The Death Pathway Of Neurons And Astrocytes Under Cerebral Ischemia Condition And The Research Of EPO Administration.

OBJECTIVE To investigate the exact death pathway of neurons and astrocytes under continuous cerebral ishemia by observation of morphologic cellular changes and the influence of EPO administration on cells during the whole process, which may help to develop a new therapy for cerebral ischemia.METHODS: 78 male SD rats were selected and randomly divided into 5 groups which are sham-opteration group, electron microscope control group, immunohistochemistry experiment group, electron microscope experiment group, EPO experiment group. The animal model of focal cerebral ischemia was established by nylon suture embolization and cerclage. 5000IU/kg EPO were injected into the abdominal cavity of EPO experiment groups at 30min after embolization. The brain samples of each group were obtained at 3h, 6h,12h,24h and 48h after embolizaiton respectively. Morphologic changes in HE dyeing, TdT-mediated dUTP-biotin nick end labeling (TUNEL) and glial fibrillary acidic protein (GFAP) immunohistochemistry analysis were observed. Electron microscopic technology were also used to evaluate morphologic changes as well. GFAP-TUNEL double labeled immunohistochemistry were used in samples of both sham-opteration group and experimental group.RESULTS: Microscopic observation found out that slightly loosened brain tissue and cells edema in core area could be seen after 3-hour ischemia . Astrocytes were more swollen as the time of ischemia went by. After 12-hours ischemia the structure of astrocyte changed so much that it could hardly be identified. Nucleuses were condensed. A large number of deep-dyeing neurons were observed and normal structure gradually degraded to null. Whilst in marginal area, widened intercellular space with swollen neuron and astrocyte could be seen at 3-hour ischemia. As ischemic condition continued, pericytol edema and nuclear condensation became severe and astrocyte proliferated gradually. Electron microscopic observationdisclosed that astrocyte in both core area and marginal area all became swollen with nuclear membrane cracked and chromatin leakage at 12-hour ischemia. As for neurons in core area, swelling and condensation could be seen at 3~6-hour ischemia while at 6-hour only condensation could be found out. In the marginal area, swelling and condensation could be seen at 3-6hour ischemia while at 6-hour condensation become prominent. TUNEL positive cells could be seen in both core and marginal area at 3-hour ischemia. More cells distributed in marginal area as ischemia went on while positive cell decreased in core area. TUNEL positive cells was less likely to be seen in EPO administration group than in corresponding experiment group. In brains of sham-operation group, TUNEL positive cell could be seen occasionally without specific distribution. GFAP positive cells could be seen in both core and marginal area at 3-hour ischemia. GFAP positive cells could hardly be seen in core area. Lots of GFAP positive cells could be seen in marginal area after 24-hour ischemia, and formed a boundary between core area and marginal area. GFAP-TUNEL labeled cell could hardly be seen in both experimental group and sham-operation group. CONCLUSION: Continuous ischemia can cause neuron in both core and marginal area coagulation death, but further investigation is needed to find out whether apoptosis happens there. Somehow oncosis is the only way of death for astrocytes in both areas. Administration of EPO can significantly reduce TUNEL positive cells during cerebral ischemia process.