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s2-2 NEURONAL DEATH INDUCED BY REACTIVE OXYGEN SPECIES. TAKUMI SATOH, YASUSHI ENOKIDO, HIROSHI HATANAKA, Div. of Protein Biosvn., Institute for Protein Research. Osaka Univ., Osaka, 565, Japan.

To investigate the involvement of reactive oxygen species (ROS) in neuronal apoptosis, we performed confocal and flow cytometry with a ROS-specific fluorogen, 6-carboxy-2’,7’-dichorodihydrofluorescein diacetate, di(acetoxymethy1 ester) (C-DCDHF-DA). In PC12 cells, hydrogen peroxide, lipid hydroperoxide and serum deprivation significantly increased the level of ROS. However, NGF and the exogenous expression of bcl-2, which prevented the apoptosis, did not affect the upregulation of ROS. Also, in rat cortical neurons, serum deprivation increased the level of ROS. EGF, which prevented apoptosis, did not affect the increase of ROS. These data suggest that survival factors rescue the apoptosis independently of ROS production.

S2-3 FUNCI’ION OF MICROGLIA ON THE PROCESS OF NEURONAL REPAIR KAZUYUKI NAKAJIMA. SHINICHI KOHSAKA

Dem. of Neurochem.. National Inst. of Neurosci.. Tokvo 187. Japan

Microglia are observed as ramified microglia in the normal central nervous system, and thought to be in a functionally inactive or quiescent state. However, when the brain is injured or affected by disease, ramified microglia change morphologically or transform into macrophage like shape and start to proliferate. At the same time, some cell surface antigens are expressed newly or more strongly. These reactive microglia has been thought to function as a scavenger or antigen presenting cells. Also, in vitro system have shown that microglia have the potential to produce a large variety of physiologically active substance such as cytokines, growth factors, and other materials. Some of factors have been shown to exert neurotrophic and neuroprotective effects, while others have neurotoxic effects. These facts suggest that activated microglia and these-derived physiologically active substances affect largely on the process of neuronal repair or degenerative processes in various brain pathologies. However, there is little information about the factors associated with microglial activation and/or regulator of its activation. Here, we describe the events of microglial activation and its function in the neuronal repair by using facial nerve transection system in vivo and in vitro culture system.

s2-4 NEURONAL CELL DEATH AFTER BRAIN ISCHEMIA. YASUO UCHIYAMA. Debt. of Cell Biol. & Anat. I. Osaka Univ. Med. Sch.. Osaka 565. Jawan

Neuronal death caused by brain ischemia has been believed to be necrosis. Until recently, it remains unknown whether there exist certain differences in dying processes of neurons between transient and permanent types of brain ischemia. Using the transient forebrain ischemia model of gerbils, we have shown that the delayed neuronal death (DND) in the CA1 pyramidal layer consists of 1) cell shrinkage, 2) nuclear chromatin condensation and DNA fragmentation, and 3) the formation of apoptotic bodies and heterophagocytosis by phagocytic cells. The results suggest that the DND in the CA1 pyramidal layer after transient ischemia is not necrotic but apoptotic. On the other hand, in the middle cerebral artery occulusion (MCAO) model of rats, neurons in the ischemic core of the cerebral cortex die with typical figures of necrosis appearing even at 2 to 6 hours after ischemic onset. In the penumbra region of ischemia, however, TUNEL-positive nuclei appear 1 to 3 days after ischemic onset, while the dying neurons in the region are shrunk and show increased immunoreactivity for lysosomal cathepsins. Mitochondrial profiles of these dying cells are usually intact, but cisterns of the endoplasmic reticulum are often swollen. These results indicate that the dying processes of the neuronal death in the penumbra after MCAO clearly differ not only from those of neurons by necrosis, but also from those of the CA1 pyramidal cells after transient ischemia.