Neural Repair By NT3-chitosan Via Enhancement Of Endogenous Neurogenesis After Adult Traumatic Brain Injury

Objects: Observed whether NT3-chitosan carriers promoted regeneration of new-born neurons in the injured area after adult traumatic brain injury(TBI),and clarified whether regenerated neurons intergrated functionally into the neural circuits to recover the cognitive function.Then observed whether TBI and NT3-chitosan activated endogenous neural stem cells(NSCs),and whether NT3-chitosan induced the proliferation,migration and differentiation of NSCs located in the SGZ and pPV.Finally,we explored whether NT3-chitosan improved micro-environments in the injured area.Methods: A self-developed biotissue cutterbar was used to establish the mechanical brain injury model in the adult Wistar rats,that is,to aspirate and remove the CA1 region of the hippocampus and the covering cortex.Chitosan carriers loaded with or without NT3 were immediately implanted into the lesion cavities after the operation.Rats from the sham control group were subjected to the same surgical procedure without receiving the aspiration injury.Rats from the lesion control group received no treatment.HE staining observed regeneration of brain tissues from all groups;all rats received an intra-peritoneal(i.p.)injection of BrdU(50mg/kg body weight)twice daily for consecutive 7 days after the operation,to label proliferating cells.BrdU/Tuj1 and BrdU/NeuN immunosatining detected the regeneration of new born neurons in the injured area at different time points after the operation,and meanwhile detected the regeneration of different types of neurons;immunofluorescent labeling of BrdU/Tuj1/ synapsin1 and PSD95 and immunogold labeling of MAP2 detected the synaptic formation between regenerated neurons in the injured area;MED64 planar microelectrode array recording system was used to detect the formation of functional neural circuits among new born neurons as well as between new born neurons and the host brains;BDA tracing was used to observe the rebuilt of intra-hippocampal neural circuit;Morris water-maze was used to evaluate the spatial learning and memory function of rats.To detect the proliferating cells of different brain areas,rats received the same BrdU injection 48 hrs before the sacrifice,and Brd U immunostaining observed and counted proliferated cells in the DG and pPV at 1 d,3 d,7 d,14 d and 28 d after the operation;BrdU/NeuN/GFAP staining detected the fate of proliferated cells in the DG at 28 d after the operation;Immunofluorescent staining of BrdU/Dcx and Dil labeling in vivo used to detect the migration of neuroblasts from SGZ and pPV.CD45 and CD11 b staining detected the effect of NT3-chitosan on microglia and macrophage;Real-time PCR detected the gene expression of pro-and anti-inflammatory cytokines after TBI and NT3-chitosan treatment;Reca-1 staining observed the angiogenesis in the injured area.Results: 1.BrdU/Tuj1 and BrdU/NeuN/GFAP staining showed NT3-chitosan promoted the regeneration of newly born neurons in the injured area,and inhibited gliosis.Newly born neurons contained glutamatergic and GABAergic neurons.2.Immunostaining of BrdU/Tuj1/synapsin1 and PSD95 and IEM of MAP2 showed NT3-chitosan promoted formation of synaptic contacts between regenerated neurons in the injured area.MED64 recording showed NT3-chitosan promoted the formation of functional networks among regenerated neuron as well as between regenerated neurons and the host brain.BDA tracing showed that NT3-chitosan rebuilt intra-hippocampal neural circuits.3.Morris water-maze showed NT3-chitosan induced recovery of cognitive functions after TBI and restored the properties of LTP of Schaffer collateral-CA1 synapses.4.Nestin staining showed that NT3-chitosan activated endogenous NSCs and promoted their migration into the injured area.5.BrdU staining showed that TBI increased the number of BrdU+ cells in the ipsilateral DG,and NT3-chitosan further enhanced TBI-induced the proliferation.Most of BrdU+ cells were still distributed in the neurogenic area including SGZ and GCL.BrdU+ cells of the SGZ in the NT3-chitosan group were significantly more than that in the lesion control group.A small percentage of proliferating cells were located in the non-neurogenic area,including molecular layer and hilus.6.BrdU/Dcx staining showed that NT3-chitosan increased the number of BrdU+ /Dcx+ cells and the ratio of BrdU+/Dcx+ cells,suggesting that NT3-chitosan not only increased the number of newly born NSCs in the SGZ but increased the ratio of NSCs in the proliferating cells.Brdu/GFAP/NeuN staining showed that NT3-chitosan enhanced survival of proliferating cells in the SGZ,and increased the number and ratio of newly born neurons in the GCL,indicating that NT3-chitosan promoted the neurogenesis and the fate of neural differentiation in the SGZ.7.NT3-chitosan promoted the migration of NSCs in the SGZ to the injured area.8.BrdU staining showed that NT3-chitosan enhanced TBI-induced cell proliferation in the pPV.BrdU/Dcx staining and Dil labeling showed that NT3-chitosan promoted migration of NSCs in the ipsilateral p PV.Meanwhile,Dil+ cells could be detected in the injured area,some of them expressed NSCs marker nestin,and some expressed neuroblast marker Dcx,indicating that NSCs of the pPV may be contributed to the regeneration of new born neurons in the injured area.9.CD45 and CD11 b staining showed that NT3-chitosan inhibited the infiltration of macrophages and the activation of microglia,and restored the ratio of resting and activated microglia.Real-time PCR demonstrated that NT3-chitosan inhibited the expression of pro-inflammatory cytokines TNF-? and IL-1? and increased the level of anti-inflammatory cytokines IL-10 and IL-4,indicating the anti-inflammatory effect of NT3-chitosan during neural repair after TBI.10.Reca-1 staining showed NT3-chitosan promoted angiogenesis in the injured area,and increased the micro-vessel density and diameter.Conclusions: NT3-chitosan carriers elicited the activation of endogenous NSCs in both SGZ and pPV and promoted their emigration to the injured area.By providing anti-inflammatory,pro-vascularizing and pro-neurogenic micro-environments,NT3-chitosan induced NSCs to differentiate into the mature neurons and build functional neural networks,and finally recover the brain function.This study provided the theoretical basis and experimental prove for clinical application of NT3-chitosan active biomaterial to repair the adult brain injury.