Hideaki Kanemitsu

Early recovery of protein synthesis following ischemia in hippocampal neurons with induced tolerance in the gerbil.

SummaryFollowing brief cerebral ischemia, tolerance to subsequent ischemia is induced in the hippocampal neurons. In this experiment, recovery of protein synthesis was investigated autoradiographically in gerbils with induced tolerance. The animals were subjected to single forebrain ischemia for 5 min (5-min ischemia group) or 2 min (2-min ischemia group). To observe the effect of tolerance acquisition, double forebrain ischemia (double ischemia group), 2-min ischemia followed by 5-min ischema was induced 2 days later. At various recircultion periods (90 min, 6 h, 1 day, and 4 days following ischemia), animals received a single dose of Lxxx-[2,3-3H]valine. In the 5-min ischemia group, protein synthesis in the CA1 sector was severely suppressed during the period from 90 min to 1 day of recirculation and never returned to the normal level even at 4 day of recirculation. In the 2-min ischemia group, protein synthesis recovered gradually and returned to near normal at 4 days of recirculation. On the other hand, in the double ischemia group, recovery of protein synthesis in the CA1 sector was rapid. At 1 day of recirculation, protein synthesis returned to near normal. Protein synthesis in the CA2 sector was inhibited during the 4 days of recirculation in this group. The present study revealed an early recovery of protein synthesis in the hippocampal CA1 neurons in the gerbil with induced tolerance. We suggest that recovery of protein synthesis is essential for the survival of neurons exposed to transient ischemia.

Differences in the Extent of Primary Ischemic Damage Between Middle Cerebral Artery Coagulation and Intraluminal Occlusion Models

The authors studied the differences between heat-shock/stress protein 70 (hsp70) gene expression and protein synthesis in the unilateral middle cerebral artery (MCA) microsurgical direct occlusion (Tamuras) model and the unilateral intraluminal occlusion model. In Tamuras model, expression of hsp70 mRNA and HSP70 protein and decreased protein synthesis were detected in the ischemic areas, including the ipsilateral cortex and caudate. These phenomena, however, were not observed in the areas outside the MCA territory, including the ipsilateral thalamus, hippocampus, and substantia nigra. These results were consistent among the experimental rats. In the intraluminal occlusion model, however, induction of both hsp70 mRNA and HSP70 protein and impairment of protein synthesis were noted in the areas outside the MCA territory, including the ipsilateral thalamus, hypothalamus, hippocampus, and substantia nigra, as well as in the MCA territory, including the ipsilateral cortex and caudate. These results were not consistent among the experimental rats. These different results might be due to widespread damage resulting from internal carotid artery (ICA) occlusion in the intraluminal occlusion model. Accordingly, the authors suggest that this model be called an ICA occlusion model, rather than a pure MCA occlusion model.

Modulation of Protein Kinases and Microtubule-associated Proteins and Changes in Ultrastructure in Female Rat Pituitary Cells: Effects of Estrogen and Bromocriptine:

This study focused on the intracellular signal transduction system and microtubule-associated proteins (MAPs), such as MAP-2 and Tau protein. The modulation of these proteins and their correlation with ultrastructural changes were investigated in rat pituitary prolactin (PRL) cells. Adult female Wistar rats were treated with estrogen and bromocriptine and their pituitary glands were removed for analysis of the expression of tubulin, MAP-2, Tau protein, protein kinase C (PKC), and calcium calmodulin (CaM) kinase. Western blot analysis showed that estrogen increased and bromocriptine decreased the expression of PKCα, β1, β2, CaM kinase α, β, MAP-2, and Tau protein. MAP-2 and Tau protein, which are cytosolic proteins, being translated on free ribosomes, were associated with the membrane of whirling rough endoplasmic reticulum (RER) in estrogen-treated cells and dissociated with vesiculated RER induced by bromocriptine. These results suggested that the modulation of MAP-2 and Tau protein may reflect changes of PKC and CaM kinase, and that the quantitative changes and intracellular modulation of MAPs induced by estrogen and bromocriptine, i.e., estrogen-induced association and bromocriptine-induced dissociation of MAP-2 and Tau protein with membrane of RER, may reflect the dynamics of microtubules and are associated with structural changes in the RER and changes in the synthesis and intracellular transport of PRL.

Up-regulation of c-myc gene expression following focal ischemia in the rat brain

Changes in gene expression including that of c-fos occur following cerebral ischemia. Proto-oncogenes c-myc and s-myc and oncosuppressor gene p53 are known to induce apoptosis in some types of cells, whereas proto-oncogene bcl-2 inhibits apoptosis. Possible induction of mRNAs for c-myc, N-myc, s-myc, c-fos, p53 and bcl-2 was examined following focal ischemia in the rat anterior cortex, hippocampus, thalamus and cerebellum by Northern blot analysis. Animals were decapitated 1, 2, 6, 12, and 24 hours following the left middle cerebral artery (MCA) occlusion. In sham-operated control rats, the mRNAs for c-myc, N-myc, c-fos and p53 were present in the anterior cortex, hippocampus, thalamus on both sides, and in the cerebellum, whereas those for s-myc and bcl-2 were not. The c-myc gene expression was rapidly and markedly induced by the MCA occlusion in the ipsilateral anterior cortex, hippocampus and thalamus in a time-dependent manner. In these regions, the c-fos gene expression was also induced as early as 1 hour after the MCA occlusion. The p-53 mRNA was induced in the ipsilateral hippocampus at 24 hours after MCA occlusion. In contrast, mRNAs for N-myc, s-myc and bcl-2 were not induced following MCA occlusion. These results indicate a possibility that high-level expression of the c-myc gene may be involved in the ischemic cellular events including apoptosis.

Cerebral Uric Acid, Xanthine, and Hypoxanthine after Ischemia: The Effect of Allopurinol

The existence of uric acid in mammalian brain was recently reported, but it has not yet become a consensus. The mammalian brain has been thought to lack xanthine oxidase, which catalyzes hypoxanthine to xanthine and xanthine to uric acid as the last steps of ATP degradation in other tissue. Using high-performance liquid chromatography, we performed assays for hypoxanthine, xanthine, and uric acid in rat brain after cerebral ischemia. It was confirmed that all three substances showed significant augmentation in the removed brains and that the chronological order of those increases corresponded to the order in the metabolic pathway. Allopurinol, a specific inhibitor of xanthine oxidase, significantly suppressed the increases in uric acid and xanthine, and a compensatory accumulation of hypoxanthine was observed. From these results, it was concluded that uric acid does exist in the brain, increases after ischemia, and is possibly the end product of purine degradation in the brain. Furthermore, it is suggested that xanthine oxidase exists in the brain and catalyzes the reaction from hypoxanthine to xanthine and then to uric acid. These reactions catalyzed by xanthine oxidase are considered to be a source of free radicals and may play important roles in the pathogenesis of cerebral ischemic injury.

Changes of Uric Acid Level in Rat Brain After Focal Ischemia

Abstract: Changes of uric acid level in rat cerebral hemisphere after left middle cerebral artery (MCA) occlusion were studied by reversed‐phase HPLC with electrochemical detection. Uric acid level in the normal group was 2.98 nmol/g tissue. Uric acid concentration of the left hemisphere in the left MCA‐occluded group progressively increased after occlusion, and reached a maximum value of 67.26 nmol/g tissue 24 h after ischemia. Uric acid levels in the right hemisphere remained unchanged. Uric acid concentration of the left hemisphere in sham‐operated group was 9.29 nmol/g tissue 24 h after the operation.

Allopurinol inhibits uric acid accumulation in the rat brain following focal cerebral ischemia

Uric acid (UA) in the rat brain was measured by HPLC with an electrochemical detector following focal ischemia. At 24 h after the operation, the UA level in the ischemic center was 105.47 +/- 8.39 nmol/g tissue, whereas it was 8.36 +/- 1.86 in the sham-operated group. Allopurinol, xanthine oxidase inhibitor, almost completely inhibited this UA accumulation. These data demonstrate that the UA increase in the ischemic brain is due to the xanthine oxidase reaction.

Key of induced tolerance to ischaemia in gerbil hippocampal CA1 is not at transcriptional level of hsp70 gene: in situ hybridization of hsp70 mRNA.

Hippocampal neurons, when pretreated with sublethal ischaemia, acquired tolerance to future treatments of normally lethal ischaemia. The level of transcription of the stress protein hsp70 was studied by in situ hybridization in control, ischaemic and tolerance-induced-ischaemic hippocampal neurons. Mongolian gerbils were subjected to single forebrain ischaemia for 2 min (2-min ischaemia group: sublethal) or 5 min (5-min ischaemia group: lethal). Other animals, were exposed to sublethal ischaemia for 2 min and were subjected subsequently to ischaemia for 5 min, 2 days later. Animals were sacrificed at 6, 12, 24 and 48 h following ischaemia and in situ hybridization was performed with thin cross sections including the hippocampus. Hybridization of the hsp70 probe in the control group (no ischaemia) was barely visible in the brain. In the 2-min ischaemia group, intense hybridization was seen in the CA1 sector from 6 through 24 h of recirculation but hybridization almost disappeared at 48 h of recirculation. In the 5-min and the double ischaemic groups, the intense induction in the CA1 sector occurred during the period from 6 through 48 h of recirculation. Our present study by in situ hybridization reveals high levels of transcription of hsp70 message following 5-min of ischaemia, however no protein was detectable based on our previous experiments employing immunocytochemistry. In tolerance-induced hippocampal neurons, both hsp70 message and protein are present. These results suggest that tolerance-induced neurons are modified such that the hsp70 gene is both transcribed and translated. These changes most likely give rise to the tolerance observed in the double ischaemia group.

EFFECT OF L-ARGININE AND NG-NITRO-L-ARGININE ON DELAYED NEURONAL DEATH IN THE GERBIL HIPPOCAMPUS

To assess the role of nitric oxide (NO) in cerebral ischemia, we investigated the effect of L-arginine, a substrate of NO synthase (NOS), and NG-nitro-L-arginine (L-NNA), a NOS inhibitor, on neuronal death in the CA1 hippocampal region. Seventy-two Mongolian gerbils were used in the study. Both carotid arteries were occluded for 4 min to induce forebrain ischemia. Temporal muscle temperature was strictly maintained at 37.5 +/- 0.3 degrees C during the ischemia. L-arginine (10 and 100 mg kg-1) or L-NNA (1, 10 and 100 mg kg-1) was administered intraperitoneally 4 times: 30 min before, 3 h, 6 h and 24 h after induction of ischemia. Four days after ischemic insult, the animals were perfusion-fixed, and the neuronal densities in the medial, middle and lateral CA1 subfield were estimated. Average neuronal cell density of the control group was 2-3 mm in each subfield. L-arginine at doses of 10 and 100 mg kg-1 did not prevent neuronal death. L-NNA at doses of 1 and 10 mg kg-1 did not protect neuronal cells from ischemia either. However, in ischemia gerbils treated with 100 mg kg-1 L-NNA, the average neuronal cell density in the lateral CA1 subfield was 54.4 +/- 19.1, L-NNA (100 mg kg-1) significantly (p < 0.05) reduced the occurrence of neuronal death in the lateral CA1 subfield. The present results suggest that NO plays an important role in the development of neuronal injury after global ischemia.

Changes of superoxide dismutase activity and ascorbic acid in focal cerebral ischaemia in rats

Free radical reactions are supposed to cause ischaemic brain damage, and active oxygens can initiate these chains reaction. If active oxygens play important roles in ischaemic brain damage, the activity of superoxide dismutase, scavenger of superoxide anion, is supposed to decrease in ischaemic brain. The reduced form of ascorbic acid also scavenges superoxide anion. In rat middle cerebral artery focal ischaemia, we investigated the changes in superoxide dismutase activity and the concentration of reduced ascorbate up to 48 hours. Middle cerebral artery territory of each cerebral hemisphere was homogenized. The supernatant was divided into two aliquots; one was dialysed to remove ascorbate and the other was not. The enzyme activity of the dialysed specimen from the ischaemic hemisphere did not decrease within 4 h after the arterial occlusion. The activity of the dialysed specimen from the nonischaemic side remained unchanged during the examination. Reduced ascorbate levels in nondialysed samples showed similar changes to the superoxide dismutase activities in the dialysed samples. Our data suggest that ascorbic acid may exert the enzyme activity and that the enzyme activity remains at the normal level in the early phase of ischaemia despite the irreversible ischaemic changes that take place within 4 h after the onset of ischaemia.