Neonatal And Molecular Mechanisms Of Striatal And Cortical Projection Neurons In Stroke Rats

Previous studies have demonstrated that newborn striatal and cortical neurons could functionally integrate with local neural networks in adult rat brain after injury.Whether ischemia-induced newborn neurons can establish connection with other brain regions is very important to promote motor function recovery.In the present study,we determined whether these newly generated neurons could develop projections to the substantia nigra,a target of striatal projection neurons.A transient middle cerebral artery occlusion(MCAO)was used to induce focal cerebral ischemia and neurogenesis.We used 5'-bromodeoxyuridine(BrdU)and a retroviral vector expressing green fluorescent protein(GFP)combined with multiple immunostaining to label newborn neurons,and nigral microinjection of fluorogold(FG)to trace the striatonigral and corticonigral projection neurons in adult rat brain.We found that FG positive(FG+)cells could be detected in newly generated neurons(BrdU+-NeuN+ and GFP+-NeuN+)in ipsilateral striatum and cortex clearly at 12,but not 2 weeks after MCAO.The data suggested that ischemia-induced newborn neurons could form long axons that targeted to the substantia nigra and reconstruct functional striatonigral and corticonigral projection pathway,since new formed projection possess intact axonal transport from the nerve terminal to cell body.Newborn striatal neurons expressed glutamate receptor(NR2)and dopamine receptor(D2L),which provided the molecular basis for responding to the inputs from cortical glutamatergic and nigral dopaminergic projection neurons.Our data provided the first morphological evidence that newborn neurons in the striatum and cortical,non-neurogenic region,could establish new neural circuits within the basal ganglia,important pathways for the maintenance of motor function.These results help us to understand endogenous cellular mechanisms of brain repair,and suggest that increasing adult neurogenesis could be a practical strategy for enhancing the efficacy of rehabilitative therapy in stroke patients.Rehabilitative training improves striatal and cortical neurogenesis in brain after stroke.However,whether it can improve axon-regeneration of new projection neurons remains unknown,which is fundamental anatomical basis for recovering motor function.We investigated the effects of exercise on axon-regeneration of newborn projection neurons in rat brain following ischemic stroke.Rats were subjected to a transient middle cerebral artery occlusion(MCAO)to induce ischemic stroke,followed by 30 minutes of exercise training daily for 5 to 28 days after MCAO.Motor function was tested using the rotarod test at different times after MCAO.We further used fluorogold(FG)nigral injection to trace striatonigral and corticonigral projection neurons,and GFP-targeting retroviral vectors combined with FG double labeling(GFP+-FG+)to detect newborn projection neurons.GFP+-NeuN+-FG+ cells were observed in ipsilateral cortex and striatum of rats to MCAO,further suggested existence of newborn striatonigral and corticonigral projection neurons in ischemic injured brain.Exercise significantly increased the number of GFP+-FG+ neurons in ipsilateral striatum and cortex,suggesting that exercise could enhance the capacity for axon regenration of newborn projection neurons.Moreover,exercise increased dopaminergic neurons,indicated by tyrosine hydroxylase immunopositive stained cells,in the substantia nigra,a region remote from the ischemic territory.Meanwhile,we also observed that such exercise improved recovery of motor function of rats after MCAO.The results revealed that exercise effectively enhanced the capacity for axon-regeneration of newborn projection neurons in ischemic injured brains while improved motor function.These results provide important evidences to illustrate the effectiveness of rehabilitative treatment post-stroke in the clinic.DNA methylation,a major epigenetic modificator,plays important roles in regulating neural stem cell self-renew and differentiation.In the present study,we mainly investigated the effect of DNA methyltransferase(DNMT1)on adult neurogenesis induced by ischemic injury.The results showed that ischemia had no obviously effect on DNMT1 level in ipsilateral striatum,but induced DNMT1 expression in astrocytes accompanied with increased of 5-methylcyosine(5'-meC)in ischemic penumbra region.To further investigate the effect of DNMT1 on neurogenesis,we used 5-aza-2'-deoxycytidine(AZA)to reduce enzymatic active of DNMT1 in ischemic brain.The results showed AZA significantly increased newly generated BrdU+-DCX+ neural progenitors in ipsilateral striatum,and the levels of Pax6 and Olig2,transcription factors to determine neural differentation.Meanwhile,AZA reduced neuronal degeneration and infarct volume.Present results suggest DNMT1 inhibitor might be helpful for brain repair,which might provide a new therapeutic strategy to improve brain recovery after ischemic injury.Conclusion:Present results revealed that ischemia-induced striatal and cortical newborn neurons can form long axon project to the substantia nigra,restablish neural circuits within the basal ganglia,important pathways for the maintenance of motor function.We also found that exercise effectively enhanced the capacity for axon-regeneration of newborn projection neurons in ischemic injured brains while improved motor function.We further found that AZA,a DNMT1 inhibitor,could promote neural stem cell differentation and reduce brain injury,suggesting downregulation of DNMT1 might be beneficial for brain self-repair after injury.