480

Brain Research, 119 (1977) 480-486 ~.~ Elsevier/North-Holland Biomedical Piess, Amsterdam - Printed in The Netherlands

Effect of repeated cerebral ischemia on metabolitee and metabolic rate in gerbil cortex

BOGOMIR B. MR~ULJA*, W. DAVID LUST, BRANISLAVA J. MR~ULJA* and JANET V. PASSONNEAU Laboratory of Neurochemistry, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.)

(Accepted October 4th, 1976)

Multiple or repetitive ischemic episodes are characteristic of certain pathological conditions affecting the brain circulation. The question of the effects of repeated circulatory occlusion can be readily studied in the gerbil (Meriones unguiculatus). The ligation of the common carotid artery results in cerebral ischemia in 30--40 ~o of the animals due to anomalies of the circle of Willis 7. Release of the occluded artery permits circulation to be restored and, when desired, the procedure may be repeated. Our previous investigations on these animals have demonstrated that following release of a single common carotid occlusion, a range of biochemical changes continues to occur long after the reestablishment of circulation lz. These observations suggested the possibility that during recirculation the brain tissue might respond differently to a second period of ischemia. The present study has demonstrated that the biochemical reactivity of the brain after an initial 1 h ischemic episode was altered when the ischemic insult was repeated. After 1 h of recovery, a second period of ischemia introduced much greater changes than those observed after a similar single insult; conversely, after 5 and 20 h, and 1 or 2 weeks of recovery, a second period of ischemia evoked a diminished response. Mongolian gerbils, weighing 50-60 g, were purchased from Tumblebrook Farm, West Brookfield, Mass. The animals were lightly anesthetized with sodium pentobarbital (20 mg/kg, i.p.) and unilateral ischemia was produced by occluding the left common carotid artery with an aneurysm clip. To assess the effects of the inital ischemic insult, animals were frozen in liquid nitrogen following 5 or 60 min of ischemia. Only animals which exhibited positive neurological symptoms 6 of ischemia after 60 min of occlusion were used in the studies on repetitive ischemia. (It was difficult to assess symptoms without ambiguity after 5 min of ischemia.) At 1, 5 and 20 h, and 1 and 2 weeks after an initial 60 min of occlusion, some animals were frozen to study the recovery status; other groups were made ischemic for a second period of either 5 or 60 min and then frozen. * Present address: Institute of Biochemistry, School of Medicine, Belgrade, Yugoslavia.

481 The outer 2-3 mm of frozen cerebral cortex was removed in a cryostat at --20 °C, and the tissue extracted as previously described 12. The metabolites and nucleotides were measured according to the following methods: ATP, P-creatine and glucose; Lowry and PassonneauS; glycogen; Passonneau and Lauderdalela; cyclic AMP; Lust et al.lX; GABA; Passonneau et al.t4; protein; Lowry et al. 1°. The metabolic rate was estimated by the changes in energy reserves between brains frozen in situ, and brains frozen 30 sec after decapitation• The utilization of high energy phosphate (~ P) has been calculated as the sum of A glucose d- A PCr ÷ 1.4 A ATP -F 2.9 A glycogen 4. The metabolic rates measured in the contralateral cortex served as controls.

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Fig. 1. The concentrations of ATP and P-creatine in the gerbil cerebral cortex after single and repeated periods of ischemia and varying periods of recovery. A: single ischemia. C, control. 5 and 60 indicate period (rain) of ischemia. There were 20-23 animals in each group. B: 1 h recovery; R, recovery. Animals had been ischemic for 1 h, the occlusion removed for 1 h, and the ligation repeated for 5 or 60 rain. Number of animals in each group: R, 4; 5 rain, 6-8; 60 min, 6-10. C: as in B except the recovery period was 5 h. Number of animals in each group; R, 4; 5 rain, 7-8; 60 min, 8-12. D : as in B except the recovery period was 20 h. Number of animals in each group: R, 4-9; 5 rain, 6-8; 60 min, 8-12. E: as in B except the recovery period was 1 week. Number of animals in each group: R, 5; 5 min, 6; 60 rain, 5-6. F: as in B except the recovery period was 2 weeks. Number of animals in each group: R, 5; 5 rain, 6; 60 min, 5-6. Values are given as the mean d= S.E.M.

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After 5 min of unilateral ischemia, there were no significant changes in any of the metabolites measured (Figs. 1A, 2A, 3A and 4A). Following 60 rain of ischemia, concentrations of ATP and P-creatine were markedly decreased. Glucose and glycogen also decreased and there was a concomitant increase in lactate. GABA concentrations were increased 3-fold, and cyclic AMP 5-fold (Figs. 3A and 4A). Lactate concentrations remained high after 60 min of ischemia, returning to normal only after 20 h of recirculation (Fig. 2D). Glycogen levels were near normal values after 1-20 h of recirculation and were 2-fold greater than control values after 1 week. Two weeks after the ischemic period, glycogen concentrations returned to normal values (Fig. 2B-F). The concentrations of glucose in the brains were somewhat decreased after 5 and 20 h of recirculation. GABA and cyclic AMP concentrations were normal after all periods of recirculation (Figs. 3B-F and 4B-F). In contrast to a single unilateral ligature of 5 min duration, a second 5 rain episode (imposed after an initial 60 min of ischemia and 60 rain of recirculation) significantly altered the levels of metabolites. It is of interest that the magnitude of the effect was the same whether the second insult was 5 or 60 min duration. The levels of

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ATP and P-creatine were reduced to 4.5 i 0.8 and 7.2 -4- 1.1 nmoles/mg protein and 3.7 ~: 0.6 and 8.1 :/: 1.2 nmoles/mg protein after 5 and 60 min of repeated ischemia, respectively (Fig. 1B). Similarly, the reductions in glucose and glycogen concentrations and increases in lactate were comparable whether the second ischemic period was 5 or 60 min (Fig. 2B). The levels of cyclic AMP were also similar after both periods of a repeated insult (Fig. 4B). In contrast, concentrations of GABA were substantially greater after 60 min than after 5 min of repeated ischemia (Fig. 3B). In contrast to the increased sensitivity to a second period of ischemia after 1 h of recirculation, the responses were decreased after longer periods of recovery. At all longer recovery periods tested (5 and 20 h; 1 and 2 weeks) a second period of occlusion of 5 min caused no changes in the levels of metabolites (Fig. 1C-F, Fig. 2C-F, Fig. 3C-E, Fig. 4C-F). When the second period of occlusion was of 60 min duration, there were decreases in the concentrations of ATP and P-creatine (Fig. 1C-F). However, these changes were considerably smaller than those observed after either a single 1 h period ofischemia, or a second period ofischemia following 1 h of recovery. There were decreases in glycogen concentrations after 60 min of a second occlusion at each

484 recovery period tested (Fig. 2C-F). Lactate increased after a second occlusion at all recovery periods examined. There were no significant changes in glucose concentration. GABA concentrations were not different after both periods of occlusion after each recovery period longer than 1 h (Fig. 3C-E). The concentrations of cyclic A M P in the 20 h recovery group increased significantly after either 5 or 60 rain of a second occlusion; however, neither of these responses were as great as seen after 1 h recovery. After one week of recovery no changes in cyclic A M P concentrations were seen after a second 1 h occlusion; the increases seen after 2 weeks of recovery were not significant (Fig. 4C-F). The metabolic rate was assessed by the changes in ~ P equivalents following decapitation as described above. In the contralateral cortex the metabolic rate was 212 nmoles/mg protein/min (Table I), a value in good agreement with those reported by others2,L The rate did not vary from that of control animals (sham-operated). After 1 h of recirculation, the metabolic rate was slightly increased. An even greater increase in metabolic rate of the cerebral cortex was observed by Watanabe and Passonneau 15, 1 h after stab wound injury. At 5 and 20 h after recirculation the metabolic rate decreased to 60 and 20 °/o of the control value, respectively. The metabolic rate after i

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485 TABLE I Metabolic rate o f cerebral cortex in control and postischemic gerbils

The metabolic rate of gerbil cerebral cortex was estimated by the differences in energy reserves in brains frozen in situ or 30 sec after decapitation. The utilization of ~ P has been calculated as the sum of A glucose q- A PCr q- 1.4 A ATP ÷ 2.9 A glycogen. Control metabolic rates were measured in the contralateral cortex. nmoles ,~ P/mg protein/rain

Control 1 h recovery 5 h recovery 20 h recovery

212 224 124 46

20 h recovery following 1 h of ischemia was much lower than that measured 24 h after stab wound injury 15. The ischemic insult appears to be far more pervasive in its effect on cerebral metabolism. The observed changes in metabolic rates may in part account for the differing responses to a second ischemic insult. At 1 h of recirculation, when metabolic rate in the brain is slightly higher than normal, the brain shows an increased sensitivity to a second period of ischemia; after longer periods of recovery, when the metabolic rate is decreased from normal values, the brain appears to be less susceptible to a further ischemic insult. Earlier investigations suggested that the brain cannot survive an ischemic insult of greater than 7-10 min 16. However, Hossman and Sato 5 have shown that signs of functional recovery such as spontaneous E E G activity and energy metabolism appear after 1 h of complete cerebral ischemia. Studies in our laboratory indicated that after 1 h ischemia, the subsequent restoration of energy metabolites is essentially complete; however, biochemical recovery is diminished after 3 h ischemia 12. Consequently, an initial 1 h period of ischemia was chosen to study the effects of repeated carotid occlusion. Two distinct postischemic periods were recognized in the present study. After the relatively short period of recirculation of 1 h, the effect of a second insult was evident even after 5 min of occlusion. After 5 h-2 weeks of recirculation, a second period of ischemia had relatively little effect on the concentrations of metabolites and putative neurotransmitters in the brain. The periods of hyper- and hyporeactivity correspond to both the neurological symptoms and the biochemical estimates of metabolic events. The signs of neurological infarction, originally described by K a h n 6, were evident during the 1 h period of initial ischemia, as were the biochemical changes in energy reserves and putative neurotransmitters. These symptoms were pronounced when a second ligation of 5 or 60 min followed 1 h recovery after the initial 60 rain occlusion. The biochemical changes in the cortex were most evident at this time. The neurological symptoms during a second period of ischemia were diminished after longer periods of recovery, as were the changes in the biochemical parameters measured in the cerebral cortex. After 1 or 2 weeks of recovery, there were no clinical signs of infarction during 5 min and minimal neurological manifestations of injury during 60 min of a second

486 ischemia period. Thus the neurological symptoms show close correspondence to the biochemical signs of ischemia. There is evidence to support the idea of the development of tolerance in the brain to metabolic stress. Tolerance to hypoxia was increased in rats subjected to repeated cardiac arrest caused by h y p o t h e r m i a 1. Similarly, rats which survived one exposure to anoxia were able to withstand much longer periods of anoxia a second time z. In the present study, the metabolic rate of the brain was slightly elevated 1 h after a 60 rain ischemic insult a n d subsequently depressed. The changes in metabolic rate coincide with the increased sensitivity to a second ischemic period at 1 h a n d the diminished response at later time intervals of recirculation. Such observations have relevance to clinical circumstances where repeated ischemic conditions may be involved.

1 Andjus, R. K., Some mechanisms of mammalian tolerance to low body temperatures, dormancy and survival, XXlllrd Syrup. Soc. exp. BioL, 23 (1969) 351-394. 2 Brunner, E. A., Passonneau, J. V. and Molstad, C., The effect of volatile anaesthetics on levels of metabolites and on metabolic rate in brain, J. Neurochem., 18 (1971) 2301-2316. 3 Dahl, N. A. and Balfour, W. M., Prolonged anoxic survival due to anoxia pre-exposure: brain ATP, lactate, and pyruvate, Amer. J. Physiol., 207 (1974) 452-456. 4 Gatfield, P. D., Lowry, O. H., Schulz, D. W. and Passonneau, J. V., Regional energy reserves in mouse brain and changes with ischemia and anaesthesia, J. Neurochem., 13 (1966) 185-195. 5 Hossman, K.-A. and Sato, K., Effect of ischemia on the function of the sensorimotor cortex in cat, Electroenceph. clin. Neurophysiol., 30 (1971) 534-545. 6 Kahn, K. M. D., The natural course of experimental cerebral infarction in the gerbil, Neurology (Minneap.), 22 (1975) 510-515. 7 Levine, S. and Pigne, H., Effects of ischemia and other procedures on the brain and retina of thc gerbil (Meriones unguiculatus), Exp. Neurol., 16 (1966) 255-262. 8 Lowry, O. H. and Passonneau, J. V., A Flexible System of Enzymatic Analysis, Academic Press, New York, 1972. 9 Lowry, O. H., Passonneau, J. V., Hasselberger, F. K. and Schulz, D. W., Effet of ischemia on known substrates and cofactors of the glycolytic pathway in brain, J. biol. Chem., 239 (1964) 18-30. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Lust, W. D., Dye, E., Deaton, A. V. and Passonneau, J. V., A modified cyclic AMP binding assay, Analyt. Biochem., 72 (1976) 8-15. 12 Mr~ulja, B. B., Lust, W. D., Mr~ulja, B. J., Passonneau, J. V. and Klatzo, I., Post-ischemic changes, in certain metabolites following prolonged ischemia in the gerbil cerebral cortex, J. Neurochem., 26 (1966) 1099-1103. 13 Passonneau, J. V. and Lauderdale, V. R., A comparison of three methods of glycogen measurement in tissues, Analyt. Biochem., 60 (1974) 405-412. 14 Passonneau, J. V., Lust, W. D. and Crites, S. K., GABA shunt metabolites and enzymes in glioma and neuroblastoma cell lines, Trans. Amer. Soc. Neurochem., 7 (1976) 192. 15 Watanabe, H. and Passonneau, J. V., The effect of trauma on cerebral glycogen and related metabolites and enzymes, Brain Research, 66 (1974) 147-159. 16 Yashon, D., White, R. S., Taslitz, N., Wolin, L. R. and Massopust, L. C., Experimental cerebral circulatory arrest: effect on electrocortical potentials, J. Neurosurg., 32 (1970) 74-82.