Brain Research, 516 (1990) 175-179 Elsevier
175
BRES 24082
Role of the excitotoxic mechanism in the development of neuronal damage following repeated brief cerebral ischemia in the gerbil: protective effects of MK-801 and pentobarbital Hiroyuki Kato, Tsutomu Araki and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Accepted 23 January 1990) Key words: Cerebral ischemia; Repeated ischemia; Selective vulnerability; Gerbil; MK-801; Flunarizine; Pentobarbital
Pretreatment with MK-801 (an NMDA antagonist) or pentobarbital (a GABAA receptor-effector) ameliorated histopathological neuronal damage to the hippocampal CA1 subfield and to the thalamus following three 2-rain forebrain ischemia at 1-h intervals in the gerbil. Flunarizine, a calcium antagonist, failed to prevent the neuronal damage. The results suggest that the excitotoxic mechanism plays a role in the neuronal damage following repeated ischemia. A recently developed model of repeated cerebral ischemia has a unique feature 11'12'17'23. Brief and morphologically non-lethal cerebral ischemia produces neuronal damage in the selectively vulnerable regions if the insult is induced repeatedly at a certain interval. Twominute bilateral carotid artery occlusion in the gerbil causes no morphological brain damage, whereas 3 occlusions at 1-h intervals lead to severe and consistent damage to the CA1 subfield of the hippocampus and moderate damage to the thalamus and to the lateral part of the striatum 11']2. The distribution of neuronal damage is as extensive as that after single 10-min forebrain ischemia 3,n,12. Recent evidence has led to propose two hypotheses for ischemia-induced cell death: the calcium hypothesis of cell death 2°'2~ and the excitotoxic hypothesis of neuronal death 19"21. In fact, neuronal damage following transient cerebral ischemia is prevented by treatment with calcium antagonists Ll°, glutamate antagonists 8'1° or y-aminobutyric acid (GABA)-ergic agents which inhibit neuronal excitability9A3,22. The pathomechanism of the cumulative effect of repeated ischemic insults, however, is not fully understood because the succeeding insult is rendered at the stage of post-ischemic hypoperfusion 23 and depressed protein synthesis 4. We, therefore, examined the effect of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; a non-competitive N M D A antagonist), flunarizine (a calcium antagonist),
and pentobarbital (a G A B A A receptor-effector) on the neuronal damage following repeated ischemic insults to clarify to what an extent the calcium and excitotoxic mechanisms are involved in repeated ischemia. Male Mongolian gerbils, aged 13-14 weeks and weighing 60-80 g, were used. They were allowed free access to food and water. Anesthesia was induced with 2% halothane in a mixture of 70% nitrous oxide and 30% oxygen. Mid-cervical skin incision was made and bilateral common carotid arteries were gently exposed and occluded with aneurysmal clips for 2 min. Anesthesia was discontinued when the clips were in place. Three occlusions were repeated at 1-h intervals. Rectal temperature was monitored and maintained using a heating pad and a lamp. As described below, the animals which were treated with MK-801 or pentobarbital were warmed so that the body temperature was comparable to that in vehicle-treated animals. A total of 30 animals were divided into 5 groups. (1) Sham-operated (n = 5). (2) MK-801 (kindly donated by Merck & Co. Inc., Rahway, New Jersey, U.S.A.) at a dose of 3 mg/kg was administered i.p. 30 min before ischemic insults. (3) Flunarizine (Sigma Chemical Co., St. Louis, MO, U.S.A.) at a dose of 30 mg/kg was administered i.p. 30 min before ischemic insults. (4) Pentobarbital (Tokyo Kasei Industry Ltd., Tokyo, Japan) at a dose of 40 mg/kg was administered i.p. 30 min before ischemic insults and a half of the dose was additionally administered twice every hour. (5) Vehicle (2% arabic
Correspondence: H. Kato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176 TABLE I
TABLE II
Rectal temperatures of the animals
Neuronal density of the CA1 subfield of the hippocampus (/mm) and neuronal damage to the striatum and thalamus (graded 0-3)
Values are expressed as mean + S.D.
Values are expressed as mean + S.D.
Shamoperated Vehicle MK-801 Flunarizine Pentobarbital
Number Before first ischemia
Before second ischemia
Before third ischemia
5 6 7 6 6
37.3+ 0.48 37.8+ 0.17" 37.7+ 0.24* 38.1 + 0.27** 37.7+0.41"*
37.1+ 0.28 37.7 + 0.45* 37.5 + 0.26** 37.9 + 0.22* 38.0+0.28**
37.2 + 0.39 37.1 + 0.37 36,8 + 0.55 37.3 + 0.42 36.8+0.23
*P < 0.05, **P < 0.01 as compared to that before ischemia (paired t-test). Not significant between vehicle-treated and drug-treated groups (non-paired t-test).
gum in distilled water) was administered, A t 7 days of survival, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and the brains were briefly washed with heparinized saline via ascending aorta, followed by perfusion-fixation with 3.7% formaldehyde for 30 min. The brains were removed 2 - 4 h later and immersed in the same fixative until they were e m b e d d e d in paraffin. Paraffin sections 5/zm thick were taken and stained with Cresyl violet and hematoxylineosin. Stained sections were examined with a light microscope without the examiner knowing the experimental protocol. Neuronal density of the hippocampal CA1 subfield, i.e. the n u m b e r of intact pyramidal cells per 1 m m linear length of CA1, was determined according to the method of Kirino et al. 13. Neuronal damage to the thalamus and striatum was semiquantitatively graded using a 0 - 3 rating system with 0 = normal, 1 = a few n e u r o n s damaged, 2 = many neurons damaged and 3 = majority of n e u r o n s damaged. The average of left and right values was considered. Statistical significances were analyzed using the Kruskal-Wallis test, the M a n n - W h i t n e y U-test and the Student's paired and non-paired t-tests. All animals tolerated 3 ischemic insults, and death and seizures were not observed. MK-801-treated and pentobarbital-treated animals did not start moving until 2 - 4 h after ischemic insults. Behavior of flunarizine-treated animals appeared the same as vehicle-treated animals. Rectal temperatures of the animals are shown in Table I. The temperature was increased by 0.6-0.8 °C following
Fig. 1. Representative photomicrographs of the CA1 subfield of the hippocampus, a: sham-operated, b: vehicle-treated, c: MK-801treated (with complete protection), d: flunarizine-treated, e: pentobarbital-treated. CA1 pyramidal cells are preserved in a, c and e, but almost all pyramidal cells are lost in b and d. Cresyl violet staining. Bar = 100/zm.
Number Hippocampus Sham-operated Vehicle MK-801 Flunarizine Pentobarbital
5 6 7 6 6
S t r i a t u m Thalamus
234 + 21.8"* 0+ 0 13 + 6.8 1.3 + 1.47 117+118.4" 0.1+0.19 15 + 11.7 0.1 + 0.20 120 + 49.4** 0+ 0
0 + 0"* 1.9 + 0.20 0+0"* 1.9 + 0.20 0.8 + 0.26**
*P < 0.05, **P < 0.01 as compared to vehicle-treated animals (Kruskal-Wallis test and Mann-WhitneyU-test).
177
Fig. 2. Representative photomicrographs of the lateral nucleus of the thalamus, a: sham-operated, b: vehicle-treated, c: MK-801-treated. d: flunarizine-treated, e: pentobarbital-treated. Severely injured, shrunken, eosinophilic neurons are seen in b and d (arrows). No damaged neurons are seen in a, c and e. Hematoxylin-eosinstaining. Bar = 100/zm. the first ischemic insult in vehicle- and flunarizine-treated animals. The temperature of the animals whose behavior was depressed (MK-801 and pentobarbital) was, due to warming, not statistically different from that of vehicleor flunarizine-treated animals. Neuronal density of the hippocampal CA1 subfield following three ischemic insults was reduced to 6% of that of sham-operated animals (Table II). MK-801 (P < 0.05) and pentobarbital (P < 0.01) ameliorated CA1 neuronal loss following ischemic insults (Table II). Pretreatment with MK-801 exhibited complete preservation of CA1 neurons in 3 of 7 animals, but almost complete destruction in 3 animals. Pentobarbital-treated animals exhibited various degree of survival of CA1 pyramidal cells without showing complete destruction of CA1. Pretreatment with flunarizine failed to prevent CA1 cell loss. Many thalamic neurons in the ventral and lateral thalamic nuclei were consistently damaged following three ischemic insults (Table II). Treatment with MK-801 (P < 0.01) and pentobarbital (P < 0.01) prevented thalamic neuronal damage (Table II). Complete protection was observed in MK-801-treated animals. Damage in pentobarbital-treated animals was mild and confined to the ventral thalamic nucleus. Flunarizine failed to ame-
liorate thalamic neuronal damage. Striatal damage following three ischemic insults was variable ranging from grade 0 to grade 3. Lateral portion of the striatum was damaged when present. Although striatal damage was not observed in any drug-treated animal except mild damage in two hemispheres, one in an MK-801-treated animal and the other in a flunarizinetreated animal, protective effects with statistical significance were not observed (Table II). According to our previous report 11'12, conspicuous and consistent neuronal damage was observed in the CA1 subfield of the hippocampus and in the ventral and lateral locations of the thalamus following three 2-min bilateral carotid artery occlusions. The hippocampal and thalamic damage was ameliorated by pretreatment with MK-801 or pentobarbital. The two agents protects against neuronal damage following transient forebrain ischemia in the gerbil 8-1°'13'22. MK-801 noncompetitively blocks the N-methyl-oaspartate (NMDA) subtype of glutamate receptor which mediate cellular influx of calcium 24. The mechanism of protective action of pentobarbital may be central nervous system (CNS) depression or through enhancing G A B A A receptor binding TM. The major inhibitory pathways in the CNS is mediated by G A B A 7. Recent evidence suggests
178 that an excitatory neurotransmitter glutamate, which is massively released during ischemia 5, triggers a chain of reactions which lead to ischemia-induced neuronal death 21. The protective effect of MK-801, which was in an all-or-none fashion in the hippocampus and was complete in the thalamus, may be explained by this hypothesis. Protection against ischemic hippocampal damage is often an all-or-none phenomenon 22. The dose of MK-801 (3 mg/kg) which we employed completely protects against hippocampal neuronal loss following 5-min ischemia in majority of animals s. This dose was less protective for the hippocampus in our model because the insult is more severe, but thalamic damage was completely prevented. In contrast, the protective effect of pentobarbital was moderate but consistent both in the hippocampus and thalamus, exhibiting neither complete protection nor complete uneffectiveness. This suggests that the mechanism of action of pentobarbital is different from that of MK-801. Flunarizine failed to protect against ischemic neuronal damage following repeated ischemic insults. It is not likely that the uneffectiveness is because the drug level was not maintained over the period of the experiment. Flunarizine is sufficiently long-acting 14'15, and the same
dose of flunarizine also failed to prevent CA1 pyramidal cell loss following 5-min forebrain ischemia in the gerbil 2'25. These results may suggest that major calcium influx is provoked by agonist-operated calcium channels. Rectal temperature of the animal was increased by 0.6-0.8 °C after 2-min forebrain ischemia. The increase may be caused by the effect of ischemia on the CNS because such an increase was not observed in the sham-operated animals. In the present experiments, the animals which were treated with MK-801 and pentobarbital were warmed so that the temperature was comparable to that of vehicle-treated animals. Therefore, although the behavior of the MK-801- and pentobarbitaltreated animals were depressed, the cerebral protecting action of these agents is not through that of hypothermia 6A6. Although the repeated ischemic insults are induced at the stage of postischemic hypoperfusion 23 and depressed protein synthesis 4, the present results suggest that the excitotoxic mechanism also plays a major role in producing neuronal damage following repeated non-lethal ischemic insults. Because the cumulative effect of repeated ischemia is of importance to clarify the dynamics of ischemic brain injury, this model deserves further study.
1 Alps, B.J., Calder, C., Hass, W.K. and Wilson, A.D., Comparative protective effects of nicardipine, flunarizine, lidoflazine and nimodipine against ischaemic injury in the hippocampus of the Mongolian gerbil, Br. J. Pharmacol., 93 (1988) 877-883. 2 Araki, T., Kogure, K. and Nishioka, K., Comparative neuroprotective effects of pentobarbital, vinpocetine, flunarizine and ifenprodil on ischemic neuronal damage in the gerbil hippocampus, Res. Exp. Med, in press. 3 Araki, T., Kato, K. and Kogure, K., Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: distribution and time course, Acta Neurol. Scand., 80 (1989) 548-553. 4 Araki, T., Kato, H., Inoue, T. and Kogure, K., Regional impairment of protein synthesis following brief cerebral ischemia in the gerbil, Acta Neuropathol., in press. 5 Benveniste, H., Drejer, J., Schousboue, A. and Diemer, H.N., Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1373. 6 Busto, R., Dietrich, W.D., Globus, M.Y.-T., Valdes, I., Scheinberg, P. and Ginsberg, M.D., Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, J. Cereb. Blood Flow Metabol., 7 (1987) 729-738. 7 Enna, S., The GABA receptor, Humana Press, Clifton, NJ, 1983. 8 Gill, R., Foster, A.C. and Woodruff, G.N., Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343-3349. 9 Hallmayer, D., Hossmann, K.-A. and Mies, G., Low dose of barbiturates for prevention of hippocampal lesions after brief ischemic episodes, Acta Neuropathol., 68 (1985) 27-31. 10 Izumiyama, K. and Kogure, K., Prevention of delayed neuronal
death in gerbil hippocampus by ion channel blockers, Stroke, 19 (1988) 1003-1007. 11 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain Research, 479 (1989) 366-370. 12 Kato, H. and Kogure, K., Neuronal damage following nonlethal but repeated cerebral ischemia in the gerbil, Acta Neuropathol., in press, 13 Kirino, T., Tamura, A. and Sano, K., A reversible type of neuronal injury following ischemia in the gerbil hippocampus, Stroke, 17 (1986) 455-459. 14 Meuldermans, W., Hendrickx, J., Hurkmans, R., Swysen, E., Woestenborghs, R., Lauwers, W. and Heykants, J., Excretion and metabolism of flunarizine in rats and dogs, Arzneim.Forsch./Drug Res., 33 (1983) 1142-1151.. 15 Michiels, M., Hendriks, R., Knaeps, F., Woestenborghs, R. and Heykants, J., Absorption and tissue distribution of flunarizine in rats, pigs and dogs, Arzneim.-Forsch./Drug Res., 33 (1983) 1135-1142. 16 Minamisawa, H., Smith, M.-L. and Siesj6, B.K., The effect of moderate hyperthermia (39 °C) and hypothermia (35 °C) on brain damage following 10 and 15 minutes of forebrain ischemia, J. Cereb. Blood Flow Metab., 9, Suppl. 1 (1989) $261. 17 Nakano, S., Kato, H. and Kogure, K., Neuronal damage in the rat hippocampus in a new model of repeated reversible transient cerebral ischemia, Brain Research, 490 (1989) 178-180. 18 OIsen, R.W., Wong, E.H.E, Stauba, G.B., Murakami, D., King, R.G. and Fisher, J.B., Biochemical properties of the GABA/barbiturate/benzodiazepine receptor chloride ion channel complex, Adv. Exp. Med. Biol., 175 (1978) 205-219. 19 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 20 Siesj6, B.K., Cell damage in the brain: a speculative synthesis, J. Cereb. Blood Flow Metabol., 1 (1981) 155-185.
179 21 Siesj6, B.K. and Bengtsson, E, Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia and spreading depression: a unifying hypothesis, .IT.Cereb. Blood Flow Metabol., 9 (1989) 127-140. 22 Sternau, L.L., Lust, W.D., Ricci, A.J. and Racheson, R., Role for 7-aminobutyric acid in selective vulnerability in gerbils, Stroke, 20 (1989) 281-287. 23 Tomida, S., Nowak, Jr., T.S., Vass, K., Kohr, T.M. and Klatzo, I., Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemic insults, J.
Cereb. Blood Flow Metabol., 7 (1987) 773-782. 24 Wong, E.H.F., Kemp, J.A., Priestly, T., Knight, A.G., Woodruff, G.N. and Iversen, L.L., The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. Sci., 83 (1986) 7104-7108o 25 Yoshidomi, M., Hayashi, T., Abe, K. and Kogure, K., Effects of a new calcium entry blocker KB-2796 on protein synthesis and brain cell damage following ischemia, J. Cereb. Blood Flow Metabol., 9, Suppl. 1 (1989) $308.
175
BRES 24082
Role of the excitotoxic mechanism in the development of neuronal damage following repeated brief cerebral ischemia in the gerbil: protective effects of MK-801 and pentobarbital Hiroyuki Kato, Tsutomu Araki and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Accepted 23 January 1990) Key words: Cerebral ischemia; Repeated ischemia; Selective vulnerability; Gerbil; MK-801; Flunarizine; Pentobarbital
Pretreatment with MK-801 (an NMDA antagonist) or pentobarbital (a GABAA receptor-effector) ameliorated histopathological neuronal damage to the hippocampal CA1 subfield and to the thalamus following three 2-rain forebrain ischemia at 1-h intervals in the gerbil. Flunarizine, a calcium antagonist, failed to prevent the neuronal damage. The results suggest that the excitotoxic mechanism plays a role in the neuronal damage following repeated ischemia. A recently developed model of repeated cerebral ischemia has a unique feature 11'12'17'23. Brief and morphologically non-lethal cerebral ischemia produces neuronal damage in the selectively vulnerable regions if the insult is induced repeatedly at a certain interval. Twominute bilateral carotid artery occlusion in the gerbil causes no morphological brain damage, whereas 3 occlusions at 1-h intervals lead to severe and consistent damage to the CA1 subfield of the hippocampus and moderate damage to the thalamus and to the lateral part of the striatum 11']2. The distribution of neuronal damage is as extensive as that after single 10-min forebrain ischemia 3,n,12. Recent evidence has led to propose two hypotheses for ischemia-induced cell death: the calcium hypothesis of cell death 2°'2~ and the excitotoxic hypothesis of neuronal death 19"21. In fact, neuronal damage following transient cerebral ischemia is prevented by treatment with calcium antagonists Ll°, glutamate antagonists 8'1° or y-aminobutyric acid (GABA)-ergic agents which inhibit neuronal excitability9A3,22. The pathomechanism of the cumulative effect of repeated ischemic insults, however, is not fully understood because the succeeding insult is rendered at the stage of post-ischemic hypoperfusion 23 and depressed protein synthesis 4. We, therefore, examined the effect of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; a non-competitive N M D A antagonist), flunarizine (a calcium antagonist),
and pentobarbital (a G A B A A receptor-effector) on the neuronal damage following repeated ischemic insults to clarify to what an extent the calcium and excitotoxic mechanisms are involved in repeated ischemia. Male Mongolian gerbils, aged 13-14 weeks and weighing 60-80 g, were used. They were allowed free access to food and water. Anesthesia was induced with 2% halothane in a mixture of 70% nitrous oxide and 30% oxygen. Mid-cervical skin incision was made and bilateral common carotid arteries were gently exposed and occluded with aneurysmal clips for 2 min. Anesthesia was discontinued when the clips were in place. Three occlusions were repeated at 1-h intervals. Rectal temperature was monitored and maintained using a heating pad and a lamp. As described below, the animals which were treated with MK-801 or pentobarbital were warmed so that the body temperature was comparable to that in vehicle-treated animals. A total of 30 animals were divided into 5 groups. (1) Sham-operated (n = 5). (2) MK-801 (kindly donated by Merck & Co. Inc., Rahway, New Jersey, U.S.A.) at a dose of 3 mg/kg was administered i.p. 30 min before ischemic insults. (3) Flunarizine (Sigma Chemical Co., St. Louis, MO, U.S.A.) at a dose of 30 mg/kg was administered i.p. 30 min before ischemic insults. (4) Pentobarbital (Tokyo Kasei Industry Ltd., Tokyo, Japan) at a dose of 40 mg/kg was administered i.p. 30 min before ischemic insults and a half of the dose was additionally administered twice every hour. (5) Vehicle (2% arabic
Correspondence: H. Kato, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176 TABLE I
TABLE II
Rectal temperatures of the animals
Neuronal density of the CA1 subfield of the hippocampus (/mm) and neuronal damage to the striatum and thalamus (graded 0-3)
Values are expressed as mean + S.D.
Values are expressed as mean + S.D.
Shamoperated Vehicle MK-801 Flunarizine Pentobarbital
Number Before first ischemia
Before second ischemia
Before third ischemia
5 6 7 6 6
37.3+ 0.48 37.8+ 0.17" 37.7+ 0.24* 38.1 + 0.27** 37.7+0.41"*
37.1+ 0.28 37.7 + 0.45* 37.5 + 0.26** 37.9 + 0.22* 38.0+0.28**
37.2 + 0.39 37.1 + 0.37 36,8 + 0.55 37.3 + 0.42 36.8+0.23
*P < 0.05, **P < 0.01 as compared to that before ischemia (paired t-test). Not significant between vehicle-treated and drug-treated groups (non-paired t-test).
gum in distilled water) was administered, A t 7 days of survival, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and the brains were briefly washed with heparinized saline via ascending aorta, followed by perfusion-fixation with 3.7% formaldehyde for 30 min. The brains were removed 2 - 4 h later and immersed in the same fixative until they were e m b e d d e d in paraffin. Paraffin sections 5/zm thick were taken and stained with Cresyl violet and hematoxylineosin. Stained sections were examined with a light microscope without the examiner knowing the experimental protocol. Neuronal density of the hippocampal CA1 subfield, i.e. the n u m b e r of intact pyramidal cells per 1 m m linear length of CA1, was determined according to the method of Kirino et al. 13. Neuronal damage to the thalamus and striatum was semiquantitatively graded using a 0 - 3 rating system with 0 = normal, 1 = a few n e u r o n s damaged, 2 = many neurons damaged and 3 = majority of n e u r o n s damaged. The average of left and right values was considered. Statistical significances were analyzed using the Kruskal-Wallis test, the M a n n - W h i t n e y U-test and the Student's paired and non-paired t-tests. All animals tolerated 3 ischemic insults, and death and seizures were not observed. MK-801-treated and pentobarbital-treated animals did not start moving until 2 - 4 h after ischemic insults. Behavior of flunarizine-treated animals appeared the same as vehicle-treated animals. Rectal temperatures of the animals are shown in Table I. The temperature was increased by 0.6-0.8 °C following
Fig. 1. Representative photomicrographs of the CA1 subfield of the hippocampus, a: sham-operated, b: vehicle-treated, c: MK-801treated (with complete protection), d: flunarizine-treated, e: pentobarbital-treated. CA1 pyramidal cells are preserved in a, c and e, but almost all pyramidal cells are lost in b and d. Cresyl violet staining. Bar = 100/zm.
Number Hippocampus Sham-operated Vehicle MK-801 Flunarizine Pentobarbital
5 6 7 6 6
S t r i a t u m Thalamus
234 + 21.8"* 0+ 0 13 + 6.8 1.3 + 1.47 117+118.4" 0.1+0.19 15 + 11.7 0.1 + 0.20 120 + 49.4** 0+ 0
0 + 0"* 1.9 + 0.20 0+0"* 1.9 + 0.20 0.8 + 0.26**
*P < 0.05, **P < 0.01 as compared to vehicle-treated animals (Kruskal-Wallis test and Mann-WhitneyU-test).
177
Fig. 2. Representative photomicrographs of the lateral nucleus of the thalamus, a: sham-operated, b: vehicle-treated, c: MK-801-treated. d: flunarizine-treated, e: pentobarbital-treated. Severely injured, shrunken, eosinophilic neurons are seen in b and d (arrows). No damaged neurons are seen in a, c and e. Hematoxylin-eosinstaining. Bar = 100/zm. the first ischemic insult in vehicle- and flunarizine-treated animals. The temperature of the animals whose behavior was depressed (MK-801 and pentobarbital) was, due to warming, not statistically different from that of vehicleor flunarizine-treated animals. Neuronal density of the hippocampal CA1 subfield following three ischemic insults was reduced to 6% of that of sham-operated animals (Table II). MK-801 (P < 0.05) and pentobarbital (P < 0.01) ameliorated CA1 neuronal loss following ischemic insults (Table II). Pretreatment with MK-801 exhibited complete preservation of CA1 neurons in 3 of 7 animals, but almost complete destruction in 3 animals. Pentobarbital-treated animals exhibited various degree of survival of CA1 pyramidal cells without showing complete destruction of CA1. Pretreatment with flunarizine failed to prevent CA1 cell loss. Many thalamic neurons in the ventral and lateral thalamic nuclei were consistently damaged following three ischemic insults (Table II). Treatment with MK-801 (P < 0.01) and pentobarbital (P < 0.01) prevented thalamic neuronal damage (Table II). Complete protection was observed in MK-801-treated animals. Damage in pentobarbital-treated animals was mild and confined to the ventral thalamic nucleus. Flunarizine failed to ame-
liorate thalamic neuronal damage. Striatal damage following three ischemic insults was variable ranging from grade 0 to grade 3. Lateral portion of the striatum was damaged when present. Although striatal damage was not observed in any drug-treated animal except mild damage in two hemispheres, one in an MK-801-treated animal and the other in a flunarizinetreated animal, protective effects with statistical significance were not observed (Table II). According to our previous report 11'12, conspicuous and consistent neuronal damage was observed in the CA1 subfield of the hippocampus and in the ventral and lateral locations of the thalamus following three 2-min bilateral carotid artery occlusions. The hippocampal and thalamic damage was ameliorated by pretreatment with MK-801 or pentobarbital. The two agents protects against neuronal damage following transient forebrain ischemia in the gerbil 8-1°'13'22. MK-801 noncompetitively blocks the N-methyl-oaspartate (NMDA) subtype of glutamate receptor which mediate cellular influx of calcium 24. The mechanism of protective action of pentobarbital may be central nervous system (CNS) depression or through enhancing G A B A A receptor binding TM. The major inhibitory pathways in the CNS is mediated by G A B A 7. Recent evidence suggests
178 that an excitatory neurotransmitter glutamate, which is massively released during ischemia 5, triggers a chain of reactions which lead to ischemia-induced neuronal death 21. The protective effect of MK-801, which was in an all-or-none fashion in the hippocampus and was complete in the thalamus, may be explained by this hypothesis. Protection against ischemic hippocampal damage is often an all-or-none phenomenon 22. The dose of MK-801 (3 mg/kg) which we employed completely protects against hippocampal neuronal loss following 5-min ischemia in majority of animals s. This dose was less protective for the hippocampus in our model because the insult is more severe, but thalamic damage was completely prevented. In contrast, the protective effect of pentobarbital was moderate but consistent both in the hippocampus and thalamus, exhibiting neither complete protection nor complete uneffectiveness. This suggests that the mechanism of action of pentobarbital is different from that of MK-801. Flunarizine failed to protect against ischemic neuronal damage following repeated ischemic insults. It is not likely that the uneffectiveness is because the drug level was not maintained over the period of the experiment. Flunarizine is sufficiently long-acting 14'15, and the same
dose of flunarizine also failed to prevent CA1 pyramidal cell loss following 5-min forebrain ischemia in the gerbil 2'25. These results may suggest that major calcium influx is provoked by agonist-operated calcium channels. Rectal temperature of the animal was increased by 0.6-0.8 °C after 2-min forebrain ischemia. The increase may be caused by the effect of ischemia on the CNS because such an increase was not observed in the sham-operated animals. In the present experiments, the animals which were treated with MK-801 and pentobarbital were warmed so that the temperature was comparable to that of vehicle-treated animals. Therefore, although the behavior of the MK-801- and pentobarbitaltreated animals were depressed, the cerebral protecting action of these agents is not through that of hypothermia 6A6. Although the repeated ischemic insults are induced at the stage of postischemic hypoperfusion 23 and depressed protein synthesis 4, the present results suggest that the excitotoxic mechanism also plays a major role in producing neuronal damage following repeated non-lethal ischemic insults. Because the cumulative effect of repeated ischemia is of importance to clarify the dynamics of ischemic brain injury, this model deserves further study.
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