The Effect Of Increased Bbb Permeability Or Selective Microglial Depletion On Neuronal Structural Plasticity After Cerebral Ischemia

Ischemic stroke induces rapid disruption of the blood brain barrier?BBB?,and the increased BBB extravasation influences the pathological process after stroke.However,it stays unknown that how the elevated BBB leakage impact the microglial activation as well as the synaptic structure after global ischemia-reperfusion.Recently,utilizing of two-photon imaging has made it possible to investigate the dynamic change of synaptic structures.In this study,we took advantage of this forementioned technology and investigated the effect of both the BBB leakage and the activated microglia on the neuronal structural plasticity after stroke in the transgenic mice.Firstly,we studied the influence of BBB leakage on the dendritic damage and the dendritic spines plasticity after global ischemia-reperfusion by using in vivo two-photon imaging in transgenic mice expressing fluorescent proteins in neurons.Our results showed that although the mannitol induced BBB leakage did not trigger the inflammatory response or the dendritic spines turnover rate in healthy animals,it induced the de-ramification of microglia.Mannitol treatment enhanced the microglial de-ramification and aggregated the tissue inflammation after ischemia-reperfusion.More importantly,although mannitol injection did not notably affect the percentage of beaded dendrite after ischemia-reperfusion or the reversible restoration of dendritic structure,the increased BBB extravasation impacted the dendritic spines plasticity.Namely,mannitol treatment reduced the dendritic spines formation rate and increased the elimination rate,leading to a net loss of dendritic spine.Microglia are rapidly activated after the ischemia-reperfusion.However,the morphological change and the pattern of microglial proliferation under different conditions of ischemia have not been revealed.In the present study,we used intravital two-imaging,confocal imaging and high-throughput sequencing technology to study the impact of different duration of ischemia on the proliferative response and the change of microglial morphology.Results showed that during ischemia-reperfusion,rapid de-ramification of microglia was induced;whereas the restoration of their morphology and the proliferation of microglia both showed heterogeneity depending on the ischemic time.In the mice that were subjected to 30 min ischemia followed by reperfusion,de-ramified microglia were recovered to normal level after one day of reperfusion and showed no significant proliferation;however,prolonging the ischemic time to 1 hour led to significant microglial proliferation,as well as marked de-ramification after 2 days of reperfusion.Moreover,prolonged ischemia up-regulated series of inflammatory factors related to microglia,so as the activation of CSF1R pathway.By using GW2580treatment to suppress the CSF1R cascade or using LPS to accelerate the level of inflammation,we found that the proliferation of microglia was regulated by both the CSF1R cascade and the inflammatory response.Microglia participate not only in the development of neurons but also in the pruning of dendritic spines.CX3CR1^CreER/+:R26^iDTR/+transgenic mice have been used by researchers to specifically depleted the microglia.In this study,we performed specific microglial depletion in the CX3CR1^CreER/+:R26^iDTR/+transgenic mice and analyzed the effect of microglia on the neuronal structures after ischemic stroke.Our results showed that although depletion of microglia in healthy mice did not have notable effect on the structure of dendrites and on the animal's behavior,it reduced the formation rate of dendritic spines.After ischemia-reperfusion,it influenced the plasticity of dendritic spine which led to the decrease of formation rate and the increase of the elimination rate,leading to a net loss of dendritic spines.In addition,microglial depletion resulted in the decline of locomotor activity in mice.Furthermore,microglial depletion elevated the expression level of several neurotoxic factors and down-regulated the genes which participated in the restoration of the neurons;importantly,specific depletion of microglia also changed the expression of inflammatory factors and synaptic molecules.These results indicated that after global ischemic stroke,aggravating BBB disruption changed plasticity of dendritic spines and caused excessive loss of dendritic spines.Furthermore,our study suggests that transient ischemia did not caused proliferation of microglia,but prolonged ischemia?60 min?caused microglial de-ramification,induced inflammatory response,and led to significant proliferation of microglia via activating the CSF1R pathway.More importantly,our results indicated that microglia prevented both the loss of dendritic spines and behavioral deficits of mice,and helped the re-establishment of neural circuit and restoration of brain function.Our research revealed a potential risk of BBB leakage and showed the beneficial effect of microglia after stroke,and these results provide the reference for the clinical treatment of ischemic stroke.