Yoshihiko Uemura

Expression of Interleukin-1β Converting Enzyme Gene Family and bcl-2 Gene Family in the Rat Brain following Permanent Occlusion of the Middle Cerebral Artery

Recent investigations have been suggesting that some neuronal subpopulations may die via programmed cell death after focal ischemic injury. To clarify the possible roles of the genes involved in the cell-death program, this study examined the expression of three members of the interleukin-1β converting enzyme (Ice) gene family (Ice, Nedd2, and Yama/CPP32) and two members of the bcl-2 gene family (bcl-2 and bcl-x) in the rat brain after permanent occlusion of the middle cerebral artery. Northern blot analysis revealed a transient induction of Nedd2 mRNA 8 h after the ischemic insult (3.8-fold) and an increase in Yama/CPP32 mRNA 16 to 24 h after the insult (5.8-fold at 24 h), whereas the expression of Ice remained constant. The expression of bcl-2 and bcl-x remained constant after the ischemic insult. Taking into account the key role of the Ice gene family in the execution of programmed cell death, the induction of Ice gene family might play a causative role in apoptotic cell death.

Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model

Various studies have demonstrated an increase in heat shock protein 70 (HSP70) synthesis in the brain following transiently induced ischemia, suggesting a protective role for HSP70 against ischemic insult. In this study, we determined the time course of HSP70 mRNA and protein induction in rat hippocampus following ischemia using Pulsinellis four-vessel occlusion model, and suggested a protective role for HSP70 induction in limiting ischemic damage to neurons and delayed neuronal death. In Northern blotting analysis using human HSP70 DNA as a probe, the accumulation of HSP70 mRNA after 5 min ischemia became evident at 4 h, and continued until 16 h, while after 30 min ischemia, HSP70 mRNA appeared at 2 h, and continued above control level until 24 h after treatment. In immunoblot analysis using anti-HSP70 antibody, induction of HSP70 protein appeared 24 h and reached a maximum 48 h after 5 min ischemia. In immunohistochemical analysis using anti-HSP70 antibody, staining was not detected in CA1 neurons until 16 h after 5 min ischemia, but staining in CA1 gradually increased 1 day after ischemia and reached a maximum level 2 days after ischemia. Similar time profiles in the staining pattern of HSP70 protein were observed in CA3 and CA4 neuronal cells following 30 min ischemia. When rats pretreated with 5 min ischemia (non-lethal for CA1 pyramidal neurons) were exposed to a 30 min, lethal period of ischemia, 2 days after pretreatment, considerable staining of HSP70 was observed. Pretreated rats had much less neuronal damage in the CA1 sector than did rats subjected to lethal, 30 min ischemia alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine hydroxylase-like immunoreactive neurons in the striatum of the rat.

Striatal neurons exhibiting tyrosine hydroxylase-like immunoreactivity (TH-LI) were found in the adult rat. Most of these neurons had a cell body with a 10-20 microns diameter and several spiny dendrites. The number of striatal neurons with TH-LI was increased on the side ipsilateral to an electrothermic or 6-hydroxydopamine-induced lesion which had been placed in the regions around the substantia nigra.

Differential induction of mRNA species encoding several classes of stress proteins following focal cerebral ischemia in rats.

We report here the time-dependent expression of several classes of HSP mRNAs following focal cerebral ischemia in rats. HSP70, GRP78, HSP27, HSP90 and HSP47 have been reported to possess distinct functions under normal and/or stress conditions. These different classes of HSP mRNAs were differentially induced by ischemia, as determined by Northern blot analysis. Messenger RNAs of the HSP70 family proteins were induced within 4 h after ischemia and then rapidly decreased, whereas HSP27 and HSP47 mRNAs reached a maximum level of expression at 24 h and 48 h after ischemic treatment, respectively. In situ hybridization showed that the expression of inducible HSP70 mRNA was observed predominantly in regions adjacent to the ischemic core except during the early periods of ischemia. HSP27 mRNA was expressed over a broad area of the ipsilateral cerebral neocortex except for the ischemic center 24 h after ischemia. The unique induction kinetics for each HSP mRNA species may reflect their distinct roles in the brain during various physiological stresses. We will also discuss that stress proteins may be involved in the central nervous system after ischemia in two important aspects: early protection against stress and restoration of damaged lesions in the brain at later stages after ischemia.

Activation of heat shock factor 1 in rat brain during cerebral ischemia or after heat shock

Recently, many studies have demonstrated the induction of stress proteins in the mammalian nervous system under various pathological conditions. These altered genetic programs may function to protect individual cells against stressful conditions. However, little is known about the molecular mechanisms regulating these stress responses in animals. We report here the activation of a heat shock factor (HSF) in the rat brain during cerebral ischemia or after heat shock. Gel mobility shift assays revealed an increase in DNA binding activity to the heat shock element (HSE) during the early phases of ischemia. Supershift experiments using specific antisera against HSF1 and HSF2 showed that the ischemia-induced HSE-binding activity was mainly due to HSF1. In the heat-shocked brain, HSF1 was also activated, and the HSE-binding activity was higher in the cerebellum than in the cerebral cortex or hippocampus; Western blot analysis also showed that HSF1 was more abundant in the cerebellum than in the other two brain regions. Our results indicate that heat shock gene transcription is regulated by the activation of HSF1 in both cerebral ischemia and heat shock, and that different brain regions display differential sensitivities in their stress response. The cellular signals for heat shock gene transcription under in vivo pathological conditions will also be discussed.

Physiological role of Ca2+-permeable nonselective cation channel in endothelin-1-induced contraction of rabbit aorta

We previously showed a role for a nonselective cation channel (NSCC) in the ETA-dependent action of endothelin-1 in mouse fibroblast and rabbit aortic smooth-muscle cell. To clarify the physiological significance of NSCCs in endothelin-1 (ET-1)-induced vasocontraction, we examined the effects of NSCC blockers such as mefenamic acid and SK&F 96365 on the contractions of deendothelialized rabbit aortic rings induced by a low (10[-10] M) or high (10[-8] M) concentration of ET-1. Mefenamic acid (< or =10[-3] M) had little effect on the contraction induced by 45 x 10(-3) M K+ or 1 x 10(-6) M Bay K-8644 in combination with 15 x 10(-3) M K+, indicating that it does not affect voltage-operated calcium channels (VOCs) and contractile mechanisms. The contraction by a low concentration of ET-1 was abolished after removal of extracellular Ca2+, but it was reduced only to 50% by a maximally effective concentration (10[-5] M) of nifedipine, an inhibitor of L-type VOCs (L-VOC). Mefenamic acid and SK&F 96365 inhibited the ET-1-induced contraction with 50% inhibitory concentration (IC50) values of 10(-4) M and 2 x 10(-5) M, respectively, and abolished it at 10(-3) M and 10(-4) M. By contrast, nifedipine, mefenamic acid, or SK&F 96365 had little effect on the contraction by a high concentration of ET-1. The contraction induced by a low or high concentration of ET-1 was abolished by an ETA antagonist, BQ-123, but not by an ETB antagonist, BQ-788. These results demonstrate that the contraction induced by ET-1 is totally mediated exclusively by ETA, but that Ca2+ entry through NSCCs in addition to L-VOCs plays an important role in contractions induced by low concentrations of ET-1, whereas it plays only a minor role in contractions induced by high concentrations of ET-1.

The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin

The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium‐denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca2+ concentrations ([Ca2+]i) in dispersed rabbit vascular smooth muscle cells with a Ca2+ indicator fura‐2. The actions of trypsin were compared with those of thrombin. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration‐dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The trypsin‐induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mm KCl were unaffected by pretreatment with trypsin. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca2+, indicating that most of the contraction is dependent on extracellular Ca2+. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L‐type voltage‐operated Ca2+ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine‐sensitive and ‐resistant Ca2+ channels are involved in these contractions. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by its protease activity. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca2+]i, but an application of trypsin could still induce an increase in [Ca2+]i in dispersed rabbit vascular smooth muscle cells. These data suggest that in addition to activation of a thrombin receptor, trypsin can contract rabbit aortae by a proteinase‐activated receptor 2 or a novel mechanism.

Possible origins of cerebrovascular nerve fibers showing vasoactive intestinal polypeptide-like immunoreactivity: an immunohistochemical study in the dog

Changes of vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) in perivascular nerve fibers of the major cerebral arteries were examined immunohistochemically in the dog. The density of cerebrovascular nerve fibers showing VIP-LI (the average number of nerve fibers with VIP-LI in a unit area of the major cerebral arteries) was estimated, by using whole-mount preparations after extirpation of the pterygopalatine, otic or superior cervical ganglion. After pterygopalatine ganglionectomy, the density was markedly decreased in major cerebral arteries of both anterior circulation (the anterior cerebral and middle cerebral arteries) and posterior circulation (the basilar, superior cerebellar, posterior cerebral and posterior communicating arteries). After otic ganglionectomy, the density was moderately reduced in the major arteries of the posterior circulation, but was not decreased in those of the anterior circulation. After superior cervical ganglionectomy, the density was decreased markedly in the major cerebral arteries of the posterior circulation, and moderately in those of the anterior circulation. The results also indicate that the pterygopalatine, otic and superior cervical ganglia supply perivascular nerve fibers showing VIP-LI to the major cerebral arteries bilaterally with an ipsilateral dominance.

Consistent injury in the striatum of C57BL/6 mice after transient bilateral common carotid artery occlusion.

OBJECTIVEnThe recent availability of transgenic mice enables us to study the functional role of single gene products in cerebral ischemia. To establish an experimental murine model of transient forebrain ischemia, this study examined the temporal profile of ischemic neuronal damage in the striatum after bilateral common carotid artery occlusion.nnnMETHODSnC57BL/6 mice, which are frequently used for genetic manipulations, were subjected to 15-minute bilateral common carotid artery occlusion. Ischemic injury was examined (4, 8, 24, 48, and 96 h after reperfusion) by Nissl staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeling, and nuclear staining with Hoechst 33258 dye.nnnRESULTSnRegional cerebral blood flow was decreased to 11 +/- 6% of control values during the ischemic insult. Striatal injury was observed in 95% of animals examined after 15-minute bilateral common carotid artery occlusion. The number of small and medium-size neurons in the striatum was significantly (P < 0.05) decreased 8 hours after reperfusion and continued to decrease until 96 hours, whereas the number of large neurons remained constant. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeling-positive cells appeared in the dorsomedial region of the striatum 48 hours after the ischemic insult and throughout the striatum 96 hours after the ischemic insult. Brain sections stained with Hoechst 33258 dye also demonstrated apoptotic nuclei 96 hours after the ischemic insult.nnnCONCLUSIONnStriatal injury after transient forebrain ischemia is reproducible in C57BL/6 mice and is a good model to study the molecular mechanisms of ischemic injury, including delayed neuronal death, using transgenic mice.

Tyrosine hydroxylase-like immunoreactivity and catecholamine fluorescence in ciliary ganglion neurons.

In the ciliary ganglia of monkeys, dogs, cats and rats, some neuronal perikarya exhibited tyrosine hydroxylase-like immunoreactivity; neither dopamine-beta-hydroxylase-like immunoreactivity nor phenylethanolamine-N-methyltransferase-like immunoreactivity was found in perikarya. Some perikarya of ciliary ganglion neurons displayed catecholamine fluorescence; the fluorescent perikarya were significantly increased in number in dogs injected with nialamide. Thus the existence of dopamine-containing ciliary ganglion neurons was suggested.