关键词: 内皮素-B, 小胶质细胞, 脑缺血, 免疫荧光, 实时定量PCR, 免疫印迹法, 大鼠

Abstract: Objective To explore the relationship between the endothelin-B(ET-B) receptors and cerebral ischemia by studying the expression of endothelin-B receptors in the activated microglial cells. Methods The rat model of middle cerebral artery occlusion (MCAO) was established by minimal invasive craniotomy. Eighty one adult male SD rats were randomly divided into ischemia group including 2 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days and 1 week after ischemia (at each time point, EM>n/EM>=9); a sham operation group (EM>n/EM>=9) and normal control group (EM>n/EM>=9). ET-B receptor immunoreactivity was observed in activated microglial cells following MCAO by double labeling with lectin. The expression of ET-B receptors mRNA and protein were investigated by Western blotting and real-time polymerase chain reactions (RT-PCR). Results ET-B immunofluorescence was markedly induced in the activated microglial cells in both infarcted and peri-infacted zones by double labeling with lectin at the 1st day, 2nd day, 3rd day and the 1st week, especially at the 3rd day and the 1st week after MCAO. In the control and sham operated rats, microglial cells appeared and relatively unactivated and showed a lack of ET-B immunofluoresecence. ET-B mRNA and protein levels were steadily upregulated from 2 hours to 12 hours (EM>P/EM><0.05) after MCAO as compared with the controls, peaking at the 6th hours. At the 1st week, ET-B mRNA and protein levels were only marginally elevated above the control level. Conclusion A major finding of this study is the massive accumulation of activated microglia and the induced expression of ET-B receptors in activated microglial cells following MCAO. It is suggested that ETs via ET-B may act on activated microglia and play an important role in cerebral ischemic injury bearing the receptor.

Key words: Endothelin-B, Microglia, Cerebral ischemia, Immunofluorescence, Real-time PCR, Western blotting, Rat