Brain Research, 534 (1990) 299-302 Elsevier
299
BRES 24397
Effects of cycloheximide on delayed neuronal death in rat hippocampus Kazuhiro Goto 1, Atsushi Ishige 1, Kyoji Sekiguchi 1, Susumu Iizuka 1, Akira Sugimoto 1, Mitsutoshi Yuzurihara ~, Masaki Aburada 1, Eikichi Hosoya I and Kyuya Kogure 2 lTsumura Research Institute for Pharmacology, Ami, Ibaraki (Japan) and 2Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Accepted 21 August 1990)
Key words: Cerebral ischemia; Hippocampus; Selective vulnerability; Delayed neuronal death; Protein synthesis; Cycloheximide; Rat
The effect of cycloheximide, a protein synthesis inhibitor, on hippocampal selective neuronal death was morphologically studied in rats subjected to 10 min forebrain ischemia using a 4-vessel occlusion model. Neuronal damage in the hippocampal CA1 subfield 72 h after ischemic insult was dramatically decreased by the lasting inhibition of protein synthesis through consecutive administration of cycloheximide. Cycloheximide, which was administered once within the first 24 h of recirculation, showed protective action on ischemic cell necrosis and its most potent effect was observed when injected at 12 h of post-ischemia. After 36 h of recirculation, however, treatment with cycloheximide could no longer prevent cell death. The possibility is considered that hippocampal delayed neuronal death following transient ischemia is caused by abnormal protein(s). Histological studies of transient ischemic insults to the brain have demonstrated that selectively vulnerable neurons to ischemia exist 2. In particular, pyramidal neurons in the CA1 subfield of the hippocampus are most vulnerable to cerebral ischemia. Death of these neurons occurs after an interval of 1 or 2 days following recirculation, during which time no energy crisis or morphological change is observed (delayed neuronal death: DND) 1'9'13'15. Many hypotheses have been proposed to explain DND, and the mechanism for this phenomenon has yet to be elucidated. It has been reported that in gerbils subjected to 5 min forebrain ischemia, protein synthesis in the CA1 subfield is persistently inhibited until neuronal necrosis occurs, despite the entire intracerebral region being virtually restored to normal within the early period of postischemic recirculation ~7. This phenomenon has been observed in the presence of ATP and after rapid normalization of the cell's RNA-synthesizing capacity x,~°. Some researchers have discussed a possibility that prolonged disturbance of protein synthesis may play a pivotal role in the occurrence of D N D 17. On the other hand, a requirement for synthesis of protein(s) has been demonstrated in the selective death of neurons. With regard to invertebrates, it has been suggested that selective death, also called programmed cell death, of the abdominal motor neurons and interneurons in the moth results from changes in gene activation by hormones and/or trophic interactions, since cycloheximide and
actinomycin-D can prevent neuronal death 5'1s. With regard to vertebrates also, Martin et al. 11 observed in an experiment using cultures that death of sympathetic neurons in rats deprived of nerve growth factor is prevented by inhibition of R N A or protein synthesis. In the experiment reported here, we examined the relationship between D N D in the hippocampus and protein synthesis to clarify the mechanism of D N D following transient ischemic insult. Male Wistar rats, each weighing between 250 and 310 g, were subjected to transient forebrain ischemia by the method of Pulsinelli and Brierley 12 with minor modifications. In brief, on the day prior to ischemia, atraumatic strings were tied loosely around each carotid artery without interrupting carotid blood flow, and both vertebral arteries were electro-cauterized at the first cervical vertebra under pentobarbital (50 mg/kg, i.p.) anesthesia. Animals were fasted overnight, and the next day carotid arteries were exposed under 2% halothane anesthesia. On completion of the surgical procedure, halothane was discontinued to minimize its effect. Three min later, when animals showed no spontaneous movement but twitched if pain stimuli were given, bilateral carotid arteries were clamped using No. 52 Sugita temporary aneurysm clips for 10 min. Criteria for forebrain ischemia were determined as bilateral loss of righting reflex, paw extension ability and mydriasis. Furthermore, only those animals, which showed continuous loss of righting reflex over 15 min after recirculation
Correspondence: K. Goto, Tsumura Research Institute for Pharmacology, 3586 Yoshiwara, Ami-machi, Ibaraki 300-11, Japan.
0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
300 were selected. Carotid strings were removed during ischemia and restoration of carotid blood flow was verified by direct observation. Body temperature was maintained at 36.5-37.5 °C during ischemia and the early post-ischemic recovery period by means of heating mat and lamp. A total of 170 animals were used. One hundred sixty animals underwent 10 min of ischemia and were randomly assigned to 11 groups. A group of 10 animals, which underwent operation but no ischemia, served as sham-operated. For morphological observations, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) at 48, 72 or 168 h of post-ischemic survival, and their brains were perfusion-fixed with FAM (40% formaldehyde:acetic acid:methanol = 1:1:8) after briefly washing out the cephalic circulation with heparinized saline. Animals were left for 1-4 h after fixing at 4 °C, then brains were removed and further fixation was performed for 1 day at room temperature. Paraffin sections (5/am) were stained with Cresyl violet. The number of pyramidal neurons in each CA1 subfield was counted under light microscopy at a magnification of x 400. Each section was photographed, its CA1 length measured and the number of CA1 neurons per mm calculated. The mean value of CA1 pyramidal neurons per mm bilaterally was termed 'neuronal density', its value being expressed as the mean value ___ S.E.M.. Statistical comparisons were performed using Mann-Whitney's U test. With this modified method of 4-vessel occlusion, 129 of 160 animals (81%) met the criteria and 8 (5%) of the animals died within 2-3 min from respiratory failure. The other rats showed oligemia but recovered their righting reflex in the ischemic period (6 rats) or within 2-7 min after recirculation (17 rats). Cycloheximide was subcutaneously injected just after 10 min of ischemia at a dose of 2.0 mg/kg, followed by 1.0 mg/kg every 12 h being at 18 h of post-ischemia. The animals were perfusion-fixed at 72 h of post-ischemic survival. The average neuronal density of the CA1 subfield was determined to be 123.8 + 4.4/mm in 10 sham-operated animals. Whereas hardly any neurons remained in the saline-treated group (12.5 + 4.0/mm, n = 10), consecutive administration of cycloheximide showed a potent effect on neuronal cell resuscitation (93.7 + 8.6/mm, n = 9) as shown in Fig. 1. However, 11 out of 20 cycloheximide-treated animals died within 42-72 h of post-ischemia. In another experiment, cycloheximide (3.0 mg/kg, s.c.) was administered once only, at various points of postischemia. The mortality was 13% for rats treated with cycloheximide and 1 out of 20 saline-treated animals died at 2 days of post-ischemia in this study. Neuronal
Sham operation
h
Control
Cycloheximide
0
!
i
50
100
Neuronal density (/mm)
Fig. 1. Protection by consecutive administration of cycloheximide against hippocampal delayed neuronal death at 72 h of postischemia in rats subjected to 10 min forebrain ischemia. Cyeloheximide was subcutaneously injected just after recirculation at a dose of 2.0 mg/kg, followed by 1.0 mg/kg every 12 h being at 18 h of post-ischemia. Values are means + S.E.M., n = 9-10. ***P < 0.001 compared with the saline-treated group.
densities 72 h following ischemia in each group are presented in Fig. 2. In rats, treated with cycloheximide within the first 24 h of recirculation, the number of damaged neurons 72 h after ischemic insult was significantly decreased. Cycloheximide showed its most potent effect when injected at 12 h of post-ischemia (87,6 + 14.6/mm, n = 9). After 36 h of recirculation, however, treatment with cycloheximide could no longer prevent neuronal death. In animals allowed to survive for 168 h following ischemia also, the protective effect of cycloheximide, which was treated at 0 or 12 h after recirculation,
[
- q ,~!c,
Sham operation
Control
Cycloheximide
0 hr
12 hr
I **
l*
24 hr
36 hr
48 hr I
o
50
lOO
Neuronal density (/mm)
Fig. 2. Protection by single administration of cycloheximide (3.0 mg/kg, s.c.) against hippocampai delayed neuronal death at 72 h of post-ischemia in rats subjected to 10 rain forebrain ischemia. Cycloheximide was administered at various times of post-ischemia. Values are mean +_ S.E.M., n = 9-11. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the saline-treated group.
301
Sham operation
3
Control
Cycloheximide 0 hr
J
I **
I **
12 hr
0
!
i
50
100
Neuronal density ( / m m )
Fig. 3. Protection by single administration of cycloheximide (3.0 mg/kg, s.c.) at 0 or 12 h of post-ischemia against hippocampal delayed neuronal death in rats subjected to 10 min forebrain ischemia. In this experiment, the animals were perfusion-fixedafter 168 h of post-ischemic survival. Values are mean + S.E.M., n = 10-11. **P < 0.01, ***P < 0.001 compared with the saline-treated group. was confirmed as shown in Fig. 3. Although the neuronal density tended to decrease in comparison with the value at 72 h after ischemia, the more potent effect of cycloheximide injected at 12 h of post-ischemia was observed even at this point. On the other hand, the average neuronal density at 48 h following ischemia was 90.0 + ll.6/mm in the CA1 subfield of 9 saline-treated animals. The major finding of the experiment reported here was that consecutive administration of the protein synthesis inhibitor, cycloheximide, prevented hippocampal DND at 72 h following 10 min of forebrain ischemia, even if only administered once at 24 h of post-ischemia. These results suggest that protein synthesis is required for CA1 neurons to die. The possibility that cycloheximide prevented DND through its inhibitory action on lysosomal autophagy can be excluded by the fact that treatment of cycloheximide at 48 h of post-ischemia, when many CA1 neurons still remained though the average neuronal density was already significantly decreased in comparison with the value of sham-operated animals (P < 0.01), could no longer prevent neuronal death. However, we cannot rule out the possibility that hypothermia was caused by treatment of cycloheximide. Since we maintained body temperature during ischemia and the early post-ischemic recovery period but did not monitor thereafter. Busto et al. 3 have reported that a reduction of brain temperature to 30 °C for 3 h, beginning 5 min after resumption of circulation, conferred a marked protective effect of DND in the CA1 subfield. If the institution of hypothermia was delayed for 30 min, however, protection was less pronounced. Therefore, if hypothermia was caused by cycloheximide-treatment, the factor does not
seem to be the cause of the protection by cycloheximide against DND. It has been demonstrated that destruction of ischemiasensitive neurons is complete 3-4 days following ischemia 9"13. On the other hand, it has also been reported that primary dissociated cultures of sympathetic neurons prepared from the cervical ganglia eventually died after 4-5 days of lasting cycloheximide treatmentlk The animals, which were consecutively administered with cycloheximide, in our experiment were therefore sacrificed at 72 h of post-ischemic survival and the efficacy of cycioheximide was evaluated at that point. According to our results, most CA1 neurons in the saline-treated animals had already disappeared by 72 h following ischemia. We have reported here that the most potent effect of cycloheximide was observed when injected at 12 h of post-ischemia. According to the result, it seems that the synthesis of new RNA for the putative killer protein(s) was required for the development of DND. Nevertheless protein synthesis in rats at 18 h after treatment of cycloheximide (2.0 mg/kg, i.p.) was found not to be inhibited 4, the protective effect of cycloheximide was confirmed even at 168 h of post-ischemic period. Hence it appears that the transcription of the mRNA continues for not so long and newly synthesized mRNA is characterized by short life. The synthesized protein(s) might initiate the subsequent intracellular cascade for neuronal death, or be stable to account for the lag between protein synthesis and neuronal death. The question still remains as to why the synthesis of RNA and protein occurs. It has reported that almost all protein synthesis rates in the hippocampus are severely depressed by transient ischemia, especially in the CA1 subfield, it is consistently inhibited until neuronal death occurs 17. Under the circumstances, mRNA for the protein(s) may be transcribed following deprivation of neurotrophic factors 11. However, such speculation remains nebulous insofar as the existence of endogenous neurotrophic factors or trophic support to the CA1 neurons has not been proved. On the other hand, evidence has accumulated that excessive release of excitatory neurotransmitters, such as glutamate, in vulnerable areas may be a critical factor leading to neuronal death 14. It is possible that activation of signal transduction at a excitatory amino acid receptor may be able to increase the synthesis of the RNA. For example, it has been demonstrated that excitatory amino acids can increase the steady state level of c-fos mRNA in the brain 16. However, it seems unlikely that c-fos mRNA is not related to the suicide response following ischemia. Since its expression in the CA1 subfield was dramatically elevated at 72 h of post-ischemia, but not yet increased at 12 h 7. In this scheme, it is necessary to suppose the
302 existence of the other gene inducted more rapidly following ischemia. Although the mechanism regulating killer protein(s) synthesis remains u n k n o w n , the hypothesis we advance is that D N D in the hippocampus subsequent to transient ischemic insult consists of programmed cell death and is
synthesis of several proteins, including constitutive and stress proteins, were observed in the early post-ischemic period 6's. Studies to clarify more directly the existence and biological properties of killer protein(s) are currently under way.
caused by abnormal protein(s), seems to be supported by reported experiments in which recovered or enhanced
This work was supported by the Science and Technology Agency.
1 Arai, H., Passonneau, J.V. and Lust, W.D., Energy metabolism in delayed neuronal death of CA1 neurons of the hippocampus following transient ischemia in the gerbil, Metab. Brain Dis., 1 (1986) 263-278. 2 Brierley, J.B., Cerebral hypoxia. In W. Blackwood and J.A.N. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1976, pp. 43-85. 3 Busto, R., Dietrich, W.D., Globus, M.Y.-T. and Ginsberg, M.D., The importance of brain temperature in cerebral ischemic injury, Stroke, 20 (1989) 1113-1114. 4 Ch'ih, J.J., Procyk, R. and Devlin, T.M., Regulation of mammalian protein synthesis in vivo, Biochem. J., 162 (1977) 501-507. 5 Fahrbach, S.E. and Truman, J.W., Mechanisms for programmed cell death in the nervous system of a moth. In G. Bock and M. O'Connor (Eds.), Selective Neuronal Death, Wiley, New York, 1987, pp. 65-81. 6 Jacewicz, M., Kiessling, M. and Pulsinelli, W.A., Selective gene expression in focal cerebral ischemia, J. Cereb. Blood Flow Metab., 6 (1986) 263-272. 7 J#rgensen, M.B., Deckert, J., Wright, D.C. and Gehlert, D.R., Delayed c-los proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study, Brain Research, 484 (1989) 393-398. 8 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. 9 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. 10 Kogure, K., Tobita, M., Sato, H. and Onodera, H., Impairment
of protein synthesis in selectively vulnerable neurons. In M.E. Raichle and W.J. Powers (Eds.), Cerebrovascular Diseases, Raven, New York, 1987, pp. 119-126. 11 Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry, O.H., Carter, J.G. and Johnson, E.M. Jr., Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J. Cell Biol., 106 (1988) 829-844. 12 Pulsinelli, W.A. and Brierley, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 13 Pulsinelli, W.A., Brierley, J.B. and Plum, E, Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 14 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemia brain damage, Ann. Neurol., 19 (1986) 105-111. 15 Smith, M.L., Auer, R.N. and Siesj6, B.K., The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia, Acta Neuropath., 64 (1984) 319-332. 16 Szekely, A.M., Barbaccia, M.L. and Costa, E., Activation of specific glutamate receptor subtypes increases c-los protooncogene expression in primary cultures of neonatal rat cerebellar granule cells, Neuropharmacology, 26 (1987) 1779-1782. 17 Thilman, 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. 18 Truman, J.W., Cell death in invertebrate nervous systems, Annu. Rev. Neurosci., 7 (1984) 171-188.
299
BRES 24397
Effects of cycloheximide on delayed neuronal death in rat hippocampus Kazuhiro Goto 1, Atsushi Ishige 1, Kyoji Sekiguchi 1, Susumu Iizuka 1, Akira Sugimoto 1, Mitsutoshi Yuzurihara ~, Masaki Aburada 1, Eikichi Hosoya I and Kyuya Kogure 2 lTsumura Research Institute for Pharmacology, Ami, Ibaraki (Japan) and 2Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)
(Accepted 21 August 1990)
Key words: Cerebral ischemia; Hippocampus; Selective vulnerability; Delayed neuronal death; Protein synthesis; Cycloheximide; Rat
The effect of cycloheximide, a protein synthesis inhibitor, on hippocampal selective neuronal death was morphologically studied in rats subjected to 10 min forebrain ischemia using a 4-vessel occlusion model. Neuronal damage in the hippocampal CA1 subfield 72 h after ischemic insult was dramatically decreased by the lasting inhibition of protein synthesis through consecutive administration of cycloheximide. Cycloheximide, which was administered once within the first 24 h of recirculation, showed protective action on ischemic cell necrosis and its most potent effect was observed when injected at 12 h of post-ischemia. After 36 h of recirculation, however, treatment with cycloheximide could no longer prevent cell death. The possibility is considered that hippocampal delayed neuronal death following transient ischemia is caused by abnormal protein(s). Histological studies of transient ischemic insults to the brain have demonstrated that selectively vulnerable neurons to ischemia exist 2. In particular, pyramidal neurons in the CA1 subfield of the hippocampus are most vulnerable to cerebral ischemia. Death of these neurons occurs after an interval of 1 or 2 days following recirculation, during which time no energy crisis or morphological change is observed (delayed neuronal death: DND) 1'9'13'15. Many hypotheses have been proposed to explain DND, and the mechanism for this phenomenon has yet to be elucidated. It has been reported that in gerbils subjected to 5 min forebrain ischemia, protein synthesis in the CA1 subfield is persistently inhibited until neuronal necrosis occurs, despite the entire intracerebral region being virtually restored to normal within the early period of postischemic recirculation ~7. This phenomenon has been observed in the presence of ATP and after rapid normalization of the cell's RNA-synthesizing capacity x,~°. Some researchers have discussed a possibility that prolonged disturbance of protein synthesis may play a pivotal role in the occurrence of D N D 17. On the other hand, a requirement for synthesis of protein(s) has been demonstrated in the selective death of neurons. With regard to invertebrates, it has been suggested that selective death, also called programmed cell death, of the abdominal motor neurons and interneurons in the moth results from changes in gene activation by hormones and/or trophic interactions, since cycloheximide and
actinomycin-D can prevent neuronal death 5'1s. With regard to vertebrates also, Martin et al. 11 observed in an experiment using cultures that death of sympathetic neurons in rats deprived of nerve growth factor is prevented by inhibition of R N A or protein synthesis. In the experiment reported here, we examined the relationship between D N D in the hippocampus and protein synthesis to clarify the mechanism of D N D following transient ischemic insult. Male Wistar rats, each weighing between 250 and 310 g, were subjected to transient forebrain ischemia by the method of Pulsinelli and Brierley 12 with minor modifications. In brief, on the day prior to ischemia, atraumatic strings were tied loosely around each carotid artery without interrupting carotid blood flow, and both vertebral arteries were electro-cauterized at the first cervical vertebra under pentobarbital (50 mg/kg, i.p.) anesthesia. Animals were fasted overnight, and the next day carotid arteries were exposed under 2% halothane anesthesia. On completion of the surgical procedure, halothane was discontinued to minimize its effect. Three min later, when animals showed no spontaneous movement but twitched if pain stimuli were given, bilateral carotid arteries were clamped using No. 52 Sugita temporary aneurysm clips for 10 min. Criteria for forebrain ischemia were determined as bilateral loss of righting reflex, paw extension ability and mydriasis. Furthermore, only those animals, which showed continuous loss of righting reflex over 15 min after recirculation
Correspondence: K. Goto, Tsumura Research Institute for Pharmacology, 3586 Yoshiwara, Ami-machi, Ibaraki 300-11, Japan.
0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
300 were selected. Carotid strings were removed during ischemia and restoration of carotid blood flow was verified by direct observation. Body temperature was maintained at 36.5-37.5 °C during ischemia and the early post-ischemic recovery period by means of heating mat and lamp. A total of 170 animals were used. One hundred sixty animals underwent 10 min of ischemia and were randomly assigned to 11 groups. A group of 10 animals, which underwent operation but no ischemia, served as sham-operated. For morphological observations, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) at 48, 72 or 168 h of post-ischemic survival, and their brains were perfusion-fixed with FAM (40% formaldehyde:acetic acid:methanol = 1:1:8) after briefly washing out the cephalic circulation with heparinized saline. Animals were left for 1-4 h after fixing at 4 °C, then brains were removed and further fixation was performed for 1 day at room temperature. Paraffin sections (5/am) were stained with Cresyl violet. The number of pyramidal neurons in each CA1 subfield was counted under light microscopy at a magnification of x 400. Each section was photographed, its CA1 length measured and the number of CA1 neurons per mm calculated. The mean value of CA1 pyramidal neurons per mm bilaterally was termed 'neuronal density', its value being expressed as the mean value ___ S.E.M.. Statistical comparisons were performed using Mann-Whitney's U test. With this modified method of 4-vessel occlusion, 129 of 160 animals (81%) met the criteria and 8 (5%) of the animals died within 2-3 min from respiratory failure. The other rats showed oligemia but recovered their righting reflex in the ischemic period (6 rats) or within 2-7 min after recirculation (17 rats). Cycloheximide was subcutaneously injected just after 10 min of ischemia at a dose of 2.0 mg/kg, followed by 1.0 mg/kg every 12 h being at 18 h of post-ischemia. The animals were perfusion-fixed at 72 h of post-ischemic survival. The average neuronal density of the CA1 subfield was determined to be 123.8 + 4.4/mm in 10 sham-operated animals. Whereas hardly any neurons remained in the saline-treated group (12.5 + 4.0/mm, n = 10), consecutive administration of cycloheximide showed a potent effect on neuronal cell resuscitation (93.7 + 8.6/mm, n = 9) as shown in Fig. 1. However, 11 out of 20 cycloheximide-treated animals died within 42-72 h of post-ischemia. In another experiment, cycloheximide (3.0 mg/kg, s.c.) was administered once only, at various points of postischemia. The mortality was 13% for rats treated with cycloheximide and 1 out of 20 saline-treated animals died at 2 days of post-ischemia in this study. Neuronal
Sham operation
h
Control
Cycloheximide
0
!
i
50
100
Neuronal density (/mm)
Fig. 1. Protection by consecutive administration of cycloheximide against hippocampal delayed neuronal death at 72 h of postischemia in rats subjected to 10 min forebrain ischemia. Cyeloheximide was subcutaneously injected just after recirculation at a dose of 2.0 mg/kg, followed by 1.0 mg/kg every 12 h being at 18 h of post-ischemia. Values are means + S.E.M., n = 9-10. ***P < 0.001 compared with the saline-treated group.
densities 72 h following ischemia in each group are presented in Fig. 2. In rats, treated with cycloheximide within the first 24 h of recirculation, the number of damaged neurons 72 h after ischemic insult was significantly decreased. Cycloheximide showed its most potent effect when injected at 12 h of post-ischemia (87,6 + 14.6/mm, n = 9). After 36 h of recirculation, however, treatment with cycloheximide could no longer prevent neuronal death. In animals allowed to survive for 168 h following ischemia also, the protective effect of cycloheximide, which was treated at 0 or 12 h after recirculation,
[
- q ,~!c,
Sham operation
Control
Cycloheximide
0 hr
12 hr
I **
l*
24 hr
36 hr
48 hr I
o
50
lOO
Neuronal density (/mm)
Fig. 2. Protection by single administration of cycloheximide (3.0 mg/kg, s.c.) against hippocampai delayed neuronal death at 72 h of post-ischemia in rats subjected to 10 rain forebrain ischemia. Cycloheximide was administered at various times of post-ischemia. Values are mean +_ S.E.M., n = 9-11. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the saline-treated group.
301
Sham operation
3
Control
Cycloheximide 0 hr
J
I **
I **
12 hr
0
!
i
50
100
Neuronal density ( / m m )
Fig. 3. Protection by single administration of cycloheximide (3.0 mg/kg, s.c.) at 0 or 12 h of post-ischemia against hippocampal delayed neuronal death in rats subjected to 10 min forebrain ischemia. In this experiment, the animals were perfusion-fixedafter 168 h of post-ischemic survival. Values are mean + S.E.M., n = 10-11. **P < 0.01, ***P < 0.001 compared with the saline-treated group. was confirmed as shown in Fig. 3. Although the neuronal density tended to decrease in comparison with the value at 72 h after ischemia, the more potent effect of cycloheximide injected at 12 h of post-ischemia was observed even at this point. On the other hand, the average neuronal density at 48 h following ischemia was 90.0 + ll.6/mm in the CA1 subfield of 9 saline-treated animals. The major finding of the experiment reported here was that consecutive administration of the protein synthesis inhibitor, cycloheximide, prevented hippocampal DND at 72 h following 10 min of forebrain ischemia, even if only administered once at 24 h of post-ischemia. These results suggest that protein synthesis is required for CA1 neurons to die. The possibility that cycloheximide prevented DND through its inhibitory action on lysosomal autophagy can be excluded by the fact that treatment of cycloheximide at 48 h of post-ischemia, when many CA1 neurons still remained though the average neuronal density was already significantly decreased in comparison with the value of sham-operated animals (P < 0.01), could no longer prevent neuronal death. However, we cannot rule out the possibility that hypothermia was caused by treatment of cycloheximide. Since we maintained body temperature during ischemia and the early post-ischemic recovery period but did not monitor thereafter. Busto et al. 3 have reported that a reduction of brain temperature to 30 °C for 3 h, beginning 5 min after resumption of circulation, conferred a marked protective effect of DND in the CA1 subfield. If the institution of hypothermia was delayed for 30 min, however, protection was less pronounced. Therefore, if hypothermia was caused by cycloheximide-treatment, the factor does not
seem to be the cause of the protection by cycloheximide against DND. It has been demonstrated that destruction of ischemiasensitive neurons is complete 3-4 days following ischemia 9"13. On the other hand, it has also been reported that primary dissociated cultures of sympathetic neurons prepared from the cervical ganglia eventually died after 4-5 days of lasting cycloheximide treatmentlk The animals, which were consecutively administered with cycloheximide, in our experiment were therefore sacrificed at 72 h of post-ischemic survival and the efficacy of cycioheximide was evaluated at that point. According to our results, most CA1 neurons in the saline-treated animals had already disappeared by 72 h following ischemia. We have reported here that the most potent effect of cycloheximide was observed when injected at 12 h of post-ischemia. According to the result, it seems that the synthesis of new RNA for the putative killer protein(s) was required for the development of DND. Nevertheless protein synthesis in rats at 18 h after treatment of cycloheximide (2.0 mg/kg, i.p.) was found not to be inhibited 4, the protective effect of cycloheximide was confirmed even at 168 h of post-ischemic period. Hence it appears that the transcription of the mRNA continues for not so long and newly synthesized mRNA is characterized by short life. The synthesized protein(s) might initiate the subsequent intracellular cascade for neuronal death, or be stable to account for the lag between protein synthesis and neuronal death. The question still remains as to why the synthesis of RNA and protein occurs. It has reported that almost all protein synthesis rates in the hippocampus are severely depressed by transient ischemia, especially in the CA1 subfield, it is consistently inhibited until neuronal death occurs 17. Under the circumstances, mRNA for the protein(s) may be transcribed following deprivation of neurotrophic factors 11. However, such speculation remains nebulous insofar as the existence of endogenous neurotrophic factors or trophic support to the CA1 neurons has not been proved. On the other hand, evidence has accumulated that excessive release of excitatory neurotransmitters, such as glutamate, in vulnerable areas may be a critical factor leading to neuronal death 14. It is possible that activation of signal transduction at a excitatory amino acid receptor may be able to increase the synthesis of the RNA. For example, it has been demonstrated that excitatory amino acids can increase the steady state level of c-fos mRNA in the brain 16. However, it seems unlikely that c-fos mRNA is not related to the suicide response following ischemia. Since its expression in the CA1 subfield was dramatically elevated at 72 h of post-ischemia, but not yet increased at 12 h 7. In this scheme, it is necessary to suppose the
302 existence of the other gene inducted more rapidly following ischemia. Although the mechanism regulating killer protein(s) synthesis remains u n k n o w n , the hypothesis we advance is that D N D in the hippocampus subsequent to transient ischemic insult consists of programmed cell death and is
synthesis of several proteins, including constitutive and stress proteins, were observed in the early post-ischemic period 6's. Studies to clarify more directly the existence and biological properties of killer protein(s) are currently under way.
caused by abnormal protein(s), seems to be supported by reported experiments in which recovered or enhanced
This work was supported by the Science and Technology Agency.
1 Arai, H., Passonneau, J.V. and Lust, W.D., Energy metabolism in delayed neuronal death of CA1 neurons of the hippocampus following transient ischemia in the gerbil, Metab. Brain Dis., 1 (1986) 263-278. 2 Brierley, J.B., Cerebral hypoxia. In W. Blackwood and J.A.N. Corsellis (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1976, pp. 43-85. 3 Busto, R., Dietrich, W.D., Globus, M.Y.-T. and Ginsberg, M.D., The importance of brain temperature in cerebral ischemic injury, Stroke, 20 (1989) 1113-1114. 4 Ch'ih, J.J., Procyk, R. and Devlin, T.M., Regulation of mammalian protein synthesis in vivo, Biochem. J., 162 (1977) 501-507. 5 Fahrbach, S.E. and Truman, J.W., Mechanisms for programmed cell death in the nervous system of a moth. In G. Bock and M. O'Connor (Eds.), Selective Neuronal Death, Wiley, New York, 1987, pp. 65-81. 6 Jacewicz, M., Kiessling, M. and Pulsinelli, W.A., Selective gene expression in focal cerebral ischemia, J. Cereb. Blood Flow Metab., 6 (1986) 263-272. 7 J#rgensen, M.B., Deckert, J., Wright, D.C. and Gehlert, D.R., Delayed c-los proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study, Brain Research, 484 (1989) 393-398. 8 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. 9 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Research, 239 (1982) 57-69. 10 Kogure, K., Tobita, M., Sato, H. and Onodera, H., Impairment
of protein synthesis in selectively vulnerable neurons. In M.E. Raichle and W.J. Powers (Eds.), Cerebrovascular Diseases, Raven, New York, 1987, pp. 119-126. 11 Martin, D.P., Schmidt, R.E., DiStefano, P.S., Lowry, O.H., Carter, J.G. and Johnson, E.M. Jr., Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation, J. Cell Biol., 106 (1988) 829-844. 12 Pulsinelli, W.A. and Brierley, J.B., A new model of bilateral hemispheric ischemia in the unanesthetized rat, Stroke, 10 (1979) 267-272. 13 Pulsinelli, W.A., Brierley, J.B. and Plum, E, Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. Neurol., 11 (1982) 491-498. 14 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemia brain damage, Ann. Neurol., 19 (1986) 105-111. 15 Smith, M.L., Auer, R.N. and Siesj6, B.K., The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia, Acta Neuropath., 64 (1984) 319-332. 16 Szekely, A.M., Barbaccia, M.L. and Costa, E., Activation of specific glutamate receptor subtypes increases c-los protooncogene expression in primary cultures of neonatal rat cerebellar granule cells, Neuropharmacology, 26 (1987) 1779-1782. 17 Thilman, 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. 18 Truman, J.W., Cell death in invertebrate nervous systems, Annu. Rev. Neurosci., 7 (1984) 171-188.
