Brain Research, 528 (1990) 114-122
114
Elsevier BRES 15868
Neuronal damage and calcium accumulation following repeated brief cerebral ischemia in the gerbil Tsutomu Araki, Hiroyuki Kato and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 20 March 1990)
Key words: Cerebral ischemia; Repeated ischemia; Selective vulnerability; Calcium accumulation; Gerbil
We investigated the distribution of neuronal damage following brief cerebral transient ischemia and repeated ischemia at 1-h intervals in the gerbil, using light microscopy and 45Ca autoradiography as a marker for detection of ischemic damage. The animals were allowed to survive for 7 days after ischemia induced by bilateral carotid artery occlusion. Following 2-min ischemia, neuronal damage determined by abnormal calcium accumulation was not observed in the forebrain regions. Following 3-min ischemia, however, abnormal calcium accumulation was recognized only in the hippocampal CA1 sector and part of the striatum. Two 2-min ischemic insults caused extensive abnormal calcium accumulation in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the substantia nigra and the inferior colliculus. The ischemic insults were more severe than that of a single 3-min ischemia. However, three 1-min ischemic insults caused abnormal calcium accumulation only in the striatum. On the other hand, three 2-min ischemic insults caused severe abnormal calcium accumulation in the brain. The abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus. Gerbils subjected to three 3-min ischemic insults revealed most severe abnormal calcium accumulation. Marked calcium accumulation was seen not only in the above sites, but also spread in the neocortex, the septum and the hippocampal CA3 sector. Morphological study after transient or repeated ischemia indicated that the distribution and frequency of the neuronal damage was found in the sites corresponding to most of the regions of abnormal calcium accumulation. The abnormal calcium accumulation, however, was not always found in the regions such as the neocortex and the hippocampal CA3 sector where the neuronal damage was seen. The present study demonstrates that repeated ischemic insults at 1-h intervals can produce severe neuronal damage not only in the basal ganglia and the limbic system but also in the brainstem. Furthermore, they suggest that the cumulative effects after repeated ischemic insults are related to the time of ischemia or the number of episodes.
INTRODUCTION Transient c e r e b r a l ischemia causes variable brain d a m a g e according to its degree and duration. N e u r o n s k n o w n to be selectively vulnerable are found in certain regions such as neocortex, striatum, h i p p o c a m p u s and t h a l a m u s 7'19. This p h e n o m e n o n has been t e r m e d selectively n e u r o n a l vulnerability. A m o n g selectively vulnerable regions, t h e r e is a further hierarchy of susceptibility to ischemic injury 2°. H i p p o c a m p a l CA1 neurons are especially susceptible to ischemia 14'19, and the neurons are lost following 3-min ischemia in the gerbil 1. T o m i d a et al. 23 recently found that r e p e a t e d cerebral ischemia has a cumulative effect since the n e u r o n a l d a m a g e is g r e a t e r following 3 episodes of 5-min ischemia at 1-h intervals in the gerbil than 15-min ischemia p r o d u c e d as a single insult. F o r this reason, they have suggested that the presence of postischemic hypoperfusion m a y play an i m p o r t a n t role in the cumulative effect of the r e p e a t e d ischemia. On the other hand, K a t o et
al. 13 have shown that the distribution of the n e u r o n a l injury p r o d u c e d by 3 o r 5 r e p e a t e d non-lethal (2-min) ischemic insults at 1-h intervals in the gerbil was consistent with the selectively v u l n e r a b l e areas undergoing g l u t a m a t e innervation. F u r t h e r m o r e , N a k a n o et al. 17 found that the n e u r o n a l d a m a g e is most severe when non-lethal ischemia is r e p e a t e d at 1-h intervals, and is relatively mild when r e p e a t e d at shorter or longer intervals. These observations suggest that the intervals and the episodes of ischemic insult play an i m p o r t a n t factor on the ischemic brain d a m a g e . We recently found that brief transient ischemia causes a severe i m p a i r m e n t of p r o t e i n synthesis in the selectively vulnerable areas 4. T h e inhibition is especially found at an early stage of recirculation after ischemia. This suggests that even a brief ischemic insult causes metabolic alteration which persists for a few hours after ischemia, and the succeeding insult m a y injure the brain. F u r t h e r m o r e , although the m o r p h o l o g i c p a t t e r n of selective vulnerability of transient ischemia has b e e n established in
Correspondence: T. Araki, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal, Miyagi 980, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
115 detail 8'11'12'15'19, t h e r e histological
attempts
are to
o n l y a few r e p o r t s
examine
the
neuronal damage following repeated Therefore,
the purpose
about
distribution
of
b r i e f i s c h e m i a 13.
of t h e p r e s e n t
study was to
i n v e s t i g a t e t h e d i s t r i b u t i o n of n e u r o n a l d a m a g e f o l l o w i n g r e p e a t e d b r i e f c e r e b r a l i s c h e m i a in t h e gerbil. F o r this p u r p o s e , w e e m p l o y e d light m i c r o s c o p y and 45Ca a u t o r a d i o g r a p h y to d e t e c t a n d visualize i s c h e m i c n e u r o n a l damage.
MATERIALS AND METHODS
Experimental procedures Adult male Mongolian gerbils weighing 60-95 g were used. They were anesthetized with 2% halothane in a mixture of 30% 0 2 and 70% N20. Bilateral common carotid arteries were exposed and occluded with aneurysm clips, and the animals were allowed to survive for 7 days after ischemia. In order to prevent blood coagulation at the clipping site, 100 IU of heparin was injected i.p. 10 rain before bilateral carotid artery occlusion. Sham-operated animals were also investigated in the same manner except for clipping the bilateral common carotid arteries. In the present study the animals were kept on a thermostatted warming mat (at 37-38 °C) following the procedure until they began to move again; they were then given food and water ad libitum.
Sham
2 min
45Ca autoradiography Thirty-seven animals were randomly assigned to 7 groups containing 5-6 gerbils for 45Ca autoradiography. Five animals were used as the sham-operated group. The remaining 32 animals were divided into 6 experimental groups which were subjected to three 1-min (3 x 1 rain), three 2-min (3 x 2 min), three 3-min (3 x 3 min) and two 2-min (2 × 2 min) bilateral carotid artery occlusions at 1-h intervals or to a single 2-min and 3-min bilateral carotid artery occlusion. 45Ca autoradiography was performed according to the method of Sakamoto et al. 21. Seven days following ischemia , the animals were anesthetized with 2% halothane in a mixture of 30% 0 2 and 70% N20. A bolus of 100 pCi of 45CAC12 (spec. act.: 10-40 mCi/mg, Amersham) was injected i.v. The animals were anesthetized with ether at 5 h after injection of 45CAC12; their brains were quickly removed and frozen in powdered dry ice. Serial coronal sections 20-pm thick were cut from the frozen brain in a cryostat at -20 °C. Serial sections were exposed to Kodak NMC-1 film for 2 weeks, and were used for the evaluation of autoradiography.
Histopathology Thirty-one animals were randomly assigned to 6 groups containing 5-6 gerbils. Five animals used as sham-operated group. Transient brief ischemia was induced for 2 and 3 min. Three 1-min (3 × 1 min), 2-min (3 × 2 min) and 3-min (3 x 3 min) bilateral carotid artery occlusions were repeated at 1-h intervals. Seven days following ischemia, the animals were anesthetized with pentobarbital 50 mg/kg i.p. The brains were perfusion-fixed transcardially with 10% formalin for 20 min. The brains removed were immersed in the same fixative, and then embedded in paraffin. Five /zm paraffin sections were stained with Cresyl violet and hematoxylin-
3 min
2 x 2 min
Fig. 1. Representative 45Ca autoradiograms. 45CAC12was injected at 7 days after ischemia and was allowed to circulate for 5 h. Sham, sham-operated group. No abnormal calcium accumulation is noted. 2 min, 2-min ischemia. Abnormal calcium accumulation is not found in the brain. 3 min, 3-min ischemia. Abnormal calcium accumulation is noted in a small part of striatum (unilateral) and the hippocampal CA1 sector. 2 × 2 min, two episodes of 2-min ischemia. Abnormal calcium accumulation is noted in the hippocampal CA1 sector, the ventral part of thalamus and the inferior colliculus.
116
3 x l min
3 x 2 min
3 x 3 min
Fig. 2. Representative 45Ca autoradiograms. 45CAC12was injected at 7 days after ischemia and was allowed to circulate for 5 h. 3 x 1 min, 3 episodes of 1-min ischemia. Slightly abnormal calcium accumulation is noted in a small part of striatum (unilateral). 3 × 2 min, 3 episodes of 2-min ischemia. Abnormal calcium accumulation is noted in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, a small part of the substantia nigra and the inferior colliculus. 3 × 3 min, 3 episodes of 3-rain ischemia. Marked calcium accumulation is noted in the neocortex, the striatum, the septum, the hippocampal CA1 sector and CA3 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus.
eosin. The sections were examined with the light microscope, and ischemic neuronal damage was graded on a semiquantitative scale: 0, normal; 1, a few neurons damaged; 2, many neurons damaged; 3, majority of neurons damaged, according to the method of Pulsinelli et al. 19. For transient ischemia, they were examined with the light microscope and the neuronal damage in the hippocampus was evaluated by determination of neuronal density of CA1 neurons per 1 mm linear length of the stratum pyramidale. The values of the neuronal damage were expressed as the mean value + S.E.M. Statistical analysis was performed using Mann-Whitney U-test.
45Ca autoradiography
Representative 45Ca autoradiographs are shown in Figs. 1 and 2. Incidence of a b n o r m a l calcium accumula-
TABLE I Incidence of abnormal calcium accumulation in the gerbil brain 7 days following transient or repeated ischemia
n = 10 hemispheres. RESULTS
Abnormal calcium accumulation
Behavioral status
Following each clip release, gerbils squatted without moving their limbs. The squatting posture after 2- and 3-min ischemia continued for about 2 and 5 min, respectively. O n the other hand, the animals subjected to 2 × 2 min, 3 x 1 min, 3 x 2 min and 3 x 3 min ischemic insults squatted for about 5 min, 3 min, 10 min and 1 h, respectively. The animals that exhibited severe seizure after ischemia were discarded.
Frontal cortex Striatum Hippocampus CA1 sector CA3 sector Thalamus Parietal cortex Medial geniculate body Substantia nigra Inferior colliculus
Sham
2 min
3 rain
2 × 2 min
0/10 0/10
0/10 0/10
0/10 1/10
0/10 5/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10
7/10 0/10 0/10 0/10 0/10 0/10 0/10
9/10 0/10 8/10 0/10 0/10 2/10 8/10
117 TABLE II Incidence o f abnormal calcium accumulation and distribution o f neuronal damage in the gerbil brain 7 days following repeated cerebral ischemia
Neuronal damage was graded 0-3. Values are expressed as means + S.E.M. n = 10-12 for abnormal calciumaccumulationand neuronal damage.
Frontal cortex Striatum Hippocampus CA1 sector CA3 sector Thalamus Parietal cortex Medial geniculate body Substantia nigra Inferior colliculus
Abnormal calcium accumulation
Neuronal damage
3 x I rain
3 x 2 rain
3 x 3 min
3 × I rain
3 x 2 min
3 x 3 rain
0/10 5/10
0/12 12/12
12/12 12/12
0,0 + 0.0 0.3 + 0.0
1.0 + 0.4 1.9 + 0.4*
2.6 + 0.2* 2.4 + 0.2*
0/10 0/10 0/10 0/10 0/10 0/10 0/10
12/12 0/12 12/12 0/12 10/12 5/12 12/12
12/12 12/12 12/12 12/12 12/12 12/12 12/12
0.0 + 0.0 0.0 + 0.0 0,0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0
2.7 + 0.3* 1.0 + 0.4 1.3 + 0.3* 1.0 + 0.4 0.1 + 0.1 0.6 + 0.3 0.8 + 0.3
3.0 + 0.0" 3.0 + 0.0" 1.7 + 0.2* 2.0 + 0.1' 1.8 + 0.2* 1.2 +__0.2* 1.3 + 0.2*
*P < 0.01 compared with 3 × 1 min ischemia group (Mann-Whitney U-test).
tion is summarized in Tables I and II. Sham-operated gerbils showed no abnormal calcium accumulation throughout the brain. Gerbils subjected to a single 2-min ischemia also revealed no abnormal calcium accumulation. Following a single 3-rain ischemia, abnormal calcium accumulation was shown only in the hippocampal CA1 sector in 7 of 10 hemispheres and in the striatum in one of 10 hemispheres. On the other hand, gerbils subjected to 2 x 2 min ischemic insults revealed extensive abnormal calcium accumulation in the brain. Abnormal calcium accumulation was seen in the dorsolateral part of the striatum in 5 of 10 hemispheres (figure not shown), the hippocampal CA1 sector in 9 of 10 hemispheres, the ventral part of thalamus in 8 of 10 hemispheres, the substantia nigra in 2 of 10 hemispheres (figure not shown) and the small part of inferior colliculus in 8 of 10 hemispheres. Gerbils subjected to 3 x 1 min ischemic insults revealed slightly abnormal calcium accumulation in the dorsolateral part of striatum in 5 of 10 hemispheres. But, the repeated ischemia did not produce abnormal calcium accumulation in the hippocampai CA1 sector which is most vulnerable to ischemia. In all animals subjected to 3 × 2 min
TABLE IIl Neuronal densities in the single ischemia groups
Each value was expressed as means + S.E.M.
Sham-operated 2 min ischemia 3 min ischemia
n
Neuronal density (~ram)
5 5 5
237 + 8 216 + 15 85 _+28*
*P < 0.05 compared with sham-operated group (Mann-Whitney U-test).
ischemic insults, abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus and the inferior colliculus; there was also such abnormal calcium accumulation in the medial geniculate body in 10 of 12 hemispheres and the substantia nigra in 5 of 12 hemispheres. Gerbils subjected to 3 x 3 min ischemic insults revealed most severe calcium accumulation in the brain, but the pattern of distribution was similar to that of 3 x 2 min ischemic insults except for neocortex, the septum and the hippocampal CA3 sector. Abnormal calcium accumulation was increased, and widely spread in the frontal cortex, the dorsolateral part of striatum, the septum (data not shown), the hippocampal CA1 and CA3 sector, the thalamus, the parietal cortex, the medial geniculate body, the substantia nigra and the inferior colliculus in all animals. In addition, the accumulation was not found in the cerebellum which is selectively vulnerable in the 4-vessel occlusion rat model. Histopathology
Distribution of ischemic brain damage is presented in Tables II and III. In the sham-operated group, the average neuronal density of hippocampal CA1 neurons was 237 + 8/mm. A single 2-min ischemia did not produce any significant neuronal damage in the hippocampal CA1 sector. However, gerbils subjected to single 3-min ischemia revealed mild neuronal damage compared to that of 5-min ischemia 2. Gerbils subjected to 3 x 1 min ischemic insults showed no neuronal damage throughout the brain except for the striatum. In the striatum, mild neuronal damage was seen in 3 of 10 hemispheres. This extent of neuronal damage was consistent with the area of abnormal calcium accu-
118
Fig. 3. Representative photographs in the striatum, the hippocampus and the thalamus, a: striatum, b: hippocampus and thalamus, 7 days after three l-rain ischemic insults. Many neurons are intact, c: striatum, d: hippocampus and thalamus, 7 days after three 3-min ischemic insults. Severe neuronal loss is noted in the dorsolateral part of striatum, the hippocampal CA1 sector, the hippocampal CA3 sector and the thalamus. Cresyl violet stain. Bar --- 500/~m.
Fig. 4. Histological changes in the hippocampal CA1 sector, a,d: 7 days after three 1-min ischemic insults. The hippocampal CA1 neurons are intact, b,d: 7 days after three 2-min ischemic insults. The hippocampal CA1 neurons are completely destroyed, c,d: 7 days after three 3-min ischemic insults. Almost complete neuronal cell loss is noted in the hippocampal CA1 and CA3 sector. Cresyl violet stain. Bar = 100/~m.
119 mulation. Gerbils subjected to 3 x 2 min ischemic insults caused variable brain damage. The most frequently and seriously damaged regions were the hippocampal CA1 sector, followed by the dorsolateral part of striatum, the hippocampal CA3 sector, the thalamus and the neocortex. The inferior colliculus, the substantia nigra and the medial geniculate body were slightly damaged. However, for the neuronal damage in the frontal cortex, the hippocampal CA3 sector, the parietal cortex, the medial geniculate body, the substantia nigra and the inferior colliculus, there was no statistical difference between the 3 × 1 min and 3 x 2 min ischemic insults. The distribution of the neuronal damage was seen in the sites corresponding to most of the regions of abnormal calcium accumulation as shown in the 4SCa autoradiographs. However, abnormal calcium accumulation was not found in the neocortex and the hippocampal CA3 sector where histopathological neuronal damage was seen. Gerbils subjected to 3 × 3 rain ischemic insults revealed the most severe neuronal damage in the brain. The histopathological changes were more pronounced than that of 3 x 2 rain ischemic insults. The changes in the most frequently affected regions were seen in the hippocampal CA1 sector and CA3 sector, the frontal
cortex and the dorsolateral part of striatum, followed by the parietal cortex, the medial geniculate body, the thalamus, the inferior colliculus and the substantia nigra. A few damaged neurons were also seen in the septum and the superior colliculus. The distribution and frequency of the neuronal damage following 3 x 3 rain ischemic insults mainly corresponded to that of abnormal calcium accumulation as shown in the 45Ca autoradiographs. Representative photographs in the striatum, the hippocampus, the thalamus, the substantia nigra and the inferior colliculus are shown in Figs. 3-6. Severe damage to the striatum was observed following 3 x 3 min ischemic insults. In the thalamus, marked neuronal damage, often infarction with a dense infiltration of macrophages in the periphery was found after 3 x 3 min ischemic insults. Hippocampal CA1 neurons were preserved after 3 x 1 min ischemic insults. However, almost all CA1 neurons were destroyed after 3 x 2 min ischemic insults. Furthermore, 3 × 3 min ischemic insults caused severe damage in the hippocampal CA1 sector and CA3 sector. In the brainstem, the proliferation of astrocytes and microglia with eventual formation of phagocytes was seen in the pars reticulata of substantia nigra and the inferior colliculus.
Fig. 5. Histological changes in the substantia nigra, a,c: 7 days after three 1-min ischemic insults. Neurons are intact, b,d: 7 days after three 3-rain ischemic insults. Severe neuronal damage and gliosis are noted in the pars reticulata of substantia nigra. Cresyl violet stain. Bar = 100 pm.
120
Fig. 6. Histological changes in the inferior colliculus, a,c: 7 days after three 1-min ischemic insults. Many neurons are intact, b,d: 7 days after three 3-min ischemic insults. Severe neuronal damage and gliosis are noted. Hematoxylin-eosin stain. Bar = 100 ~m. DISCUSSION The present study has demonstrated that brief but repeated forebrain ischemia in the gerbil can cause severe neuronal damage not only in the basal ganglia and the limbic system but also in the brainstem. A single 2-min ischemia is non-lethal to the brain since no neuronal damage was observed in the hippocampal CA1 sector which is most vulnerable. Gerbils subjected to a single 3-min ischemia revealed only a mild neuronal damage in the hippocampal CA1 sector. However, at least 3 x 2 min ischemic insults at 1-h intervals produced severe destruction of the brain. Among those regions, the degree of the neuronal damage was different; severe in the hippocampal CA1 sector, moderate in the dorsolateral part of striatum and the thalamus, slight in the neocortex, the hippocampal CA3 sector, the inferior colliculus, the substantia nigra and the medial geniculate body. Gerbils subjected to 3 × 3 min ischemic insults at 1-h intervals also revealed most severe neuronal damage in above cited regions. In contrast, the gerbils subjected to 3 x 1 min ischemic insults at 1-h intervals revealed a slight neuronal damage only in the striatum. Therefore, this study suggests that a threshold for development of neuronal damage after repeated ischemia may exist between 1 and 2 min.
The relationship between calcium accumulation and ischemic cell change in neural tissue is not known, but several studies have suggested that the calcium accumulation is closely related to the progression of ischemic neuronal damage 5,9,1°,21. In this study, 45Ca autoradiographic observations revealed that the distribution of abnormal calcium accumulation was seen in the sites corresponding to most of the regions of the histological neuronal damage. Therefore, 45Ca autoradiographic technique seems to be a useful approach for the diagnosis of ischemic neuronal damage because of its high visibility. However, abnormal calcium accumulation was not always found in the neocortex and the hippocampal CA3 sector where the neuronal damage was seen after ischemic insults. The reason for this is presently unclear. A definite answer to the question whether the abnormal calcium accumulation reflected the histological neuronal damage must await further investigation. It is well known that certain regions such as neocortex, hippocampus, striatum, thalamus and cerebellum are selectively vulnerable 7"12"19. The present study also suggests that repeated ischemic insults can produce severe neuronal damage in selectively vulnerable regions when it is induced repeatedly at 1-h intervals. These patterns of neuronal damage after repeated ischemia are essentially the same as those following single 10-15-min ischemia in
121 the gerbil 3, and the mechanisms of ischemic neuronal damage in repeated ischemia are partly the same as those in transient ischemia. However, there are only a few reports about histological attempts to study the neuronal damage after repeated ischemia in the brainstem 13. Interestingly, we observed clear signs of degeneration of the brainstem such as the substantia nigra and inferior colliculus. This phenomenon may account for the development of infarctions but not for selective neuronal cell loss, since these are known to have a very high glycogen content in the early postischemic period TM.The neuronal injury of the brainstem, therefore, may be due to excessive lactic acid accumulation. Four possible mechanisms may account for the cumulative effect of neuronal damage produced by repeated ischemic insults. The first is that a microcirculatory disturbance may occur if the succeeding ischemic insult is induced. Tomida et al. 23 reported that postischemic hypoperfusion, which is most prominent at 1 h after 5-min ischemia in the gerbil, may play an essential role in the cumulative effect. However, it is unclear whether the postischemic hypoperfusion takes place at 1 h after even a brief ischemia, and this remains to be elucidated. Secondly, postischemic alteration in the receptor sensitivity or in the calcium conductance may have a facilitative effect on the glutamatergic excitation induced by the succeeding insult. Kato et al. 13 found that the distribution of neuronal damage produced by repeated 2-min ischemic insults in the gerbil was mainly consistent with the vulnerable areas undergoing glutamatergic innervation. In this study, gerbils subjected to repeated ischemia revealed severe neuronal damage not only in the basal ganglia and the limbic system receiving glutamatergic innervation but also in the brainstem such as the substantia nigra and inferior colliculus which have low
levels of glutamate receptors 16. This suggests that the neuronal damage to repeated ischemia may involve factors beyond the excitotoxicity of glutamate. Thirdly, an embolic occlusion may be produced by the thrombus made in the repeated clipping site. In this study, however, it is unlikely that the embolic occlusion implicates the neuronal damage distributed uniformly over the vulnerable areas. Finally, postischemic metabolic disturbance may contribute to the cumulative effect. It is well known that protein synthesis after 5-min ischemia in the gerbil is markedly depressed in the hippocampal CA1 sector where it is destined to die 6"22. We have recently demonstrated that amino acid incorporation into proteins is depressed in the vulnerable regions for several hours after brief ischemia in the gerbil 4. This suggests that even a brief ischemic insult may produce metabolic alteration which persists for several hours after ischemia. Thus, the impairment of protein synthesis may play an important role in the pathogenesis of neuronal damage after repeated brief ischemia in the gerbil. However, further studies should be performed to elucidate the link between the inhibition of protein synthesis and the ischemic neuronal damage after repeated ischemia. In conclusion, the present study indicates that repeated brief ischemic insults can cause severe neuronal damage not only in the basal ganglia and the limbic system but also in the brainstem. Furthermore, they suggest that the cumulative effect after repeated ischemic insults is related to the time of ischemia and the number of episodes. Although the detailed mechanisms of neuronal damage produced by repeated ischemic insults remain unknown, this model may facilitate further clarification of mechanisms of repeated hemodynamic transient cerebral ischemic attacks.
REFERENCES
Greenfield's Neuropathology, Arnold, London, 1976, pp. 41-85. 8 Crain, B.J., Westerkam, W.D., Harrison, A.H. and Nadler, J.V., Selective neuronal death after transient forebrain isehemia in the Mongolian gerbil: a silver impregnation study, Neuroscience, 27 (1988) 387-402. 9 Dienel, G.A., Regional accumulation of calcium in postischemic rat brain, J. Neurochem., 43 (1984) 913-925. 10 Dienel, G.A. and Pulsinelli, W.A., Uptake of radiolabeled ions in normal and ischemia-damaged brain, Ann. Neurol., 19 (1986) 465-472. 11 Hatakeyama, T., Matsumoto, M., Brengman, J.M. and Yanagihara, T., Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in gerbils, Stroke, 19 (1988) 1526-1534. 12 Jorgersen, M.B. and Diemer, N.H., Selective neuron loss after cerebral ischemia in the rat: possible role of neurotransmitter glutamate, Acta Neurol. Scand., 66 (1982) 536-546. 13 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain Research, 479 (1989) 366-370. 14 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69.
1 Araki, T. and Kogure, K., Prevention of delayed neuronal death in gerbil hippocampus by a novel vinca alkaloid derivative (Vinconate), Mol. Chem. Neuropathol., 11 (1989) 33-43. 2 Araki, T., Kogure, K. and Izumiyama, K., Prevention of ischemic neuronal damage by al-adrenoceptor agonist (Methoxamine), Acta Neurol. Scand., 80 (1989) 451-454. 3 Araki, T., Kato, H. 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 followingbrief cerebral ischemia in the gerbil, Acta Neuropathol., 79 (1990) 501-505. 5 Benveniste, H. and Diemer, N.H., Early postischemic 45Ca accumulation in rat dentate hilus, J. Cereb. Blood Flow Metab., 8 (1988) 713-719. 6 Bodsch, W., Takahashi, K., Barbier, A., Grosse Ophoff, B. and Hossmann, K.-A., Cerebral protein synthesis and ischemia, Progr. Brain Res., 63 (1985) 197-210. 7 Brierley, J.B., Cerebral hypoxia. In W. Blackwood et al. (Eds.),
122 15 Kitagawa, K., Matsumoto, M., Ninobe, M., Mikoshiba, K., Hata, R., Ueda, H., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K. and Kamada, T., Microtubule-associated protein 2 as a sensitive marker for cerebral ischemic damage - - immunohistochemical investigation of dendritic damage, Neuroscience, 31 (1989) 401-411. 16 Monaghan, D.T. and Cotman, C.R., Distribution of N-methylD-aspartate-sensitive h-[3H]glutamate-binding sites in the rat brain, J. Neurosci., 5 (1985) 2909-2919. 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 Pulsinelli, W.A., Brierley, J.B., Duffy, T., Levy, D. and Plum, F., Ischemic neuronal damage, postischemic regional blood flow, and glucose metabolism in the rat brain, J. Cereb. Blood Flow Metab., 1 (1981) 166-167. 19 Pulsinelli, W.A., Brierley, J.B. and Plum, E, Temporal profile
20 21 22
23
of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. Pulsinelli, W.A., Selective neuronal vulnerability: morphological and molecular characteristics, Progr. Brain Res., 63 (1985) 29-37. Sakamoto, N., Kogure, K., Kato, H. and Ohtomo, H., Disturbed Ca 2+ homeostasis in the gerbil hippocampus following brief transient ischemia, Brain Research, 364 (1986) 372-376. Thilmann, R., Xie, Y., Kleihues, P. and Kiessling, M., Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus, Acta Neuropathol., 71 (1986) 88-93. Tomida, S., Nowak Jr., T.S., Vass, K., Lohr, J.M. and Klatzo, I., Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemic insults, J. Cereb. Blood Flow Metab., 7 (1987) 773-782.
114
Elsevier BRES 15868
Neuronal damage and calcium accumulation following repeated brief cerebral ischemia in the gerbil Tsutomu Araki, Hiroyuki Kato and Kyuya Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 20 March 1990)
Key words: Cerebral ischemia; Repeated ischemia; Selective vulnerability; Calcium accumulation; Gerbil
We investigated the distribution of neuronal damage following brief cerebral transient ischemia and repeated ischemia at 1-h intervals in the gerbil, using light microscopy and 45Ca autoradiography as a marker for detection of ischemic damage. The animals were allowed to survive for 7 days after ischemia induced by bilateral carotid artery occlusion. Following 2-min ischemia, neuronal damage determined by abnormal calcium accumulation was not observed in the forebrain regions. Following 3-min ischemia, however, abnormal calcium accumulation was recognized only in the hippocampal CA1 sector and part of the striatum. Two 2-min ischemic insults caused extensive abnormal calcium accumulation in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the substantia nigra and the inferior colliculus. The ischemic insults were more severe than that of a single 3-min ischemia. However, three 1-min ischemic insults caused abnormal calcium accumulation only in the striatum. On the other hand, three 2-min ischemic insults caused severe abnormal calcium accumulation in the brain. The abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus. Gerbils subjected to three 3-min ischemic insults revealed most severe abnormal calcium accumulation. Marked calcium accumulation was seen not only in the above sites, but also spread in the neocortex, the septum and the hippocampal CA3 sector. Morphological study after transient or repeated ischemia indicated that the distribution and frequency of the neuronal damage was found in the sites corresponding to most of the regions of abnormal calcium accumulation. The abnormal calcium accumulation, however, was not always found in the regions such as the neocortex and the hippocampal CA3 sector where the neuronal damage was seen. The present study demonstrates that repeated ischemic insults at 1-h intervals can produce severe neuronal damage not only in the basal ganglia and the limbic system but also in the brainstem. Furthermore, they suggest that the cumulative effects after repeated ischemic insults are related to the time of ischemia or the number of episodes.
INTRODUCTION Transient c e r e b r a l ischemia causes variable brain d a m a g e according to its degree and duration. N e u r o n s k n o w n to be selectively vulnerable are found in certain regions such as neocortex, striatum, h i p p o c a m p u s and t h a l a m u s 7'19. This p h e n o m e n o n has been t e r m e d selectively n e u r o n a l vulnerability. A m o n g selectively vulnerable regions, t h e r e is a further hierarchy of susceptibility to ischemic injury 2°. H i p p o c a m p a l CA1 neurons are especially susceptible to ischemia 14'19, and the neurons are lost following 3-min ischemia in the gerbil 1. T o m i d a et al. 23 recently found that r e p e a t e d cerebral ischemia has a cumulative effect since the n e u r o n a l d a m a g e is g r e a t e r following 3 episodes of 5-min ischemia at 1-h intervals in the gerbil than 15-min ischemia p r o d u c e d as a single insult. F o r this reason, they have suggested that the presence of postischemic hypoperfusion m a y play an i m p o r t a n t role in the cumulative effect of the r e p e a t e d ischemia. On the other hand, K a t o et
al. 13 have shown that the distribution of the n e u r o n a l injury p r o d u c e d by 3 o r 5 r e p e a t e d non-lethal (2-min) ischemic insults at 1-h intervals in the gerbil was consistent with the selectively v u l n e r a b l e areas undergoing g l u t a m a t e innervation. F u r t h e r m o r e , N a k a n o et al. 17 found that the n e u r o n a l d a m a g e is most severe when non-lethal ischemia is r e p e a t e d at 1-h intervals, and is relatively mild when r e p e a t e d at shorter or longer intervals. These observations suggest that the intervals and the episodes of ischemic insult play an i m p o r t a n t factor on the ischemic brain d a m a g e . We recently found that brief transient ischemia causes a severe i m p a i r m e n t of p r o t e i n synthesis in the selectively vulnerable areas 4. T h e inhibition is especially found at an early stage of recirculation after ischemia. This suggests that even a brief ischemic insult causes metabolic alteration which persists for a few hours after ischemia, and the succeeding insult m a y injure the brain. F u r t h e r m o r e , although the m o r p h o l o g i c p a t t e r n of selective vulnerability of transient ischemia has b e e n established in
Correspondence: T. Araki, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal, Miyagi 980, Japan. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
115 detail 8'11'12'15'19, t h e r e histological
attempts
are to
o n l y a few r e p o r t s
examine
the
neuronal damage following repeated Therefore,
the purpose
about
distribution
of
b r i e f i s c h e m i a 13.
of t h e p r e s e n t
study was to
i n v e s t i g a t e t h e d i s t r i b u t i o n of n e u r o n a l d a m a g e f o l l o w i n g r e p e a t e d b r i e f c e r e b r a l i s c h e m i a in t h e gerbil. F o r this p u r p o s e , w e e m p l o y e d light m i c r o s c o p y and 45Ca a u t o r a d i o g r a p h y to d e t e c t a n d visualize i s c h e m i c n e u r o n a l damage.
MATERIALS AND METHODS
Experimental procedures Adult male Mongolian gerbils weighing 60-95 g were used. They were anesthetized with 2% halothane in a mixture of 30% 0 2 and 70% N20. Bilateral common carotid arteries were exposed and occluded with aneurysm clips, and the animals were allowed to survive for 7 days after ischemia. In order to prevent blood coagulation at the clipping site, 100 IU of heparin was injected i.p. 10 rain before bilateral carotid artery occlusion. Sham-operated animals were also investigated in the same manner except for clipping the bilateral common carotid arteries. In the present study the animals were kept on a thermostatted warming mat (at 37-38 °C) following the procedure until they began to move again; they were then given food and water ad libitum.
Sham
2 min
45Ca autoradiography Thirty-seven animals were randomly assigned to 7 groups containing 5-6 gerbils for 45Ca autoradiography. Five animals were used as the sham-operated group. The remaining 32 animals were divided into 6 experimental groups which were subjected to three 1-min (3 x 1 rain), three 2-min (3 x 2 min), three 3-min (3 x 3 min) and two 2-min (2 × 2 min) bilateral carotid artery occlusions at 1-h intervals or to a single 2-min and 3-min bilateral carotid artery occlusion. 45Ca autoradiography was performed according to the method of Sakamoto et al. 21. Seven days following ischemia , the animals were anesthetized with 2% halothane in a mixture of 30% 0 2 and 70% N20. A bolus of 100 pCi of 45CAC12 (spec. act.: 10-40 mCi/mg, Amersham) was injected i.v. The animals were anesthetized with ether at 5 h after injection of 45CAC12; their brains were quickly removed and frozen in powdered dry ice. Serial coronal sections 20-pm thick were cut from the frozen brain in a cryostat at -20 °C. Serial sections were exposed to Kodak NMC-1 film for 2 weeks, and were used for the evaluation of autoradiography.
Histopathology Thirty-one animals were randomly assigned to 6 groups containing 5-6 gerbils. Five animals used as sham-operated group. Transient brief ischemia was induced for 2 and 3 min. Three 1-min (3 × 1 min), 2-min (3 × 2 min) and 3-min (3 x 3 min) bilateral carotid artery occlusions were repeated at 1-h intervals. Seven days following ischemia, the animals were anesthetized with pentobarbital 50 mg/kg i.p. The brains were perfusion-fixed transcardially with 10% formalin for 20 min. The brains removed were immersed in the same fixative, and then embedded in paraffin. Five /zm paraffin sections were stained with Cresyl violet and hematoxylin-
3 min
2 x 2 min
Fig. 1. Representative 45Ca autoradiograms. 45CAC12was injected at 7 days after ischemia and was allowed to circulate for 5 h. Sham, sham-operated group. No abnormal calcium accumulation is noted. 2 min, 2-min ischemia. Abnormal calcium accumulation is not found in the brain. 3 min, 3-min ischemia. Abnormal calcium accumulation is noted in a small part of striatum (unilateral) and the hippocampal CA1 sector. 2 × 2 min, two episodes of 2-min ischemia. Abnormal calcium accumulation is noted in the hippocampal CA1 sector, the ventral part of thalamus and the inferior colliculus.
116
3 x l min
3 x 2 min
3 x 3 min
Fig. 2. Representative 45Ca autoradiograms. 45CAC12was injected at 7 days after ischemia and was allowed to circulate for 5 h. 3 x 1 min, 3 episodes of 1-min ischemia. Slightly abnormal calcium accumulation is noted in a small part of striatum (unilateral). 3 × 2 min, 3 episodes of 2-min ischemia. Abnormal calcium accumulation is noted in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus, the medial geniculate body, a small part of the substantia nigra and the inferior colliculus. 3 × 3 min, 3 episodes of 3-rain ischemia. Marked calcium accumulation is noted in the neocortex, the striatum, the septum, the hippocampal CA1 sector and CA3 sector, the thalamus, the medial geniculate body, the substantia nigra and the inferior colliculus.
eosin. The sections were examined with the light microscope, and ischemic neuronal damage was graded on a semiquantitative scale: 0, normal; 1, a few neurons damaged; 2, many neurons damaged; 3, majority of neurons damaged, according to the method of Pulsinelli et al. 19. For transient ischemia, they were examined with the light microscope and the neuronal damage in the hippocampus was evaluated by determination of neuronal density of CA1 neurons per 1 mm linear length of the stratum pyramidale. The values of the neuronal damage were expressed as the mean value + S.E.M. Statistical analysis was performed using Mann-Whitney U-test.
45Ca autoradiography
Representative 45Ca autoradiographs are shown in Figs. 1 and 2. Incidence of a b n o r m a l calcium accumula-
TABLE I Incidence of abnormal calcium accumulation in the gerbil brain 7 days following transient or repeated ischemia
n = 10 hemispheres. RESULTS
Abnormal calcium accumulation
Behavioral status
Following each clip release, gerbils squatted without moving their limbs. The squatting posture after 2- and 3-min ischemia continued for about 2 and 5 min, respectively. O n the other hand, the animals subjected to 2 × 2 min, 3 x 1 min, 3 x 2 min and 3 x 3 min ischemic insults squatted for about 5 min, 3 min, 10 min and 1 h, respectively. The animals that exhibited severe seizure after ischemia were discarded.
Frontal cortex Striatum Hippocampus CA1 sector CA3 sector Thalamus Parietal cortex Medial geniculate body Substantia nigra Inferior colliculus
Sham
2 min
3 rain
2 × 2 min
0/10 0/10
0/10 0/10
0/10 1/10
0/10 5/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10
0/10 0/10 0/10 0/10 0/10 0/10 0/10
7/10 0/10 0/10 0/10 0/10 0/10 0/10
9/10 0/10 8/10 0/10 0/10 2/10 8/10
117 TABLE II Incidence o f abnormal calcium accumulation and distribution o f neuronal damage in the gerbil brain 7 days following repeated cerebral ischemia
Neuronal damage was graded 0-3. Values are expressed as means + S.E.M. n = 10-12 for abnormal calciumaccumulationand neuronal damage.
Frontal cortex Striatum Hippocampus CA1 sector CA3 sector Thalamus Parietal cortex Medial geniculate body Substantia nigra Inferior colliculus
Abnormal calcium accumulation
Neuronal damage
3 x I rain
3 x 2 rain
3 x 3 min
3 × I rain
3 x 2 min
3 x 3 rain
0/10 5/10
0/12 12/12
12/12 12/12
0,0 + 0.0 0.3 + 0.0
1.0 + 0.4 1.9 + 0.4*
2.6 + 0.2* 2.4 + 0.2*
0/10 0/10 0/10 0/10 0/10 0/10 0/10
12/12 0/12 12/12 0/12 10/12 5/12 12/12
12/12 12/12 12/12 12/12 12/12 12/12 12/12
0.0 + 0.0 0.0 + 0.0 0,0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0
2.7 + 0.3* 1.0 + 0.4 1.3 + 0.3* 1.0 + 0.4 0.1 + 0.1 0.6 + 0.3 0.8 + 0.3
3.0 + 0.0" 3.0 + 0.0" 1.7 + 0.2* 2.0 + 0.1' 1.8 + 0.2* 1.2 +__0.2* 1.3 + 0.2*
*P < 0.01 compared with 3 × 1 min ischemia group (Mann-Whitney U-test).
tion is summarized in Tables I and II. Sham-operated gerbils showed no abnormal calcium accumulation throughout the brain. Gerbils subjected to a single 2-min ischemia also revealed no abnormal calcium accumulation. Following a single 3-rain ischemia, abnormal calcium accumulation was shown only in the hippocampal CA1 sector in 7 of 10 hemispheres and in the striatum in one of 10 hemispheres. On the other hand, gerbils subjected to 2 x 2 min ischemic insults revealed extensive abnormal calcium accumulation in the brain. Abnormal calcium accumulation was seen in the dorsolateral part of the striatum in 5 of 10 hemispheres (figure not shown), the hippocampal CA1 sector in 9 of 10 hemispheres, the ventral part of thalamus in 8 of 10 hemispheres, the substantia nigra in 2 of 10 hemispheres (figure not shown) and the small part of inferior colliculus in 8 of 10 hemispheres. Gerbils subjected to 3 x 1 min ischemic insults revealed slightly abnormal calcium accumulation in the dorsolateral part of striatum in 5 of 10 hemispheres. But, the repeated ischemia did not produce abnormal calcium accumulation in the hippocampai CA1 sector which is most vulnerable to ischemia. In all animals subjected to 3 × 2 min
TABLE IIl Neuronal densities in the single ischemia groups
Each value was expressed as means + S.E.M.
Sham-operated 2 min ischemia 3 min ischemia
n
Neuronal density (~ram)
5 5 5
237 + 8 216 + 15 85 _+28*
*P < 0.05 compared with sham-operated group (Mann-Whitney U-test).
ischemic insults, abnormal calcium accumulation was found in the dorsolateral part of striatum, the hippocampal CA1 sector, the thalamus and the inferior colliculus; there was also such abnormal calcium accumulation in the medial geniculate body in 10 of 12 hemispheres and the substantia nigra in 5 of 12 hemispheres. Gerbils subjected to 3 x 3 min ischemic insults revealed most severe calcium accumulation in the brain, but the pattern of distribution was similar to that of 3 x 2 min ischemic insults except for neocortex, the septum and the hippocampal CA3 sector. Abnormal calcium accumulation was increased, and widely spread in the frontal cortex, the dorsolateral part of striatum, the septum (data not shown), the hippocampal CA1 and CA3 sector, the thalamus, the parietal cortex, the medial geniculate body, the substantia nigra and the inferior colliculus in all animals. In addition, the accumulation was not found in the cerebellum which is selectively vulnerable in the 4-vessel occlusion rat model. Histopathology
Distribution of ischemic brain damage is presented in Tables II and III. In the sham-operated group, the average neuronal density of hippocampal CA1 neurons was 237 + 8/mm. A single 2-min ischemia did not produce any significant neuronal damage in the hippocampal CA1 sector. However, gerbils subjected to single 3-min ischemia revealed mild neuronal damage compared to that of 5-min ischemia 2. Gerbils subjected to 3 x 1 min ischemic insults showed no neuronal damage throughout the brain except for the striatum. In the striatum, mild neuronal damage was seen in 3 of 10 hemispheres. This extent of neuronal damage was consistent with the area of abnormal calcium accu-
118
Fig. 3. Representative photographs in the striatum, the hippocampus and the thalamus, a: striatum, b: hippocampus and thalamus, 7 days after three l-rain ischemic insults. Many neurons are intact, c: striatum, d: hippocampus and thalamus, 7 days after three 3-min ischemic insults. Severe neuronal loss is noted in the dorsolateral part of striatum, the hippocampal CA1 sector, the hippocampal CA3 sector and the thalamus. Cresyl violet stain. Bar --- 500/~m.
Fig. 4. Histological changes in the hippocampal CA1 sector, a,d: 7 days after three 1-min ischemic insults. The hippocampal CA1 neurons are intact, b,d: 7 days after three 2-min ischemic insults. The hippocampal CA1 neurons are completely destroyed, c,d: 7 days after three 3-min ischemic insults. Almost complete neuronal cell loss is noted in the hippocampal CA1 and CA3 sector. Cresyl violet stain. Bar = 100/~m.
119 mulation. Gerbils subjected to 3 x 2 min ischemic insults caused variable brain damage. The most frequently and seriously damaged regions were the hippocampal CA1 sector, followed by the dorsolateral part of striatum, the hippocampal CA3 sector, the thalamus and the neocortex. The inferior colliculus, the substantia nigra and the medial geniculate body were slightly damaged. However, for the neuronal damage in the frontal cortex, the hippocampal CA3 sector, the parietal cortex, the medial geniculate body, the substantia nigra and the inferior colliculus, there was no statistical difference between the 3 × 1 min and 3 x 2 min ischemic insults. The distribution of the neuronal damage was seen in the sites corresponding to most of the regions of abnormal calcium accumulation as shown in the 4SCa autoradiographs. However, abnormal calcium accumulation was not found in the neocortex and the hippocampal CA3 sector where histopathological neuronal damage was seen. Gerbils subjected to 3 × 3 rain ischemic insults revealed the most severe neuronal damage in the brain. The histopathological changes were more pronounced than that of 3 x 2 rain ischemic insults. The changes in the most frequently affected regions were seen in the hippocampal CA1 sector and CA3 sector, the frontal
cortex and the dorsolateral part of striatum, followed by the parietal cortex, the medial geniculate body, the thalamus, the inferior colliculus and the substantia nigra. A few damaged neurons were also seen in the septum and the superior colliculus. The distribution and frequency of the neuronal damage following 3 x 3 rain ischemic insults mainly corresponded to that of abnormal calcium accumulation as shown in the 45Ca autoradiographs. Representative photographs in the striatum, the hippocampus, the thalamus, the substantia nigra and the inferior colliculus are shown in Figs. 3-6. Severe damage to the striatum was observed following 3 x 3 min ischemic insults. In the thalamus, marked neuronal damage, often infarction with a dense infiltration of macrophages in the periphery was found after 3 x 3 min ischemic insults. Hippocampal CA1 neurons were preserved after 3 x 1 min ischemic insults. However, almost all CA1 neurons were destroyed after 3 x 2 min ischemic insults. Furthermore, 3 × 3 min ischemic insults caused severe damage in the hippocampal CA1 sector and CA3 sector. In the brainstem, the proliferation of astrocytes and microglia with eventual formation of phagocytes was seen in the pars reticulata of substantia nigra and the inferior colliculus.
Fig. 5. Histological changes in the substantia nigra, a,c: 7 days after three 1-min ischemic insults. Neurons are intact, b,d: 7 days after three 3-rain ischemic insults. Severe neuronal damage and gliosis are noted in the pars reticulata of substantia nigra. Cresyl violet stain. Bar = 100 pm.
120
Fig. 6. Histological changes in the inferior colliculus, a,c: 7 days after three 1-min ischemic insults. Many neurons are intact, b,d: 7 days after three 3-min ischemic insults. Severe neuronal damage and gliosis are noted. Hematoxylin-eosin stain. Bar = 100 ~m. DISCUSSION The present study has demonstrated that brief but repeated forebrain ischemia in the gerbil can cause severe neuronal damage not only in the basal ganglia and the limbic system but also in the brainstem. A single 2-min ischemia is non-lethal to the brain since no neuronal damage was observed in the hippocampal CA1 sector which is most vulnerable. Gerbils subjected to a single 3-min ischemia revealed only a mild neuronal damage in the hippocampal CA1 sector. However, at least 3 x 2 min ischemic insults at 1-h intervals produced severe destruction of the brain. Among those regions, the degree of the neuronal damage was different; severe in the hippocampal CA1 sector, moderate in the dorsolateral part of striatum and the thalamus, slight in the neocortex, the hippocampal CA3 sector, the inferior colliculus, the substantia nigra and the medial geniculate body. Gerbils subjected to 3 × 3 min ischemic insults at 1-h intervals also revealed most severe neuronal damage in above cited regions. In contrast, the gerbils subjected to 3 x 1 min ischemic insults at 1-h intervals revealed a slight neuronal damage only in the striatum. Therefore, this study suggests that a threshold for development of neuronal damage after repeated ischemia may exist between 1 and 2 min.
The relationship between calcium accumulation and ischemic cell change in neural tissue is not known, but several studies have suggested that the calcium accumulation is closely related to the progression of ischemic neuronal damage 5,9,1°,21. In this study, 45Ca autoradiographic observations revealed that the distribution of abnormal calcium accumulation was seen in the sites corresponding to most of the regions of the histological neuronal damage. Therefore, 45Ca autoradiographic technique seems to be a useful approach for the diagnosis of ischemic neuronal damage because of its high visibility. However, abnormal calcium accumulation was not always found in the neocortex and the hippocampal CA3 sector where the neuronal damage was seen after ischemic insults. The reason for this is presently unclear. A definite answer to the question whether the abnormal calcium accumulation reflected the histological neuronal damage must await further investigation. It is well known that certain regions such as neocortex, hippocampus, striatum, thalamus and cerebellum are selectively vulnerable 7"12"19. The present study also suggests that repeated ischemic insults can produce severe neuronal damage in selectively vulnerable regions when it is induced repeatedly at 1-h intervals. These patterns of neuronal damage after repeated ischemia are essentially the same as those following single 10-15-min ischemia in
121 the gerbil 3, and the mechanisms of ischemic neuronal damage in repeated ischemia are partly the same as those in transient ischemia. However, there are only a few reports about histological attempts to study the neuronal damage after repeated ischemia in the brainstem 13. Interestingly, we observed clear signs of degeneration of the brainstem such as the substantia nigra and inferior colliculus. This phenomenon may account for the development of infarctions but not for selective neuronal cell loss, since these are known to have a very high glycogen content in the early postischemic period TM.The neuronal injury of the brainstem, therefore, may be due to excessive lactic acid accumulation. Four possible mechanisms may account for the cumulative effect of neuronal damage produced by repeated ischemic insults. The first is that a microcirculatory disturbance may occur if the succeeding ischemic insult is induced. Tomida et al. 23 reported that postischemic hypoperfusion, which is most prominent at 1 h after 5-min ischemia in the gerbil, may play an essential role in the cumulative effect. However, it is unclear whether the postischemic hypoperfusion takes place at 1 h after even a brief ischemia, and this remains to be elucidated. Secondly, postischemic alteration in the receptor sensitivity or in the calcium conductance may have a facilitative effect on the glutamatergic excitation induced by the succeeding insult. Kato et al. 13 found that the distribution of neuronal damage produced by repeated 2-min ischemic insults in the gerbil was mainly consistent with the vulnerable areas undergoing glutamatergic innervation. In this study, gerbils subjected to repeated ischemia revealed severe neuronal damage not only in the basal ganglia and the limbic system receiving glutamatergic innervation but also in the brainstem such as the substantia nigra and inferior colliculus which have low
levels of glutamate receptors 16. This suggests that the neuronal damage to repeated ischemia may involve factors beyond the excitotoxicity of glutamate. Thirdly, an embolic occlusion may be produced by the thrombus made in the repeated clipping site. In this study, however, it is unlikely that the embolic occlusion implicates the neuronal damage distributed uniformly over the vulnerable areas. Finally, postischemic metabolic disturbance may contribute to the cumulative effect. It is well known that protein synthesis after 5-min ischemia in the gerbil is markedly depressed in the hippocampal CA1 sector where it is destined to die 6"22. We have recently demonstrated that amino acid incorporation into proteins is depressed in the vulnerable regions for several hours after brief ischemia in the gerbil 4. This suggests that even a brief ischemic insult may produce metabolic alteration which persists for several hours after ischemia. Thus, the impairment of protein synthesis may play an important role in the pathogenesis of neuronal damage after repeated brief ischemia in the gerbil. However, further studies should be performed to elucidate the link between the inhibition of protein synthesis and the ischemic neuronal damage after repeated ischemia. In conclusion, the present study indicates that repeated brief ischemic insults can cause severe neuronal damage not only in the basal ganglia and the limbic system but also in the brainstem. Furthermore, they suggest that the cumulative effect after repeated ischemic insults is related to the time of ischemia and the number of episodes. Although the detailed mechanisms of neuronal damage produced by repeated ischemic insults remain unknown, this model may facilitate further clarification of mechanisms of repeated hemodynamic transient cerebral ischemic attacks.
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