Brain Research, 586 (1992) 121-124 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
121
BRES 25254
Protection of rat hippocampus against ischemic neuronal damage by pretreatment with sublethal ischemia Y o n g Liu, H i r o y u k i K a t o , N a o k i N a k a t a a n d K y u y a K o g u r e Department of Neurology, Institute of Brain Diseases, Tohoku Unh,ersity School of Medicine, Sendal (Japan) (Accepted 31 March 1992)
Key words: Cerebral ischemia; Hippocampus; lschemic brain damage; Heat shock protein; Tolerance; Stress response; Rat
We examined whether preconditioning with sublethal ischemia protects against neuronal damage following subsequent lethal ischemic insults. Forebrain ischemia for 3 min in Wistar rats increased heat shock protein-70 immunoreactivity in the hippocampal CAI subfield but produced no neuronal damage. Preconditioning with 3 rain of ischemia followed by 3 days of reperfusion protected against hippocampal CA! neuronal damage followin$ 6 ;~ml 8 rain of ischemia but not damage after 10 min of ischemia. The result strongly suggests that stress response induced by sublethal ischemia protects against ischemic brain damage.
Even a few minutes of cerebral ischemia produces necrosis of certain neuronal populations in both humans and experimental animals ~'m7'18.These selectively vulnerable neurons are found in the hippocampus, striatum, thalamus, certain layers of cerebral corte~, and the cerebellar cortex ~s'2°'22. The CAI subfield of the hippocampus is especially susceptible to ischemia and neurons are lost following only 3 rain of ischemia in gerbils s. Cells exposed to various types of stress acquire resistance or tolerance to subsequent lethal insults. Cells become resistant to hyperthermia following exposure to heat, ethanol, hypoxia and heavy metals 4'n. Retinal neurons exposed to elevated temperature greatly enhance their tolerance to light-induced retinal injury ~. Elevated temperature before ischemia also enhances hippocampal CA1 neuronal survival after subsequent lethal ischemia in rats and gerbils 3'~°. Various harmful forms of stress such as heat, ischemia, trauma, seizures, physical or chemical agents make cells produce a family of proteins referred to as the heat shock proteins (HSPs) 2'12'~3'19.The induction of tolerance has been reported to be correlated with the systhesis of HSPs II. However, the mechanism of the protective effects of preliminary exposure to stress is not fully
understood. We, therefore, examined whether brief and sublethal ischemia which induces HSP synthesis protects against subsequent ischemic insults. For this purpose, we used a forebrain ischemia model in rats and induced various periods of ischemia 3 days after pretreatment with sublethal ischemia. We used male adult Wistar rats (Nippon Clea, Tokyo, Japan) weighing 260-300 g. Forebrain ischemia was induced by 4-vessel occlusion as described by Puisinelli et al. I~, Rats were anesthetized with pentobarbital (50 mg/kg i.p.) and the vertebral arteries were electrocauterized. On the following day, common carotid arteries were exposed under anesthesia with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide. The arteries were then occluded with aneurysm clips. Carotid artery blood flow was restored by releasing the clips following 3 min of occlusion. Three days later, the arteries were again occluded for 6, 8 and 10 rain. Animals in single ischemia groups received sham operations followed by the same periods of ischemia. Animals were also subjected to single 3 min of ischemia or sham operations. Rectal temperature was maintained at close to 37.0°C during and after ischemia. Cessation of electroencephalogram (EEG) activity was confirmed during ischemic insults. Behav-
Correspondence: Y. Liu, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan. Fax: 81-22-272-5818.
122 ior during and after ischemia was also carefully observed. One week after the second ischemia, the brains were perfusion-fixed with 4% paraformaladehyde in 0.1 M phosphate buffer (pH 7.4). Paraffin sections, 5 /zm thick, were prepared and stained with Cresyl violet and hematoxylin-eosin. The sections were examined with a light microscope and the neuronal density of the hippocampal CAI subfield was determined in a blinded fashion. Statistical significance was analyzed using the KruskaI-Wallis test followed by Williams-Wilcoxon rank sum test, and the Mann-Whitney U-test. For immunohistochemical staining of 70-kDa heatshock protein (HSP70), the brains were perfusion-fixed with 4% paraforamaladehyde in phosphate-buffered saline (PBS) 3 days after 3 rain of ischemia or sham operation and immersed in 30% sucrose. Frozen sections, 20 #m thick, were prepared stained for HSP70 using a mouse monoclonal antibody raised against HSP70 (Amersham, RPN 1197) and a Vectastain elite ABC kit (Vector Laboratories) as described by Vass et al. '1. Each group contained 4 animals. Neuronal density of the hippocampal CAI subfield is shown in Fig. 1. TIle CAI neuronal density of sham-openltcd animals was 168 + 6.4/ram (n = 5). Single 3 rain of ischemia caused no reduction in the neuronal density (168 + 13.9/ram; n - 10), Sham-operation followed by 6, 8 and 10 rain of ischemia produced moderate to severe reductions in the number of CAI pyramidal cells depending on the length of ischomia, which was 31 ± 17,5 (n ~ 9), 21 ± 17,2 (n ~, 7) and 14 ± 8.l/ram (n ~ 6), respectively, By contrast, CAI pyramidal cells wore preserved in animals subjected to pretreatment with 3 rain of ischemia. The neuronal density in 6.rain and 8-rain subgroups was 154 ± 19,6 (n ~ 11) and 88 ± 56.8/mm (n = 9), respectively. The neuronal density of 55 :t: 55.8/mm (n = 10) in the 10rain double ischemia subgroup was not significantly different from that in single 10.rain ischemia group, Immunohistochemical staining revealed definite increase in HSP70 in neurons in the hippocampal CA I subfield 3 days after 3 rain of cerebral ischemia (Fig. 2). The expression of HSP70 was seen in the whole CAI subfield in 4 hippocampi and was restricted to the CAla and b subregions in 4 hippocampi, The present result clearly demonstrated that proconditioning with sublethal ischemia, which permits recovery without any morphological damage but induces HSP70 synthesis, protects the brain against subsequent lethal ischemic insults. Three-min ischemia produced no reductions in the hippocampal CAI pyramidal neurons and therefore is a sublethal insult, More than 80% of the CAI pyramidal cells were destroyed following 6 rain of ischomia, but more than 90% of the
neurons survived following preconditioning. Eight-min ischemia destroyed 87% of the CA1 neurons, but more than 50% of the neurons survived following preconditioning. However, the preconditioning did not protect against neuronal damage following 10 min of ischemia which destroyed more than 90% of the CA1 neurons. Thus, the protection offered by HSP70 induction was not absolutely powerful. The intervening period between ischemic insults is a critical factor as is the duration of the ischemia itself. We chose the 3-min ischemia and 3-day interval as the conditioning insult and intervening wait, respectively, because of our previous results. We reported that single 3-rain ischemia in rats does not produce any neuronal damage in the hippocampal CAI sector but produces cumulative neuronal damage when repeated at l-h intervals ~4, and that gerbils pretreated with sublethal ischemia acquire resistance to subsequent lethal length of ischemia rendered 1-7 days later s. We confirmed that EEG became isoeloctric during isehemia and that animals were unresponsive to stimuli and the pupils were dilated and unresponsive to light during ischemia in all animals used in this study. Therefore, the protective effects are not the result of incomplete ischomia although the second ischemic insults wore induced four days after ¢loctrocoagulation of the vertebral arteries, We also monitorod and mainrained rectal temperature at 37,0°C during and up to 2 h after ischomia, The protection, therefor0, is not caused by hypothermia, Recently, the same protective phenomenon, 'ischemic tolerance', was reported in gorbils. Gerbils
sham 3min sluml ~Smln )min*6mln sham+Stain
3mln+Smln st~ml* IOmin ,train ~ IOmin !
0
I00
200 {/nun)
Fig, I, Neuronal density (/ram) of the CAI subfield of the hippocampus, The neuronal density is not reduced following 3 rain of ischemia but is significantly reduced following 6, 8 and l0 rain of ischemia. Preconditioning with 3 rain of ischemia resulted in significant preservation of CAI neurons following subsequent 6 and 8 rain of ischemia, * P < 0,05, * * P < 0.01 vs. corresponding single-ischemia subgroups (Mann-Whitney U-test). *# P < 0.01 vs. sham operation (Williams-Wilcoxon rank sum test).
123
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*
•
:"
qD
.
°.
Fill. 2. Immunohistochemtcal stainin8 of 70-kDa heat shock protein (HSPT0) in rat hippocampus 3 days after sham operation (a) and 3 rain of ischemia (b), Note conspicuous HSP70 immunoreactivity in CA1 following ischemia (b), Bar ~. 0,5 ram.
subjected to 3-5 min of forebrain isehemia 1-4 days after 2 min of sublethal ischemia do not develop neuronal damage in the hippocampal CAI sector although a single ischemic insult always leads to severe hippocampal CAI neuronal loss 5'8'9. Furthermore, exposure to hyperthermia protects the brain against subsequent lethal ischemia in rats and gerbils3'm°. However, the mechanism of tolerance induction is not fully understood although a role of stress response is suggested. The tolerance takes plar'e coincidentally with heat shock/stress protein induction in the surviving neurons t.s. Experimental evidence indicates that sublethal isehemia induces conspicuous HSP70 gene expression s,n6 as shown in this study. Reports showing that HSP70 expression following 10 min of ischemia in gerbils is minimal in CA1 neurons which are destined to die but intense in surviving CA3 and dentate gyrus neurons 2n support this view. However, the tolerance phenomenon may not explained solely by HSP70 be-
cause Kirino et al, s reported that the temporal profile of the protective effects was not the same as that of HSP70 induction, and others also have shown that the pattern of HSP70 expression after ischemia has a poor correlation with histologic outcome ¢''n5. In conclusion, the present result indicates that even sublethal ischemia induces tolerance to subsequent lethal length of ischemia when such ischemia is stressful enough to induce HSP synthesis. Further study is warranted because the elucidation of the mechanism may provide a clue for the treatment and prevention of sti'oke in man. The authors would like to thank Ms. Rumiko Tanaka for her excellent technical assistance.
References 1 Barbe, M.F., Tytell, M., Grower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 (1988) 1817-1820.
2 Brown, I.R., Rush, S. and Ivy, G.O., Induction of heat shock gene at the site of tissue injury in the rat brain, Neuron, 2 (1989) 1559- ! 564. 3 Chopp, M., Chen, H., Ho, K.-L., Dereski, M.O., Brown, E., Hetzel, F.W. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat, Neurology, 39 (1989) 1396-1398. 4 Gerner, E.W. and Schneider, M.J., Induced thermal resistance in hela cells, Nature, 256 (1975) 500-502. 5 Kato, H., Liu, Y., Araki, T. and Kogure, K., Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects, Brain Res., 553 ( 1991) 238-242. 6 Kiessling, M., Dienel, G.A., Jacewicz, M. and Pulsinelli, W.A., Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis, J. Cereb. Blood Flow Metab., 6 (1986) 642-649. 7 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982)57-69. 8 Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to i~hemia in gerbil hippocampal neurons, J. Cereb. Blood Flow Metab., I I (1991) 299-307. 9 Kitagawa, K., Matsumoto, M., Tagaya. M., Hata, R., Ueda. H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K. and Kamada, T., 'lschemic tolerance' phenomenon found in the brain, Brain Rt,s., 528 (1990) 21-24. 10 Kitagawa, K., Matsumoto, M., Tagaya, M., Kuwabara, K., Hata, R., Handa, N., Fukunaga, R., Kimura, K. and Kamata, T., Hyperthermia-induced neuronal protection against ischemic injury in gerbils, J. Cereb. Blood Flow Metab., 11 (1991) 449-452. II Li, G.C. and Werb, Z., Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese
hamster fibroblasts, Proc. Natl. Acad. Sci. USA, 79 (1982) 32183222. 12 Lindquist, S., The heat-shock response, Annu. Rel'. Biochem., 55 (1986) 1151-1191. 13 Lowenstein, D.H., Gonzalez, M., Simon, R.P. and Sharp, F., The pattern of 72-kDa heat stock protein-like immunoreactivity in the rat brain following fluorothyi-induced status epilepticus, Brain Res., 531 (1990) 173-182. 14 Nakano, S., Kato, H. and Kogure, K., Neuronal damage in the rat hippocampus in a new model of repeated reversible transient cerebral ischemia, Brain Res., 490 (1989) 178-180. 15 Nowak, Jr., T.S., Synthesis of a stress protein following transient ischemia in the gerbil, J. Neurochern., 45 (1985) 1635-1641. 16 Nowak, Jr., T.S., Localization of 70-kDa stress protein mRNA induction in gerbil brain after ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 432-439. 17 Petito, C.K., Feldmann, E., Pulsinelli, W.A. and Plum, F., Delayed hippocampaldamagein humansfollowing cardiorespiratory arrest, Neurology, 37 (1987) 128! - 1286. 18 Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. NeuroL, I 1 (1982) 491-498. 19 Simon, R.P., Cho, H., Gwinn, R. and Lowenstein, D.H., The temporal profile of 72-kDa heat-shock protein expression following global ischemia, J. Neurosci., 11 (1991)881-889. 20 Smith, M.-L., Auer, R.N. and Siesj6, B.K., The density and distribution of ischemic brain injury in the rat following 2-10 rain of forebrain ischemia, Acta Neuropathol., 64 (1984) 319-332. 21 Vass, K., Welch, W.J. and Nowak, Jr., T.S., Localization of 70-kDa stress protein induction in gerbil brain after ischemia, Acta Neuropathol., 77 (1988) 128-135. 22 Wieloch, T., Neurochemical correlates to selective neuronal vulnerability, Frog. Brain Res,, 63 (1985) 69-85,
121
BRES 25254
Protection of rat hippocampus against ischemic neuronal damage by pretreatment with sublethal ischemia Y o n g Liu, H i r o y u k i K a t o , N a o k i N a k a t a a n d K y u y a K o g u r e Department of Neurology, Institute of Brain Diseases, Tohoku Unh,ersity School of Medicine, Sendal (Japan) (Accepted 31 March 1992)
Key words: Cerebral ischemia; Hippocampus; lschemic brain damage; Heat shock protein; Tolerance; Stress response; Rat
We examined whether preconditioning with sublethal ischemia protects against neuronal damage following subsequent lethal ischemic insults. Forebrain ischemia for 3 min in Wistar rats increased heat shock protein-70 immunoreactivity in the hippocampal CAI subfield but produced no neuronal damage. Preconditioning with 3 rain of ischemia followed by 3 days of reperfusion protected against hippocampal CA! neuronal damage followin$ 6 ;~ml 8 rain of ischemia but not damage after 10 min of ischemia. The result strongly suggests that stress response induced by sublethal ischemia protects against ischemic brain damage.
Even a few minutes of cerebral ischemia produces necrosis of certain neuronal populations in both humans and experimental animals ~'m7'18.These selectively vulnerable neurons are found in the hippocampus, striatum, thalamus, certain layers of cerebral corte~, and the cerebellar cortex ~s'2°'22. The CAI subfield of the hippocampus is especially susceptible to ischemia and neurons are lost following only 3 rain of ischemia in gerbils s. Cells exposed to various types of stress acquire resistance or tolerance to subsequent lethal insults. Cells become resistant to hyperthermia following exposure to heat, ethanol, hypoxia and heavy metals 4'n. Retinal neurons exposed to elevated temperature greatly enhance their tolerance to light-induced retinal injury ~. Elevated temperature before ischemia also enhances hippocampal CA1 neuronal survival after subsequent lethal ischemia in rats and gerbils 3'~°. Various harmful forms of stress such as heat, ischemia, trauma, seizures, physical or chemical agents make cells produce a family of proteins referred to as the heat shock proteins (HSPs) 2'12'~3'19.The induction of tolerance has been reported to be correlated with the systhesis of HSPs II. However, the mechanism of the protective effects of preliminary exposure to stress is not fully
understood. We, therefore, examined whether brief and sublethal ischemia which induces HSP synthesis protects against subsequent ischemic insults. For this purpose, we used a forebrain ischemia model in rats and induced various periods of ischemia 3 days after pretreatment with sublethal ischemia. We used male adult Wistar rats (Nippon Clea, Tokyo, Japan) weighing 260-300 g. Forebrain ischemia was induced by 4-vessel occlusion as described by Puisinelli et al. I~, Rats were anesthetized with pentobarbital (50 mg/kg i.p.) and the vertebral arteries were electrocauterized. On the following day, common carotid arteries were exposed under anesthesia with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide. The arteries were then occluded with aneurysm clips. Carotid artery blood flow was restored by releasing the clips following 3 min of occlusion. Three days later, the arteries were again occluded for 6, 8 and 10 rain. Animals in single ischemia groups received sham operations followed by the same periods of ischemia. Animals were also subjected to single 3 min of ischemia or sham operations. Rectal temperature was maintained at close to 37.0°C during and after ischemia. Cessation of electroencephalogram (EEG) activity was confirmed during ischemic insults. Behav-
Correspondence: Y. Liu, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendal 980, Japan. Fax: 81-22-272-5818.
122 ior during and after ischemia was also carefully observed. One week after the second ischemia, the brains were perfusion-fixed with 4% paraformaladehyde in 0.1 M phosphate buffer (pH 7.4). Paraffin sections, 5 /zm thick, were prepared and stained with Cresyl violet and hematoxylin-eosin. The sections were examined with a light microscope and the neuronal density of the hippocampal CAI subfield was determined in a blinded fashion. Statistical significance was analyzed using the KruskaI-Wallis test followed by Williams-Wilcoxon rank sum test, and the Mann-Whitney U-test. For immunohistochemical staining of 70-kDa heatshock protein (HSP70), the brains were perfusion-fixed with 4% paraforamaladehyde in phosphate-buffered saline (PBS) 3 days after 3 rain of ischemia or sham operation and immersed in 30% sucrose. Frozen sections, 20 #m thick, were prepared stained for HSP70 using a mouse monoclonal antibody raised against HSP70 (Amersham, RPN 1197) and a Vectastain elite ABC kit (Vector Laboratories) as described by Vass et al. '1. Each group contained 4 animals. Neuronal density of the hippocampal CAI subfield is shown in Fig. 1. TIle CAI neuronal density of sham-openltcd animals was 168 + 6.4/ram (n = 5). Single 3 rain of ischemia caused no reduction in the neuronal density (168 + 13.9/ram; n - 10), Sham-operation followed by 6, 8 and 10 rain of ischemia produced moderate to severe reductions in the number of CAI pyramidal cells depending on the length of ischomia, which was 31 ± 17,5 (n ~ 9), 21 ± 17,2 (n ~, 7) and 14 ± 8.l/ram (n ~ 6), respectively, By contrast, CAI pyramidal cells wore preserved in animals subjected to pretreatment with 3 rain of ischemia. The neuronal density in 6.rain and 8-rain subgroups was 154 ± 19,6 (n ~ 11) and 88 ± 56.8/mm (n = 9), respectively. The neuronal density of 55 :t: 55.8/mm (n = 10) in the 10rain double ischemia subgroup was not significantly different from that in single 10.rain ischemia group, Immunohistochemical staining revealed definite increase in HSP70 in neurons in the hippocampal CA I subfield 3 days after 3 rain of cerebral ischemia (Fig. 2). The expression of HSP70 was seen in the whole CAI subfield in 4 hippocampi and was restricted to the CAla and b subregions in 4 hippocampi, The present result clearly demonstrated that proconditioning with sublethal ischemia, which permits recovery without any morphological damage but induces HSP70 synthesis, protects the brain against subsequent lethal ischemic insults. Three-min ischemia produced no reductions in the hippocampal CAI pyramidal neurons and therefore is a sublethal insult, More than 80% of the CAI pyramidal cells were destroyed following 6 rain of ischomia, but more than 90% of the
neurons survived following preconditioning. Eight-min ischemia destroyed 87% of the CA1 neurons, but more than 50% of the neurons survived following preconditioning. However, the preconditioning did not protect against neuronal damage following 10 min of ischemia which destroyed more than 90% of the CA1 neurons. Thus, the protection offered by HSP70 induction was not absolutely powerful. The intervening period between ischemic insults is a critical factor as is the duration of the ischemia itself. We chose the 3-min ischemia and 3-day interval as the conditioning insult and intervening wait, respectively, because of our previous results. We reported that single 3-rain ischemia in rats does not produce any neuronal damage in the hippocampal CAI sector but produces cumulative neuronal damage when repeated at l-h intervals ~4, and that gerbils pretreated with sublethal ischemia acquire resistance to subsequent lethal length of ischemia rendered 1-7 days later s. We confirmed that EEG became isoeloctric during isehemia and that animals were unresponsive to stimuli and the pupils were dilated and unresponsive to light during ischemia in all animals used in this study. Therefore, the protective effects are not the result of incomplete ischomia although the second ischemic insults wore induced four days after ¢loctrocoagulation of the vertebral arteries, We also monitorod and mainrained rectal temperature at 37,0°C during and up to 2 h after ischomia, The protection, therefor0, is not caused by hypothermia, Recently, the same protective phenomenon, 'ischemic tolerance', was reported in gorbils. Gerbils
sham 3min sluml ~Smln )min*6mln sham+Stain
3mln+Smln st~ml* IOmin ,train ~ IOmin !
0
I00
200 {/nun)
Fig, I, Neuronal density (/ram) of the CAI subfield of the hippocampus, The neuronal density is not reduced following 3 rain of ischemia but is significantly reduced following 6, 8 and l0 rain of ischemia. Preconditioning with 3 rain of ischemia resulted in significant preservation of CAI neurons following subsequent 6 and 8 rain of ischemia, * P < 0,05, * * P < 0.01 vs. corresponding single-ischemia subgroups (Mann-Whitney U-test). *# P < 0.01 vs. sham operation (Williams-Wilcoxon rank sum test).
123
A';"
*
•
:"
qD
.
°.
Fill. 2. Immunohistochemtcal stainin8 of 70-kDa heat shock protein (HSPT0) in rat hippocampus 3 days after sham operation (a) and 3 rain of ischemia (b), Note conspicuous HSP70 immunoreactivity in CA1 following ischemia (b), Bar ~. 0,5 ram.
subjected to 3-5 min of forebrain isehemia 1-4 days after 2 min of sublethal ischemia do not develop neuronal damage in the hippocampal CAI sector although a single ischemic insult always leads to severe hippocampal CAI neuronal loss 5'8'9. Furthermore, exposure to hyperthermia protects the brain against subsequent lethal ischemia in rats and gerbils3'm°. However, the mechanism of tolerance induction is not fully understood although a role of stress response is suggested. The tolerance takes plar'e coincidentally with heat shock/stress protein induction in the surviving neurons t.s. Experimental evidence indicates that sublethal isehemia induces conspicuous HSP70 gene expression s,n6 as shown in this study. Reports showing that HSP70 expression following 10 min of ischemia in gerbils is minimal in CA1 neurons which are destined to die but intense in surviving CA3 and dentate gyrus neurons 2n support this view. However, the tolerance phenomenon may not explained solely by HSP70 be-
cause Kirino et al, s reported that the temporal profile of the protective effects was not the same as that of HSP70 induction, and others also have shown that the pattern of HSP70 expression after ischemia has a poor correlation with histologic outcome ¢''n5. In conclusion, the present result indicates that even sublethal ischemia induces tolerance to subsequent lethal length of ischemia when such ischemia is stressful enough to induce HSP synthesis. Further study is warranted because the elucidation of the mechanism may provide a clue for the treatment and prevention of sti'oke in man. The authors would like to thank Ms. Rumiko Tanaka for her excellent technical assistance.
References 1 Barbe, M.F., Tytell, M., Grower, D.J. and Welch, W.J., Hyperthermia protects against light damage in the rat retina, Science, 241 (1988) 1817-1820.
2 Brown, I.R., Rush, S. and Ivy, G.O., Induction of heat shock gene at the site of tissue injury in the rat brain, Neuron, 2 (1989) 1559- ! 564. 3 Chopp, M., Chen, H., Ho, K.-L., Dereski, M.O., Brown, E., Hetzel, F.W. and Welch, K.M.A., Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat, Neurology, 39 (1989) 1396-1398. 4 Gerner, E.W. and Schneider, M.J., Induced thermal resistance in hela cells, Nature, 256 (1975) 500-502. 5 Kato, H., Liu, Y., Araki, T. and Kogure, K., Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects, Brain Res., 553 ( 1991) 238-242. 6 Kiessling, M., Dienel, G.A., Jacewicz, M. and Pulsinelli, W.A., Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis, J. Cereb. Blood Flow Metab., 6 (1986) 642-649. 7 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982)57-69. 8 Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to i~hemia in gerbil hippocampal neurons, J. Cereb. Blood Flow Metab., I I (1991) 299-307. 9 Kitagawa, K., Matsumoto, M., Tagaya. M., Hata, R., Ueda. H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K. and Kamada, T., 'lschemic tolerance' phenomenon found in the brain, Brain Rt,s., 528 (1990) 21-24. 10 Kitagawa, K., Matsumoto, M., Tagaya, M., Kuwabara, K., Hata, R., Handa, N., Fukunaga, R., Kimura, K. and Kamata, T., Hyperthermia-induced neuronal protection against ischemic injury in gerbils, J. Cereb. Blood Flow Metab., 11 (1991) 449-452. II Li, G.C. and Werb, Z., Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese
hamster fibroblasts, Proc. Natl. Acad. Sci. USA, 79 (1982) 32183222. 12 Lindquist, S., The heat-shock response, Annu. Rel'. Biochem., 55 (1986) 1151-1191. 13 Lowenstein, D.H., Gonzalez, M., Simon, R.P. and Sharp, F., The pattern of 72-kDa heat stock protein-like immunoreactivity in the rat brain following fluorothyi-induced status epilepticus, Brain Res., 531 (1990) 173-182. 14 Nakano, S., Kato, H. and Kogure, K., Neuronal damage in the rat hippocampus in a new model of repeated reversible transient cerebral ischemia, Brain Res., 490 (1989) 178-180. 15 Nowak, Jr., T.S., Synthesis of a stress protein following transient ischemia in the gerbil, J. Neurochern., 45 (1985) 1635-1641. 16 Nowak, Jr., T.S., Localization of 70-kDa stress protein mRNA induction in gerbil brain after ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 432-439. 17 Petito, C.K., Feldmann, E., Pulsinelli, W.A. and Plum, F., Delayed hippocampaldamagein humansfollowing cardiorespiratory arrest, Neurology, 37 (1987) 128! - 1286. 18 Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. NeuroL, I 1 (1982) 491-498. 19 Simon, R.P., Cho, H., Gwinn, R. and Lowenstein, D.H., The temporal profile of 72-kDa heat-shock protein expression following global ischemia, J. Neurosci., 11 (1991)881-889. 20 Smith, M.-L., Auer, R.N. and Siesj6, B.K., The density and distribution of ischemic brain injury in the rat following 2-10 rain of forebrain ischemia, Acta Neuropathol., 64 (1984) 319-332. 21 Vass, K., Welch, W.J. and Nowak, Jr., T.S., Localization of 70-kDa stress protein induction in gerbil brain after ischemia, Acta Neuropathol., 77 (1988) 128-135. 22 Wieloch, T., Neurochemical correlates to selective neuronal vulnerability, Frog. Brain Res,, 63 (1985) 69-85,
