The Effect Of Bcl-2 On The Projection Of Striatum Neonatal Neurons After Cerebral Ischemia In Adult Rats

According to extensive recent reports,neurogenesis exists in some areas of adult mammalian brain throughout the life,including the subventricular zone(SVZ)and the subgranular zone(SGZ)of hippocampal dentate gyrus(DG).In addition,neurogenesis can also be stimulated by pathological injury in several special regions such as striatum,cortex and CA1 of hippocampus.Those endogenous neural stem cells or neural progenitors in the brain can be stimulated to generate newborn neurons replacing injured ones,which is thought to be one way of brain self-repair.Meanwhile,many protective factors can be activated for repairing injured brain.And the expression of various growth factors,DNA repair factors and anti-apoptotic proteins are upregulated at early stage of reperfusion to decrease apoptosis and promote brain repair.One of them is anti-apoptotic protein Bcl-2,which plays an important role in the development of the central nervous system and the process of neurodegenerative disease.Except for the classic anti-apoptotic function,Bcl-2 can promote axon growth of cultured neurons in vitro,enhance ischemia-induced neurogenesis and the differentiation and maturation of newborn neurons in vivo.However,it is still not known yet whether Bcl-2 can promote.neurite development of those newborn neurons in adult brain.To investigate the effect of Bcl-2 on the formation of projection of newborn strital neurons to the substantia nigra in the rat brain after ischemia injury,we intraventricularly injected human Bcl-2-expressive plasmid(pBcl-2)or control plasmid(pEGFP)into rat brains to overexpress Bcl-2 protein.Cerebral ischemia was induced by middle cerebral artery occlusion(MCAO).5'-bromo-2'-deoxyuridine(BrdU)was used to label dividing cells.Retrograde tracer fluorogold(FG)was stereotaxicly injected into the ipsilateral substantia nigra pars reticulata(SNr)at 11 weeks of reperfusion,and FG signals were detected in the ipsilateral cortex and striatum under microscopy with UV excitation.Immunohistochemical staining combined with confocal laser scanning was used to observe newborn projection neurons.The results were as follows:1.Detection of EGFP expression at 12 weeks of reperfusion in ischemic rat brainTo explore the effect of Bcl-2 overexpression on the formation of ischemia-induced strital newborn projection cells,we detected the expression of enhanced green fluorescence protein(EGFP)in the brain.The results showed that EGFP positive cells could not be observed in the ipsilateral or contralateral SVZ,the same as in bilateral cortex and striatum.It indicated that the expression duration of Bcl-2 plasmid or EGFP plasmid was limited to a short period,which was less than 12 weeks.2.The effect of Bcl-2 overexpression on infarct region at 12 weeks following MCAOBy CV staining and image analysis process,we found that few infarct regions could be observed in the ipsilateral striatum and were mainly located in the lateral side,which possessed a distinct boundary with non-infarct region.The infarct area of striatum reduced significantly compared with that at 1 to 4 weeks post MCAO.Moreover,the nissle bodies were stained darkly and packed densely and irregularly.In addition,there was no obvious difference between pBcl-2 group and pEGFP group.The situations in the cortex were similar.These results indicated that the effect of Bcl-2 overexpression on the repair of infarct region might be time-dependent.To further investigate the possible reasons for the reduction of infarct area,we labeled GFAP immunohistochemically to reveal changes on the astrocyte.The results showed that there was also a distinct boundary between two kinds of GFAP positive cells,which was similar to CV staining.Meanwhile,overexpression of Bcl-2 did not change the distribution of GFAP positive cells significantly.In addition,GFAP positive cells in the infarct region exhibited apparent morphological features of active astrocytes,suggesting that reactive astrocytes might be involved in the repair of infarct region under long-time course.3.Overexpression of Bcl-2 promoted the formation of strital newborn projection neurons to ipsilateral substantia nigra pars reticulata in ischemic rat brainAs was mentioned above,to explore the effect of Bcl-2 overexpression on the formation of strital newborn projection neurons,we injected retrograde tracer FG into ipsilateral substantia nigra pars reticulata stereotaxicly to label strital projection neurons.By ultraviolet microscope,we observed the injection site and FG expression regions(cortex and striatum)to assure the accuracy of injection site.On this base,we further observed that the newborn cells(BrdU+)mainly concentrated in the infarct region,most of which were newborn mature neurons(BrdU+-NeuN+ or BrdU+-MAP-2+).Moreover,some of newborn neurons at periphery of the infarct region had been characterized by mature projection newrons(BrdU+-FG+-NeuN+ or BrdU+-FG+-MAP-2+).Furthermore,all of the projection cells(FG+)possessed features of mature neurons(NeuN+ or MAP-2+).The results indicated that some newborn neurons gradually became mature after a period of development and projected to the ipsilateral substantia nigra pars reticulata.By statistics analysis,we also found that the number of BrdU+ cells and BrdU+-FG+ cells,a kind of newborn projection neurons,increased remarkably in pBcl-2 group(506.0± 197.3 and 114.6±28.0)compared with pEGFP group(117.7±16.2 and 21.8±4.3),respectively.The results indicated that overexpression of Bcl-2 post ischemia could promote the projection development of strital newborn neurons to substantia nigra pars reticulata significantly.Summary:1.Both the overexpression of Bcl-2 and the repair effect on ischemic area are time-effective in the striatum and cortex of MCAO rat brain.2.Bcl-2 overexpression enhances neurogenesis and promotes development of the projection of strital newborn neurons to ipsilateral substantia nigra pars reticulata in the adult rat brain after ischemia injury.Conclusion:Bcl-2 overexpression promotes the formation of newborn strital projection neurons to ipsilateral substantia nigra pars reticulata in the rat brain after ischemia injury.