact mechanism of this enhancement may lead to the discovery of new targets for therapeutic intervention.

253. Tymianski, M., et al., Characterization of neuroprotection from excitotoxicity by moderate and profound hypothermia in cultured cortical neurons unmasks a temperature-insensitive component of glutamate neurotoxicity. J Cereb Blood Flow Metab, 1998. 18(8): p. 848-67. Although profound hypothermia has been used for decades to protect the human brain from hypoxic or ischemic insults, little is known about the underlying mechanism. We therefore report the first characterization of the

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effects of moderate (30 degrees C) and profound hypothermia (12 degrees to 20 degrees C) on excitotoxicity in cultured cortical neurons exposed to excitatory amino acids (EAA; glutamate, N-methyl-D-aspartate [NMDA], AMPA, or kainate) at different temperatures (12 degrees to 37 degrees C). Cooling neurons to 30 degrees C and 20 degrees C was neuroprotective, but cooling to 12 degrees C was toxic. The extent of protection depended on the temperature, the EAA receptor agonist employed, and the duration of the EAA challenge. Neurons challenged briefly (5 minutes) with all EAA were protected, as were neurons challenged for 60 minutes with NMDA, AMPA, or kainate. The protective effects of hypothermia (20 degrees and 30 degrees C) persisted after rewarming to 37 degrees C, but rewarming from 12 degrees C was deleterious. Surprisingly, however, prolonged (60 minutes) exposures to glutamate unmasked a temperature-insensitive component of glutamate neurotoxicity that was not seen with the other, synthetic EAA; this component was still mediated via NMDA receptors, not by ionotropic or metabotropic non-NMDA receptors. The temperature-insensitivity of glutamate toxicity was not explained by effects of hypothermia on EAA-evoked [Ca2+]i increases measured using high- and low-affinity Ca2+ indicators, nor by effects on mitochondrial production of reactive oxygen species. This first characterization of excitotoxicity at profoundly hypothermic temperatures reveals a previously unnoticed feature of glutamate neurotoxicity unseen with the other EAA, and also suggests that hypothermia protects the brain at the level of neurons by blocking, rather than slowing, excitotoxicity.

254. Budd, S.L. and D.G. Nicholls, Mitochondria, calcium regulation, and acute glutamate excitotoxicity in cultured cerebellar granule cells. J Neurochem, 1996. 67(6): p. 2282-91. Exposure of cultured cerebellar granule cells to 100 microM glutamate plus glycine in the absence of Mg2+ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca2+ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca2+ concentration), and extensive cell death. Glutamate-evoked Ca2+ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca2+ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little 45Ca2+ and maintaining a low bulk cytoplasmic free Ca2+ concentration determined by fura-2. It is concluded that mitochondrial Ca2+ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca2+ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca2+ accumulation in a microdomain accessible to the mitochondria.

255. Nicholls, D.G. and S.L. Budd, Mitochondria and neuronal glutamate excitotoxicity. Biochim Biophys Acta, 1998. 1366(1-2): p. 97-112. The role of mitochondria in the control of glutamate excitotoxicity is investigated. The response of

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cultured cerebellar granule cells to continuous glutamate exposure is characterised by a transient elevation in cytoplasmic free calcium concentration followed by decay to a plateau as NMDA receptors partially inactivate. After a variable latent period, a secondary, irreversible increase in calcium occurs (delayed calcium deregulation, DCD) which precedes and predicts subsequent cell death. DCD is not controlled by mitochondrial ATP synthesis since it is unchanged in the presence of the ATP synthase inhibitor oligomycin in cells with active glycolysis. However, mitochondrial depolarisation (and hence inhibition of mitochondrial calcium accumulation) without parallel ATP depletion (oligomycin plus either rotenone or antimycin A) strongly protects the cells against DCD. Glutamate exposure is associated with an increase in the generation of superoxide anion by the cells, but superoxide generation in the absence of mitochondrial calcium accumulation is not neurotoxic. While it is concluded that mitochondrial calcium accumulation plays a critical role in the induction of DCD we can find no evidence for the involvement of the mitochondrial permeability transition.

256. Castilho, R.F., M.W. Ward, and D.G. Nicholls, Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells. J Neurochem, 1999. 72(4): p. 1394-401. On exposure to glutamate, cultured rat cerebellar granule cells undergo a delayed Ca2+ deregulation (DCD), which precedes and predicts cell death. We have previously shown that mitochondria control the sensitivity of the neurons to DCD. Mitochondrial depolarization by rotenone/oligomycin before glutamate addition is strongly neuroprotective, and the indication is therefore that mitochondrial Ca2+ loading leads to a delayed loss of bioenergetic function culminating in DCD and cell death. In this report it is shown that superoxide (O2.-) generation in intact cells, monitored by oxidation of hydroethidine to ethidium, was enhanced by glutamate only when mitochondria were polarized. Production of superoxide was higher in the subset of cells undergoing DCD. In the presence of rotenone and oligomycin, addition of glutamate did not result in increased superoxide generation. Menadione-generated superoxide enhances the DCD of cells exposed to glutamate; in contrast, glutamate-induced DCD was potently inhibited by the presence of the cell-permeant antioxidant manganese(III) tetrakis(4-benzoic acid) porphyrin. An inverse correlation is observed between the cytoplasmic free Ca2+ maintained in individual cells in the presence of glutamate and the ability of these cells to restore basal Ca2+ when NMDA receptors are inhibited and mitochondrial Ca2+ is released. It is concluded that mitochondrial Ca2+ accumulation and reactive oxygen species each contribute to DCD, probably related to damage to a process controlling Ca2+ efflux from the cell.

257. Schubert, D. and D. Piasecki, Oxidative glutamate toxicity can be a component of the excitotoxicity cascade. J Neurosci, 2001. 21(19): p. 7455-62. Along with ionotropic and metabotropic glutamate receptors, the cystine/glutamate antiporter x(c)(-) may play a critical role in CNS pathology. High levels of extracellular glutamate inhibit the import of cystine, resulting in the depletion of glutathione and a form of cell injury called oxidative glutamate toxicity. Here we show that a portion of the cell death associated with NMDA receptor-initiated excitotoxicity can be caused by oxidative glutamate toxicity. In primary mouse cortical neurons the cell death resulting from the short-term application of 10 &mgr;m glutamate can be divided into NMDA and NMDA receptor-independent phases.

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The NMDA receptor-independent component is associated with high extracellular glutamate and is inhibited by a variety of reagents that block oxidative glutamate toxicity. These results suggest that oxidative glutamate toxicity toward neurons lacking functional NMDA receptors can be a component of the excitotoxicity-initiated cell death pathway.

258. Moghaddam, B., et al., Glucocorticoids mediate the stress-induced extracellular accumulation of glutamate. Brain Res, 1994. 655(1-2): p. 251-4. The hippocampal damage caused by stress has been attributed to an increased glutamatergic tone brought about by secretion of glucocorticoids. Although exposure to stress has been shown to increase the outflow of glutamate, direct involvement of glucocorticoid in this phenomenon has not been examined. The present study demonstrates that adrenalectomy attenuates the stress-induced outflow of glutamate in the hippocampus and prefrontal cortex and that glucocorticoid replacement abolishes this attenuation.

259. Tombaugh, G.C., et al., Glucocorticoids exacerbate hypoxic and hypoglycemic hippocampal injury in vitro: biochemical correlates and a role for astrocytes. J Neurochem, 1992. 59(1): p. 137-46. The acute secretion of glucocorticoids is critical for responding to physiological stress. Under normal circumstances these hormones do not cause acute neuronal injury, but they have been shown to enhance ischemic and seizure-induced neuronal injury in the rat brain. Using fetal rat hippocampal cultures, we asked whether hypoxic and hypoglycemic cell damage in vitro could be exacerbated by direct exposure to corticosterone (CORT). Each of these insults alone damaged neuronal cells, whereas 4-6 h of hypoxic treatment could damage age-matched astrocytes if glucose was reduced or omitted. Ischemic-like injury to both cell types could be attenuated by pretreatment with high (30 mM) glucose. Exposure to 100 nM CORT did not affect cell viability under control conditions but enhanced both hypoxic and hypoglycemic neuronal injury. In both cases, pretreatment with high glucose abolished this CORT-mediated synergy. In astrocyte cultures, CORT exacerbated both hypoxic and hypoglycemic injury and this effect was also attenuated by high-glucose pretreatment. Identical 24-h CORT treatment caused a 13% reduction in glucose uptake in astrocytes and a 38% reduction in glycogen content, without affecting the level of intracellular glucose. Thus, CORT could endanger both neurons and astrocytes in mixed hippocampal cultures and this effect emerged only under conditions of substrate depletion. The metabolic disruption in astrocytes by CORT further suggests that the ability of CORT to exacerbate neuronal injury may be due in part to impaired glial cell function.

260. Suchecki, D., P.A. Tiba, and S. Tufik, Paradoxical sleep deprivation facilitates subsequent corticosterone response to a mild stressor in rats. Neurosci Lett, 2002. 320(1-2): p. 45-8. The pituitary-adrenal responsiveness to a mild stressor was assessed in rats that were deprived of paradoxical sleep (PS) and in controls that were not deprived. Animals were either individually- or group-deprived for 96 h and hormone levels were assessed at 0, 5, 20 or 60 min after a saline injection+novelty and compared with rats which were not deprived. Both types of PS deprivation resulted in elevated adrenocorticotropin levels at 0 min, which peaked at 5 min in all animals. Individually-deprived rats exhibited the highest corticosterone (CORT) levels at 0 min. Peak levels were higher and occurred earlier in PS-deprived than in control rats (5 vs. 20 min,

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respectively). At 20 min, CORT levels had already returned to unstressed levels in PS-deprived rats, but not in control rats. These data indicate that PS deprivation induces facilitation of the adrenocortical response to a mild stressor, but do not suggest that PS deprivation changes the negative feedback sensitivity to CORT.

261. Hairston, I.S., et al., Sleep deprivation elevates plasma corticosterone levels in neonatal rats. Neurosci Lett, 2001. 315(1-2): p. 29-32. Plasma corticosterone (CORT) levels were measured after short periods of sleep deprivation in rats at postnatal days 12, 16, 20, and 24. There was an age-dependent increase in basal CORT levels and sleep deprivation significantly elevated CORT at all ages compared to non-sleep deprived controls. The levels of CORT after sleep deprivation in P16, P20 and P24 animals were similar, resulting in an age-dependent decrease of the magnitude of the response. Sleep deprived P12 animals had lower levels of CORT. However, the observed response to sleep deprivation suggests that sleep loss is a significant stressor at this age. These observations suggest that younger animals are more sensitive to the effects of mild sleep deprivation than older ones.

262. Maelicke, A., et al., Physostigmine and neuromuscular transmission. Ann N Y Acad Sci, 1993. 681: p. 140-54. Single channel studies carried out in cultured rat myoballs and cultured hippocampal neurons, and ion flux studies performed on Torpedo electrocyte membrane vesicles, showed that physostigmine (Phy), a well-established acetylcholinesterase inhibitor, interacts directly with nicotinic acetylcholine receptors (nAChR). Low concentrations (0.1 microM) of Phy activate the receptor integral channel, whereas higher concentrations blocked the channel in its opened state. In contrast to channel activation by acetylcholine (ACh) and classical cholinergic agonists, however, Phy was capable of activating the nAChR channel even when the ACh binding sites were blocked by competitive antagonists, such as alpha-neurotoxins and d-tubocurarine, or when the nAChR was desensitized by preincubation with high concentrations of ACh. The binding site at which Phy binds and activates the nAChR was mapped. It was located within the N-terminal extracellular region of the alpha-polypeptide, in close proximity to the binding site of the natural transmitter. These data identify a novel binding site at nAChRs from many species and tissues that may be involved in receptor regulatory processes.

263. Aiello-Malmberg, P., et al., Effects of morphine, physostigmine and raphe nuclei stimulation on 5-hydroxytryptamine release from the cerebral cortex of the cat. Br J Pharmacol, 1979. 65(4): p. 547-55. 1. The release of 5-hydroxytryptamine (5-HT) from the cerebral cortex and caudate nucleus of brainstem-transected cats and from the cerebral cortex of rats anaesthetized with urethane was determined by radioenzymatic and biological assay. 2. The stimulation of nucleus linearis intermedius of raphe doubles the basal 5-HT release in the caudate nucleus and increases it 3 fold in the cerebral cortex. The effects of the electrical stimulation of the raphe are potentiated by chlorimipramine. 3. Brain 5-HT release is greatly increased by morphine hydrochloride (6 mg/kg i.v.) and by physostigmine (100 microgram/kg i.v.), but not by DL-DOPA (50 mg/kg i.v.). 4. It is suggested that the 5-HT releasing action of physostigmine can contribute to some of its pharmacological effects such as the analgesic effect so far

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attributed exclusively to its indirect cholinomimetic activity. 5. The 5-HT releasing action of physostigmine seems unrelated to its anticholinesterase activity.

264. Anden, N.E., Effects of oxotremorine and physostigmine on the turnover of dopamine in the corpus striatum and the limbic system. J Pharm Pharmacol, 1974. 26(9): p. 738-40.

265. Choi, J.Y., J.C. Morris, and C.Y. Hsu, Aging and cerebrovascular disease. Neurol Clin, 1998. 16(3): p. 687-711. Aging influences cerebrovascular disease expression by a variety of mechanisms. Age-related changes in cerebral autoregulation, cellular metabolism, the blood-brain barrier, and autonomic function may leave the cerebrovascular system vulnerable to injury. Certain cerebrovascular disease, such as atrial fibrillation, watershed infarctions, carotid artery atherosclerosis, cerebral hemorrhages, subdural hematomas, and transient global amnesia manifest in the elderly. Vascular dementia and white matter disease are better understood with newer neuroimaging studies, careful neuropsychological and histopathologic examinations. Atherosclerosis and cerebral amyloid angiopathy may have larger roles than previously understood in Alzheimer's disease.

266. Banks, W. and A. Kastin, Aluminum increases permeability of the blood brain barrier to labeled DSIP and ß-endorphin: possible implications for senile and dialysis dementia. Lancet, 1983: p. 1227-1229.

267. Sinton, C.M., et al., Stressful manipulations that elevate corticosterone reduce blood brain barrier permeability to pyridostigmine in the rat. Toxicology and Applied Pharmacology, 2000. 165: p. 99-105.

268. Telang, F.W., et al., Pyridostigmine, a carbamate acetylcholinesterase AChE inhibitor and reactivator, is used prophylactically against chemical warfare agents. Nucl Med Biol, 1999. 26(2): p. 249-50.

269. Jones, K.H., et al., Subchronic effects following a single sarin exposure on blood-brain and blood-testes barrier permeability, acetylcholinesterase, and acetylcholine receptors in the central nervous system of rat: a dose-response study. J Toxicol Environ Health A, 2000. 61(8): p. 695-707. Subchronic neurotoxic effects of sarin (O-isopropyl methylphosphonofluoridate) treatment at various doses in male Sprague Dawley rats were studied. The animals were treated with a single intramuscular (im) injection of 0.01, 0.1, 0.5, or 1 x LD50 (100 microg/kg). The animals were maintained for 90 d thereafter. [3H]Hexamethonium iodide was used to monitor the changes in blood-brain barrier (BBB) permeability in cortex, brainstem, midbrain, and cerebellum. Brainstem exhibited a significant decrease (approximately 58% of control) in uptake of [3H]hexamethonium iodide at 1 x LD50 dose. No significant changes were observed in BBB permeability in cortex, midbrain, and cerebellum at any dose. Plasma butyrylcholinesterase (BChE) activity remained unchanged, reflecting recovery of the enzyme activity from the initial inhibition following single exposure of 1 x LD50 sarin. Acetylcholinesterase (AChE) activity in the cortex remained inhibited (approximately 29%), whereas in the brainstem

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there was an increase (approximately 20%) at 1 x LD50 dose of sarin. The m2-selective muscarinic acetylcholine receptor (m2-mAChR) ligand binding was inhibited significantly at 1 x LD50 in the cortex, whereas brainstem showed significantly increased (approximately 45%) ligand binding at 1 x LD50 dose. Nicotinic acetylcholine receptor (nAChR), on the other hand, showed a biphasic response in ligand binding in the cortex with a decrease (approximately 30%) at 0.01 x LD50 but an increase (approximately 40%) at 1 x LD5O. Brainstem did not show any significant change in nAChR ligand binding. These results suggest that single exposure of sarin could lead to changes that may play an important role in neuropathological abnormalities in the central nervous system.

270. Lallement, G., et al., Heat stress, even extreme, does not induce penetration of pyridostigmine into the brain of guinea pigs. Neurotoxicology, 1998. 19(6): p. 759-66. Stress due to forced swimming was recently shown to allow penetration of pyridostigmine (PYR) into the brain of mice. Accordingly, it was suggested that in troops exposed to emotional stress under conditions of war, as during the Gulf War, the BBB may have unexpectedly become permeable to PYR thus leading to an increased frequency of CNS symptoms. In this study, the entry of PYR into the brain was investigated in guinea pigs subjected to different heat stress levels. In a first group, guinea pigs were maintained at room temperature for 2 hours, their core temperature remaining stable at about 39.8 degrees C. In a second group, animals were placed in a climatic chamber in order to keep their core temperature at 41.5 degrees C for 2 hours. In a third group, animals were subjected to a high ambient temperature (42.6 degrees C) during about 2 hours and developed heatstroke symptoms, their core temperature progressively increasing and reaching around 44.3 degrees C. In each group, the stress of the animals was assessed by measuring the increase of plasma cortisol level. PYR (0.2 mg/kg, s.c.) was injected 90 minutes after beginning the experiment. Penetration of the drug into the brain was examined by measurement of acetylcholinesterase (AChE) activity in the cortex, the striatum and the hippocampus of the animals 30 minutes after PYR administration. A passage of this drug into the brain was also evaluated autoradiographically after i.v. injection of tritiated PYR 90 minutes after the beginning of the experiment (100 microCi/animal). Whatever the group examined, no entry of PYR into the CNS could be detected. Exposure to an ambient temperature at 42.6 degrees C for 2 hours resulted by itself in a partial inhibition of cerebral AChE activity. Our results, which agree with previous data obtained in humans exposed to heat stress, are opposite to the recent research showing a central passage of PYR in mice following a forced swim stress test. This demonstrated that the penetration of PYR into the brain of rodents under stress depends on the experimental conditions used (animal species, nature of the stressor, etc.). Extrapolations to humans of results primarily obtained in rodents about central passage of a drug under stress must thus be done very carefully.

271. Friedman, A., et al., Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response. Nat Med, 1996. 2(12): p. 1382-5. Pyridostigmine, a carbamate acetylcholinesterase (AChE) inhibitor, is routinely employed in the treatment of the autoimmune disease myasthenia gravis. Pyridostigmine is also recommended by most Western armies for use as

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pretreatment under threat of chemical warfare, because of its protective effect against organophosphate poisoning. Because of this drug's quaternary ammonium group, which prevents its penetration through the blood-brain barrier, the symptoms associated with its routine use primarily reflect perturbations in peripheral nervous system functions. Unexpectedly, under a similar regimen, pyridostigmine administration during the Persian Gulf War resulted in a greater than threefold increase in the frequency of reported central nervous system symptoms. This increase was not due to enhanced absorption (or decreased elimination) of the drug, because the inhibition efficacy of serum butyryl-cholinesterase was not modified. Because previous animal studies have shown stress-induced disruption of the blood-brain barrier, an alternative possibility was that the stress situation associated with war allowed pyridostigmine penetration into the brain. Here we report that after mice were subjected to a forced swim protocol (shown previously to simulate stress), an increase in blood-brain barrier permeability reduced the pyridostigmine dose required to inhibit mouse brain AChE activity by 50% to less than 1/100th of the usual dose. Under these conditions, peripherally administered pyridostigmine increased the brain levels of c-fos oncogene and AChE mRNAs. Moreover, in vitro exposure to pyridostigmine increased both electrical excitability and c-fos mRNA levels in brain slices, demonstrating that the observed changes could be directly induced by pyridostigmine. These findings suggest that peripherally acting drugs administered under stress may reach the brain and affect centrally controlled functions.

272. Tian, H., et al., Neither forced running nor forced swimming affect acute pyridostigmine toxicity or brain-regional cholinesterase inhibition in rats. Toxicology, 2002. 176(1-2): p. 39-50. Stress-induced change in the distribution of the drug pyridostigmine (PYR) has been proposed as a contributing factor to unexplained illnesses in Persian Gulf War veterans. We evaluated the effects of two stress models, forced running and forced swimming, on acute PYR (30 mg/kg, p.o.) toxicity and cholinesterase (ChE) inhibition in the blood and selected brain regions of young adult male Sprague-Dawley rats (6 weeks of age). Plasma corticosterone levels were measured at 0, 1 and 3 h after termination of forced swimming or forced running to confirm the induction of stress. PYR was given either immediately before stress (15 min swimming; 20 min running) or immediately after stress (15 min swimming; 90 min running) and cholinergic toxicity and ChE inhibition were evaluated at 1, 2 or 4 h after PYR exposure. Additionally, rats were subjected to either swimming (15 min) or running (90 min) stress, anesthetized, injected with horseradish peroxidase (HRP, 100 mg/kg, transcardial) and brain-regional HRP activity measured as an indicator of altered blood-brain barrier integrity. Both forced swimming and forced running resulted in significant elevations of plasma corticosterone levels. PYR caused cholinergic toxicity at all time-points evaluated. Swimming and running stress had little influence on expression of PYR-induced toxicity, however. Blood ChE activity was generally inhibited 77-91% at 1-4 h after PYR, but rats pretreated with PYR prior to forced swimming showed lesser inhibition (64%) 1 h after dosing, possibly because of swimming-induced hypothermia and delayed absorption of the drug. Minimal changes in ChE activity were noted in frontal cortex, cerebellum and hippocampus following PYR exposure (maximal inhibition 28%), and neither swimming nor running stress affected the degree of inhibition. Neither stress model increased HRP accumulation in any brain region. The

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results suggest that stress associated with forced running or forced swimming has little effect on acute PYR toxicity, entry of PYR into the brain or PYR-induced brain-regional ChE inhibition.

273. Cook, W.O., et al., Comparison of effects of anatoxin-a(s) and paraoxon, physostigmine and pyridostigmine on mouse brain cholinesterase activity. Toxicon, 1988. 26(8): p. 750-3. Anatoxin-a(s), an alkaloid neurotoxin from the freshwater cyanobacterium, Anabaena flos-aquae NRC-525-17, was compared to paraoxon, physostigmine and pyridostigmine for effects on brain cholinesterase after i.p. injection into Balb/c mice. The duration of clinical signs in mice injected with anatoxin-a(s) persisted longer than in mice given the carbamates and was comparable with that of paraoxon. Anatoxin-a(s) did not inhibit brain cholinesterase activity suggesting that this toxin is unable to cross the blood-brain barrier.

274. Lin, J., J.J. Freeman, and J.W. Kosh, Antagonism of acute physostigmine and neostigmine toxicity in mice by hemicholinium-3. Res Commun Chem Pathol Pharmacol, 1987. 56(1): p. 137-40. Hemicholinium-3 (HC-3) was administered intraperitoneally to mice concurrently with the intraperitoneal administration of physostigmine or neostigmine. HC-3 increased the LD50 values for both physostigmine and neostigmine but did not alter the effect on brain ACh levels produced by these agents. Since HC-3 does not cross the blood brain barrier after intraperitoneal administration, the antidoting action of HC-3 is peripherally mediated and does not solely involve an inhibition of ACh synthesis. The increase in brain acetylcholine caused by neostigmine was related to a reduction in acetylcholinesterase activity, providing evidence that intraperitoneally administered neostigmine crosses the blood-brain barrier.

275. Kamikawa, T., et al., Effects of coenzyme Q10 on exercise tolerance in chronic stable angina pectoris. Am J Cardiol, 1985. 56(4): p. 247-51. The effects of coenzyme Q10(CoQ10) on exercise performance were studied in 12 patients, average age 56 years, with stable angina pectoris. The study involved a double-blind, placebo-controlled, randomized, crossover protocol, using multistage treadmill exercise tests. CoQ10(150 mg/day in 3 daily doses) was administered orally for 4 weeks, tended to reduce anginal frequency from 5.3 +/- 4.9 to 2.5 +/- 3.3 attacks for 2 weeks and nitroglycerin consumption from 2.6 +/- 2.8 to 1.3 +/- 1.7 tablets for 2 weeks compared with patients receiving the placebo, but the reduction was not statistically significant. Exercise time increased from 345 +/- 102 seconds with placebo to 406 +/- 114 seconds during CoQ10 treatment (p less than 0.05). The time until 1 mm of ST-segment depression occurred increased from 196 +/- 76 seconds with placebo to 284 +/- 104 seconds during CoQ10 treatment (p less than 0.01). During the exercise test, ST-segment depression, heart rate and pressure-rate product at the same and at the maximal workload showed no significant difference between patients after placebo and CoQ10 administration. The average CoQ10 plasma concentration increased from 0.95 +/- 0.48 microgram/ml to 2.20 +/- 0.98 microgram/ml after CoQ10 treatment. This increase was significantly related to the increase in exercise duration (r = 0.68, p less than 0.001). Only 1 patient had a loss of appetite, but continued therapy. This study suggests that CoQ10 is a safe and promising treatment for angina pectoris.

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276. Overvad, K., et al., Coenzyme Q10 in health and disease. Eur J Clin Nutr, 1999. 53(10): p. 764-70. The literature concerning the importance of coenzyme Q10 in health and disease has been reviewed. Usual dietary intake together with normal in vivo synthesis seems to fulfil the demands for Q10 in healthy individuals. The importance of Q10 supplementation for general health has not been investigated in controlled experiments. The literature allows no firm conclusions about the significance of Q10 in physical activity. In different cardiovascular diseases, including cardiomyopathy, relatively low levels of Q10 in myocardial tissue have been reported. Positive clinical and haemodynamic effects of oral Q10 supplementation have been observed in double-blind trials, especially in chronic heart failure. These effects should be further examined. No important adverse effects have been reported from experiments using daily supplements of up to 200 mg Q10 for 6-12 months and 100 mg daily for up to 6 y.

277. Thorsteinsdottir, B., et al., No difference in ubiquinone concentration of muscles and blood in fibromyalgia patients and healthy controls. Clin Exp Rheumatol, 1998. 16(4): p. 513-4.

278. Lister, R.E., An open, pilot study to evaluate the potential benefits of coenzyme Q10 combined with Ginkgo biloba extract in fibromyalgia syndrome. J Int Med Res, 2002. 30(2): p. 195-9. An open, uncontrolled study was undertaken to measure the subjective effects of coenzyme Q10 combined with a Ginkgo biloba extract in volunteer subjects with clinically diagnosed fibromyalgia syndrome. Anecdotal reports from patients with fibromyalgia syndrome have claimed benefits from the use of these supplements. The aim of this study was to determine if these reports could be substantiated in a pilot clinical trial. Patient questioning had determined that poor quality of life was a major factor in the condition and a quality-of-life questionnaire was used to measure potential benefit. Subjects were given oral doses of 200 mg coenzyme Q10 and 200 mg Ginkgo biloba extract daily for 84 days. Quality of life was measured, using the well-validated Dartmouth Primary Care Cooperative Information Project/World Organization of Family Doctors (COOP/WONCA) questionnaire that measures seven different subjective responses, at 0-, 4-, 8-, and 12-week intervals. The subjects were asked for an overall self-rating at the end of the study. A progressive improvement in the quality-of-life scores was observed over the study period and at the end, the scores showed a significant difference from those at the start. This was matched by an improvement in self-rating with 64% claiming to be better and only 9% claiming to feel worse. Adverse effects were minor. A controlled study is now planned.

279. Sneed, P.A., Treatment of fibromyalgia with ubiquinone 10 and succinic acid. Official Gazette of the United States Patent and Trademark Office Patents, 2002. 1255(3): p. No Pagination. A method is described for using a combination of ubiquinone 10 and succinic acid in the treatment of human patients afflicted with fibromyalgia to alleviate one or more symptoms associated with that disease state. Fibromyalgia positive patients treated buccally, sublingually or by oral ingestion administration of ubiquinone 10 and succinic acid enjoy a reduction in clinical symptoms of the disease.

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280. Krieger, A.C. and N.S. Redeker, Obstructive sleep apnea syndrome: its relationship with hypertension. J Cardiovasc Nurs, 2002. 17(1): p. 1-11. Respiratory disturbance during sleep, in its most common form, obstructive sleep apnea, is a prevalent condition in the general population. During the past several years, researchers have investigated a possible link between obstructive sleep apnea and hypertension. This article discusses the available epidemiologic and clinical evidence supporting this link. The possible mechanisms leading to the development of hypertension in patients with obstructive sleep apnea will also be discussed, as well as the effects of therapeutic interventions on arterial blood pressure. Last, the clinical importance of such interaction, which may lead to excessive cardiovascular morbidity and mortality, will be addressed.

281. Blankfield, R.P. and S.J. Zyzanski, Bilateral leg edema, pulmonary hypertension, and obstructive sleep apnea: a cross-sectional study. J Fam Pract, 2002. 51(6): p. 561-4. This study was undertaken to clarify whether pulmonary hypertension is a useful marker for underlying obstructive sleep apnea in patients with edema. Twenty-eight ambulatory adults with bilateral leg edema and a normal echocardiogram were enrolled. Sixteen subjects had pulmonary hypertension, and 12 subjects had normal pulmonary artery pressures. Spirometry, pulse oximetry on room air, and polysomnography were obtained for each subject. Ten of 16 (63%) pulmonary hypertension subjects and 9 of 12 (75%) nonpulmonary hypertension subjects had obstructive sleep apnea (P =.48). Eleven of 16 (69%) pulmonary hypertension subjects and 11 of 12 (92%) nonpulmonary hypertension subjects were obese (P =.20). If these results are generalizable, obstructive sleep apnea is frequently associated with bilateral leg edema and obesity, regardless of the presence of pulmonary hypertension. Thus, especially in obese patients, bilateral leg edema may be a useful clinical marker for underlying obstructive sleep apnea.

282. Yamakawa, H., et al., Pulmonary hypertension in patients with severe obstructive sleep apnea. Psychiatry Clin Neurosci, 2002. 56(3): p. 311-2. Thirty-seven patients (35 men and two women) with obstructive sleep apnea-hypopnea syndrome (OSAHS) without any known cardiovascular and lung diseases were examined by Doppler echocardiography. Eight of the 37 (21.6%) patients experienced daytime pulmonary hypertension (PH), and all of them had severe OSAHS with an apnea-hypopnea index of > 30. The study suggested that one-third of patients with severe OSAHS had daytime PH.

283. Lee, A.G., et al., Sleep apnea and intracranial hypertension in men. Ophthalmology, 2002. 109(3): p. 482-5. PURPOSE: To investigate sleep apnea as an associated finding in idiopathic intracranial hypertension (IIH) in men. DESIGN: Multicenter, retrospective, noncomparative interventional case series. METHODS: Retrospective review of all men with the diagnosis of IIH seen within the last 5 years at three tertiary care academic ophthalmologic institutions. Cases with sleep apnea (SA) and IIH were identified and reviewed. RESULTS: Thirty-two cases of IIH in men were reviewed. Six cases with SA met the modified Dandy criteria for the diagnosis of IIH. Of these six patients, one received acetazolamide alone, four received acetazolamide and continuous positive airway pressure (CPAP), and one was treated with CPAP alone. All patients had preserved central acuity (20/20 or better in both eyes), enlarged blind spots, and optic disc edema in both eyes. Five patients had normal visual fields after treatment,

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and one patient had residual visual field loss. Three patients had normal optic nerve examinations, with resolution of the optic disc edema at last follow-up. After resolution of the optic disc edema, these three patients were maintained on CPAP but discontinued acetazolamide. Two patients had persistent but improved papilledema and are under continued treatment with acetazolamide and CPAP. One patient had optic disc pallor in both eyes and is stable. CONCLUSIONS: SA was a common finding in men meeting the modified Dandy criteria for IIH in adults. Treatment of sleep apnea with nocturnal oxygenation may improve the signs and symptoms of IIH in affected men.

284. Blankfield, R.P., A.A. Tapolyai, and S.J. Zyzanski, Left ventricular dysfunction, pulmonary hypertension, obesity, and sleep apnea. Sleep Breath, 2001. 5(2): p. 57-62. The purpose of this study was to determine the frequency of central and obstructive sleep apnea in adult patients who have echocardiographic evidence of left ventricular dysfunction and pulmonary hypertension. Subjects with left ventricular dysfunction, pulmonary hypertension (pulmonary artery systolic pressure >30 mm Hg) and no lung disease were evaluated for risk factors associated with pulmonary hypertension. Of eight eligible adults, six completed the study. Subjects were from suburban and inner city family practices. Spirometric assessment, pulse oximetry on room air, rheumatologic evaluation, polysomnography, and additional history were taken. All six subjects had sleep apnea (apnea-plus-hypopnea index, or AHI, > or = 20): obstructive, central, or mixed. All were obese, and almost all the subjects had a restrictive pattern on spirometry, which is consistent with obesity. All had a pulmonary artery systolic blood pressure of 35 mm Hg or greater. None had daytime hypoxemia or collagen vascular disease, and none had ever used appetite suppressants. This study found a strong association between pulmonary hypertension and obstructive or central sleep apnea in obese patients with congestive heart failure (CHF). We propose that a pulmonary artery systolic pressure of 35 mm Hg or greater in ambulatory patients with CHF may signify an increased risk of sleep apnea.

285. Silverberg, D.S., A. Iaina, and A. Oksenberg, Treating obstructive sleep apnea improves essential hypertension and quality of life. Am Fam Physician, 2002. 65(2): p. 229-36. About one half of patients who have essential hypertension have obstructive sleep apnea, and about one half of patients who have obstructive sleep apnea have essential hypertension. A growing body of evidence suggests that obstructive sleep apnea is a major contributing factor in the development of essential hypertension. Despite many patients with obstructive sleep apnea having clear symptoms of the disorder, an estimated 80 to 90 percent of cases are undiagnosed. When physicians routinely seek the diagnosis of obstructive sleep apnea by asking patients (especially those with hypertension) three basic sleep-related questions about snoring, excessive daytime sleepiness and reports of witnessed apneic events, the number of cases diagnosed and treated increases by about eightfold. Eliminating snoring and occurrences of apneic-hypopneic episodes will dramatically improve patients' quality of sleep and eliminate excessive daytime sleepiness, which has a detrimental effect on general functioning. Increased alertness will reduce the likelihood that patients will be involved in motor vehicle crashes. In most studies in which blood pressure was measured following treatment for obstructive sleep apnea, daytime and nighttime blood pressure levels were found to decrease significantly.

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This decrease in blood pressure may also reduce the likelihood of cardiovascular complications. The key to the diagnosis of obstructive sleep apnea is physician knowledge about the disorder. The dramatic improvement in quality of life that occurs when patients are successfully treated for obstructive sleep apnea makes detecting and treating this disorder imperative.

286. Chaska, B.W., Obstructive sleep apnea and essential hypertension--is there a link? Am Fam Physician, 2002. 65(2): p. 182, 185-6.

287. Alchanatis, M., et al., Daytime pulmonary hypertension in patients with obstructive sleep apnea: the effect of continuous positive airway pressure on pulmonary hemodynamics. Respiration, 2001. 68(6): p. 566-72. BACKGROUND: Limited information exists regarding the development of pulmonary hypertension in patients with obstructive sleep apnea (OSA) in the absence of lung and heart comorbidity. OBJECTIVES: The aims of this study were to investigate whether OSA patients without any other cardiac or lung disease develop pulmonary hypertension, and to assess the effect of continuous positive airway pressure (CPAP) treatment on pulmonary artery pressure (P(PA)). METHODS: Twenty-nine patients aged 51 +/- 10 years with OSA and 12 control subjects were studied with pulsed-wave Doppler echocardiography for estimation of P(PA) before and after 6-month effective treatment with CPAP. RESULTS: A significantly higher mean P(PA) was found in OSA patients as compared to control subjects (17.2 +/- 5.2 vs. 12.1 +/- 1.9 mm Hg, p < 0.001). Six out of the 29 OSA patients had mild pulmonary hypertension (P(PA) > or = 20 mm Hg). Significant differences were observed between pulmonary hypertensive and normotensive OSA patients with respect to age (62 +/- 4 vs. 48 +/- 15 years, respectively, p < 0.05), body mass index (41 +/- 7 vs. 32 +/- 4 kg/m(2), p < 0.02) and daytime P(a)O(2) (81 +/- 9 vs. 92 +/- 9 mm Hg, p < 0.05). CPAP treatment was effective in reducing mean P(PA) in both groups of pulmonary hypertensive and normotensive OSA patients (decreases in P(PA) from 25.6 +/- 4.0 to 19.5 +/- 1.5 mm Hg, p < 0.001; from 14.9 +/- 2.2 to 11.5 +/- 2.0 mm Hg, respectively, p < 0.001). CONCLUSIONS: A proportion (20.7%) of OSA patients without any other lung or heart disease and characterized by older age, greater obesity and lower daytime oxygenation develop mild pulmonary hypertension which has been partially or completely reversed after 6-month CPAP treatment. In conclusion, OSA alone constitutes an independent risk factor for the development of pulmonary hypertension.

288. Richert, A., K. Ansarin, and A.S. Baran, Sleep apnea and hypertension: pathophysiologic mechanisms. Semin Nephrol, 2002. 22(1): p. 71-7. This article reviews the pathophysiology of hypertension (HTN) in obstructive sleep apnea (OSA). The article is divided into 3 sections. The first section describes epidemiologic studies of the relationship of sleep-related breathing disorders, including OSA, to HTN and argues that OSA contributes to the genesis of HTN. The second section describes the known immediate physiologic consequences of 3 components of OSA that may contribute to the genesis of persistent systemic HTN. The 3 components are (1) the large negative intrathoracic pressure changes associated with OSA, (2) intermittent hypoxemia, and (3) arousal from sleep. The last section reviews current physiologic models of essential HTN

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genesis and attempts to integrate them with the suspected HTN-generating aspects of OSA. In its summary, the authors conclude that OSA contributes to the genesis of HTN and advise physicians not to ignore the contribution of frequently comorbid non-OSA factors, such as obesity, to the genesis of OSA-related HTN.

289. Lavie, P., et al., Obstructive sleep apnea and hypertension: from correlative to causative relationship. J Clin Hypertens (Greenwich), 2001. 3(5): p. 296-301. Sleep-disordered breathing, manifested by repetitive episodes of partial or complete cessation of breathing during sleep associated with brief arousal and autonomic activation, is estimated to affect as many as 4% of adult men and 2% of adult women. Studies conducted during the 1980s revealed a strong association between sleep-disordered breathing and hypertension. The results of these early studies, which relied on relatively small samples of patients, have been confirmed in recent years by large-scale epidemiologic studies that are controlled for all possible confounding factors. This paper reviews the evidence suggesting a causative relationship between hypertension and disordered breathing in sleep. The authors discuss the possible underlying mechanisms of the two entities and address the clinical implications of this relationship. They conclude by recommending a proactive approach to the diagnosis of breathing disorders in sleep, in order to prevent the cardiovascular sequelae of this syndrome.

290. von Kanel, R., et al., The hypercoagulable state in sleep apnea is related to comorbid hypertension. J Hypertens, 2001. 19(8): p. 1445-51. OBJECTIVE: Obstructive sleep apnea (OSA) is associated with increased prevalence of atherosclerotic disease. A hypercoagulable state thought to underly atherosclerosis has been described in both OSA and systemic hypertension. We wondered about the respective contribution of apnea and hypertension to a hypercoagulable state. DESIGN: Eighty-seven subjects with symptoms suggestive of OSA, mean age 47 years (range 32-64 years), underwent polysomnography and blood pressure (BP) screening. OSA was diagnosed when respiratory disturbance index (RDI) > or = 15. Subjects having systolic BP (SBP) > 140 mmHg and/or diastolic BP (DBP) > 90 mmHg were classified as having hypertension. Three hypercoagulability markers were measured: thrombin/antithrombin III complex (TAT), fibrin D-dimer (DD), and von Willebrand factor antigen (vWF:ag). RESULTS: Analysis of variance and multiple linear regression were performed on the following four subject groups: (1) normotensive non-apneics (n = 19), (2) normotensive apneics (n = 38), (3) hypertensive non-apneics (n = 11), and (4) hypertensive apneics (n = 19). OSA (groups 2 and 4) had no significant main effect on hemostasis. Hypertensives (groups 3 and 4) had higher plasma levels of TAT (median/inter-quartile range, 148/59-188 versus 77/53-108 pmol/l; P = 0.009) and of DD (376/265-721 versus 303/190-490 ng/ml; P = 0.040) than normotensives (groups 1 and 2). Across all subjects, SBP was the only significant predictor of TAT (P = 0.001) and of DD (P = 0.004), whereas DBP was the only significant predictor of vWF:ag (P = 0.029). These findings persisted even after controlling for gender, age, body mass index, RDI, mean SaO2, and hematocrit. CONCLUSION: Hypercoagulability in OSA is mediated by comorbid hypertension and might account for high cardiovascular morbidity in OSA in general.

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291. Sanders, M.H., Article reviewed: Association of sleep-disordered breathing, sleep apnea, and hypertension in a large community-based study. 2000. 1(4): p. 327-328.

292. Otsuka, K., [Nasal CPAP treatment and hypertension and altered cardiovascular variability associated with obstructive sleep apnea (OSA)]. Nippon Rinsho, 2001. 59(5): p. 983-91. In the past 5 years several epidemiologic studies have demonstrated that sleep-related breathing disorders are an independent risk factor for hypertension, probably resulting from a combination of repetitive episodes of hypoxia, hypercapnea, arousals, and a striking surge in sympathetic excitation, and altered baroreflex control during sleep. Obstructive sleep apnea (OSA) may lead to the cardiac arrhythmias and myocardial ischemia and it is a possible risk factor for stroke. We confirmed that nasal CPAP has been shown to lower blood pressure in some hypertensive OSA patients. Early recognition and treatment of sleep-apnea may improve cardiovascular function.

293. Narkiewicz, K. and V.K. Somers, Obstructive sleep apnea as a cause of neurogenic hypertension. Curr Hypertens Rep, 1999. 1(3): p. 268-73. Abnormalities in neural circulatory control may contribute importantly to the hypertensive state. The sympathetic nervous system in particular is a key mechanism for increasing blood pressure. Patients with obstructive sleep apnea have increased sympathetic activity. Obesity or other coexisting disease states do not explain the heightened sympathetic drive. This review examines the evidence linking sleep apnea with hypertension and the possible role of excessive sympathetic drive as a mediator of higher blood pressure in sleep apnea. Abnormalities in reflex circulatory control that could act to increase sympathetic activity in sleep apnea are also discussed.

294. Shiomi, T., et al., Primary pulmonary hypertension with central sleep apnea: sudden death after bilevel positive airway pressure therapy. Jpn Circ J, 2000. 64(9): p. 723-6. An obese 23-year-old man with sleep-disordered breathing and primary pulmonary hypertension (PPH) had been administered oral beraprost sodium, anticoagulant warfarin, and home oxygen therapy, at another hospital as treatment for the PPH, but he had not experienced any symptomatic improvement. The patient had a body mass index of 32.4kg/m2, and complained of fatigue, shortness of breath on exertion, excessive daytime sleepiness, and snoring. Arterial blood gas analysis showed a PaO2 and a PaCO2 of 70.9 and 31.2mmHg, respectively. A polysomnographic study revealed central sleep apnea with an apnea-hypopnea index (AHI) of 29.7episodes/h. The patient showed improvement of daytime sleepiness after starting nocturnal nasal bilevel positive airway pressure (BiPAP) therapy for the central sleep apnea, but his pulmonary hypertension, measured in the daytime, worsened. The patient died suddenly while walking to the bathroom in the morning 1 month after initiation of BiPAP therapy. It is necessary to consider the possibility of sudden death when nasal BiPAP therapy is given to a PPH patient with central sleep apnea.

295. Hudgel, D.W., Beyond systemic hypertension: understanding cardiac dysfunction in obstructive sleep apnea. Respiration, 2000. 67(4): p. 360-1.

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296. Salo, T.M., et al., Comparison of autonomic withdrawal in men with obstructive sleep apnea syndrome, systemic hypertension, and neither condition. Am J Cardiol, 2000. 85(2): p. 232-8. Obstructive sleep apnea syndrome is characterized by obesity, nocturnal breathing abnormalities, arterial hypertension, and an increased number of cardiovascular events. Sympathetic activity is increased during nocturnal apneic episodes, which may mediate the cardiovascular complications of sleep apnea. We studied 15 male subjects with obstructive sleep apnea syndrome and associated hypertension, 54 subjects with mild to moderate essential hypertension, and 25 healthy normotensive men. Cardiovascular autonomic control was assessed using frequency domain measures of heart rate variability (HRV) during a controlled breathing test and during orthostatic maneuver. Compared with normotensive and hypertensive groups, total power and low- and high-frequency components of HRV during controlled breathing were significantly (analysis of variance, p<0.0001) lower in the obstructive sleep apnea syndrome. During the orthostatic maneuver, the change in total power of HRV was different between the 3 groups (analysis of variance, p = 0.004). The total power of HRV tended to increase in the normotensive (4.11+/-12.29 ms2) and in hypertensive (2.31+/-12.65 ms2) groups, but decreased (1.13+/-1.23 ms2) in the hypertensive group with obstructive sleep apnea syndrome. According to multivariate regression analysis, age and sleep apnea were the major independent determinants of HRV. This study found that an abnormal response to autonomic nervous tests characterizes hypertension in overweight subjects with obstructive sleep apnea syndrome. This could be due to autonomic withdrawal or supersaturation of the end-organ receptors by excessive and prolonged sympathetic stimulation. Our results also show the reduced response of orthostatic maneuver and controlled breathing in the hypertensive group compared with the normotensive group.

297. Willis, R.A., et al., The effect of ethanol and/or food restriction on coenzyme Q in liver in rats. Mol Aspects Med, 1997. 18 Suppl: p. S205-11. The individual and combined effects of ethanol and lovastatin on rats and their prevention by supplemental coenzyme Q10 (CoQ10) was studied. The ethanol and lovastatin findings are reported elsewhere. This paper focuses on the food restriction which occurred in rats fed 35% of energy as ethanol and those control rats pair-fed to the 35% of energy as ethanol group. Six groups of rats received 35% of energy as ethanol (with or without lovastatin and/or CoQ10 treatment). One group served as a 0% ethanol ad libitum control and one 0% ethanol control group was pair-fed to the 35% ethanol group. Rats receiving 35% of energy as ethanol and their pair-fed controls consumed 83% of the energy/day consumed by the ad libitum controls. This was consistent regardless of lovastatin or CoQ10 treatment. Weight gains were 84% of control. The energy reduction was consistently associated with a substantial (48%+) increase in liver CoQ9 concentrations. Reports by others of associations between food restriction and increased longevity in rodents has focused on a decrease in oxidant damage in tissues of food restricted animals. The increase in CoQ levels in the food restricted animals would result in an increase in antioxidant protection and might explain the observed increases in longevity.

298. Silvestre-Aillaud, P., et al., Could coenzyme Q10 and L-carnitine be a treatment for diabetes secondary to 3243 mutation of mtDNA? Diabetologia, 1995. 38(12): p. 1485-6.

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299. Kucharska, J., et al., Deficit of coenzyme Q in heart and liver mitochondria of rats with streptozotocin-induced diabetes. Physiol Res, 2000. 49(4): p. 411-8. Mitochondrial dysfunction and oxidative stress participate in the development of diabetic complications, however, the mechanisms of their origin are not entirely clear. Coenzyme Q has an important function in mitochondrial bioenergetics and is also a powerful antioxidant. Coenzyme Q (CoQ) regenerates alpha-tocopherol to its active form and prevents atherogenesis by protecting low-density lipoproteins against oxidation. The aim of this study was to ascertain whether the experimentally induced diabetes mellitus is associated with changes in the content of endogenous antioxidants (alpha-tocopherol, coenzymes Q9 and Q10) and in the intensity of lipoperoxidation. These biochemical parameters were investigated in the blood and in the isolated heart and liver mitochondria. Diabetes was induced in male Wistar rats by a single intravenous injection of streptozotocin (45 mg x kg(-1)), insulin was administered once a day for 8 weeks (6 U x kg(-1)). The concentrations of glucose, cholesterol, alpha-tocopherol and CoQ homologues in the blood of the diabetic rats were increased. The CoQ9/cholesterol ratio was reduced. In heart and liver mitochondria of the diabetic rats we found an increased concentration of alpha-tocopherol, however, the concentrations of CoQ9 and CoQ10 were decreased. The formation of malondialdehyde was enhanced in the plasma and heart mitochondria. The results have demonstrated that experimental diabetes is associated with increased lipoperoxidation, in spite of the increased blood concentrations of antioxidants alpha-tocopherol and CoQ. These changes may be associated with disturbances of lipid metabolism in diabetic rats. An important finding is that heart and liver mitochondria from the diabetic rats contain less CoQ9 and CoQ10 in comparison with the controls. We suppose that the deficit of coenzyme Q can participate in disturbances of mitochondrial energy metabolism of diabetic animals.

300. Liou, C.W., et al., Correction of pancreatic beta-cell dysfunction with coenzyme Q(10) in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome and diabetes mellitus. Eur Neurol, 2000. 43(1): p. 54-5.

301. Kishi, T., et al., Bioenergetics in clinical medicine. XI. Studies on coenzyme Q and diabetes mellitus. J Med, 1976. 7(3-4): p. 307-21. The activity of the succinate dehydrogenase-coenzyme Q10 reductase from 120 diabetic patients was significantly lower (P less than 0.001) and the per cent deficiency was significantly higher (P less than 0.001) than that of the controls. The diabetes of 37 patients was controlled by diet; the enzyme activity was lower (P less than 0.001) and the deficiency was higher (P less than 0.02) than for controls. In decreasing effectiveness, Dymelor, Glyburide, Phenformin and Tolazamide inhibited the COQ10-enzyme, NADH-oxidase. Tolbutamide, Glypizide, and Chlorpropamide were noninhibitory to succinoxidase and NADH-oxidase. Patients receiving Tolazamide and Phenformin showed a higher incidence (P less than 0.001 to P less than 0.05) of COQ10-deficiency than patients controlled by diet or normal controls. Certain diabetic patients controlled by diet may have a deficiency of COQ10 which may be enhanced by the inhibition by certain commonly used antidiabetic drugs of COQ10-enzymes. A deficiency of COQ10 in the pancreas could impair bioenergetics, the generation of ATP, and the biosynthesis of insulin.

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302. Iwasaki, Y., N. Wakata, and M. Kinoshita, Parkinsonism induced by pyridostigmine. Acta Neurol Scand, 1988. 78(3): p. 236.

303. Iwasaki, Y., M. Kinoshita, and N. Wakata, [A case report of pyridostigmine induced parkinsonism]. Rinsho Shinkeigaku, 1988. 28(6): p. 662-4.

304. Gothoni, P., M. Lehtinen, and M. Fincke, Drugs for Parkinson's disease reduce tremor induced by physostigmine. Naunyn Schmiedebergs Arch Pharmacol, 1983. 323(3): p. 205-10. The effects of anticholinergic and dopaminergic drugs used for Parkinson's disease were studied on the tremor induced by physostigmine (0.3-3.0 mg/kg) in rats. For the measurement of tremor a new electronic device was employed. Atropine (0.3-1.2 mg/kg) and biperiden (0.01-1.0 mg/kg) reduced the physostigmine-induced tremor in a dose-related manner and could abolish it. Biperiden was less potent than atropine. Methylatropine in a dose of 1.2 mg/kg slightly inhibited the tremor. Amantadine (0.3-3.0 mg/kg) reduced the tremor but only to a certain degree. Bromocriptine (0.1-10.0 mg/kg) reduced it in a manner that was not dose-related. Pimozide potentiated the tremor in the dose of 0.2 mg/kg but not in larger doses. At the onset of the tremor, a small decrease in rectal temperature occurred. The hypothermia lasted significantly longer than the tremor. Neither the anticholinergic nor the dopaminergic anti-Parkinson drugs altered the hypothermic effect of physostigmine. The results show that those anti-Parkinson drugs, which act by increasing the dopaminergic activity can counteract the tremor induced by physostigmine. However, these drugs are clearly less active than th anticholinergic anti-Parkinson drugs.

305. Friedrich, M.J., Pesticide study aids Parkinson research. Jama, 1999. 282(23): p. 2200.

306. Semchuk, K.M., E.J. Love, and R.G. Lee, Parkinson's disease and exposure to agricultural work and pesticide chemicals. Neurology, 1992. 42(7): p. 1328-35. This population-based case-control study of 130 Calgary residents with neurologist-confirmed idiopathic Parkinson's disease (PD) and 260 randomly selected age- and sex-matched community controls attempted to determine whether agricultural work or the occupational use of pesticide chemicals is associated with an increased risk for PD. We obtained by personal interviews lifetime occupational histories, including chemical exposure data, and analyzed the data using conditional logistic regression for matched sets. In the univariate analysis, a history of field crop farming, grain farming, herbicide use, or insecticide use resulted in a significantly increased crude estimate of the PD risk, and the data suggested a dose-response relation between the PD risk and the cumulative lifetime exposure to field crop farming and to grain farming. However, in the multivariate analysis, which controlled for potential confounding or interaction between the exposure variables, previous occupational herbicide use was consistently the only significant predictor of PD risk. These results support the hypothesis that the occupational use of herbicides is associated with an increased risk for PD.

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307. Ritz, B. and F. Yu, Parkinson's disease mortality and pesticide exposure in California 1984-1994. Int J Epidemiol, 2000. 29(2): p. 323-9. BACKGROUND: In the last two decades reports from different countries emerged associating pesticide and herbicide use with Parkinson's disease (PD). California growers use approximately 250 million pounds of pesticides annually, about a quarter of all pesticides used in the US. METHODS: We employed a proportional odds mortality design to compare all cases of PD recorded as underlying (1984-1994) or associated causes (1984-1993) of death occurring in California with all deaths from ischaemic heart disease (ICD-9 410-414) during the same period. Based on pesticide use report data we classified California counties into several pesticide use categories. Agricultural census data allowed us to create measures of percentage of land per county treated with pesticides. Employing logistic regression models we estimated the effect of pesticide use controlling for age, gender, race, birthplace, year of deaths, and education. RESULTS: Mortality from PD as the underlying cause of death was higher in agricultural pesticide-use counties than in non-use counties. A dose response was observed for insecticide use per county land treated when using 1982 agricultural census data, but not for amounts of restricted pesticides used or length of residency in a country prior to death. CONCLUSIONS: Our data show an increased PD mortality in California counties using agricultural pesticides. Unless all of our measures of county pesticide use are surrogates for other risk factors more prevalent in pesticide use counties, it seems important to target this prevalent exposure in rural California in future studies that use improved case finding mechanisms and collect pesticide exposure data for individuals.

308. Taylor, M.C., et al., Paraoxonase polymorphisms, pesticide exposure and Parkinson's disease in a Caucasian population. J Neural Transm, 2000. 107(8-9): p. 979-83. Parkinson's disease (PD) has been associated with exposure to pesticides and oxidative injury. The involvement of paraoxonase in both pesticide metabolism and lipid peroxidation suggests that it may play a role in the pathogenesis of PD. We examined the frequency of polymorphic alleles of the PON1 and PON2 genes in a sample of caucasian subjects with PD. The frequency distribution of these genotypes did not differ significantly between patients and controls, including those who had reported exposure to pesticides.

309. Berger, A., Parkinson's disease linked with pesticide. Bmj, 2000. 321(7270): p. 1175A.

310. Adam, D., Pesticide use linked to Parkinson's disease. Nature, 2000. 408(6809): p. 125.

311. Betarbet, R., et al., Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat Neurosci, 2000. 3(12): p. 1301-6. The cause of Parkinson's disease (PD) is unknown, but epidemiological studies suggest an association with pesticides and other environmental toxins, and biochemical studies implicate a systemic defect in mitochondrial complex I. We report that chronic, systemic inhibition of complex I by the lipophilic pesticide, rotenone, causes highly selective nigrostriatal dopaminergic degeneration that is associated behaviorally with hypokinesia and rigidity.

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Nigral neurons in rotenone-treated rats accumulate fibrillar cytoplasmic inclusions that contain ubiquitin and alpha-synuclein. These results indicate that chronic exposure to a common pesticide can reproduce the anatomical, neurochemical, behavioral and neuropathological features of PD.

312. Helmuth, L., Neuroscience. Pesticide causes Parkinson's in rats. Science, 2000. 290(5494): p. 1068.

313. Karen, D.J., et al., Striatal dopaminergic pathways as a target for the insecticides permethrin and chlorpyrifos. Neurotoxicology, 2001. 22(6): p. 811-7. Because insecticide exposure has been linked to both Parkinsons disease and Gulf War illness, the neurotoxic actions of pyrethroid and organophosphate insecticides on behavior and striatal dopaminergic pathways were investigated in C57BL/6 mice treated with permethrin (three i.p. doses at 0.2-200 mg/kg) or chlorpyrifos (three s.c. doses at 25-100 mg/kg) over a 2-week period. Permethrin altered maximal [3H]dopamine uptake in striatal synaptosomes from treated mice, with changes in Vmax displaying a bell-shaped curve. Uptake was increased to 134% of control at a dose of 1.5 mg/kg. At higher doses of PM (25 mg/kg), dopamine uptake declined to a level significantly below that of control (50% of control at 200 mg/kg, P < 0.01). We also observed a small, but statistically significant decrease in [3H]dopamine uptake by chlorpyrifos, when given at a dose of 100 mg/kg. There was no significant effect on the Km for dopamine transport. Evidence of cell stress was observed in measures of mitochondrialfunction, which were reduced in mice given high-end doses of chlorpyrifos and permethrin. Although cytotoxicity was not reflected in decreased levels of striatal dopamine in either 200 mg/kg PM or 100 mg/kg CPF treatment groups, an increase in dopamine turnover at 100 mg/kg CPF was indicated by a significant increase in titers of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid. Both permethrin and chlorpyrifos caused a decrease in open field behavior at the highest doses tested. Although frank Parkinsonism was not observed, these findings confirm that dopaminergic neurotransmission is affected by exposure to pyrethroid and organophosphorus insecticides, and may contribute to the overall spectrum of neurotoxicity caused by these compounds.

314. Ambrani, L.M. and M.H. Van Woert, Modification of the tremorigenic activity of physostigmine. Br J Pharmacol, 1972. 46(2): p. 344-7.

315. Ambani, L.M., M.H. Van Woert, and M.B. Bowers, Jr., Physostigmine effects on phenothiazine-induced extrapyramidal reactions. Arch Neurol, 1973. 29(6): p. 444-6.

316. Haley, R.W., et al., Evaluation of neurologic function in Gulf War veterans. A blinded case-control study. Jama, 1997. 277(3): p. 223-30. OBJECTIVE: To determine whether Gulf War-related illnesses are associated with central or peripheral nervous system dysfunction. DESIGN: Nested case-control study. PARTICIPANTS: Twenty-three veterans with factor analysis-derived syndromes (the cases), 10 well veterans deployed to the Gulf War (the deployed controls), and 10 well veterans not deployed to the Gulf War (the nondeployed controls). METHOD: With investigators blinded to group identities, participants underwent objective neurophysiological, audiovestibular,

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neuroradiological, neuropsychological, and blood tests. MAIN OUTCOME MEASURES: Evidence of neurologic dysfunction. RESULTS: Compared with the 20 controls, the 23 cases had significantly more neuropsychological evidence of brain dysfunction on the Halstead Impairment Index (P=.01), greater interside asymmetry of the wave I to wave III interpeak latency of brain stem auditory evoked potentials (P=.02), greater interocular asymmetry of nystagmic velocity on rotational testing, increased asymmetry of saccadic velocity (P=.04), more prolonged interpeak latency of the lumbar-to-cerebral peaks on posterior tibial somatosensory evoked potentials (on right side, P=.03, and on the left side, P=.005), and diminished nystagmic velocity after caloric stimulation bilaterally (P values range from .02 to .04). Cases (n=5) with syndrome 1 ("impaired cognition") were the most impaired on brain stem auditory evoked potentials (P=.005); those (n=13) with syndrome 2 ("confusion-ataxia") were the most impaired on the Halstead Impairment Index (P=.006), rotational testing (P=.01), asymmetry of saccadic velocity (P=.03), and somatosensory evoked potentials (P< or =.01); and those (n=5) with syndrome 3 ("arthro-myo-neuropathy") were the most impaired on caloric stimulation (P< or =.01). CONCLUSIONS: The 3 factor-derived syndromes identified among Gulf War veterans appear to represent variants of a generalized injury to the nervous system.

317. Hom, J., R.W. Haley, and T.L. Kurt, Neuropsychological correlates of Gulf War syndrome. Arch Clin Neuropsychol, 1997. 12: p. 531-544.

318. McDowell, I., Alzheimer's disease: insights from epidemiology. Aging (Milano), 2001. 13(3): p. 143-62. While a complete understanding of the pathogenesis of Alzheimer's disease (AD) remains elusive, many conclusions can be drawn from the numerous epidemiological studies undertaken to date. Prevalence and incidence estimates show consistency, following a roughly exponential pattern with a doubling of both parameters roughly every five years after age 65. Roughly 7% of the population aged 65 and over has AD. The clinical course of the disease is reasonably well established and mortality rates rise with increasing levels of cognitive deficit. Four risk factors for AD are firmly established: increasing age, the presence of the apolipoproteinE-epsilon4 allele, familial aggregation of cases, and Down's syndrome. Numerous other associations have been shown in some studies, but not in others. For example, women generally appear at higher risk than men, as do people with lower levels of education; depression is probably prodromal; head injury is an established risk factor, and may interact with the apoE gene; several occupational exposures appear hazardous, and exposure to aluminum in the water supply confers excess risk. Hypertension and other vascular symptoms appear to predispose to AD, which is now seen as nosologically closer to vascular dementia than was previously believed. Several apparently protective factors have been identified, although preventive trials based on these have so far shown minimal effectiveness. The use of non-steroidal anti-inflammatory drugs to treat arthritis is associated with a reduced risk of AD, as is estrogen use by post-menopausal women. Physical activity appears beneficial, as does a diet with high levels of vitamins B6, B12 and folate. while red wine in moderate quantities appears protective. This review concludes with a discussion of the strengths and limitations of current epidemiological methods for studying Alzheimer's disease.

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319. Snowdon, D.A., et al., Linguistic ability in early life and cognitive function and Alzheimer's disease in late life. Findings from the Nun Study. Jama, 1996. 275(7): p. 528-32. OBJECTIVE--To determine if linguistic ability in early life is associated with cognitive function and Alzheimer's disease in late life. DESIGN--Two measures of linguistic ability in early life, idea density and grammatical complexity, were derived from autobiographies written at a mean age of 22 years. Approximately 58 years later, the women who wrote these autobiographies participated in an assessment of cognitive function, and those who subsequently died were evaluated neuropathologically. SETTING--Convents in the United States participating in the Nun Study; primarily convents in the Milwaukee, Wis, area. PARTICIPANTS--Cognitive function was investigated in 93 participants who were aged 75 to 95 years at the time of their assessments, and Alzheimer's disease was investigated in the 14 participants who died at 79 to 96 years of age. MAIN OUTCOME MEASURES--Seven neuropsychological tests and neuropathologically confirmed Alzheimer's disease. RESULTS--Low idea density and low grammatical complexity in autobiographies written in early life were associated with low cognitive test scores in late life. Low idea density in early life had stronger and more consistent associations with poor cognitive function than did low grammatical complexity. Among the 14 sisters who died, neuropathologically confirmed Alzheimer's disease was present in all of those with low idea density in early life and in none of those with high idea density. CONCLUSIONS--Low linguistic ability in early life was a strong predictor of poor cognitive function and Alzheimer's disease in late life.

320. Head injury linked to increased risk of Alzheimer's disease. FDA Consum, 2001. 35(1): p. 8.

321. Plassman, B.L., et al., Documented head injury in early adulthood and risk of Alzheimer's disease and other dementias. Neurology, 2000. 55(8): p. 1158-66. BACKGROUND: The association between antecedent head injury and AD is inconsistent. OBJECTIVE: To examine the association between early adult head injury, as documented by military hospital records, and dementia in late life; and to evaluate the interaction between head injury and APOE epsilon4 as risk factors for dementia. METHODS: The study had a population-based prospective historical cohort design. It included men who were World War II Navy and Marine veterans, and were hospitalized during their military service with a diagnosis of either a nonpenetrating head injury or another unrelated condition. In 1996 to 1997, military medical records were abstracted to document the occurrence and details of closed head injury. The entire sample was then evaluated for dementia and AD using a multistage procedure. There were 548 veterans with head injury and 1228 without head injury who completed all assigned stages of the study. The authors estimated risk of dementia, specifically AD, using proportional hazards models. RESULTS: Both moderate head injury (hazard ratio [HR] = 2.32; CI = 1.04 to 5.17) and severe head injury (HR = 4.51; CI = 1.77 to 11.47) were associated with increased risk of AD. Results were similar for dementia in general. The results for mild head injury were inconclusive. When the authors stratified by the number of APOE epsilon4 alleles, they observed a nonsignificant trend toward a stronger association between AD and head injury in men with more epsilon4 alleles. CONCLUSIONS:

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Moderate and severe head injuries in young men may be associated with increased risk of AD and other dementias in late life. However, the authors cannot exclude the possibility that other unmeasured factors may be influencing this association.

322. SoRelle, R., Head injuries linked to Alzheimer's disease. Circulation, 2000. 102(18): p. E9036-7.

323. Sobel, R.K., Alzheimer's first blow. Head injuries raise risk. US News World Rep, 2000. 129(18): p. 54.

324. Norris, F.H., et al., Trial of oral physostigmine in amyotrophic lateral sclerosis. Clin Pharmacol Ther, 1993. 54(6): p. 680-2. We evaluated a double-blind, placebo-controlled, and double-crossover trial of oral physostigmine salicylate for a 9-month period in 13 of 25 patients with sporadic amyotrophic lateral sclerosis (ALS). A large dropout rate of 48% was secondary to eight deaths and four exclusions attributed to the incapability to swallow the tablets (physostigmine) and capsules (lecithin) or to attend the clinic. Parameters used for assessment of the drug efficacy included body weight, ALS score, Jamar grip strength, forced vital capacity, and maximum voluntary ventilation. It revealed slight benefit in reduced loss of grip strength compared with the pretrial and placebo periods. However, the rates of decline for body weight, ALS score, forced vital capacity, maximum voluntary ventilation, and megascore did not differ significantly between the pretrial, placebo, and physostigmine periods. We therefore concluded that overall no significant alteration in the clinical course was gained by oral physostigmine therapy in the 13 patients with ALS who were included in this study.

325. Aquilonius, S.M., et al., Cholinesterase inhibitors lack therapeutic effect in amyotrophic lateral sclerosis. A controlled study of physostigmine versus neostigmine. Acta Neurol Scand, 1986. 73(6): p. 628-32. Seven patients with amyotrophic lateral sclerosis participated in a double-blind cross-over trial of oral physostigmine and neostigmine (10 and 45 mg/day, respectively, for 3 days). Six of the patients were also given intravenous injections (1 and 1.5 mg, respectively) of the drugs in an open trial. No significant effects on muscle strength or neurophysiological parameters were observed.

326. Backman, S.B., et al., Different properties of the bradycardia produced by neostigmine and edrophonium in the cat. Can J Anaesth, 1996. 43(7): p. 731-40. PURPOSE: The bradycardia produced by neostigmine and edrophonium was examined according to its relation to cholinesterase inhibition and to its sensitivity to block by muscarinic receptor antagonists. For comparison, the ability of muscarinic antagonists to block the bradycardia produced by electrical stimulation of the vagus nerve was determined. METHODS: Cats were anaesthetized, vagotomized and propranolol-treated. Heart rate was continuously recorded. Erythrocyte cholinesterase activity of arterial blood was measured using a radiometric technique. The right vagus nerve was isolated for electrical stimulation. The muscarinic antagonists used were atropine, glycopyrrolate, pancuronium, gallamine, and AFDX-116. RESULTS: Neostigmine produced a dose-dependent decrease in cholinesterase activity which reached a plateau at a cumulative dose of 0.16 mg.kg-1 (ED50 0.009 +/- 0.003 mg.kg-1). Neostigmine produced a dose-

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dependent decrease in heart rate with the dose-response relationship (ED50 0.1 +/- 0.01 mg.kg-1; P = 0.0006) shifted to the right of that for the inhibition of cholinesterase activity. In contrast to the anticholinesterase effect, the bradycardic effect did not reach a plateau and continued to increase even at doses at which the cholinesterase inhibition was maximal. The maximal decrease in heart rate when the heart was still in sinus rhythm was by 81 +/- 13 bpm (49 +/- 7% of baseline), which was produced by a dose of 0.32 mg.kg-1. Edrophonium produced dose-dependent decreases in cholinesterase activity and heart rate, which were highly correlated (correlation coefficient r = 0.99, P < 0.0001). The ED50 of the reduction in heart rate (0.9 +/- 0.18 mg.kg-1) and cholinesterase activity (0.89 +/- 0.12 mg.kg-1) produced by edrophonium were similar. Moreover, the reduction in heart rate and cholinesterase activity produced by edrophonium reached a plateau at the same dose (6.4 mg.kg-1). At this dose, heart rate decreased by 22 +/- 2 bpm (14.6 +/- 0.9% of baseline). Compared to the bradycardia produced by stimulation of the vagus nerve, that produced by neostigmine was blocked by muscarinic antagonists at significantly lower doses while that produced by edrophonium was blocked at similar doses. CONCLUSIONS: The neostigmine-induced bradycardia is poorly correlated with cholinesterase inhibition compared to that produced by edrophonium, and has a higher sensitivity to muscarinic receptor antagonists compared to that produced by edrophonium or vagus nerve stimulation. These results are consistent with the hypothesis that the neostigmine-induced bradycardia is, in part, the result of neostigmine directly activating cholinergic receptors within the cardiac parasympathetic pathway. The bradycardia produced by edrophonium may be accounted for solely by an anticholinesterase action.

327. Baraka, A., Safe reversal. 2. Atropine-neostigmine mixture: an electrocardiographic study. Br J Anaesth, 1968. 40(1): p. 30-6.

328. Baraka, A., Safe reversal. 1. Atropine followed by neostigmine. An electrocardiographic study. Br J Anaesth, 1968. 40(1): p. 27-9.

329. Beebe, D.S., S.J. Shumway, and R. Maddock, Sinus arrest after intravenous neostigmine in two heart transplant recipients. Anesth Analg, 1994. 78(4): p. 779-82.

330. Bramich, N.J., J.A. Brock, and G.D. Hirst, Potentiation by neostigmine of responses to vagal nerve stimulation in the sinus venosus of the toad. Auton Neurosci, 2000. 82(3): p. 109-14. The effects of the cholinesterase inhibitor neostigmine on the responses to vagus nerve stimulation of isolated sinus venosus/atrial preparations of the toad, Bufo marinus, were examined. In control solutions, trains of stimuli applied to the vagus nerve led to a decrease in heart rate that was susceptible to muscarinic receptor blockade. Membrane potential recordings made from sinus venosus cells showed that the responses to trains of stimuli, delivered at frequencies of less than 10 Hz, were little changed by the addition of neostigmine. However, the responses to longer trains of stimuli at 10 Hz (30 versus 10 s) were potentiated and the nature of the membrane potential changes was altered. The results suggest that, due to the activity of cholinesterases, acetylcholine (ACh) released from parasympathetic nerves normally has little access to the muscarinic receptors in the pacemaker region, which are linked to potassium channels.

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331. Cavaliere, F., et al., [Effectiveness of low-dose neostigmine in the treatment of sinus tachycardia during aortocoronary bypass]. Minerva Anestesiol, 1993. 59(7-8): p. 361-5. Thirteen patients, who underwent surgery for myocardial revascularization, were given neostigmine, 0.25-1 mg i.v., in order to control intraoperative sinus tachycardia. Three minutes after the administration, the drug was effective in all patients, by decreasing the heart rate and improving the indices of the risk of myocardial ischemia. Seven minutes later the heart rate decreased further. In 5 patients out of 13, short periods of bradycardia were observed during surgical compression or dislocation of the heart. As cardiac rate decreased, P and T waves and ST, QT, and QTc intervals lengthened without reaching pathological levels. Three minutes after neostigmine, the stroke volume was unchanged in spite of the decrease of the heart rate; consequently cardiac index decreased. Seven minutes later a slight increase of the stroke volume balanced the further decrease of the heart rate; so cardiac index did not change any more. Arterial pressure decreased slightly after neostigmine whereas systemic vascular resistance was unaffected. Also central venous pressure did not change while pulmonary capillary wedge pressure showed a small increase 10 minutes after neostigmine. Finally airway resistance did not change significantly.

332. Constable, P.D., et al., Atrial fibrillation associated with neostigmine administration in three cows. J Am Vet Med Assoc, 1990. 196(2): p. 329-32. Three cattle with gastrointestinal disease were given neostigmine as part of the treatment for postsurgical ileus. All 3 cattle developed atrial fibrillation either before or during neostigmine administration, and 2 converted to normal sinus rhythm after cessation of treatment.

333. Dowdy, E.G., Effect of neostigmine on cardiac rhythm in digitalized dogs. Guest discussion. Anesth Analg, 1971. 50(6): p. 1088-9.

334. Fuchs-Buder, T., et al., Antagonism of vecuronium-induced neuromuscular block in patients pretreated with magnesium sulphate: dose-effect relationship of neostigmine. Br J Anaesth, 1999. 82(1): p. 61-5. We have investigated the dose-effect relationship of neostigmine in antagonizing vecuronium-induced neuromuscular block with and without magnesium sulphate (MgSO4) pretreatment. Neuromuscular block was assessed by electromyography with train-of-four (TOF) stimulation. First, we determined neostigmine-induced recovery in patients pretreated with MgSO4 (group A) or saline (group B) (n = 12 each). The height of T1, 5 min after neostigmine, was 43 (7)% in group A and 65 (6)% in group B (P < 0.01). Respective values after 10 min were 59 (7)% and 83 (5)% (P < 0.01). TOF ratio, 5 min after neostigmine, was 29 (6)% in group A and 29 (5)% in group B. Respective values after 10 min were 38 (11)% and 51 (7)% (P < 0.01). To gain insight into the mechanisms leading to delayed recovery after MgSO4, we calculated assisted recovery, defined as neostigmine-induced recovery minus mean spontaneous recovery. Spontaneous recovery was assessed in another 24 patients. Patients in group C received MgSO4/vecuronium and patients in group D vecuronium only (n = 12 each). Five minutes after neostigmine, assisted recovery was 22 (7)% in the MgSO4 pretreated patients and 28 (6)% in controls (P < 0.05). Ten minutes after

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neostigmine, values were 24 (7)% and 22 (6)%. Maximum assisted recovery was not influenced by MgSO4 pretreatment (27 (6)% in group A and 32 (6)% in group B) and time to maximum effect was comparable between groups: 6 (4-10) min and 7 (5-8) min, respectively. We conclude that neostigmine-induced recovery was attenuated in patients treated with MgSO4. This was mainly a result of slower spontaneous recovery and not decreased response to neostigmine.

335. Eldor, J. and D.Z. Frankel, Use of low-dose neostigmine intravenously in the treatment of supraventricular tachycardia: an immediate bradycardic effect. Resuscitation, 1989. 18(1): p. 103-10. Neostigmine was first used 50 years ago to treat sinus tachycardia and paroxysmal auricular tachycardia. Then there were reports of successful treatment by neostigmine of other forms of supraventricular tachycardias. However, reports of sudden death using neostigmine for reversal of neuromuscular blockade at the end of an operation, which were not properly treated with atropine abandoned the use of neostigmine as an antiarrhythmic drug. Low-dose neostigmine intravenously was used in the treatment of supraventricular tachycardia in three patients described herein. It gave an immediate bradycardic effect in all three patients. The use of a low-dose neostigmine intravenously for an immediate treatment of supraventricular tachycardia is a novel suggestion. It has to be further evaluated and compared to the conventional drugs used like digoxin, verapamil, propranolol or esmolol.

336. Ivankovic, A.D., et al., Effect of neostigmine on cardiac rhythm in digitalized dogs. Anesth Analg, 1971. 50(6): p. 1079-88.

337. Kadoya, T., et al., Development of rapid atrial fibrillation with a wide QRS complex after neostigmine in a patient with intermittent Wolff-Parkinson-White syndrome. Br J Anaesth, 1999. 83(5): p. 815-8. We report the case of a 67-yr-old man with intermittent Wolff-Parkinson-White (WPW) syndrome in whom neostigmine produced life-threatening tachyarrhythmias. The patient was scheduled for microsurgery for a laryngeal tumour. When he arrived in the operating room, the electrocardiogram showed normal sinus rhythm with a rate of 82 beat min-1 and a narrow QRS complex which remained normal throughout the operative period. On emergence from anaesthesia, the sinus rhythm (87 beat min-1) changed to atrial fibrillation with a rate of 80-120 beat min-1 and a normal QRS complex. We did not treat the atrial fibrillation because the patient was haemodynamically stable. Neostigmine 1 mg without atropine was then administered to antagonize residual neuromuscular block produced by vecuronium. Two minutes later, the narrow QRS complexes changed to a wide QRS complex tachycardia with a rate of 110-180 beat min-1, which was diagnosed as rapid atrial fibrillation. As the patient was hypotensive, two synchronized DC cardioversions of 100 J and 200 J were given, which restored sinus rhythm. No electrophysiological studies of anticholinesterase drugs have been performed in patients with WPW syndrome. We discuss the use of these drugs in this condition.

338. Kjellberg, M. and T. Tammisto, Heart-rate changes after atropine and neostigmine given for the reversion of muscle paralysis. Acta Anaesthesiol Scand, 1970. 14(3): p. 203-10.

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339. Klingenmaier, C.H., et al., Reversal of neuromuscular blockade with a mixture of neostigmine and glycopyrrolate. Anesth Analg, 1972. 51(3): p. 468-72.

340. Pratila, M.G. and V. Pratilas, A case of tachydysrhythmia. Refractory to propranolol and responsive to neostigmine. Anaesthesia, 1977. 32(10): p. 1017-9. A case of atrial fibrillation, with a fast ventricular response, which developed under enflurane anaesthesia is described in a patient previously treated with digitalis and propranolol. The intravenous administration of propranolol was ineffective whereas that of neostigmine and atropine, 2 hours later, was successful and reduced the ventricular rate to normal values.

341. Raymond, C.W., Letter: Neostigmine methysulfate in the treatment of cardiac arryhthmia induced by perphenazine-amitriptyline. Can Med Assoc J, 1976. 114(2): p. 102-7.

342. Rosner, V., E.R. Kepes, and F.F. Foldes, The effects of atropine and neostigmine on heart rate and rhythm. Recommendation for their use to reverse residual neuromuscular block. Br J Anaesth, 1971. 43(11): p. 1066-74.

343. Schultheis, L.W., et al., High dose neostigmine treatment of malignant sinus tachycardia. Pacing Clin Electrophysiol, 1997. 20(5 Pt 1): p. 1369-72. Sinus tachycardia caused by circulating catecholamines in the setting of congestive heart failure may impair systemic perfusion because of decreased diastolic filling time. We report the case of a patient with Wolff-Parkinson-White syndrome with angina and cardiogenic shock who improved dramatically following administration of neostigmine. Cardiac output, blood pressure, and stroke volume increased as heart rate was reduced. A previous attempt at heart rate control, in the same patient, using a low dose beta-antagonist, precipitated hemodynamic collapse. The remarkable recovery of our patient suggests that acetylcholinesterase inhibitors may warrant further investigation in patients with severe sinus tachycardia.

344. Tetzlaff, J.E. and M.J. Choban, Asystole following neostigmine administration during carotid sinus stimulation. J Clin Anesth, 1990. 2(3): p. 212-3.

345. Wetterslev, J., et al., Split-dose atropine versus glycopyrrolate with neostigmine for reversal of gallamine-induced neuromuscular blockade. Acta Anaesthesiol Scand, 1991. 35(5): p. 398-401. The effects of a split-dose of atropine sulphate versus a single dose of glycopyrrolate given with neostigmine for the reversal of gallamine-induced neuromuscular blockade were studied in 55 patients undergoing gynaecological surgery. The patients were randomized to receive either a single dose of glycopyrrolate (7 micrograms.kg-1) or two doses of atropine (8 micrograms.kg-1 each), given with an interval of 1 min. There were no differences between the two methods with respect to percentage heart rate changes, salivation or arousal time. Four patients demonstrated cardiac arhythmias in the atropine group, whereas none occurred in the glycopyrrolate group (P less than 0.05). It is concluded that a split-dose of atropine has similar

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chronotropic effects to a single dose of glycopyrrolate for the reversal of gallamine-induced neuromuscular blockade. However, the finding of a higher incidence of cardiac arrhythmias in the atropine group suggests that this reversal regime should be reserved for patients without cardiac disease.

346. Bigger, J.T., Jr., et al., Is physostigmine effective for cardiac toxicity of tricyclic antidepressant drugs. Jama, 1977. 237(13): p. 1311.

347. Das, P.K. and S.K. Bhattacharya, Studies on the effect of physostigmine on experimental cardiac arrhythmias in dogs. Br J Pharmacol, 1972. 44(3): p. 397-403.

348. Dysken, M.W. and D.S. Janowsky, Dose-related physostigmine-induced ventricular arrhythmia: case report. J Clin Psychiatry, 1985. 46(10): p. 446-7. Physostigmine, a centrally-acting cholinesterase inhibitor, has been shown to have potential as a treatment for primary degenerative dementia. In an experiment to test its effects, i.v. physostigmine caused the onset of unifocal premature contractions and runs of bigeminy in an 85-year-old man.

349. Elonen, E., Effect of beta-adrenoceptor blocking drugs, physostigmine, and atropine on the toxicity of doxepin in mice. Med Biol, 1975. 53(4): p. 231-7. Large doses of doxepin given intravenously to animals cause tachyarrhythmias, and still higher doses lead to a progressive and, finally, lethal bradycardia. The effect of pretreatment with five different beta-adrenoceptor blocking drugs (propranolol, alprenolol, practolol, metoprolol or tolamolol), p physostigmine, or atropine on these toxic actions of doxepin was investigated. Mice were sedated with diazepam. Doxepin was injected i.v. 0.1 mg every 15 sec until death. ECG was recorded at 10 sec after every injection. All five beta-blockers injected i.p. 30 min before doxepin inhibited the doxepin-induced tachyarrhythmias. None of the drugs prevented or postponed the death of mice. Large doses of beta-blockers dose-dependently enhanced the doxepin-induced bradycardia and accelerated death. The cardioselective beta-blocking drugs practolol and metoprolol proved less active in enhancing bradycardia than the third cardio-selective drug, tolamolol, and non-selective propranolol and alprenolol. This difference may have resulted from properties other than beta-blockade since practolol and metoprolol lack the "cardiodepressant" and local anaesthetic properties. Since physostigmine and atropine did not modify the doxepin effects the anticholinergic property may not be important in the severe cardiotoxic effects of doxepin.

350. Goldberger, A.L. and G.P. Curtis, Immediate effects of physostigmine on amitryptyline-induced QRS prolongation. J Toxicol Clin Toxicol, 1982. 19(5): p. 445-54. Prior studies have suggested that physostigmine may be useful in reversing QRS prolongation due to amitriptyline toxicity. To investigate this question, we devised a pharmacologic model in rabbits utilizing an initial intravenous bolus of amitriptyline (4-6 mg) followed by a constant amitriptyline infusion (0.2-0.4 mg/min) empirically titered to maintain the QRS at 50% or more of control value for at least 5 min. Intravenous physostigmine (2 mg) sufficient to produce muscle fasciculations and significant (P less than 0.01) slowing of sinus rate was then administered to six animals. No significant

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change in QRS duration was noted at 1, 3, and 5 min intervals following physostigmine. Although no immediate antidotal effect of physostigmine on amitriptyline-induced QRS prolongation could be demonstrated, these results do not exclude a possible interaction between the membrane effects of the tricyclic antidepressants (and related agents) and the vagal branch of the autonomic nervous system.

351. James, T.N., F. Urthaler, and R.K. Lewis, Termination of "ventricular" arrhythmias from digoxin by selective production of complete atrioventricular block with physostigmine in the dog. J Pharmacol Exp Ther, 1979. 211(3): p. 561-70. In previous studies we have come to doubt that ventricular rhythms of an automatic nature will arise spontaneously from the peripheral Purkinje system. In 20 anesthetized dogs, digoxin was administered i.v. (0.1-1.0 mg/kg) and in 12 dogs by selectively perfusing the atrioventricular (AV) node artery (2 ml; 40 microgram/ml). We obtained the following results. First, selective pharmacological production of complete AV block (acetylcholine or physostigmine) interrupts the "ventricular" arrhythmias considered characteristic of digitalis intoxication.Second, digitalis arrhythmias are difficult to produce when this type of complete heart block had been previously established. Third, abolition of ventricular arrhythmias by selective pharmacological production of heart block can be reversed (i.e., the arrhythmia restored) with atropine. Fourth, rapid pacing of the ventricles during complete heart block in dogs poisoned with digitalis can eventually induce ventricular arrhythmias, but not quickly. We interpret that these digitalis arrhythmias originated within the acetylcholine-sensitive portion of the AV node-His bundle region.

352. Janowsky, D.S., et al., Effects of physostigmine on pulse, blood pressure, and serum epinephrine levels. Am J Psychiatry, 1985. 142(6): p. 738-40. In this study, the infusion of physostigmine in 14 patients with affective disorder who were pretreated with methscopolamine caused significant and often profound increases in the patients' epinephrine levels, pulse rates, and blood pressure. Since physostigmine is being used experimentally in the treatment of elderly subjects who have Alzheimer's disease, these cardiovascular effects may have clinical importance.

353. Kulig, K. and B.H. Rumack, Physostigmine and asystole. Ann Emerg Med, 1981. 10(4): p. 228-20.

354. Maister, A.H., Atrial fibrillation following physostigmine. Can Anaesth Soc J, 1983. 30(4): p. 419-21. The side effects of physostigmine, a tertiary amine anticholinesterase agent commonly used to treat postanaesthetic depression caused by a large number of drugs, are thought to be fairly mild. A case is reported in which atrial fibrillation followed a total dose of 2 mg of this drug to a patient who was slow in recovering from a general anaesthetic. The arrhythmia was self-limiting and required no therapy. Indications and precautions for the use of physostigmine are discussed.

355. Pentel, P. and C.D. Peterson, Asystole complicating physostigmine treatment of tricyclic antidepressant overdose. Ann Emerg Med, 1980. 9(11): p. 588-90. Physostigmine is a commonly used therapy for the anticholinergic manifestations of

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tricyclic antidepressant (TCA) overdose. We describe two patients with TCA toxicity who developed asystole following the administration of physostigmine to treat seizures.

356. Schmidt, W. and K. Lang, Life-threatening dysrhythmias in severe thioridazine poisoning treated with physostigmine and transient atrial pacing. Crit Care Med, 1997. 25(11): p. 1925-30. OBJECTIVES: To describe clinical, electrocardiographic, and blood chemistry findings in a case of high-dosage thioridazine self-poisoning, focusing on the cellular mechanisms of the cardiovascular toxicity. DESIGN: Case report and literature review. SETTING: Intensive care unit (ICU) of a district hospital in Germany. PATIENT: A 68-yr-old male patient admitted to the ICU for treatment of a severe thioridazine intoxication. INTERVENTIONS: Prevention of absorption (gastric lavage), mechanical ventilation, fluids, alkalinization, catecholamines, drugs (physostigmine, neostigmine), direct current cardioversion/defibrillation, and transient pacemaker (atrial stimulation). MEASUREMENTS AND MAIN RESULTS: Central nervous, cardiovascular, and gastrointestinal systems indicated the adverse side effects of thioridazine intoxication over a period of 9 days. During high toxic thioridazine plasma concentrations (6061 to 6480 ng/mL), a life-threatening crisis occurred due to malignant ventricular arrhythmias followed by bradycardia (e.g., sinus node arrest). The electrocardiogram showed delays in all parts of the conduction system of the heart, including prolonged repolarization for several days, which disappeared completely when thioridazine plasma concentrations were within the therapeutic range. CONCLUSIONS: An individual therapeutic approach is needed in cases of thioridazine overdose. The primary aim is to stabilize the cardiac rhythm and the circulation.

357. Schulz, M. and A. Schmoldt, Successful physostigmine treatment of acute dothiepin intoxication. Pharmazie, 1994. 49(8): p. 614. We report on a severe intoxication with the tricyclic antidepressant dothiepin. A treatment with a continuous infusion of physostigmine was successful and improved the ventricular arrhythmia. Dothiepin and its active metabolites were determined in plasma and urine by HPLC.

358. Seitz, R., M. Wehr, and N. Pallmert, [Arrhythmias in poisoning with tricyclic antidepressive agents. Successful treatment with physostigmine and sodium]. Dtsch Med Wochenschr, 1982. 107(28): p. 1096-9. A 26-year-old female patient was admitted 30 minutes after suicidal poisoning with 750 mg amitriptyline (Saroten), 4800 mg dibenzepine (Noveril retard) and 2100 mg thioridazine (Melleril). Life threatening disturbances of cardiac rhythm occurred in addition to coma, pulmonary insufficiency requiring assisted ventilation, and hypotension. Physostigmine only caused temporary improvement and the arrhythmias only resolved completely after additional administration of NaCl.

359. Snyder, B.D., L. Blonde, and W.R. McWhirter, Reversal of amitriptyline intoxication by physostigmine. Jama, 1974. 230(10): p. 1433-4.

360. Tobis, J. and B.N. Das, Cardiac complications in amitriptyline poisoning. Successful treatment with physostigmine. Jama, 1976. 235(14): p. 1474-6.

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361. Wright, S.P., Usefulness of physostigmine in imipramine poisoning. A dramatic response in a child resistant to other therapy. Clin Pediatr (Phila), 1976. 15(12): p. 1123-8.

362. Weisdorf, D., et al., Physostigmine for cardiac and neurologic manifestations of phenothiazine poisoning. Clin Pharmacol Ther, 1978. 24(6): p. 663-7. Psychotropic drugs such as the phenothiazine neuroleptics and tricyclic antidepressants are known to cause electrocardiographic abnormalities as well as a central anticholinergic syndrome. Physostigmine is known to reverse the central muscarinic anticholinergic manifestations by inhibition of the enzyme cholinesterase. An unusual case of trifluoperazine overdose, in which the patient presented with cardiac arrhythmias and a central anticholinergic syndrome, is presented. Treatment with physostigmine reversed the central anticholinergic syndrome as well as the electrocardiographic abnormalities. Effects of phenothiazines on altering cardiac status are also discussed.

363. Dorner, G., et al., Further evidence of teratogenic effects apparently produced by neurotransmitters during brain differentiation. Endokrinologie, 1977. 70(3): p. S326-330.

364. Dorner, G., K. Hecht, and G. Hinz, Teratopsychogenetic effects apparently produced by nonphysiological neurotransmitter concentrations during brain differentiation. Endokrinologie, 1976. 68(1): p. 1-5.

365. Dorner, G. and G. Hinz, Apparent effects of neurotransmitters on sexual differentiation of the brain without mediation of sex hormones. Endokrinologie, 1978. 71(1): p. S104-108.

366. Landauer, W., Cholinomimetic teratogens: Studies with chicken embryos. Teratology, 1975. 12: p. 125-146.

367. Al-Shekhlee, A., N. Robin, and H.J. Kaminski, Pyridostigmine-induced microcephaly. Neurology, 2001. 56(11): p. 1606-7.

368. Cowan, D.N., et al., The risk of birth defects among children of Persian Gulf War veterans. N Engl J Med, 1997. 336(23): p. 1650-6. BACKGROUND: There has been suspicion that service in the Persian Gulf War affected the health of veterans adversely, and there have been claims of an increased rate of birth defects among the children of those veterans. METHODS: We evaluated the routinely collected data on all live births at 135 military hospitals in 1991, 1992, and 1993. The data base included up to eight diagnoses from the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) for each birth hospitalization, plus information on the demographic characteristics and service history of the parents. The records of over 75,000 newborns were evaluated for any birth defect (ICD-9-CM codes 740 to 759, plus neoplasms and hereditary diseases) and for birth defects defined as severe on the basis of the specific diagnoses and the criteria of the Centers for Disease Control and Prevention. RESULTS: During the study period, 33,998 infants were born to Gulf War veterans and

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41,463 to non-deployed veterans at military hospitals. The overall risk of any birth defect was 7.45 percent, and the risk of severe birth defects was 1.85 percent. These rates are similar to those reported in civilian populations. In the multivariate analysis, there was no significant association for either men or women between service in the Gulf War and the risk of any birth defect or of severe birth defects in their children. CONCLUSIONS: This analysis finds no evidence of an increase in the risk of birth defects among the children of Gulf War veterans.

369. Penman, A.D., R.S. Tarver, and M.M. Currier, No evidence of increase in birth defects and health problems among children born to Persian Gulf War Veterans in Mississippi. Mil Med, 1996. 161(1): p. 1-6. The Department of Veterans' Affairs, Jackson, Mississippi, and the Mississippi State Department of Health conducted a collaborative investigation of an apparent increase in the numbers of birth defects and other health problems among children born to veterans of two Mississippi National Guard units who had served in the Persian Gulf War. The medical records of all children conceived by and born to veterans of the two units after deployment were reviewed; observed numbers of birth defects and other health problems were compared with expected numbers using rates from birth defect surveillance systems and previous surveys. The total number of all types of birth defects was not greater than expected, but whether the number of specific birth defects was greater than expected could not be determined. The frequency of premature birth, low birth weight, and other health problems appeared similar to that in the general population.

370. Araneta, M.R., et al., Birth defects prevalence among infants of Persian Gulf War veterans born in Hawaii, 1989-1993. Teratology, 2000. 62(4): p. 195-204. BACKGROUND: Gulf War veterans (GWVs) have expressed concern about possible teratogenic exposures. However, epidemiologic studies on birth defects prevalence among their progeny have been limited to military hospitals, anomalies diagnosed among newborns, or self-reported data. To measure the prevalence of selected birth defects among infants of GWVs and nondeployed veterans (NDVs) in Hawaii, using birth defects surveillance records. METHODS: Personal identifiers of 684,645 GWVs and 1,587,102 NDVs and their families were matched against birth certificate records of 99,545 live births reported to the State of Hawaii Department of Health between 1989 and 1993 to identify births to military personnel. These births were matched with records from the Hawaii Birth Defects Program. RESULTS: A total of 17,182 military infants (3,717 GWV infants and 13,465 NDV infants) were identified. Of these, 367 infants (2.14/100 live births) were identified with one or more of 48 major birth defects diagnoses. The prevalence of the 48 birth defects were similar for GWV and NDV infants during the prewar and postwar periods, and among GWV infants who were conceived before and after the Gulf war. CONCLUSIONS: The results must be interpreted with caution because of the small number of affected infants in each birth defects category. This study demonstrated the feasibility of measuring birth defects prevalence among military infants through multiple data linkage. Further, it included live births to parents who had separated from the military, births in civilian hospitals, and birth defects diagnosed through the first year of life.

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371. Doyle, P., E. Roman, and N. Maconochie, Birth defects among children of Persian Gulf War veterans. N Engl J Med, 1997. 337(16): p. 1175-6.

372. Zhou, Y.X., et al., Detection of the organophosphorus nerve agent sarin by a competitive inhibition enzyme immunoassay. Arch Toxicol, 1995. 69(9): p. 644-8. Two artificial antigens, NalphaNepsilon-di(O,O-diisopropyl) phosphoryl L-lysine (DIP)- bovine serum albumin (BSA) conjugate (DIP-BSA) and DIP-KLH (keyhole limpet hemocyanin), were synthesized. Antibodies against sarin (O-isopropyl methylphosphonofluoridate) were obtained after immunization of rabbits with DIP-KLH conjugate. A competitive inhibition enzyme immunoassay (CIEIA) was developed to detect the organophosphorus nerve agent sarin. The antibody solutions could be inhibited by sarin as low as 10(-6) mol/l, and the standard curve was linear over 3 orders of magnitude. The coefficients of intraassay and interassay variation of this method were 5.4-6.2% (n = 11) and 8.0-9.5% (n = 6) at a sarin concentration range of 10(-3)-10(-6) mol/l, respectively. The recovery of sarin in water samples at the concentration of 5 x 10(-5) mol/l was in the range of 96.8-102.5%. The specificity of the antiserum was assessed by comparing the inhibition induced by sarin with soman, Vx, isopropyl alcohol and isopropyl methyl phosphonic acid. The results showed that less than 5 mmol/l soman, 2 mmol/l Vx, 16 mmol/l isopropyl alcohol and 8 mmol/l isopropyl methyl phosphonic acid did not influence the determination of sarin in water samples.

372.5 Kang H, Magee C, Mahan C, et al. Pregnancy outcomes among U.S. Gulf War veterans: a population-based survey of 30,000 veterans. Ann Epidemiol. 2001;11:504-11. PURPOSE: We evaluated an association between veterans' Gulf War service and reported adverse pregnancy outcomes. METHODS: We conducted a health survey in which selected reproductive outcomes of a population-based sample of 15,000 Gulf War veterans representing four military branches and three unit components (active, reserve, and National Guard) were compared to those of 15,000 non-Gulf veteran controls. RESULTS: Male Gulf veterans, compared with their non-Gulf veteran controls, reported a significantly higher rate of miscarriage (odds ratio [OR] = 1.62; 95% confidence interval [CI] = 1.32-1.99). Female Gulf veterans also reported more miscarriages than their respective controls, although their excess was not statistically significant (OR= 1.35; CI = 0.97-1.89). Both men and women deployed to the Gulf theater reported significant excesses of birth defects among their liveborn infants. These excess rates also extended to the subset of "moderate to severe" birth defects [males: OR= 1.78 (CI = 1.19-2.66); females: OR = 2.80 (CI = 1.26-6.25)]. No statistically significant differences by deployment status were found among men or women for stillbirths, pre-term deliveries or infant mortality. CONCLUSION: The risk of veterans reporting birth defects among their children was significantly associated with veteran's military service in the Gulf War. This observation needs to be confirmed by a review of medical records to rule out possible reporting bias.

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