The Role Of Pannexinl/HDAC9 Expression Change In Mice’s Brain After Cerebral Ischemic Stroke

BackgroudAccording to the global epidemiological survey of stroke,cerebral stroke is the second leading cause of death after ischaemic heart disease.Ischemic stroke is the most common type among all cerebrovascular diseases.Because of its high disability and mortality rate,cerebral stroke makes a severe thread to the health of human being and brings a heavy burden to all the countries,especailly the low-income and middle-income countries.However,the treatment of ischemic stroke is still very limited and there are no effective drugs discovered to improve the injury after ischmia in clinic.Therefore,making a further study of the mechanism of ischemic stroke and searching the appropriate and potential therapeutic target are becoming the study hotspots.Pannexinl,abundantly expressed in the central nervous system of mammals,is considered as a membrane channels rather than a gap junction recently.Some experiments have proved that Pannexinl was related to the ischmia,however,the expression of Pannexinl dhring ischemia is lack of furher studying.Thus,this study will focus on discovering the expression pattern of Pannexinl after stroke,which is expected to provide the theoretical basis for studying the role and mechanism of Pannexinl during ischemic stroke.Recently some researches found that Histone Deacetylation(HDAC),a part of epigenetics,played an important role in the mechanism of ischemic stroke.Moreover,HD AC inhibitors could provide the neuroprotection against the injury after ischemia.The family of HD AC is very large and includes many subtypes,and their mechanism in ischmia is different.The role of some subtypes of HD AC has been studied.However,the role of HDAC9 in ischmia has not been studied further.Therefore,we will study the expression of HDAC9 after ischemic stroke in order to explore its relationship to ischemia,which is expected to provide the theoretical basis for studying the role and mechanism of HDAC9 during ischemic stroke.Chapter 1 The role of Pannexinl expression change in mice’s brain after cerebral ischemic strokeObjectiveTo study the expression of Pannexinl at different parts of the brain and at different time point after ischemia and to further disclose the possible association between HDAC9 and ischemic stroke.MethodHealthy male C57BL/6 mice were randomly divided into 2 groups:sham operation group and MCAO group.Injury models of left middle cerebral artery were prepared in MCAO group following in Koizumi’s method.The skin of the neck was cut vertically and then suture immediately in Sham group.According to different time points after MCAO operation(4h,8h,12h,1d,3d,7d and 14d),the ipsilesional cortex and the contralesional cortex was collected.Immunofluorescence was performed to investigate the distribution of Pannexinl in the brain,and its relationship with MAP2 and GFAP on day 3 after MCAO.Western blot and qRT-PCR were applied to analyze the expression of Pannexinl in two group.Results1.Immunofluorescence showed that Pannexinl was expressed more around the infarction after stroke.2.Double immunofluorescent staining:there was co-localization between Pannexinl and MAP2 around the cerebral infarction,but not between Pannexinl and GFAP.3.Western blot and qRT-PCR:(1)Compared with the contralesional cortex and the healthy cortex respectively,Pannexinl was expressed more abundantly in the ipsilesional cortex of MCAO(all p<0.05).The expression of mRNA detected by qRT-PCR coincided with that of Pannexinl protein.(2)The expression of Pannexinl at the ipsilesional cortex began to increase after ischemia,and reached a peak on day3,then decrease gradually,but still remained a higher level on day 12.(3)There was no change of Pannexin2 on its expression after cerebral infarction(P>0.05).Conclusion1.The increasing expression and the change of Pannexinl may be closely related to the cerebral ischemia injury.2.Pannexin1 is mainly expressed in neurons rather than in astrocytes after ischemia in the brain.3.Pannexin2 may not participate in the pathology of ischemic stroke with Pannexin1.Chapter 2 The Role of HDAC9 expression change in mice’ brain after cerebral ischemic strokeObjectiveTo study the expression and the change of subcellular localization of HDAC9 in male mice after cerebral ischemic injury and to further disclose the possible association between HDAC9 and ischemic stroke.MethodHealthy male C57BL/6 mice were randomly divided into 2 groups:sham operation group and MCAO group.Injury models of left middle cerebral artery were prepared in MCAO group following in Koizumi’s method.The skin of the neck was cut vertically and then suture immediately in Sham group.Immunofluorescence was performed to investigate the change of subcellular localization on day 3 after MCAO.Western blot and qRT-PCR were applied to analyze the expression of HDAC9 in different parts of the brain.Results1.Immunofluorescence showed that HDAC9 was expressed more in cytoplasm but less in nucleus around the infarction after stroke.2.Compared with other parts of the brain,HDAC9 was expressed more abundantly in the ipsilesional cortex of MCAO(all p<0.05).The expression of mRNA detected by qRT-PCR coincided with that of HDAC9 protein.ConclusionThe increasing expression and the subcellular change of HDAC9 may be closely related to the cerebral ischemia injury,and HDAC9 may participate in the pathophysiological duration of ischemic stroke.