Expression Of Endothelins In The Activated Microglial Cells Following Cerebral Ischemia In The Adult Rats

Objective To explore the biological function of endothelins in the activated microglial cells by observing the morphological changes of microglia in the infracted, peri-infracted (penumbral) zones and the control brain areas, and studying the expression of endothelins in the activated microglial cells after occlusing middle cerebral artery at rats. It's providing an important theory and experiment basis for the clinical cerebrovascular disease's prevention.Methods The rat model of middle cerebral artery occlusion (MCAO) was established by minimal invasive craniotomy. The Eighty one adult male rats were randomly divided into ischemia group including 2h,6h,12h, 1d,2d,3d and 1w after ischemia (at each time point n=9); a sham operation group (n=9) and normal control group (n=9). The change of the microglia was studied via immunochemistry with anti-OX-42 antibody and lectin-labled affinity histochemistry methods. ETs immunoreactivity was observed in activated microglial cells following MCAO by double labeling with lectin. Neuronal change and cellular apoptosis was showed by Nissl's staining and TUNEL assay. The expression of ETs mRNA and protein was investigated by real time-polymerase chain reactions (RT-PCR) and enzyme linked immunosorbent assay (ELISA).Results After focal cerebral ischemia, cerebral ischemia regions shows some apoptotic cells.Ramified resting microglial cells was observed in the peripheral control zones of ischemic cortex and contralateral cortex, the same as the control. The penumbral zone has a large number of of activated microglia with a large spherical cell body and short processes, like amoeboid microglia. Activated microglia increased gradually and peaked at 3d～1w after MCAO. ETs immunoreactive activated microglia co-labeled with lectin occurred in large numbers in the penumbral zones at 3d and 1w after MCAO. Resting microglial cells showed lack of ETs immunoreactive. Some pycnosis and degenerating cells were observed in the ischemic cortex by Nissl's staining, peaking at 3d and 1w after MCAO. The TUNEL assay showed a large number of the apoptotic cells in the penumbral zone. Real time PCR analysis showed that ET-1 and ET-3 mRNA level of ischemic cortex increased significantly (p＜0.05) compared with the contralateral cortex and sham operation group, peaked at 2h and 12h after MCAO. ELISA analysis also shows that ET-1 and ET-3 protein level of ischemic cortex increased significantly compared with the contralateral cortex and sham group (p＜0.05), peaking at 1d after ischemic exposure.Conclusions①Microglial cells have a large number of accumulation and activation in the penumbral zone after focal cerebral ischemia. Activated microglia may be relative with slow neuronal injury.②Activated microglia expressed ETs which becomed an important source of cells, such as embryonic and early postnatal amoeboid microglia.ETs possibly through autocrine or paracrine involvement the pathological process of cerebral ischemia.