The effects of various methods of sacrifice and of ethanol on adenosine levels in selected areas of rat brain

Journal of Neuroscience Method~, 25 (1988) 243-249 243 Elsevier

NSM 00853

The effects of various methods of sacrifice and of ethanol on adenosine levels in selected areas of rat brain

Mike Clark and M. Saeed D a r

Department of Pharmacology, East Carolina University, School of Medicine, Greentqlle, NC 27834 ( U. S, A.)

(Received 11 November 1987) (Revised 22 February 1988)

(Accepted 1l April 1988)

Key words: Adenosine; Brain area; High performance liquid chromatography; Ethanol; Focused microwave; Hypoxia

The effect of acute ethanol on adenosine content in four motor areas of the male Sprague-Dawley rat brain was investigated using HPLC-fluorescence detection. Since basal adenosine levels are difficult to assess due to extremely rapid turnover of adenosine, four different methods of sacrifice were also evaluated for adenosine measurement. The rank order for best results in measuring adenosine content with the various methods of sacrifice was: focused microwave irradiation > decapitation into liquid nitrogen > immersion into liquid nitrogen > decapitation. These differences probably reflect differences in degree of hypoxia and postmortem anoxia, factors well known to elevate adenosine, associated with the sacrifice method. Focused microwave irradiation of appropriate duration was found to be the best method of sacrifice and the results probably most closely reflect true basal adenosine levels. No significant alteration in adenosine content in any brain region examined was observed due to ethanol administration.

Introduction

Over the last decade, much data have been collected which strongly suggest a neuromodula- tory role for adenosine in the CNS (Phillis and Wu, 1981; Dunwiddie, 1985; Marangos and Boulenger, 1985). Adenosine is believed to exert its actions via receptors located on the extracellu- lar surface of neuronal membranes (Marangos and Boulenger, 1985). Two subtypes of cell surface receptors are thought to produce the physiological responses to exogenously administered adenosine and adenosine agonists. The A1 subtype is inhibi- tory to adenylate cyclase and has nanomolar affinity, whereas the A 2 subtype is stimulatory to the cyclase and has micromolar affinity for adenosine.

Correspondence: M.S. Dar, Department of Pharmacology, East Carolina University, School of Medicine, Greenville, NC 27834, U.S.A.

Both the A 1 and A 2 subtypes are competitively antagonized by methylxanthines such as theophyl- line and caffeine. Indeed, it is becoming well accepted that these methylxanthines exert their CNS excitation by blocking the action of endoge- nous adenosine at its cell surface receptors (Phillis and Wu, 1981; Stone, 1981). Many of the behavioral actions of adenosine bear striking resem- blance to those of ethanol, e.g. sedation, ataxia, decreased spontaneous motor activity and a bi- phasicity of some behavioral effects. Additionally, both adenosine and ethanol share anticonvulsant, hypothermic and analgesic properties.

We have been interested in studying the in- volvement of central adenosine in the CNS effects of ethanol. A possible role for adenosine in the CNS effects of ethanol was first suggested by Dar et al. (1983). Since then, other laboratories have confirmed and extended their findings (Proctor and Dunwiddie, 1984; Gordon et al., 1986). Fol- lowing recent, additional evidence for a role of

0165-0270//88/$03.50 <':" 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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brain adenosine in the mediation of motor dis- turbing effects of ethanol (Dar and Wooles, 1986; Clark and Dar, 1988), we decided to further in- vestigate the possible mechanism(s) of adenosine mediation. One possible mechanism could be an ethanol-induced increase in the levels of adenosine with a resultant motor impairment. The present study, therefore, deals with the measurement of adenosine levels and the effect of ethanol on it in specific brain areas (motor cortex, striatum, brain stem, and cerebellar cortex), which are believed to be involved in normal motor coordination and motor activity. Additionally, having encountered difficulties in measuring true basal adenosine levels, a problem reported as well by others (Winn et al., 1979; Wojcik and Neff, 1982), we examined the effect of four methods of sacrifice on adenosine levels in these brain areas of rats treated with either saline or ethanol.

Materials and Methods

Male Sprague-Dawley rats (170-210 g; Charles River, Research Triangle Park, NC) were used in all experiments. The animals were housed 3 per cage on a 12-h l ight/12-h dark, day-night cycle with free access to food and water. To eliminate any possible diurnal variation in ethanol metabolism or in the levels of adenosine, all rats were treated with ethanol /sal ine and killed between 07.00 h and 12.00 h.

Drug treatments To, keep the drug treatment regimen in our

laboratory uniform with concurrent behavioral studies, the rats received a saline pretreatment (0.5 ml /100 g b. wt.) followed 10 min later by a saline or ethanol treatment (1.0 ml /100 g b. wt.). All administrations were by i.p. injection. Ethanol solution was prepared in saline. The treatment groups used were: (1) sa l ine+ saline, and (2) saline + ethanol (1.5 g/kg) .

Methods of sacrifice Fifteen minutes after the second injection, rats

were killed by one of four methods. This experi- mental design in which animals were sacrificed 15'

min post a single ethanol injection was consistent with our behavioral study reported elsewhere (Clark and Dar, 1988) as well as with our objec- tive to look for a possible functional relationship between ethanol-induced motor incoordination and any change in adenosine levels in specific brain areas. The dose of ethanol used was the same as used in the behavioral studies which produced a marked motor incoordination 15 min post ethanol injection.

(l) Decapitation into liquid nitrogen. The animal 's head was allowed to drop directly into liquid nitrogen below the guillotine and removed from it after temperature equilibration. The head was then thawed at 4 ° C to remove the brain which was immediately dissected and the tissue samples prepared from each of the four brain areas.

(2) Immersion into liquid nitrogen. The rat was placed into a mesh wire restrainer and then im- mersed head-first into liquid nitrogen until temperature equilibration. The animal was thawed at 4 °C until the brain could be removed. The tissues were processed immediately after the brain re- moval and dissection of areas.

(3) Focused microwave irradiation. The rats were killed by focused microwave irradiation (6.5 s, 1.33 kW, 2450 MHz; General Medical Engineer- ing Co., Litton System 70/50, model BN-K2, Peabody, MA). The brain was rapidly removed, frozen on sifted dry ice and subsequently dissected for brain areas and tissue sample preparation.

(4) Decapitation. The rats were killed by decapitation. The brain was rapidly removed and dissected on a glass plate chilled with ice. The tissue samples were immediately prepared.

At least 4 animals from each treatment group were killed by each method.

Tissue sample preparation All brains were dissected on a glass plate chilled

with ice. The cerebellar cortex, striatum, motor cortex and brain stem were removed as quickly as possible according to the scheme outlined by Glowinski and Iversen (1966) and placed in centrifuge tubes containing ice-cold 0.1 M perchloric acid. The motor cortex was a cerebral cortical area found to be rich in pyramidal cells

(via separate histological studies; data not shown) and consisted of a section approximately 5 m m wide above the anterior-most hippocampus and posterior-most striatum. The tubes containing the tissues were kept in ice and tissues were homoge- nized (Polytron, setting 7, 12 s) in approximately 10 vols. of 0.1 M perchloric acid. Each sample was mixed and centrifuged (8000 g) for 10 min. The supernatants were stored at - 7 0 °C until assayed (within 5 days). The resultant pellet was resus- pended in distilled water (same volume as for homogenization) and used for protein determination (Lowry et al., 1951).

Adenosine measurement

A modification of the assay by HPLC-fluorescence detection reported by Wojcik and Neff (1982) was used for adenosine measurement. The perchlorate supernatant was diluted 1 :100 with distilled water and 1 ml of each sample was placed in a 1.5 ml conical vial with 20 /~1 of chloroacetaldehyde (ICN Biomedicals, Inc., Plainview, NY) subsequently added for precolumn derivatization and to give a final concentration of 135 mM (optimal amount found). The vials were capped, vortexed and placed in boiling water for 30 rain to complete the conversion of adenosine to the fluorescent compound 1,N6-ethenoadenosine. The HPLC system consisted of a 5 #m reverse-phase column (Cts/~Bondapak, 4 m m x 15 cm, Waters Associates, Milford, MA) and an au- tomated injector (WISP 710B, Waters Associates). The column was connected to a fluorescence de- tector (McPherson Model FL-749, Acton, MA) with High Sensitivity Attachment and excitation monochromator set at 270 nm (uncorrected) and a seconda~ filter with a cut-off below 360 nm. The mobile phase consisted of 0.05 M sodium acetate buffer (pH 5.0) to which was added sodium oc- tylsulfonate 1 mM as an ion-pairing reagent (Pfaltz and Bauer, Waterbury, CT) and 3% acetonitrile. Isocratic separation of adenosine was accom- plished at ambient temperature (22-24 ° C) and at a flow rate of 1.5 ml /min . The ion-pairing reagent hastened the elution of anionic compounds such as cAMP. ATP, but delayed the elution of cationic fluorescent ethenoadenosine derivative, thereby markedly improving the resolution of the latter.

245

A B

i )l"l "

"6

_=

c D

i I I i ,i

Fig. 1. Chromatograms of derivatized adenosine. A: adenosine standard. B: cerebellar cortical tissue sample. C: adenosine standard treated with adenosine deaminase (cf. A). D: cerebellar cortical tissue sample treated with adenosine deaminase (cf. B). Retention time for the adenosine peak was about 13 min.

The peak for adenosine (1,N6-ethenoadenosine) in tissue samples was identified by comparison with the peak due to standard 1,N6-ethenoadeno - sine (Sigma, St. Louis, MO; data not shown) as well as with the peak due to ethenoadenosine converted from the standard adenosine (Sigma, St, Louis, MO; Fig. 1A). In addition, further con- firmation of the peak identity was carried out by the absence of adenosine peak (Fig. 1C, D) after the destruction of adenosine (standard as well as in tissue samples) by incubation with adenosine deaminase (EC 3.5.4.4 Sigma, St. Louis, MO Cat No. A-1030), pH 7.5 at room temperature for 30 min prior to derivatization with chloroacetaldehyde. The concentration of adenosine in the tissue samples was calculated by external standard quan- titation method using Data Module (Model 720B; Waters Associates). The derivatized adenosine (not ethenoadenosine) was used as the standard. The adenosine values were expressed as n m o l / m g tissue protein. The yield of adenosine derivatization reaction (average 90%) was calculated by carrying out a series of separate reactions in which increas-

246

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/ / /

j / •

J , , / /

/ J

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~ f ' J I I J t t I I I ~o 40 6o 80 1~ 1~,0 14o leo 18o 21~

Fig. 2. Correlation between peak height and standard adenosine concentration (pmol).

Statistics Adenosine levels in each brain area were com-

pared between treatment groups and between methods of sacrifice by two-way analysis of vari- ance (ANOVA, Crunch version 3; Crunch Soft- ware Corp., Oakland, CA) with Newman-Keuls post hoc comparisons (Crunch) for differences in methods of sacrifices. One-way analysis of vari- ance (Crunch) was applied to test each method of sacrifice for differences between treatment groups if the two-way ANOVA implied an interaction. Differences in adenosine content were considered statistically significant at P < 0.05.

ing amounts of chloroacetaldehyde were used and by comparing the peak heights due to equimolar concentrations of commercial ethenoadenosine and the adenosine reaction product. The yield of adenosine derivatization reaction was measured in aqueous medium (0.001 M HC104). We also determined 80 fmol as the lowest detection limit for adenosine and observed linearity between the de- tector response (peak height) and the adenosine concentration (from 0.1 to 200 pmol; Fig, 2). The concentrations of tissue adenosine we determined were within this linear range.

Results

Table I shows the concentration of adenosine in four specific brain areas i.e. motor cortex, striatum, brain stem and cerebellar cortex, in each treatment group as well as in each method of sacrifice. Since adenosine levels are extremely sensitive to the degree of hypoxia and postmortem anoxia, the method and the duration of sacrifice of the animals as well as the subsequent harvesting of brain and the processing of the brain tissues are

TABLE I

EFFECTS OF VARIOUS METHODS OF SACRIFICE AND OF ETHANOL ON ADENOSINE LEVELS IN SELECTED BRAIN AREAS OF THE RAT

Brain area Treatment Adenosine (nmol /mg protein)

Decapitation into Immersion into Focussed microwave a Decapitation liquid nitrogen liquid nitrogen irradiation

Cerebellar Saline + saline 5.96 _+ 0.67 b (8) 8.16 ± 0.57 (13) 0.408 + 0.068 (4) 11.61 _+ 1.11 (6) cortex Saline + ethanol c 5.18 + 0.44 (6) 10.69 ± 1.02 * (8) 0.307 + 0.028 (10) 14.11 ± 0.50 (6)

Striatum Saline + saline 5.70 ± 0.39 (9) 6.72 ± 0.78 (13) 0.464 +_ 0.096 (5) 14.16 ± 1.19 (6) Saline + ethanol 4.83 + 0.41 (7) 6.63 ± 0.69 (8) 0.263 + 0.033 (7) 15.86 + 0.85 (5)

Motor Saline + saline 4.05 + 0.29 (9) 3.76 ± 0.24 (12) 0.231 ± 0.037 (6) 14.67 ± 1.74 (4) cortex Saline + ethanol 3.59 ± 0.26 (7) 5.45 ± 0.57 ** (8) 0.227 +_ 0.026 (8) 13.46 ± 0~58 (6)

Brain Saline + saline 3.46 _+ 0.46 (7) 5.38 ± 0.44 (13) 0.211 ± 0.035 4 5.76 ± 0.08 (4) stem Saline + ethanol 4.29 ± 0.20 (5) 6.70 ± 1.17 (8) 0.214 ± 0.029 (10) 8.38 ± 0.97 * (51)

a 1.33 kW, 6.5 s. b Values are expressed as mean + S.E.M. Numbers in parentheses represent the number of rats used.

Ethanol = 1.5 g/kg, i.p. given 10 ruin after saline and animals sacrificed by one of the four methods 15 rain post ethanol. * P < 0.05 compared to saline + saline group.

* * P < 0.05 compared to saline + saline group.

very important variables. We observed lowest values for adenosine in all brain areas of saline + saline-treated animals killed by the focused microwave irradiation method compared to the other three methods. Decapitation was found to be least satisfactory because of relatively very high adenosine levels observed compared to the other three methods. Post hoc comparisons show P < 0.01 in all brain areas for decapitation vs the other three methods of sacrifice with the single exception that no significant difference was observed for decapitation vs. immersion into liquid nitrogen in the brain stem. The effect of ethanol on adenosine levels was not significant in any of the four areas of animals killed either by decapitation into liquid nitrogen method or by focused microwave irradiation (Table I). There was, however, an ethanol-induced increase in the levels of adenosine in the cerebellar cortex (df = 1, F = 5.54, P < 0.05) and the motor cortex (dr = 1, F = 9.55, P < 0.01) in animals killed by immersion into liquid nitrogen and in the brainstem (df = 1, F = 5.70, P < 0.05) of decapitated animals (Table I).

Discussion

The present study confirmed earlier reports (Winn et al., 1979; Wojcik and Neff, 1982) that brain adenosine levels vary with the method of animal sacrifice and the tissue collection. The lowest values were associated with those methods of sacrifice which result in fast arrest of enzymatic activity and least autolytic changes postmortem. Adenosine levels in the rat striatum reported by Wojcik and Neff (1982) were lower (0.1 nmol /mg protein) compared to ours (0.464 nmo l /mg protein) by focused microwave irradiation (Table I). Recently, Yanik and Radulovacki (1987) reported striatal adenosine levels of 1.4 nmol /mg protein in rats killed by focused microwave irradiation, which are 14 times higher compared to those reported by Wojcik and Neff (1982) and about 3 times higher than ours (Table I). Still, Criswell et al. (1986) reported striatal adenosine levels of about 18 nmol /mg protein in rats killed also by focused microwave irradiation. The best method

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of sacrifice, based on our data, was the focused microwave irradiation which resulted in 0.464 _+ 0.068 n m o l / m g protein of striatal adenosine (Ta- ble I). Thus our striatal adenosine values were markedly lower than those reported by Criswell et al. (1986) and by Yanik and Radulovacki (1987), but were higher than those reported by Wojcik and Neff (1982). Although focused microwave irradiation was the method of killing in the studies cited above, the actual irradiation conditions, however, were different: 3.5 kW, 2.5 s (Criswell et al., 1986); 1.5 kW, 6 s (Yanik and Radulovacki, 1987); 6.0 kW, 2.5 s (Wojcik and Neff, 1982) compared to 1.33 kW, 6.5 s used by us.

The lower levels of tissue adenosine reported by Wojcik and Neff (1982) could be due obviously to the 6 kW output microwave unit used by these investigators which was highest among the above cited studies. However, none of the above investigators have stated in their reports the percent conversion of adenosine to ethenoadenosine: and therefore, the extent of adenosine derivatization and the adequacy of chloroacetaldehyde is not known. Lower tissue adenosine values reported by Wojcik and Neff (1982), using the decapitation method as compared to ours by the same method, may reflect an inadequate derivatization of tissue adenosine. The proportionate increase of striatal adenosine (decapitation vs focused microwave) reported by these investigators (46 times) was simi- lar to ours (31 times). However, the proportionate increase in the pons-medulla (brain stem) was 2 vs 27 times in ours and in the cerebellum (cerebellar cortex) 2.5 vs 28 times in our case respectively, indicating perhaps the marked difference in the amount of chloroacetaldehyde used. The higher adenosine values reported by Yanik and Radulo- vacki (1987) compared to ours under nearly iden- tical irradiation conditions could be explained perhaps due to incomplete tissue fixation. Based on our pilot data (not shown), in brains which still looked pink after irradiation, the tissues were not completely fixed and in fact gave adenosine values as high as those reported by these investigators. We, however, subsequently assayed only those brains which looked cooked (light brown) after irradiation. The higher striatal values (18 nmol /mg protein) by Criswell et al. (1986) probably indicate

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an inadequate exposure time since a shorter exposure time (1.33 kW, 2.5 s; data not shown) gave 15 nmol /mg protein of striatal adenosine.

The differences in adenosine levels that we report may be due to differences in the hypoxic conditions induced by various killing methods. It is well accepted that brain adenosine levels are increased quite rapidly during cerebral hypoxia and hypercapnia postmortem (Nordstrom et al., 1977; Winn et al., 1979). Wojcik and Neff (1982) also suggested that hypercapnia rapidly increased brain adenosine concentrations. However, these authors did not check for the possible fall in p O 2 in the container used to expose rats to CO2. Therefore, the increased adenosine content they reported may have been due to hypoxia as well as hypercapnia.

The adenosine levels (Table I) using focused microwave irradiation may or may not represent true basal levels because probably no ideal method for such measurement of adenosine in specific brain areas is presently available. Nevertheless, irradiation at a higher kW (Wojcik and Neff, 1982), appeared to be the best method of arresting enzymatic activity and to minimize autolytic changes in adenosine levels post mortem. The values obtained as such may thus be reasonably close to the true basal adenosine levels or at least may provide information regarding the trends in the basal adenosine levels under different experi- mental conditions.

The results of the present study indicate that ethanol did not change basal adenosine levels in the specific brain areas. This is in agreement with the findings of Volicer et al. (1977). These investigators studied the effect of acute ethanol on the cAMP system in the rat brain and concluded that the effects of ethanol on the cAMP system were most likely due to an alteration of adenosine metabolism or on neurotransmitter release. How- ever, they did observe ethanol-induced decreased adenosine levels with time after decapitation, i.e. during hypoxia as compared to saline controls. Although we found no change in adenosine content in ethanol vs. control rats, this would not necessarily rule out a role for adenosine in ethanol's CNS functions. The determination of adenosine levels alone would not reflect the dy-

namic biochemical processes such as synthesis a n d / o r metabolism or even release a n d / o r up- take. Therefore, it is important that the levels of the major metabolites of adenosine be measured to have some insight into the synthesis, metabolism, and indirectly about adenosine's turnover in the presence or absence of ethanol. The observed increase in adenosine levels due to ethanol treatment of animals killed by immersion into liquid nitrogen and decapitation methods was less con- vincing since these were the least sensitive methods and gave the highest values. Even if these changes in adenosine levels due to ethanol in selective areas of brain are true, it is not unusual and is well known for ethanol to effect differently the different areas of the brain (Bacopoutos et al., 1978; Kalant and Woo, 1981). It is also known that there are regional variations in adenosine release that may reflect a differential distribution of neurons (or perhaps glia) specifically involved in adenosine release (Potter and White, 1980; Wojcik and Neff, 1982).

Acknowledgement

This work was supported in part by North Carolina Alcoholism Research Authority Grant 8406. The authors thank Mrs. Linda Pope for her expert word processing in the preparation of this manuscript.

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Documents

The effects of various methods of sacrifice and of ethanol on adenosine levels in selected areas of rat brain