Experimental Study In The Signals That Regulate Neurogenesis Trigged By Ischemia-reperfusion In The Area Of Dentate Gyrus Of Adult Rats

Background and objection:It is well established that the generation of new neurons continues throughout adulthood in the DG in many species of vertebrates . Reportedly, such pathological states as lesion and ischemia may result in hippocampal DG neurogenesis. After transient forebrain ischemia, newly generated neurons migrate and incorporate into functional synaptic circuitry, which provides a possible therapeutic strategy for ischemic injury repair. The identification of intracellular signaling events that regulate the rate of ischemia-induced progenitor cell proliferation is therefore of significant interest.Src, a cytoplasmic non receptor tyrosine kinase, activated by Ca2+ flue implicated in regulating diverse cellular responses including proliferation, differentiation, migration in number of model systems. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated neurogenesis in the adult DG and its underlying mechanism. Nimodipine, a dihydropyridine calcium channel blocker, Because it has some selectivity for cerebral vasculature, nimodipine's mainly used in cerebral ischemic stroke to prevent cerebral vasospasm and resultant ischemia,However,the effects of Nimodipine on neurogenesis trigged by brain ischemia has not been explored.Methods:1. To investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated neurogenesis in the adult DG and its underlying mechanism: The models with cerebral ischemia-reperfusion were induced using four-vessel occlusion as described previously and received 10 min ischemia. Rats were administrated with Src kinase inhibitor SU6656 or ERK inhibitor U0126 using a microinjector into bilateral cerebral ventricle. Immunohistochemistry of Brdu were conduced to detect proliferation of neuronal progenitors in the dentate gyrus. Western blotting was operated to detect expression of p-Src, Src, p-Raf, Raf, p-ERK,ERK, p-CREB CREB. Sections were processed for staining with Toluidine blue (Nissl staining) for histological assessment of damage.2. To observe effects of Nimodipine on neurogenesis in the dentate gyrus of the adult rat trigged by ischemia-reperfusion and investigate its molecular mechanism :The models with cerebral ischemia-reperfusion were induced using four-vessel occlusion as described previously and received 10 min ischemia. Rats in the group of Nimodipine were administrated intraperitoneally with Nimodipine, and the group of depressing p-ERK were administrated with U0126 using a microinjector into bilateral cerebral ventricle. Immunohistochemistry of Brdu were conduced to detect proliferation of neuronal progenitors in the dentate gyrus. Western blotting was operated to detect expression of ERK and p-ERK.Results:1. Src kinase activated continuously in the DG 24 h and 72h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72h of reperfusion; U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf; Activation of Src but not ERK promote delayed neuronal death of CA1 region2. Nimodipine not only inhibited neurogenesis but also expression of p-ERK in the area of dentate gyrus after ischemia-reperfusion. There was no distinction in neurogenesis of dentate gyrus between the group of U0126 and the group of U0126 combined with Nimodipine.Conclusions:1. Src kinase promotes hippocampal neurogenesis via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia.2. Pretreated Nimodipine inhibit neurogenesis in region of dentate gyrus by down-regulate p-ERK after ischemia-reperfusion.