Yanagihara, Takehiko

The effect of cerebral anoxia on leucine incorporation into proteins of various subcellular fractions from rabbit brain was studied by using a previously reported in vitro model. Although the decrease in leucine incorporation was observed earlier in nuclei and microsomes, other fractions also demonstrated a rapid decrease. These results were quite different from the pattern of leucine incorporation observed in a type of reversible suppression produced with diphenylhydantoin.

Amino acid incorporation into proteins of rabbit brain slices was studied during the recovery stage after various anoxic periods in an in vitro experimental model. There was a distinct difference between anoxic periods up to five minutes and those beyond ten minutes. In the former, inhibition of amino acid incorporation was of lesser degree and was reversible. On the other hand, more than 50% inhibition was observed following anoxic periods of 30 minutes and this inhibition was irreversible. Amino acid incorporation into proteins was more sensitive to anoxia than was amino acid transport into brain slices.

A combined autoradiographic and immunohistochemical method was used to correlate the extent of focal cerebral ischemia and morphologic ischemic damage following unilateral carotid occlusion in 16 gerbils for 5-30 minutes. Immunohistochemical lesions detectable by the reaction for microtubule-associated proteins 1 and 2 were visible in the subiculum-CA1 and CA2 regions of the hippocampus and layer III/IV of the cerebral cortex after 5 minutes of ischemia (n = 4). Local blood flow was promptly reduced but still heterogeneous after 10 minutes of ischemia (n = 4); local blood flow in immunohistochemical lesions was less than 5 ml/100 g/min except in highly vulnerable regions, where flow values of 5-15 ml/100 g/min were observed. After 15 minutes of ischemia (n = 4) local blood flow in less vulnerable regions including the thalamus and caudoputamen also declined to less than 5 ml/100 g/min, and immunohistochemical lesions became visible in those regions after 30 minutes of ischemia (n = 4). On the other hand, many brain regions tolerated local blood flow of less than 5 ml/100 g/min without ischemic damage. The present study demonstrates that selective tissue vulnerability during progressive cerebral ischemia depends on the degree of hypoperfusion and on factors inherent to neurons in various brain regions.

In order to compare the difference in the vulnerability of macromolecular syntheses, protein, RNA, and lipid syntheses were studied with ischemic brain tissue three hours following unilateral carotid ligation in gerbils. Precursor incorporation was measured in various subcellular fractions following in vitro incorporation with brain slices. There was marked inhibition of protein synthesis, but RNA and phospholipid syntheses showed little or no change. On the basis of available information on rapid deterioration of polysomal system for polypeptide synthesis, a hypothesis was proposed that messenger ribonucleic acid (RNA) at the polysomal level is promptly affected in this pathophysiologic condition.

A previously described in vitro model for cerebral anoxia has been modified to ensure vitality of the rabbit brain slices. The oxygen or nitrogen atmosphere was provided by a continuous gas flow system. Amino acid incorporation into proteins was studied during recovery periods up to 60 minutes. An initial decrease of incorporation and subsequent recovery were observed after an anoxic period of five minutes. On the other hand, a marked decrease and only partial recovery were seen after anoxic periods of 15 minutes or longer. No consistent change was observed in acid-soluble radioactivity. This in vitro model appears to be feasible for various biochemical studies of cerebral anoxia.

Background and Purpose Proto-oncogene activation and induction of heat shock protein (HSP) occur in response to various stimuli to brain, but the role in neuronal survival after cerebral ischemia remains uncertain. We compared the extent of insults and induction of c- fos and c- jun gene products (c-FOS and c-JUN) as well as HSP in ischemic and postischemic gerbil brains immunohistochemically. Methods Common carotid arteries of Mongolian gerbils were occluded for 5 or 15 minutes and recirculated for 0 minutes to 7 days. Antibodies for c-FOS, c-JUN, and HSP 70 were used for immunohistochemistry, and positive reactions were semiquantitatively analyzed. The presence of ischemic and postischemic lesions was ascertained with an antibody for microtubule-associated proteins. Results After ischemia for 15 minutes and reperfusion, c-FOS was induced promptly after 1 to 6 hours in pyramidal cells of the CA3 and CA4 regions, while c-JUN became visible in the same areas after recirculation for 4 to 48 hours. HSP 70 was detected after recirculation for 24 hours in the CA3 region. In layers I and II of the cerebral cortex, c-FOS and c-JUN peaked at 3 hours and HSP 70 at 96 hours. Induction of these proteins was absent or negligible in the areas that developed ischemic or postischemic lesions, including the subiculum-CA1 and CA1 regions of the hippocampus and layers III/IV and Vb/VI of the cerebral cortex. After shorter ischemia for 5 minutes and reperfusion, c-FOS and c-JUN were rapidly induced at 15 minutes to 1 hour except for the subiculum-CA1 and CA1 regions of the hippocampus. Induction of HSP 70 did not occur for 24 hours and was noted only in the hippocampus. Conclusions Induction of c-FOS and c-JUN occurred in the areas surviving after transient cerebral ischemia, but the extent of induction and the latent period varied depending on the duration of the insult and the location. In the areas with ischemic or postischemic damage detected by loss of the reaction for microtubule-associated proteins, the induction of c-FOS and c-JUN was either absent or minimal, suggesting that active induction of those immediate early gene products occurred early in surviving neurons. On the other hand, the induction of HSP 70 did not occur until reperfusion for 24 hours and actively occurred only in the areas with earlier induction of c-FOS and/or c-JUN, suggesting that the induction of HSP 70 occurred in neurons that survived to that point, but it did not participate in early responses for neuronal survival after global cerebral ischemia.

A method was developed to predict the severity of cerebral ischemia before permanent occlusion of a common carotid artery in gerbils by observing the diameter and appearance of the artery after temporary occlusion and observing clinical signs after permanent occlusion. The severity of cerebral ischemia was confirmed by a sensitive immunohistochemical method and measurement of focal cerebral blood flow after 30 minutes' ischemia. All gerbils with greater than 40% reduction of the diameter and a white arterial margin distal to temporary occlusion developed severe neurologic signs following permanent occlusion, but no gerbils with reduction of less than 30% and a red arterial margin developed neurologic signs. With the cumulative neurologic score, gerbils could be divided into classes with no, mild, moderate, and severe symptoms, mostly after 10 minutes. Severely symptomatic gerbils were identified in 3 minutes. Extensive ischemic damage was observed in severely symptomatic gerbils, but no immunohistochemical lesion was detected in mildly symptomatic gerbils. Cerebral blood flow was markedly reduced in severely symptomatic gerbils but more selectively reduced in the cortical structures of moderately symptomatic gerbils. This prediction method is useful for investigating early cerebral ischemia and for evaluating the effectiveness of pharmacologic agents.

We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.