Neuroscience Letters, 146 (1992) 75-78 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
75
NSL 09043
Barbiturate promotes post-ischemic reaggregation of polyribosomes in gerbil hippocampus P e t r a B o n n e k o h , T o s h i h i k o K u r o i w a l, U t e Oschlies a n d K o n s t a n t i n - A l e x a n d e r H o s s m a n n
Max-Planck-lnstitutefor NeurologicalResearch, Department of Experimental Neurology, Cologne (FRG) (Received 22 May 1992; Revised version received 13 July 1992; Accepted 29 July 1992)
Key words: Transientforebrain ischemia; Selectivevulnerability; Polyribosome; Mongolian gerbil; Hippocampus; Delayed neuronal death; Barbiturate A brief period of cerebral ischemia is followed by severe inhibition of protein synthesis which is slowly reversed in the resistant but not in the selectivelyvulnerable regions of the brain. Inhibition occurs at the translational level, as evidenced by the disaggregation of ribosomes into monosomes. In order to evaluate the importance of this disturbance for the evolution of ischemic injury, the effect of the neuroprotective drug, pentobarbital, on ribosomal aggregation was studied in gerbils subjected to 5 min bilateral carotid artery occlusion. Pentobarbital (50 mg/kg, i.p.) was applied shortly after the ischemia, and the aggregational state of ribosomes was investigated by electron microscopy after recirculation times ranging from 15 min to 1 day. Pentobarbital treatment did not prevent the initial post-ischemicdisaggregation but promoted the subsequent reaggregation in the selectivelyvulnerable neurons. This suggests that post-ischemicapplication of barbiturates exerts its beneficialeffect by reversing the post-ischemic block of ribosomal reaggregation in vulnerable regions.
A brief period of transient global ischemia of the brain results in prolonged inhibition of protein synthesis [1, 21, 22] although energy metabolism is restored within a few minutes [14]. As shown by quantitative autoradiography of amino acid incorporation into brain proteins, anatomical structures that recover from the ischemic episode resume protein synthesis after a few hours whereas in selectively vulnerable regions such as the hippocampal CA1 subfield protein synthesis is not or only transiently and incompletely restored [1, 21, 22]. A comparison of the autoradiographic findings with electron microscopical investigations of the aggregational state of ribosomes shows a close correlation. With the beginning of recirculation polyribosomes disaggregate into monosomes in all regions in which protein synthesis is suppressed. After a few hours new polyribosomes are formed in the resistant but not in the vulnerable structures of the brain [14]. This and in vitro assays of ribosomal fractions [2] suggest that the post-ischemic inhibition of protein synthesis is due to an inhibition of
Correspondence: K.-A. Hossmann, Max-Planck-Institut for Neurologische Forschung, Gleuelerstr. 50, D-5000 KSln 41, FRG. ~Present address: Medical Research Institute, Tokyo Medical and Dental University,Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan.
m R N A translation at the polypeptide chain initiation step. Barbiturates are known to reverse the post-ischemic suppression of protein synthesis and to prevent neuronal death in the selectively vulnerable regions [23]. Therefore, the question arose whether barbiturates act by preventing the initial disaggregation or by promoting the subsequent reaggregation of ribosomes. The study included 12 adult gerbils (Meriones unguiculatus) of either sex weighing 50-80 g. Prior to the experiments the animals had free access to water and food. Anesthesia was induced by 3% halothane and then continued with 1.5% halothane in a gas mixture o f 70% nitrogen and 30% oxygen. Rectal temperature was kept close to 37.5°C by a feed-back controlled heating system. The c o m m o n carotid arteries were exposed on both sides by a midline cervical incision and occluded for 5 min with miniature Biemer clips (FD562, Aesculap, Tuttlingen, F R G ) . After the ischemic period the clips were released and the anesthesia was discontinued. Pentobarbital (50 mg/kg) was injected intraperitoneaUy immediately after vascular release, and the animals were allowed to survive for 15 min, 1, 2, 8 and 24 h, respectively. Successful reversal of the post-ischemic suppression of protein synthesis with this drug dosage has been shown in previous studies [8, 23]. After the respective recirculation
76 times animals were reanesthetized and transcardially perfused with 2.5% glutaraldehyde/2% paraformaldehyde. The brains were carefully removed, postfixed with the same fixative and processed for electron microscopy. Electron micrographs were taken from 50 neurons of the frontal cortex, the dentate gyrus and the CA1 and CA3 sectors of hippocampus. An index of mean ribosomal aggregation (A.I.) was calculated by classifying each neuron as follows: 1.0 -- normal aggregation, 0.5 = partial disaggregation and 0 = complete disaggregation of cytoplasmic polyribosomes. The results obtained were compared with a previous series of experiments in which similar measurements were carried out in untreated gerbils [14]. Control group. Non-ischemic control animals exhibited cytosolic polyribosomal complexes consisting of 5 6 ribosomes in all cortical and hippocampal neurons (CA1 and CA3 sector, dentate gyrus, Fig. 1). The aggregational index was 1.0 in all regions investigated (Fig. 2). Barbiturate treated ischemic group. The electron microscopic investigation revealed mostly neurons with either normal aggregation or complete disaggregation of ribosomes, but there were several neurons in which both polysomes and monoribosomes were present. Post-is-
chemic barbiturate treatment did not prevent the initial post-ischemic disaggregation in either cortex, the CA1 and CA3 sector or the dentate gyrus: after 15 min of recirculation most cytoplasmic polysomes were disaggregated into monoribosomes (Fig. 1), and the aggregational index was close to zero (Fig. 2). An almost complete disaggregation was still observed after 2 h of recirculation, but at 8 h following barbiturate therapy reaggregation was present in all regions including the CA1 sector (Fig. 2). One day after post-ischemic treatment, the aggregational index had returned to control in all areas of the brain (Fig. 1). Previous studies of protein synthesis using autoradiographic and electron microscopic techniques have consistently demonstrated that global cerebral ischemia of as short as 5 min produces a global disaggregation of ribosomes and a global inhibition of protein synthesis that lasts for several hours although energy metabolism is rapidly resumed [1, 8, 13, 14, 22]. Remarkably, inhibition of protein synthesis and disaggregation of ribosomes is completely reversible in resistant areas, but not in the vulnerable regions. In particular, CA1 sector of hippocampus exhibits only transient incomplete recovery, followed by secondary suppression before cell death ensues [1, 21, 22]. This suggests that inhibition of proteins synthesis is an epiphenomenon or even the actual cause of selective vulnerability of hippocampal neurons. Barbiturates belong to the increasing number of drugs that have been found to alleviate hippocampal injury [8, 11, 12, 18, 19]. Other drugs with similar effects are glutaA I
Cortex
A I
-° 1
,°° l
1
/
oJ
Fig. 1. Electron micrographs of ribosomes in cortical and hippocampal CA I neurons of control animals (A,D) and after different postischemic recirculation times (B,E, 15 min; C,F, 24 h). In the control animal the neuronal cytoplasm contains mainly polysomes (arrows; A: cortical neuron; D: CA1 neuron). After recirculation of 15 min ribosomes are completelydispersed into monosomes (arrowheads; B: cortex; E: CA1 sector). Both regions present total reaggregation into polysomes (arrows) 24 h after ischemia (C: cortex; F: CA1 sector). Original magnification x 9,400.
oJ 2
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2l hours
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075 050]
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CA1
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24 hours
Gdent
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0
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0 251
025
0-'
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, isch~mia 1
, #--e-K,~-, 2 8 2/~h . . . .
ischemmio 1
; //-T--//-m 8 24 hours
Fig. 2. Aggregation index (A.I.) of ribosomes in neurons from cortex, hippocampal CA1 and CA3 sectors and dentate gyrus after postischemic treatment with barbiturates. Note reaggregation of ribosomes not only in the resistant areas but also in the selectivelyvulnerableCA1 sector.
77 mate receptor antagonists o f the n o n - N M D A type [3, 16], serotonin antagonists [5], adenosine or opioid agonists [4, 7], anticonvulsants [20], non-steroidal anti-inf l a m m a t o r y drugs [15] and inhibitors o f peroxidation [6] or protein kinase C activity [9]. Some o f these drugs maintain their therapeutic efficiency when applied after the ischemic impact [3, 5, 8, 11, 12, 16] and the therapeutical w i n d o w o f these substances varies between a b o u t 40 min for short-acting barbiturates [12] and several hours for the A M P A - r e c e p t o r antagonist N B Q X [16]. The present d e m o n s t r a t i o n that barbiturate does not prevent post-ischemic disaggregation but is able to reverse this disturbance, m a y provide an explanation for this p h e n o m e n o n . R i b o s o m e s are k n o w n to disaggregate when the rate o f polypeptide chain elongation and termination exceeds that o f initiation. U n d e r physiological conditions protein synthesis is mainly controlled by the speed o f polypeptide chain initiation (for review see ref. 10). The degree o f ribosomal aggregation is therefore an indirect indicator o f the protein synthesis rate. The c o m plete disaggregation o f polysomes in all parts o f the brain after the ischemic impact is in line with the previously observed global inhibition o f a m i n o acid i n c o r p o r a t i o n into brain proteins and suggests that the mechanism o f this disturbance is at the level o f initiation. Barbiturates reverse the disturbance o f protein synthesis in the vulnerable areas by p r o m o t i n g reaggregation o f ribosomes and, in consequence, o f polypeptide chain initiation. This m a y explain that they - and possibly other neuroprotective drugs - are able to prevent ischemic injury when applied at some time after the ischemic impact [12, 16]. In conclusion, the results o f the present investigation suggest that post-ischemic inhibition o f protein synthesis is due to translational disturbances at the polypeptide chain initiation step. Pentobarbital p r o m o t e s the reversal o f this disturbance in the selectively vulnerable neurons and thereby contributes to the prevention o f post-ischemic delayed neuronal death. The authors wish to t h a n k Mrs. M. H a h m a n n and Mrs. F. K i e r d o r f for their excellent secretarial assistance. We thank Mrs. I. Mtihlh6ver and Mr. B H u t h for the artwork. 1 Bodsch, W., Takahashi, K., Barbier, A., GroBe Ophoff, B. and Hossmann, K.-A., Cerebral protein synthesis and ischemia. In K. Kogure, K.-A. Hossmann, B.K. Siesj6 and F.A. Welsh (Eds.), Progress in Brain Research, Vol. 63, Elsevier, Amsterdam, 1985, pp. 197-210. 2 Cooper, H.K., Zalewska, T., Kawakami, S. and Hossmann, K.-A., The effects of ischemia and recirculation on protein synthesis in the brain, J. Neurochem., 28 (1977) 929 934. 3 Diemer, N.H., Johansen, F.F. and Jorgensen, M.B., N-Methyl-D-
aspartate and non-N-methyl-D-aspartate antagonists in global cerebral ischemia, Stroke, 21 (1990) 39-42. 4 Dux, E., Fastbom, J., Ungerstedt, U., Rudolphi, K. and Fredholm, B.B., Protective effect of adenosine and a novel xanthine derivative propentofylline on the cell damage after bilateral carotid occlusion in the gerbil hippocampus, Brain Res., 516 (1990) 248-256. 5 Fujikura, H., Kato, H., Nakano, S. and Kogure, K., A serotonin S-2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil, Brain Res., 494 (1989) 387390. 6 Hall, E.D. and Pazara, K.E., Quantitative analysis of effects of Kappa-opioid antagonists on postischemic hippocampal CA 1 neuronal necrosis in gerbils, Stroke, 19 (1988) 1008-1012. 7 Hall, E.D., Pazara, K.E. and Braughler, J.M., 21-Aminosteroid lipid peroxidation inhibitor U74006F protects against cerebral ischemia in gerbils, Stroke, 19 (1988) 997-1002. 8 Hallmayer, J., Hossmann, K.-A. and Mies, G., Low dose of barbiturates for prevention of hippocampal lesions after brief ischemic episodes, Acta Neuropathol., 68 (1985) 27 31. 9 Hara, H., Onodera, H., Yoshidomi, M., Matsuda, Y. and Kogure, K., Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat, J. Cereb. Blood Flow Metab., l0 (1990) 646-653. 10 Jagus, J., Anderson, W.F. and Safer, B., The regulation of initiation of mammalian protein synthesis, Prog. Nucl. Acid Res., 25 (1981) 127-185. 11 Kirino, T., Tamura, A. and Sano, K., A reversible type of neuronal injury following ischemia in the gerbil, Stroke, 17 (1986) 455-459. 12 Kuroiwa, T., Bonnekoh, E and Hossmann, K.-A., Therapeutic window of CAI neuronal damage defined by an ultrashort-acting barbiturate after brain ischemia in gerbils, Stroke, 21 (1990) 14891493. 13 Munekata, K. and Hossmann, K.-A., Effect of 5 minute ischemia on regional pH and energy state of the gerbil brain: relation to selective vulnerability of the hippocampus, Stroke, 8 (1987) 412417. 14 Munekata, K., Hossmann, K.-A., Xie, Y., Seo, K. and Oschlies, U., Selective vulnerability of hippocampus: ribosomal agregation, protein synthesis and tissue pH. In W.J. Powers and M.E. Raichle (Eds.), Cerebrovascular Diseases, Raven, New York, 1987, pp. 107-117. 15 Sasaki, T., Nakagomi, T., Kirino, T., Tamura, A., Noguchi, M., Saito, I. and Takakura, K., Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA-1 sector, Stroke, 19 (1988) 1399 1403. 16 Sheardown, M.J., Nielsen, E.O., Hansen, A.J., Jacobsen, E and Honore, T., 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline: a neuroprotectant for cerebral ischemia, Science, 247 (1990) 571-574. 17 Smith, C.B., Deibler, G.E., Eng, N., Schmidt, K. and Sokoloff, L., Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation, Proc. Natl. Acad. Sci. USA, 85 (1988) 9341-9345. 18 Smith, D.S., Rehncrona, S. and Siesj6, B.K., Barbiturates as protective agents in brain ischemia and as free radical scavengers in vitro, Acta Physiol. Scand. (Suppl.), 492 (1980) 129-134. 19 Spetzler, R.F. and Hadley, M.N., Protection against cerebral ischemia: the role of barbiturates, Cerebrovasc. Brain Metab. Rev., 1 (1989) 212-229. 20 Taft, W.C., Cliford, G.L., Blair, R.E. and De Lorenzo. R.J., Phenytoin protects against ischemia-produced neuronal cell death, Brain Res., 483 (1989) 143 148.
78 21 Thilmann, R., Xie, Y., Kleihues, P. and Kiessling, M., Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus, Acta Neuropathol., 60 (1986) 88 93. 22 Widmann, R., Kuroiwa, T., Bonnekoh, P. and Hossmann, K.-A., [~4C]Leucine incorporation into brain proteins in gerbils after tran-
sient ischemia: relationship to selective vulnerability of hippocampus, J. Neurochem., 56 (1991) 789-796. 23 Xie, Y., Seo, K. and Hossmann, K.-A., Effect of barbiturate treatment on post-ischemic protein biosynthesis in gerbil brain, J. Neurol. Sci., 92 (1989) 317 328.
75
NSL 09043
Barbiturate promotes post-ischemic reaggregation of polyribosomes in gerbil hippocampus P e t r a B o n n e k o h , T o s h i h i k o K u r o i w a l, U t e Oschlies a n d K o n s t a n t i n - A l e x a n d e r H o s s m a n n
Max-Planck-lnstitutefor NeurologicalResearch, Department of Experimental Neurology, Cologne (FRG) (Received 22 May 1992; Revised version received 13 July 1992; Accepted 29 July 1992)
Key words: Transientforebrain ischemia; Selectivevulnerability; Polyribosome; Mongolian gerbil; Hippocampus; Delayed neuronal death; Barbiturate A brief period of cerebral ischemia is followed by severe inhibition of protein synthesis which is slowly reversed in the resistant but not in the selectivelyvulnerable regions of the brain. Inhibition occurs at the translational level, as evidenced by the disaggregation of ribosomes into monosomes. In order to evaluate the importance of this disturbance for the evolution of ischemic injury, the effect of the neuroprotective drug, pentobarbital, on ribosomal aggregation was studied in gerbils subjected to 5 min bilateral carotid artery occlusion. Pentobarbital (50 mg/kg, i.p.) was applied shortly after the ischemia, and the aggregational state of ribosomes was investigated by electron microscopy after recirculation times ranging from 15 min to 1 day. Pentobarbital treatment did not prevent the initial post-ischemicdisaggregation but promoted the subsequent reaggregation in the selectivelyvulnerable neurons. This suggests that post-ischemicapplication of barbiturates exerts its beneficialeffect by reversing the post-ischemic block of ribosomal reaggregation in vulnerable regions.
A brief period of transient global ischemia of the brain results in prolonged inhibition of protein synthesis [1, 21, 22] although energy metabolism is restored within a few minutes [14]. As shown by quantitative autoradiography of amino acid incorporation into brain proteins, anatomical structures that recover from the ischemic episode resume protein synthesis after a few hours whereas in selectively vulnerable regions such as the hippocampal CA1 subfield protein synthesis is not or only transiently and incompletely restored [1, 21, 22]. A comparison of the autoradiographic findings with electron microscopical investigations of the aggregational state of ribosomes shows a close correlation. With the beginning of recirculation polyribosomes disaggregate into monosomes in all regions in which protein synthesis is suppressed. After a few hours new polyribosomes are formed in the resistant but not in the vulnerable structures of the brain [14]. This and in vitro assays of ribosomal fractions [2] suggest that the post-ischemic inhibition of protein synthesis is due to an inhibition of
Correspondence: K.-A. Hossmann, Max-Planck-Institut for Neurologische Forschung, Gleuelerstr. 50, D-5000 KSln 41, FRG. ~Present address: Medical Research Institute, Tokyo Medical and Dental University,Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan.
m R N A translation at the polypeptide chain initiation step. Barbiturates are known to reverse the post-ischemic suppression of protein synthesis and to prevent neuronal death in the selectively vulnerable regions [23]. Therefore, the question arose whether barbiturates act by preventing the initial disaggregation or by promoting the subsequent reaggregation of ribosomes. The study included 12 adult gerbils (Meriones unguiculatus) of either sex weighing 50-80 g. Prior to the experiments the animals had free access to water and food. Anesthesia was induced by 3% halothane and then continued with 1.5% halothane in a gas mixture o f 70% nitrogen and 30% oxygen. Rectal temperature was kept close to 37.5°C by a feed-back controlled heating system. The c o m m o n carotid arteries were exposed on both sides by a midline cervical incision and occluded for 5 min with miniature Biemer clips (FD562, Aesculap, Tuttlingen, F R G ) . After the ischemic period the clips were released and the anesthesia was discontinued. Pentobarbital (50 mg/kg) was injected intraperitoneaUy immediately after vascular release, and the animals were allowed to survive for 15 min, 1, 2, 8 and 24 h, respectively. Successful reversal of the post-ischemic suppression of protein synthesis with this drug dosage has been shown in previous studies [8, 23]. After the respective recirculation
76 times animals were reanesthetized and transcardially perfused with 2.5% glutaraldehyde/2% paraformaldehyde. The brains were carefully removed, postfixed with the same fixative and processed for electron microscopy. Electron micrographs were taken from 50 neurons of the frontal cortex, the dentate gyrus and the CA1 and CA3 sectors of hippocampus. An index of mean ribosomal aggregation (A.I.) was calculated by classifying each neuron as follows: 1.0 -- normal aggregation, 0.5 = partial disaggregation and 0 = complete disaggregation of cytoplasmic polyribosomes. The results obtained were compared with a previous series of experiments in which similar measurements were carried out in untreated gerbils [14]. Control group. Non-ischemic control animals exhibited cytosolic polyribosomal complexes consisting of 5 6 ribosomes in all cortical and hippocampal neurons (CA1 and CA3 sector, dentate gyrus, Fig. 1). The aggregational index was 1.0 in all regions investigated (Fig. 2). Barbiturate treated ischemic group. The electron microscopic investigation revealed mostly neurons with either normal aggregation or complete disaggregation of ribosomes, but there were several neurons in which both polysomes and monoribosomes were present. Post-is-
chemic barbiturate treatment did not prevent the initial post-ischemic disaggregation in either cortex, the CA1 and CA3 sector or the dentate gyrus: after 15 min of recirculation most cytoplasmic polysomes were disaggregated into monoribosomes (Fig. 1), and the aggregational index was close to zero (Fig. 2). An almost complete disaggregation was still observed after 2 h of recirculation, but at 8 h following barbiturate therapy reaggregation was present in all regions including the CA1 sector (Fig. 2). One day after post-ischemic treatment, the aggregational index had returned to control in all areas of the brain (Fig. 1). Previous studies of protein synthesis using autoradiographic and electron microscopic techniques have consistently demonstrated that global cerebral ischemia of as short as 5 min produces a global disaggregation of ribosomes and a global inhibition of protein synthesis that lasts for several hours although energy metabolism is rapidly resumed [1, 8, 13, 14, 22]. Remarkably, inhibition of protein synthesis and disaggregation of ribosomes is completely reversible in resistant areas, but not in the vulnerable regions. In particular, CA1 sector of hippocampus exhibits only transient incomplete recovery, followed by secondary suppression before cell death ensues [1, 21, 22]. This suggests that inhibition of proteins synthesis is an epiphenomenon or even the actual cause of selective vulnerability of hippocampal neurons. Barbiturates belong to the increasing number of drugs that have been found to alleviate hippocampal injury [8, 11, 12, 18, 19]. Other drugs with similar effects are glutaA I
Cortex
A I
-° 1
,°° l
1
/
oJ
Fig. 1. Electron micrographs of ribosomes in cortical and hippocampal CA I neurons of control animals (A,D) and after different postischemic recirculation times (B,E, 15 min; C,F, 24 h). In the control animal the neuronal cytoplasm contains mainly polysomes (arrows; A: cortical neuron; D: CA1 neuron). After recirculation of 15 min ribosomes are completelydispersed into monosomes (arrowheads; B: cortex; E: CA1 sector). Both regions present total reaggregation into polysomes (arrows) 24 h after ischemia (C: cortex; F: CA1 sector). Original magnification x 9,400.
oJ 2
8
AI
/ ~
/
/"--'/ i ischemio l
2l hours
CA3
075 050]
ool !
\ i Jschemia 1
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CA1
8
24 hours
Gdent
°°1
0
2
75
050
0 251
025
0-'
0
, isch~mia 1
, #--e-K,~-, 2 8 2/~h . . . .
ischemmio 1
; //-T--//-m 8 24 hours
Fig. 2. Aggregation index (A.I.) of ribosomes in neurons from cortex, hippocampal CA1 and CA3 sectors and dentate gyrus after postischemic treatment with barbiturates. Note reaggregation of ribosomes not only in the resistant areas but also in the selectivelyvulnerableCA1 sector.
77 mate receptor antagonists o f the n o n - N M D A type [3, 16], serotonin antagonists [5], adenosine or opioid agonists [4, 7], anticonvulsants [20], non-steroidal anti-inf l a m m a t o r y drugs [15] and inhibitors o f peroxidation [6] or protein kinase C activity [9]. Some o f these drugs maintain their therapeutic efficiency when applied after the ischemic impact [3, 5, 8, 11, 12, 16] and the therapeutical w i n d o w o f these substances varies between a b o u t 40 min for short-acting barbiturates [12] and several hours for the A M P A - r e c e p t o r antagonist N B Q X [16]. The present d e m o n s t r a t i o n that barbiturate does not prevent post-ischemic disaggregation but is able to reverse this disturbance, m a y provide an explanation for this p h e n o m e n o n . R i b o s o m e s are k n o w n to disaggregate when the rate o f polypeptide chain elongation and termination exceeds that o f initiation. U n d e r physiological conditions protein synthesis is mainly controlled by the speed o f polypeptide chain initiation (for review see ref. 10). The degree o f ribosomal aggregation is therefore an indirect indicator o f the protein synthesis rate. The c o m plete disaggregation o f polysomes in all parts o f the brain after the ischemic impact is in line with the previously observed global inhibition o f a m i n o acid i n c o r p o r a t i o n into brain proteins and suggests that the mechanism o f this disturbance is at the level o f initiation. Barbiturates reverse the disturbance o f protein synthesis in the vulnerable areas by p r o m o t i n g reaggregation o f ribosomes and, in consequence, o f polypeptide chain initiation. This m a y explain that they - and possibly other neuroprotective drugs - are able to prevent ischemic injury when applied at some time after the ischemic impact [12, 16]. In conclusion, the results o f the present investigation suggest that post-ischemic inhibition o f protein synthesis is due to translational disturbances at the polypeptide chain initiation step. Pentobarbital p r o m o t e s the reversal o f this disturbance in the selectively vulnerable neurons and thereby contributes to the prevention o f post-ischemic delayed neuronal death. The authors wish to t h a n k Mrs. M. H a h m a n n and Mrs. F. K i e r d o r f for their excellent secretarial assistance. We thank Mrs. I. Mtihlh6ver and Mr. B H u t h for the artwork. 1 Bodsch, W., Takahashi, K., Barbier, A., GroBe Ophoff, B. and Hossmann, K.-A., Cerebral protein synthesis and ischemia. In K. Kogure, K.-A. Hossmann, B.K. Siesj6 and F.A. Welsh (Eds.), Progress in Brain Research, Vol. 63, Elsevier, Amsterdam, 1985, pp. 197-210. 2 Cooper, H.K., Zalewska, T., Kawakami, S. and Hossmann, K.-A., The effects of ischemia and recirculation on protein synthesis in the brain, J. Neurochem., 28 (1977) 929 934. 3 Diemer, N.H., Johansen, F.F. and Jorgensen, M.B., N-Methyl-D-
aspartate and non-N-methyl-D-aspartate antagonists in global cerebral ischemia, Stroke, 21 (1990) 39-42. 4 Dux, E., Fastbom, J., Ungerstedt, U., Rudolphi, K. and Fredholm, B.B., Protective effect of adenosine and a novel xanthine derivative propentofylline on the cell damage after bilateral carotid occlusion in the gerbil hippocampus, Brain Res., 516 (1990) 248-256. 5 Fujikura, H., Kato, H., Nakano, S. and Kogure, K., A serotonin S-2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil, Brain Res., 494 (1989) 387390. 6 Hall, E.D. and Pazara, K.E., Quantitative analysis of effects of Kappa-opioid antagonists on postischemic hippocampal CA 1 neuronal necrosis in gerbils, Stroke, 19 (1988) 1008-1012. 7 Hall, E.D., Pazara, K.E. and Braughler, J.M., 21-Aminosteroid lipid peroxidation inhibitor U74006F protects against cerebral ischemia in gerbils, Stroke, 19 (1988) 997-1002. 8 Hallmayer, J., Hossmann, K.-A. and Mies, G., Low dose of barbiturates for prevention of hippocampal lesions after brief ischemic episodes, Acta Neuropathol., 68 (1985) 27 31. 9 Hara, H., Onodera, H., Yoshidomi, M., Matsuda, Y. and Kogure, K., Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat, J. Cereb. Blood Flow Metab., l0 (1990) 646-653. 10 Jagus, J., Anderson, W.F. and Safer, B., The regulation of initiation of mammalian protein synthesis, Prog. Nucl. Acid Res., 25 (1981) 127-185. 11 Kirino, T., Tamura, A. and Sano, K., A reversible type of neuronal injury following ischemia in the gerbil, Stroke, 17 (1986) 455-459. 12 Kuroiwa, T., Bonnekoh, E and Hossmann, K.-A., Therapeutic window of CAI neuronal damage defined by an ultrashort-acting barbiturate after brain ischemia in gerbils, Stroke, 21 (1990) 14891493. 13 Munekata, K. and Hossmann, K.-A., Effect of 5 minute ischemia on regional pH and energy state of the gerbil brain: relation to selective vulnerability of the hippocampus, Stroke, 8 (1987) 412417. 14 Munekata, K., Hossmann, K.-A., Xie, Y., Seo, K. and Oschlies, U., Selective vulnerability of hippocampus: ribosomal agregation, protein synthesis and tissue pH. In W.J. Powers and M.E. Raichle (Eds.), Cerebrovascular Diseases, Raven, New York, 1987, pp. 107-117. 15 Sasaki, T., Nakagomi, T., Kirino, T., Tamura, A., Noguchi, M., Saito, I. and Takakura, K., Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA-1 sector, Stroke, 19 (1988) 1399 1403. 16 Sheardown, M.J., Nielsen, E.O., Hansen, A.J., Jacobsen, E and Honore, T., 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline: a neuroprotectant for cerebral ischemia, Science, 247 (1990) 571-574. 17 Smith, C.B., Deibler, G.E., Eng, N., Schmidt, K. and Sokoloff, L., Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation, Proc. Natl. Acad. Sci. USA, 85 (1988) 9341-9345. 18 Smith, D.S., Rehncrona, S. and Siesj6, B.K., Barbiturates as protective agents in brain ischemia and as free radical scavengers in vitro, Acta Physiol. Scand. (Suppl.), 492 (1980) 129-134. 19 Spetzler, R.F. and Hadley, M.N., Protection against cerebral ischemia: the role of barbiturates, Cerebrovasc. Brain Metab. Rev., 1 (1989) 212-229. 20 Taft, W.C., Cliford, G.L., Blair, R.E. and De Lorenzo. R.J., Phenytoin protects against ischemia-produced neuronal cell death, Brain Res., 483 (1989) 143 148.
78 21 Thilmann, R., Xie, Y., Kleihues, P. and Kiessling, M., Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus, Acta Neuropathol., 60 (1986) 88 93. 22 Widmann, R., Kuroiwa, T., Bonnekoh, P. and Hossmann, K.-A., [~4C]Leucine incorporation into brain proteins in gerbils after tran-
sient ischemia: relationship to selective vulnerability of hippocampus, J. Neurochem., 56 (1991) 789-796. 23 Xie, Y., Seo, K. and Hossmann, K.-A., Effect of barbiturate treatment on post-ischemic protein biosynthesis in gerbil brain, J. Neurol. Sci., 92 (1989) 317 328.
