Effect Of Expression Of HSP22 And Neuroprotection Of Brain Ischemic Preconditioning After Cerebral Ischemia/Reperfusion In Gerbils

ObjectiveIschemic stroke severely harm the health of human being because of its higher morbidity,crippling rate,and mortality.The pathophysiologic mechanisms of cerebral ischemia damage are very complicated.It is still unclear that the mechanisms involve changes of many interrelated genes and proteins and their complicated association. Brain ischemic preconditioning(BIP) refers to that the brain is produced marked tolerance to lately lethal ischemia after one or multiple times sublethal ischemia.The essence of BIP is to promote synthesis of new proteins by arousing the mechanism of endogenous neuroprotection and prevent or attenuate ischemic cascade response which is produced by cerebral ischemia.HSP22 is a new member of mammal small heat shock proteins(sHSPs) family.HSP22 is expressed predominatly in muscle and brain tissues.It possesses molecular chaperone activity,modulators of apoptosis,and so on.In our experiments,we set up a gerbil model of bilateral carotid artery occlusion(BCAO) and intervene it by BIP in two days before BCAO.In one hand,we observe the nerve tissue ultrastructural change,the change of brain tissue nerve cell apoptosis,and the change of HSP22,Bcl-2,Bax gene mRNA and protein expression and active caspases-3 protein expression in gerbil model of bilateral carotid artery occlusion after BIR In the other hand,we observe the change of MAP2,GFAP gene mRNA and protein expression,the co-localization between HSP22 and MAP2 of cellular skeleton in neurons,and the co-localization between HSP22 and GFAP of cellular skeleton in astrocytes in gerbil model of bilateral carotid artery occlusion after BIP by confocal Immunofluorographs and immunogold double-labeling of electron microscopy.We further research the effect of expression of HSP22 and neuroprotection of brain ischemic preconditioning after cerebral ischemia/reperfusion in gerbilsMethods1.Experimental animal model preparation and grouping.120 healthy male gerbils were divided into control group,sham operation group,ischemia/reperfution (I/R) group,and ischemic preconditioning(IPC) group randomly.In I/R group and IPC group there were three subgroup-1d subgroup,3d subgroup and 7d subgroup-according to the time after I/R.In control group and sham operation group there were 18 gerbils;In I/R group and IPC group there were 12 gerbils in 1d subgroup and 7d subgroup,and 18 gerbils in 3d subgroup.Gerbil models of bilateral carotid artery occlusion(BCAO) were established by occluding both carotid arteries for 10 min.Double 2-minute periods of preconditioning ischemia,which were induced with a 1-day interval,were induced in two days before gerbils models of BCAO in IPC group.2.Observe the effect of BIP on the nerve tissue ultrastructural change by transmission electron microscopy.3.Detect the effect of BIP on the brain tissue nerve cellular apoptosis by TUNEL staining.4.Observe the co-localization between HSP22 and MAP2 of cytoskeleton in neurons,and the co-localization between HSP22 and GFAP of cytoskeleton in astrocytes after BIP by confocal Immunofluorographs.5.Observe the co-localization between HSP22 and MAP2 of cytoskeleton in neurons,and the co-localization between HSP22 and GFAP of cytoskeleton in astrocytes after BIP by immunogold double-labeling of electron microscopy.6.Detect the effect of BIP on the brain tissue HSP22,Bcl-2,Bax,MAP2,GFAP gene mRNA expression by RT-PCR method.7.Detect the effect of BIP on the brain tissue HSP22,Bcl-2,Bax,caspase-3, MAP2,GFAP protein expression by Western blot method. Results1.The nerve tissue ultrastructural change of gerbil forebrain cortexIn control group and sham operation group,the nerve cell was complete in gerbil forebrain cortex.In 3d subgroup of I/R group,cell nucleus vague of membrane,fewer crest and external membrane rupture of mitochondria and mild extension of rough endoplasmic reticulum were observed under transmission electron microscopy.In ischemic preconditioning group,the above-mentioned ultrastructural impairment of nerve cell was much lower than that of I/R group.2.TUNEL stain of gerbil forebrain cortexIn control group and sham operation group,TUNEL-positive cells almost were not seen in gerbil forebrain cortex.In 1d subgroup of I/R group,there was several TUNEL-positive cells.In 3d and 7d subgroup of I/R group,there were many apoptotic cells.With the prolong of time,the number of apoptotic cells was increased. In 1d,3d and 7d subgroup of IPC group,TUNEL-positive cells were much less than that of I/R group in gerbil forebrain cortex(p＜0.05).3.Confocal ImmunofluorographsIn perikaryon and dendrite of all cortical neurons,an extensive overlap of HSP22 immunoreactivity(red) and MAP2 immunoreactivity(green) were were observed under confocal Immunofluorographs microscopy.In all cortical astrocytes,an extensive overlap of HSP22 immunoreactivity(red) and GFAP immunoreactivity (green) were observed under confocal immunofluorographs microscopy.4.Immunogold labelingIn perikaryon and dendrite of all cortical neurons,an extensive overlap of the immunogold granules of HSP22(10nm) and the immunogold granules of MAP2(15nm) were observed under electron microscopy.In cytoplasm and prominency of all cortical astrocytes,an extensive overlap of the immunogold granules of HSP22(10nm) and the immunogold granules of GFAP(15nm) were observed under electron microscopy.5.The expression of HSP22 gene mRNA and protein In 3d subgroup of IPC group,HSP22mRNA expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group,HSP22 protein expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).6.The expression of Bcl-2,Bax gene mRNA and proteinIn 3d subgroup of IPC group,Bcl-2mRNA expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group, the expression of Bcl-2 protein was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 3d subgroup of I/R group,Bax mRNA expression was much higher than that of IPC group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of I/R group,the expression of Bax protein was much higher than that of IPC group in gerbil forebrain cortex(p＜0.05).There was no distinct difference in gerbil forebrain cortex Bax mRNA expression between IPC group and contral group in 3d subgroup(p＞0.05).There was no distinct difference in gerbil forebrain cortex Bax protein expression between IPC group and contral group in 1d,3d,7d subgroup (p＜0.05).7.The expression of active caspase-3 proteinIn 1d,3d,7d subgroup of IPC group,the expression of active caspase-3 protein was much lower than that of I/R group in gerbil forebrain cortex(p＜0.05).8.The expression of MAP2 gene mRNA and proteinIn 3d subgroup of IPC group,MAP2mRNA expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group,MAP2 protein expression was much higher than that of I/R group in gerbil forebrain cortex(p＜0.05).9.The expression of GFAP gene mRNA and proteinIn 3d subgroup of IPC group,GFAPmRNA expression was much lower than that of I/R group in gerbil forebrain cortex(p＜0.05).In 1d,3d,7d subgroup of IPC group, GFAP protein expression was much lower than that of I/R group in gerbil forebrain cortex(p＜0.05). Conclusions1.BIP can significantly ease the damage of nerve cells ultrastructural organization and decrease the nerve cell apoptosis of gerbil forebrain cortex after I/R. it can obviously increase the mRNA of anti-apoptosis factor Bcl-2 and protein expression and decrease active caspase-3 protein expression.However,there was no distinct difference in gerbil forebrain cortex the mRNA of pro-apoptosis factor Bax and protein expression.2.BIP can significantly increase the mRNA of HSP22 and protein expression of gerbil forebrain cortex after I/R.3.BIP can significantly increase the mRNA of nerve cytoskeleton protein MAP2 and protein expression of gerbil forebrain cortex after I/R;HSP22 and MAP2 are co-located in perikaryon and dendrite of gerbil forebrain cortical neurons4.BIP can significantly decrease the mRNA of astrocyte cytoskeleton protein GFAP and protein expression;HSP22 and GFAP are co-located in cytoplasm and prominency of gerbil forebrain cortical astrocytes.