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Hyperpyrexia and head trauma Final, 1975-1983

6. PERFORMING ORG. REPORT NUMBER

S, "AUTHOR(q) 8. CONTRACT OR GRANT NUMBER(@)

Thomas A. Rudy, Ph.D. N00014-75-C-0939

.. PERFORMING ORGANIZATION NAME AND ADDRESS i0. PROGRAM ELEMENT. PROJECT, TASKAREA 6 WORK UNIT NUMBERS

' University of Wisconsin, School ofPharmacy, 425 N. Charter Street,Madison. WI 53706

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r-4 Physiology Program (Code 441), Office of 9 March 1984. Naval Research, 800 N. Quincy Street, 13. NUMBER OF PAGES

eel Arlington, VA 22217 154. MONITORING AGENCY NAWE & ADDRESS(II dflerent from Controlling Office) IS. SECURITY CLASS. (of this report)

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18. SUPPLEMENTARY NOTES

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19. KEY WORDS (Continue on reverse side It necessary and identify by block number)

Head trauma, intraventricular hemorrhage, fever, thermoregula-tion, prostaglandins, norepinephrine, serotonin, carbamyl-choline, hypothalamus, preoptic area

2O ABSTRACT (Continue on reverse aide it necessary aid identify by block number)

SUnilateral mechanical lesions of the anterior hypothalamic/pre-'. optic (AH/PO) region of the rat were found to produce immediate.I. pyrexia. The pyrexia was generated by the coordinated activationLAJ of heat gain and heat retention effectors. Its magnitude was not.j.- -strongly affected by ambient temperature, and the plateau level of

ep L pyrexia was well defended in the face of forced perturbations ofcore temperature. Iyrexia could be prevented and reversed by -

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- the prostaglandin synthesis inhibitor, indomethacin. Intraven-tricular injection of fresh blood or serum derived from bloodwhich had been incubated under sterile conditions at 370 C forfrom 2 hours to 21 days produced pyrexia in cats. Pretreatmentof the cats with indomethacin prevented the pyrexia produced bythe serums but including indomethacin in the incubating blood didnot. These results indicate that prostaglandins are importantlyinvolved in the production of pyrexia by AlH/P trauma and by in-traventricular bleeding. Studies of the central nervous systemsite of action of prostaglandins in the production of pyrexiausing a microinjection ','mapping" method showed that the AH/POregion is the sole site of action in the upper portion of the ratbrain./ Similar studies of the posterior portion of the rat brainhave ' dvealed that there probably exists at least one lower brainsteW site where prostaglandins can act to produce pyrexia. In-jections of prostaglandins into the rat spinal subarachnoid spacedid not produce increases in core temperature.

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X. W.

HYPERPYREXIA AND HEAD TRAUMA

FINAL REPORT

by

Thomas A. Rudy, Responsible Investigator

(For the period 1975-1983)

Supported by

Office of Naval Research .-..Contract N00014-75-C-0939 r ' ',F

University of Wisconsin

Madison, Wisconsin 53706

Distoribuiom/ .

- Availability COdesiAvail and/or

4#Dist Pncial

Reproduction in whole or in part is permitted

for any purpose of the United States Government

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TABLE OF CONTENTS

Part Page

I. Publications

A. Papers and book chapters ......................... 3

B. Abstracts and presentations ...................... 4

II. Personnel supported .................................. 6

III. Overview of contract accomplishments

A. Pyrexia produced by acute mechanical destructionof brain tissue ...... ............. ............... 7

B. Pyrexia produced by simulated intraventricularhemorrhage ....................................... 9

C. Central nervous system site of pyrogenic actionof prostaglandins ................................ 10

'SD. Evaluation of the pyrogenic effect of leukotrienes

C4 D4 and E4 .................................... 12

E. Involvement of supraspinal and spinal neurotrans-mitters in thermoregulation and fever(1) Supraspinal neurotransmitters ............... 13

(2) Spinal neurotransmitters .................... 15

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I. Publications

A. Papers and book chapters

(1) Williams, J. W., Rudy, T. A., Yaksh, T. L. and Viswanathan,C. T. An extensive exploration of the rat brain for sitesmediating prostaglandin-induced hyperthermia. Brain Re-search, 120: 251-262 (1977).

(2) Komiskey, H. L. and Rudy, T. A. Serotonergic influenceson brainstem thermoregulatory pathways in the cat. BrainResearch, 134: 297-315 (1977).

(3) Rudy, T. A., Williams, J. and Yaksh, T. L. Antagonism byindomethacin of neurogenic hyperthermia produced by uni-lateral puncture of the anterior hypothalamic/preopticregion. J. Physiol., 272: 721-736 (1977).

(4) Rudy, T. A. and Yaksh, T. L. Hyperthermic effects of mor-phine: Set point manipulation by a direct spinal action.Br. J. Pharmacol., 61: 91-96 (1977).

(5) Rudy, T. A. and Yaksh, T. L. Elevation of the setpointfor thermoregulation by intrahypothalamic injection ofcarbamylcholine in the rhesus monkey. IN Cooper, K. E.,Lomax, P. and Schonbaum, E. (eds.), Drugs, Biogenic Aminesand Body Temperature, Karger, Basel, pp. 26-30 (1977).

(6) Rudy, T. A., Westergaard, J. and Yaksh, T. L. Hyperthermiaproduced by simulated intraventricular hemorrhage in thecat. Exptl. Neurol., 58: 296-310 (1978).

(7) Ackerman, D. and Rudy, T. A. Thermoregulatory character-istics of neurogenic hyperthermia in the rat. J. Physiol.,307: 59-70 (1980).

(8) Rudy, T. A. Studies of fever associated with cerebraltrauma and intracranial hemorrhage in experimental animals.IN Lipton, J. M. (ed.), Fever, Raven Press, New York,pp. 165-175 (1980).

(9) Rudy, T. A. Pathogenesis of fever associated with cerebraltrauma and intracranial hemorrhage. IN Cox, B., Lomax, P.,Milton, A. S. and Schonbaum, E. (eds.), Thermoregulatorymechanisms and their therapeutic implications, Karger,Basel, pp. 75-81 (1980).

(10) LoPachin, R. and Rudy, T. A. An improved method for chroniccatheterization of the spinal subarachnoid space of the rat.Physiol. Behav., 27: 559-561 (1981).

(11) LoPachin, R. and Rudy, T. A. The effect of intrathecalsympathomicetic agents on neural activity in the lumbarsympathetic chain of rats. Brain Research, 224: 195-198(1981).

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(12) LoPachin, R. and Rudy, T. A. The thermoregulatory effectsof noradrenaline, serotonin and carbachol injected into therat spinal subarachnoid space. J. Physiol., 333: 511-529 (1982).

(13) LoPachin, R. and Rudy, T. A. Sites and mechanism of actionfor the effects of intrathecal noradrenaline on thermoregu-lation in the rat. J. Physiol., 341: 527-544 (1983).

(14) O'Rourke, S. T. and Rudy, T. A. Intracerebroventricularand preoptic injections of leukotrienes C , D and E inthe rat: Lack of febrile effect. Brain Research, 295:283-288 (1984k in press).

B. Abstracts and presentations

(1) Williams, J. W. and Rudy, T. A. An extensive neuroana-tomical mapping of the rat brain for sites which mediateprostaglandin-induced hyperthermia. Neurosci. Abstracts,1: 417 (1975).

(2) Rudy, T. A. and Komiskey, H. L. Functionally antagonisticthermoregulatory effects of 5-hydroxytryptamine within theanterior hypothalamus and preoptic area of the cat. Fed.Proc., 35: 530 (1976).

(3) Rudy, T. A., Williams, J. W. and Yaksh, T. L. Hyperthermiaevoked by acute mechanical damage to the hypothalamus andits antagonism by indomethacin. Neurosci. Abstracts, 2:731 (1976).

N (4) Rudy, T. A. Thermoregulatory effects of 5-hydroxytrypta-mine injected into the brainstem of the cat: Observationof two anatomically distinct sites of action. Paper readat the Third International Symposium on the Pharmacology ofThermoregulation, Banff, Canada, 1976.

(5) Ackerman, D. and Rudy, T. A. The effect of ambient temper-ature on the hyperthermia evoked by acute mechanical damageto the hypothalamus. Ncurosci. Abstracts, 3: 393 (1977).

*>. (6) LoPachin, R. and Rudy, T. A. The effects on body tempera-ture of norepinephrine and 5-hydroxytryptamine injectedinto the rat spinal subarachnoid space. Fed. Proc., 38:756 (1979).

(7) Rudy, T. A. Pathogenesis of fever associated with cerebraltrauma and intracranial hemorrhage. Paper read at the FourthInternational Symposium on the Pharmacology of Thermoregu-lation, Oxford, England, 1979.

(8) Rudy, T. A. Studies of fever associated with cerebraltrauma and intracranial hemorrhage in experimental animals.Paper read at the First International Symposium on Fever,Dallas, Texas, 1979.

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(9) LoPachin, R. and Rudy, T. A. Spinal cord monoaminergicand cholinergic mechanisms in thermoregulation. Pharma-cologist, 22: 184 (1980).

(10) Rudy, T. A. Enhanced pyrogenicity of decomposed versusfresh blood injected intraventricularly in the cat. Fed..* Proc., 39: 991 (1980).

(11) LoPachin, R. and Rudy, T. A. Sites of action for the effectsof intrathecal norepinephrine on thermoregulation. Neuro-sci. Abstracts, 7: 855 (1980).

% 4 (12) Minnich, J. L., Sugiyama, H., LoPachin, R. and Rudy, T. A.Effects of botulinum neurotoxin directly on the CNS:Hypothermia and death. Neurosci. Abstracts, 7: 55 (1980).

(13) Rudy, T. A. and Gollman, H. M. The role of cyclooxygenaseproducts in fever elicited by intraventricular injectionof serum derived from aged blood. Fed. Proc., 1984 (inpress).

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II. Personnel supported

This contract provided full or partial salary support for thefollowing individuals:

. Tony L. Yaksh (postdoctoral associate)

* ,Jerome L. Westergaard (specialist)

Joseph C. Yeung (graduate student)

Bruce Cornwell (specialist)

Richard LoPachin (graduate student)

Patricia Koch (specialist)

Deborah Ackerman (graduate student)

Stephen T. O'Rourke (graduate student)

Harold Gollman (graduate student)

Contract funds were also used to compensate the following Univer--'I sity of Wisconsin undergraduate students for part time clerical or

laboratory work related to the goals of the contract:

Michael Cain

Karin Gast

Sung-Ping.Huang

Jennifer Ondrajka

Robert Plant

.- Sandra White

*Patricia Hick

Stanley Tam

Dennis Hu

Ronald Epperson

Diane Boszhardt

Vicki Post

Jackie Wilson

,., Domenick Garzone

Gordon Meyer

*Patricia Duffy

"..a Keith Zelhafer

Debra FinleyDebra Brown

Julia Simon

Roger Hess

V Ron Paulson4..

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III. Overview of Contract Accomplishments

The general goal of the work supported by this contract was togain a better understanding of the pathophysiological basis of thepyrexias which sometimes follow cranio-cerebral trauma. A reviewof the clinical and experimental literature suggested that these"trauma-induced fevers" could be due to destruction of or injury tocerebral tissue, particularly that of the hypothalamus, and/or tothe effect of blood entering the cerebral ventricles or subarach-noid spaces. A specific goal, therefore, was to attempt to developanimal models of trauma-induced fevers in which hyperthermia wouldbe elicited by intentional lesioning of the hypotlalamus or by in-troduction of blood into the cerebral ventricles. These modelswould then be used to study the fundamental mechanisms throughwhich the pyrexias were produced, with a particular emphasis onthe possible involvement of arachidonic acid metabolites (e.g.,prostaglandins) and certain CNS neurotransmitters. The followingnarrative summarizes the results obtained from studies supportedby the contract. Details can be obtained from the publications(appended) stemming from these studies.

A. Pyrexia produced by acute mechanical destruction of braintissue (Papers #3 and #7).

Our first accomplishment was to develop a model in the rat ofthe acute pyrexia produced by rapidly developing injury to theanterior hypothalamic/preoptic(AH/Po) region of the brain. In thismodel, an 18 ga stainless steel guide tube was permenently im-planted just-above one side of the AH/PO region, and the tube wasoccluded with a solid stylet the same length as the guide. Toproduce a lesion, the stylet was removed and replaced with one 6 mmlonger than the guide. Insertion of the longer stylet producedinstantaneous destruction of the medial AH/PO region on one side.It was shown in a series of more than 80 rats that such lesioningproduced with nearly 100% reliability a pyrexia which began immed-

. iately, reached its peak magnitude (mean peak magnitude = +2.30c)within 30 to 90 minutes and lasted 8 to 16 hours. The pyrexia wasnot a consequence of convulsive activity, increased motor activityor behavioral excitability. The fact that a unilateral injurycould produce pyrexia suggested to us that at least some types oftrauma-induced fevers did not depend upon a disinhibition of brain-

*stem structures caudal to the lesion which control thermogenesisand/or upon destruction of AH/PO neurons responsible for heat dissi-pation. The hypothalamus is bilaterally redundant, both anatomicallyand functionally. A lesion on one side only is therefore highlyunlikely to result in a disinhibition of heat gain or a loss of theability to dissipate heat.

It was also demonstrated that pretreatment of the rats with theprostaglandin synthesis inhibitor, indomethacin, would reduce themagnitude of the pyrexia in a dose dependent fashion. In fact, adose of 15 mg/kg given 1 hour before lesioning virtually abolishedthe effect (88% reduction in the 6-hour Fever Index). It was alsofound that injection of indomethacin in a rat already made pyrexicby lesioning cuased body temperature to return rapidly to the pre-

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lesioning level. As a result of other work supported by thecontract (see below), we knew that prostaglandins injected intothe AH/PO region of the rat elicit hyperthermia. It was alsoknown that brain tissue can synthessize prostaglandins in responseto injury. We therefore formulated the hypothesis that unilateralAH/PO injury caused pyrexia through the release of prostaglandinsfrom the injured tissue. These prostaglandins were posited toact on surviving AH/PO tissue on the lesioned side and/or by diffusionto the intact AH/PO tissue on the side contralateral to the lesion.

The "disinhibition/loss of function" hypothesis has often beencited in the literature as a speculative explanation of trauma-induced hyperthermia. As indicated above, our data argue againstthe involvement of such a mechanism in at least some trauma-inducedfevers. Another frequently mentioned explanation of trauma-inducedfevers is the "neurogenic" hypothesis. In general terms, this hy-pothesis suggests that neurons in the tissue surrounding the lesionare rendered hyperirritable by non-specific processes such asmechanical displacement, edema or ischemia. This, in turn, wasspeculated to result in persistant activation of neuronal systemscontrolling heat gain, with a resultant rise in core temperature.It is noteworthy that our own hypothesis is not totally incompat-ible with the neurogenic hypothesis. Rather, we have simply sub-stituted specific mediators (prostaglandins) for the non-specificeffects associated with the neurogenic hypothesis. However, thereare significant differences between the two hypotheses in regardto how the pyrexia is effected. Pyrexia produced by intracerebralinjection of prostaglandins is generated and maintained by a coor-dinated modulation of thermogenic and heat retentive effectors,the magnitude of the hyperthermia is not strongly affected byvariations in ambient temperature, and the elevated temperature is

- defended vigorously in the face of thermal stress. Effector coor-dination during a hyperthermia caused by the intractable stimulationof heat gain associated with the neurogenic hypothesis should bepoor, the magnitude of the hyperthermia should be strongly affectedby ambient temperature, and the elevated core temperature shouldnot be well defended. We therefore carried out experiments tocharacterize the regulatory properties of pyrexias produced by uni-lateral mechanical AH/PO lesions in rats. In these studies we(a) observed the thermoregulatory effector activities which were

" responsible for generating the pyrexia, (b) observed the thermoreg-ulatory reactions elicited by forced elevation and depression ofcore temperature during a pyrexic episode and (c) observed theeffect of ambient temperature on pyrexia magnitude. The resultsindicated that the pyrexia produced by unilateral AH/PO puncturein the rat is generated through a coordinated effort of heat gainand heat retentive effectors, that the magnitude of the pyrexia isnot strongly altered by changes in ambient temperature and that theplateau level of hyperthermia is defended vigorously. The resultsthus strongly support our contention that the lesion-induced pyrexiaswere mediated by prostaglandin release and not by a "neurogenic"mechanism involving continuous irritative activation of heat gain.A clinical implication of our findings is that prostaglandin syn-

othesis inhibitors, given in adequate dosage, might well be of bene-fit.'in some cases of trauma-induced hyperthermia in humans.

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B. Pyrexia produced by simulated intraventricular hemorrhage(Paper #6 and Abstracts #10 and # 13).

As indicated earlier, there is reason to believe that somepyrexias associated with cranio-cerebral trauma are dependent uponintraventricular bleeding rather than tissue damage for theirgenesis. Whole blood contains or releases during the clotting pro-cess several potentially pyrogenic substances and some known py-rogens, among them the prostaglandins. Prostaglandins of the "E"type injected intraventricularly or directly into the rostral hypo-

*' thalamus or the preoptic region produce a rapidly developing py-rexia. The latter structures are situated close to the ependymain the walls of the rostroventral aspect of the third cerebralventricle. Thus, the third ventricular region olfers a likelysite of action if, in fact, blood can elicit a v r by acting from

.. within the ventricular spaces.

Our first objective was to develop an anim model of pyrexiaassociated with ventricular bleeding. The sec 2 nbjective was toascertain the approximate location of the reacti- region of the

'QV ventricular system. To accomplish these objectives, we implantedcats with stainless steel guide cannulae with their tips situatedin either the dorsal or ventral aspect of the third ventricle. Aftera 10 day recovery period, each animal was subjected to two experi-ments which were separated by minimum interval of 7 days. The firstconsisted of a control injection of 500 ul of a sterile, pyrogen-free artificial cerebrospinal fluid and the second, a similar in-jection of a fresh, non-anticoagulated sample of the animal's ownvenous blood.-Four hours after the blood injection, the animalswere killed and the distribution of blood in the ventricles deter-mined.

The results showed that inject ons of blood often elicited apyrexic response whereas the control injections did not. Becauseof misplacement of several guide cannulae and because of the de-velopment of scar tissue around some of the guide cannula tips,many of the blood injections distributed in ways other than had beenintended. This was fortunate because it permitted us to determinethat pyrexia developed after blood injection only when the bloodhad reached the rostroventral aspect of the third ventricle. Evenlarge amounts of blood reaching other ventricular spaces failed to

*i produce pyrexia. Thus, the AH/PO region is the likely site ofaction vis-a-vis the pyrogenic effect of intraventricular blood.

In subseqgent work, it was shown that blood which had been in-cubated at 37 C under sterile conditions for periods up to 21 dayswas more pyrogenic than fresh blood. The idea to test aged bloodwas derived from clinical reports which suggested that fevers pro-duced by breakthrough into a ventricle of the reliquified contentsof old encapsulated hematomas were more intense than those produced

* *by bleeding directly into a ventricle. We were also able to showthat the particulate-free supernatant liquid derived by centrifu-gation of aged bloods was pyrogenic and that the pyrogenicity in-creased with incubation time. Supernatants derived from blood aged

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15 days had the maximum pyrogenicity, but those derived fromblood aged 6 days were nearly as active. Thus, some soluble py-rogen accumulates in the fluid portion of a clot as it ages understerile conditions. Thus far, we have not carried out the experi-ments required to identify the pyrogen. However, we have evaluatedthe possible role of prostaglandins or other cyclo-oxygenase pro-ducts. In these experiments, cats were given a systemic injectionof indomethacin 1 hour prior to the intraventricular injection ofsupernatants which were known to be pyrogenic. These cats did not

-. -develop a pyrexia. However, supernatants derived from blood which" had been incubated in the presence of a high concentration of indo-

methacin (25 ug/ml) were pyrogenic when injected in cats which hadnot received a systemic indomethacin injection. As it is known thatindomethacin prevents prostaglandin synthesis but does not preventthe pyrogenic effect of preformed prostaglandins, the results ofthis study indicate that supernatant-induced pyrexia is not due tothe presence of prostaglandins in the supernatant. However, thedata also indicate that prostaglandins are somehow involved in themediation of the pyrexia. Our working hypothesis is that a sub-stance present in the supernatant acts on ependymal cells and/oron periventricular brain tissue to evoke the release of prosta-glandins within the tissue and/or into the cerebrospinal fluid.

The possibility that the pyrogen in the supernatants may beendotoxin has been examined (Rudy, T. A., unpublished), but the re-sults are inconclusive. Limulus Amebocyte Lysate (LAL) assay of

-* supernatants derived from blood aged from 2 hours to 21 days showedthat the supernatant volume that we n(- use routinely to producepyrexia (200 il) contained from 36 to _9 pg of LAL-reactive material.This is a small amount but, as some endotoxins are, strongly pyrogenic,the possibility that endotoxin might be responsible for the pyro-genicity of the supernatants canno-. be dismissed. On the otherhand, because of the large number and diverse nature of substancespresent in the supernatants, there is the possibility that the LAL-

*m reactive material we detected is not endotoxin. Obviously, furtherexperiments need to be done to assess the possible contribution ofendotoxin. It is noteworthy, however, that our aged bloods andsupernatants have been shown to be sterile and that our controls forpreventing contamination of these products with endotoxin are rigid.Also, we have carried out experiments to ascertain at what point theIAL-reactive material enters the incubation system. We are con-

*vinced that, if the LAL-reactive material is endotoxin, that endo-toxin was present in the cat's blood at the time it was drawn. Thatthe blood of healthy animals can routinely contain small amounts ofendotoxin is supported by several literature reports.

The possibility that the pyrogenic material present in bloodand in supernatants derived from incubated blood may be 5-hydroxy-tryptamine or acetylcholine is discussed in later sections of thisreport.

C. Central nervous system site of pyrogenic action of prosta-glandins (Paper #1).

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As has been mentioned in preceding sections, we have hypothe-sized that tome trauma-induced fevers are due to the action ofprostaglandins on neuronal elements within the AH/Po region. Atthe time this contract began (1975), 'it was well established inthe literature that prostaglandins E and E caused hyperthermiawhen injected intraventricularly. Il had aIso been shown thatinjections of these prostaglandins directly into the AH/PO regionproduced a similar effect. However, it had not been demonstratedthat the AH/PO region was the only CNS region where prostaglandinscan act to cause hyperthermia; very few areas other than theAH/PO region had been examined. We therefore carried out a "mapping"study in which small amounts of PGE (50-100 ug in 1 ul) were in-jected into various sites in the rai brain rostral to the medulla.The results are described in detail in Paper #1. In brief, it wasfound, after an examination of 272 sites, that all of the siteswhere PGE1 injection produced a temperature rise were clustered inthe AH/PO region. It was concluded that the AH/PO region is animportant and probably the only supramedullary site of PGE1 actionin the rat brain. This finding forms the basis for our contentionthat prostaglandins released by hypothalamic trauma or by bleedinginto the third ventricle cause hyperthermia by an action within theAH/PO region.

Subsequent to the completion of the study mentioned above,several reports appeared in the literature which strongly suggestedthe existence of an extrahypothalamic site of action for pyrogens/prostaglandins in the production of fever. The most convincingof these was the finding that total bilateral ablation of the AH/POregion in rherus monkeys did not reduce the pyrogenic effect ofsystemically injected pyrogens or intraventricularly injected pros-taglandins. We were also aware from a survey of the literaturethat pontine lesions in humans and in cats commonly resulted inhyperthermia, and it seemed possible that prostaglandins releasedfrom injured pontine tissue might be acting at a lower brain stem(or spinal cord) prostaglandin-responsive site to produce a temper-ature rise. We therefore undertook a study whose goal was to as-certain the location of the extrahypothalamic site of action, if,indeed, such a locus of action exists. We expect that two publica-tions describing this work will be submitted this year.

The CNS region explored in this study ranged from the spinalcord (lumbar level) to the lower midbrain. Although the lower mid-brain had been examined in the study described in Paper #1, thenumber of injection sites was small, and it was decided that amore extensive exploration of this area would be desirable. Thespinal cord response to prostaglandins was examined first. The cordwas considered a viable potential site of action because we hadshown in ancillary experiments (Paper #4) that morphine injected in-to the spinal subarachnoid space could produce a large core temper-ature increase in the rat and that this increase was a regulated one(i.e., functionally equivalent to a true fever). This effect waslikely due to a biasing within the cord of thermal input from skin,deep body or spinal cord thermodetector units such that a false"cold" signal was transmitted to supraspinal structures involved in

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thermoregulation. We reasoned that it was possible that othersubstances, e.g., prostaglandins, might alter thermoregulationthrough a direct action -n the cord. However, extensive studiesin which a wide range of doses of PGE1 and PGE 2 were injected viaan indwelling spinal catheter into the lumbar, thoracic and cer-vical subarachnoid spaces revealed no consistent hyperthermiceffect. The only consistent, dose-related effect produced by theseprostaglandins was a core temperature fall elicited by lumbarlevel injections.

We next examined the reactivity of sites in the rat brainsituated between the spinomedullary junction and frontal planeP-2 in the atlas of Pelligrino et al. We have thus far tested theability of 100 ng of PGE injected into 445 different sites in theposterior aspect of the iat brain to elicit a temperature rise.The vast majority of these sites were unreactive. However, injec-tions into about 30 sites evoked a pyrexic response. Most of thesereactive sites are clustered within two reasonably distinct regionswhich we prefer not to identify precisely until we publish the datalater this year. We have focussed our attention on that regionwhich responds most consistently. PGE1 injections into this areaproduce a hyperthermic effect which is about half as large asthat produced by injection of an equal amount of PGE into the AH/POregion. The response begins immediately after injection and hasthe time course of an AH/PO injection. The response is reproduciblewithin a given rat, both within a session and between sessions.Saline injections had no effect on body temperature. There is noindication the temperature rises are due to convulsions, enhancedmotor activity or behavioral excitability.

The major impediment to pronouncing the above mentioned locusan authentic extrahypothalamic site of prostaglandin action isthat it borders on a subarachnoid space. Injected PGE might thusdiffuse via the CSF to the AH/PO region and act there to effect apyrexic response. Evidence against this possibility gained thusfar is (a) the injection volume is small and the response beginsimmediately, (b) injections at numerous other loci bordering onsubarachnoid spaces or on ventricular spaces have not produced py-rexia, (c) injections of 100 ng of PGE1 in 1 ul directly into thefourth ventricle, aquaduct or the cisterna magna do not produce py-rexia, (d) injections of 1 ul of a very concentrated dye solutioninto the putative reactive region give no indication of diffusionto the AH/PO region. However, to prove beyond doubt that transportto the AH/PO region is not responsible for the effect, we feel itis necessary to show that injections into the putative reactive lo-cus are effective in rats in which the AH/PO region has been de-stroyed bilaterally. We have developed a method for producing sucha lesion reproducibly and have shown that lesioned rats are unre-sponsive to PGE injected into the third ventricle or into the sub-arachnoid space-beneath the lesioned area. At this writing, we arepreparing to test injections of PGE1 into the putative reactiveregion in rats with AH/PO lesions.

D. Evaluation of the pyrogenic effect of leukotrienes C4 , D4and E4 (Paper #14).

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13

These experiments dealt with the possibility that arachidonicacid metabolites other than prostaglandins might be pyrogenic.The metabolites of interest to us are leukotrienes C4 , D and EThese are recently identified arachidonic acid products ahich aiisevia the lipoxygenase pathway rather than the more familiar cyclo-oxygenase pathway. They exhibit numerous biological activitiesand, like the prostaglandins, are very likely involved in inflamma-

* -" tion. None of the lipoxygenase products have been examined for py-rogenic activity, perhaps in part because traditional antipyreticshave been thought to be specific inhibitors of the cyclo-oxygenaseenzyme. However, recent reports that aspirin and indomethacin caninhibit some lipoxygenases suggest that antipyretics may not be asselective for the cyclo-oxygenase pathway as had been believed. Thus,a direct action of lipoxygenase products in fever production is apossibility. Furthermore, some lipoxygenase products can enhancethe generation of prostaglandins and other cyclo-oxygenase productsand might thus be involved in fever generation through this indirectmechanism. In view of this information, it seemed that an examin-ation of lipoxygenase products for pyrogenic activity would beworthwhile.

The details of the experiments are presented in Paper #14. Insummary, we injected 1 ug doses of leukotriene C , D and E intothe third cerebral ventricle of rats which had bien thown tA de-velop pyrexia following similar injections of 1 ug of PGE1. Wealso examined the effect of bilateral injections of 400 ng of eachleukotriene into the AH/PO region in rats in which bilateral injec-tions of 40 ng of PGE1 had been shown to produce pyrexia. Noneof the leukotriene in~ections produced a significant increase inbody temperature. Dye studies demonstrated that the injectioncannulae had been placed correctly. At the completion of the study,the leukotriene samples used were assayed for biological activityusing the guinea pig tracheal strip method. It was found that thesamples were still highly active. We conclude that the leukotri-enes tested are not likely to be involved in the production of feverby cerebral trauma or, for that matter, in any other type of fever.

E. Involvement of supraspinal and spinal neurotransmittersin thermoregulation and fever.

(1) Supraspinal neurotransmitters (Papers #2 and #5).

An oft cited model of thermoregulation devised by R. D.Myers indicates that serotonergic synapses in the rostral hypothal-amus are involved in the mediation of the thermogenic and heat re-tentive activity initiated by pyrogens. These conclusions were basedon microinjection studies in monkey and cat in which 5-HT injectedinto the AH/PO region caused very long lasting hyperthermias. Theselong lasting effects were, we felt, incompatible with the knownrapid disposition of 5-HT in brain tissue. We also noted that someworkers had reported that intraventricular injections of 5-HT in

.-. * the cat produced a fall rather than a rise in body temperature.Further, no one had demonstrated that the 5-HT-induced temperaturerise reported by Myers was mediated by 5-HT acting on specific 5-HT

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14

receptors. We therefore carried out a study in cats in which 5-HTwas injected in various doses into numerous sites within theAH/PO region. The responses were noted, and the experiments werethen repeated after systemic injection of indomethacin or aftersystemic administration or injection into the 5-HT microinjectionsites of the 5-HT antagonist, methysergide. The results showedthat (a) 5-HT injections into the anterior hypothalamus producedshort lasting initial temperature rises which were often followedby prolonged secondary hyperthermias, (b) injections of 5-HT intopreoptic sites produced short lasting temperature falls which wereoften followed by prolonged hyperthermias, (c) both the initialrise and the initial fall produced by 5-HT could be prevented bysystemically injected methysergide or by merthysergide injectedinto the 5-HT microinjection site, (d) all of the delayed, pro-longed hyperthermias were blocked by indomethacin pretreatment,whereas the initial, short lasting temperature changes were un-affected. These findings indicate that the initial, short lastingfalls and rises were produced by 5-HT acting on specific 5-HT re-ceptors and thus presumably reflect a neurotransmitter action of5-HT in the AH/PO. The delayed and long lasting rises, however,were probably due to prostaglandin release. The data support Myers'suggestion that 5-HT synapses in the anterior hypothalamus couldbe involved in the mediation of pyrogen induced heat gain and heatretention. However, they also indicate that the 5-HT synapses inthe preoptic region could well be involved in heat dissipationrather than in heat gain. It might seem reasonable to suggest that5-HT released from brain tissue as a consequence of cerebral traumaor 5-HT released from platelets in cases of intraventricular bleed-ing could act at the anterior hypothalamic synapses involved inheat gain to produce pyrexia. However, our earlier mentioned workshows that indomethacin blocks pyrexias produced by unilateralAH/PO trauma or by intraventricular injection of blood. It is thusunlikely that an action of 5-HT on specific 5-HT receptors is in-volved in the mediation of these pyrexias. It remains possible,however, that 5-HT released by tissue damage or ventricular bleedingcause3 brain tissue to release prostaglandins which would, in turn,produce pyrexia. Thus, an involvement of 5-HT in trauma-inducedfevers cannot be completely discounted.

The previously mentioned model of thermoregulation adduced byR. D. Myers indicates that, in addition to 5-HT synapses, there arecholinergic synapses in the AH/PO region which are involved in theactivation of heat gain effectors. This aspect of the model wasbased on microinjection studies in the monkey in which cholinergicagonists were injected into the AK/PO region and were found to pro-duce a temperature rise. We carried out similar studies using thecholinergic agonist, carbamylcholine (CCh) and were able to repro-duce Myers' results(i.e., core temperature increased). However,we found that the rise was not due to unregulated thermogenesis.Instead, CCh evoked a well regulated temperature increase that wasfunctionally equivalent to a true fever. Thus, cholinergic synapsesin the AH/PO region may not be involved exclusively in heat gain.Rather, these synapses seem to be part of the integration systemresponsible for generation of the setpoint for thermoregulation.

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15

Of more specific relevance to this contract is our additionalfinding that the hyperthermia produced by CCh was completely pre-vented by systemic injection of the muscarinic cholinergic antag-onist, atropine, whereas sodium salicylate (900 mg p.o.) or indo-methacin (10 mg/kg i.m.) had no effect on the pyrexia. This in-dicates that the CCh effect was caused by an action on AH/PO mus-carinic receptors and that prostaglandin release was not at allinvolved in the response. Therefore, since indomethacin blockstrauma-induced fevers in both of our experimental models, it doesnot seem likely that acetylcholine released from injured tissueor by some substance present in intraventricular blood can beimportantly involved in the pyrexias associated with these models.

(2) Spinal neurotransmitters (Papers #11, #12 and #13).

The final series of experiments supported by the contractwas designed to evaluated the roles of spinal neurotrasmittersin thermoregulation, with the notion that their involvement infever production would be studied later. The contract was termin-ated before these latter studies could be carri ed out. At the timethese studies were undertaken, the role of srinal neurotransmittersin temperature regulation was totally unexplored. It was thoughtthat these neurotransmitters must be of importance because thecord is involved in the upward transfer of sensory information fromperipheral and deep body thermodetectors and the downward transferof impulses controlling the level of activity of thermoregulatoryeffectors. In addition, the cord itself possesses a population ofthermodetect6r units whose thermoregulatory "potency' is equivalentto that of the thermodetectors in the AH/PO region. The activityof these spinal detectors could well be modulated by serotonergicand/or noradrenergic fibers descending from the brainstem or bycholinergic units intrinsic to the cord.

The reader is referred to the two major publication resultingfrom these studies (Papers #12 and #13) for details of the methodsand results. In brief overview, it was found that injection ofnorepinephrine (NE), 5-HT and CCh into the lumbar spinal subarach-noid space of rats produced dose dependent thermoregulatory effects.NE produced a transient rise followed by a prolonged fall, 5-HTand CCh produced hyperthermia only. Studies relating to the siteand mechanism of action of NE in producing the biphasic core temp-erature change indicated that the transient rise was probably dueto leakage of NE from the subarachnoid space into the general circ-ulation, with subsequent stimulation of heat production by activa-tion of peripheral non-shivering thermogenic mechanisms. The pro-longed temperature fall produced by NE was shown to be a directspinal effect of the drug. Further studies revealed that the fallwas probably due to an inhibitory effect of NE on sympathetic pre-ganglionic neurons located in the intermediolateral cell columnof the cord, with a resultant inhibition of peripheral vasoconstric-tion and non-shivering thermogenesis. The hyperthermic effectsproduced by 5-HT and CCh were also demonstrated to be consequencesof direct actions on the spinal cord, but studies of the sites of

.* action and the mechanisms of action have not be carried out.

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