Effect Of Microglial Proton Channel Protein Hv1 On Neuronal Injury And Functional Reconstruction After Spinal Cord Injury

Objective:Spinal cord injury(SCI)is a common disease of the spinal cord with high disability and mortality,which can lead to serious neurological dysfunction.SCI results in severe neuronal injury encompassing neuronal apoptosis and pyroptosis.However,the temporal and spatial distribution patterns of apoptosis and pyroptosis in neuron after SCI and the underlying mechanism of neuronal pyroptosis are still unclear.Voltage-gated proton channel(Hv1)is specifically expressed in microglia in the central nervous system.In the ischemic brain injury,the microglial Hv1 channel contributes to the generation of reactive oxygen species(ROS)that cause neuronal damage.However,the pathologic effects of microglia Hv1 channel on SCI and the underlying mechanisms need to be further explored.This study aims to explore the spatiotemporal distribution characteristics of pyroptosis and apoptosis of neurons and the role of microglial Hv1 channel in neuronal damage and functional reconstruction after SCI.Methods:Firstly,wild-type mice were used to establish SCI model.The spatial and temporal distribution of apoptosis(TUNEL)and pyroptosis(Gasdermin D,GSDMD)were observed and microglia activation was detected by immunofluorescence staining at 1,3 and 7 days after surgery.BMS score was used to conduct behavioral evaluation on 1,3,7,14 and 28 days after SCI to assess the neurological function recovery.Then,SCI models were established in Hv1 knockout(Hv1-/-)mice and wild type mice,respectively.Neuronal pyroptosis and apoptosis,expression of inflammasome and inflammatory factor,microglial activation and ROS generation,myelin and axon changes,and behavior detection of neurological function were analyzed by using immunofluorescence,western blot and RTPCR technology.Furthermore,oxygen-glucose deprivation/reoxygenation(OGD/R)treatment of neuronal cell line PC 12 cells was used to simulate SCI model.The effects of ROS inhibitor N-acetylcysteine(NAC)on neuronal pyroptosis,apoptosis and inflammasome were observed by using immunofluorescence,western blot,ELISA and flow cytometry.Results:The model of SCI was successfully established and verified by LFB staining and behavior tests.After SCI,apoptosis(TUNEL/DAPI)and pyroptosis(GSDMD/DAPI)were detected by double fluorescence staining.We found that TUNEL-positive cells were mainly located closer to the injury core than GSDMD-positive cells.Then,we performed a doublelabeling for GSDMD/TUNEL with NeuN,respectively.The results showed that the neuronal apoptosis peaked one day after injury,then gradually decreased,and almost no neuronal apoptosis was observed at day 7.However,neuronal pyroptosis peaked 3 days after injury.In addition,microglia were obviously activated and neural function was impaired.Compared with wild-type mice,Hv1-/-mice showed significantly reduced neuronal apoptosis and pyroptosis,remarkably decreased expression of pyroptosis pathway proteins NLRP3 inflammasome and inflammatory factors.Then,microglial phenotype and ROS production were detected.The results demonstrated that compared with wild-type mice,the activation of microglia was inhibited,anti-inflammatory microglia phenotype(CD206/IBA-1)was down-regulated,pro-inflammatory microglia phenotype(CD16/32/IBA-1)was up-regulated,and ROS production was decreased in the Hv1-/-mice group.In addition,the detection of myelin sheath and axon-related proteins(myelin:MBP,MAG;axon:NF-L,NF-H)suggested that Hv1 knockout could alleviate myelin and axon damage.The behavioral(BMS score)results verified that the neurological damage in Hv1-/-mice was less than that in wild-type mice.Furthermore,the ROS production of PC12 cells after OGD/R was detected by DCFHDA.The results indicated that ROS production was significantly increased after OGD/R,which was decreased using ROS inhibitor NAC.The results of scanning electron microscope showed that after OGD/R,PC 12 cells presented a typical morphology of pyroptosis,with obvious cell swelling and the emergence of large bubbles from the plasma membrane.Immunofluorescence and western blotting results indicated that pyroptosis of neurons significantly increased at 6h and 24h after OGD/R.TUNEL staining and flow cytometry detection of apoptosis showed that after OGD/R,neuronal apoptosis increased at 3h but decreased at 24h,which further confirmed that neuronal pyroptosis occurred later than neuronal apoptosis,and the duration of neuronal pyroptosis was longer than apoptosis.NAC can simultaneously inhibit pyroptosis and apoptosis of neurons after OGD/R.In addition,NAC can significantly reduce the expression of NLRP3 inflammatory body,IL-18 secretion and caspase-1 activity in neurons after OGD/R.Conclusion:The spatiotemporal patterns of neuronal pyroptosis and apoptosis after SCI were different.Deficiency of microglial Hv1 channel can reduce the pyroptosis and apoptosis of neurons and the expression of pyroptosis pathway proteins NLRP3 inflammasome,so as to attenuate the damage of myelin sheath and axon and promote the recovery of nerve function,which may be mediated by inhibiting the activation of microglia and the generation of ROS.