Effect Of Microglial Voltage-gated Proton Channel Hv1 Deficiency On Ischemic White Matter Injury Induced By Chronic Hypoperfusion In Mice And The Underlying Mechanism

Objective: Vascular cognitive impairment(VCI)is one of the main causes for cognitive impairment and is often caused by chronic cerebral hypoperfusion.In response of chronic ischemia,the innate immune cell microglia can be activated and produce lots of biological mediators.The voltage-gated proton channel,Hv1,is selectively expressed in microglia and is required for NOX-dependent ROS generation in the brain.The aim of this study is to explore the role of microglia Hv1 in the white matter lesion induced by chronic hypoperfusion.Methods: In order to mimic white matter lesion induced by chronic hypoperfusion,we employed a mouse model of bilateral carotid artery stenosis(BCAS).The cerebral blood flow during operation was detected by transcranial Doppler monitor.The expression of MBP and MAG were measured by immunofluorescence at 3,7,14,28 days after operation.LFB staining was used to observe the disarrangement and disappearance of the myelinated fiber.Transmission eletromicroscopy was adopted to examine the myelin sheath thickness,morphological change and the wrapped nerve fiber change.Eight-arm maze test was used to measure the behavioral impairment.Moreover,immunofluorescence was used to observe the dynamic changes of oligodendrocytes,oligodendrocyte precursor cells(OPCs)and microglia.Western blot was used to detect the expression of Hv1 and Nox2 in corpers callosum.In addition,conditional Hv1 knock-out in microglia was utilized to examine the roles of Hv1 in the microglial activation,reactive oxygen species(ROS)and proinflammatory cytokines generation,oligodendrocyte lineage proliferation and differentiation,degree of white matter lesion and cognitive impairment.The primary cultured microglia from wild-type or Hv1-/-mice were co-cultured with OPCs,the production of ROS and proinflammatory cytokines in microglia and the differentiation of OPCs were measured after OGD treatment.Results: At day 28 after operation,the results of LFB staining,transmission eletromicroscopy and behavioral test confirmed that this model was successfully established.LFB staining showed the disarrangement and disappearance of the myelinated fiber in mice of BCAS group.Transmission eletromicroscopy demonstrated that myelin sheath became thinner in mice after BCAS compared to sham ones.Eight-arm maze test verified the impaired working memory after chronic hypoperfusion.Immunofluorescence staining of the oligodendrocyte lineage revealed that the number of mature oligodendrocytes decreased while the OPCs numbers increased.Microglia showed obvious activation and elevated density.The expression of Hv1 and Nox2 upregulated significantly after chonic hypoperfusion.Hv1 knockout inhibited the activation of microlgia,down-regulated the expression of ROS,Nox2,TNF-? and IL-1? in white matter compared to the wild-type mice.Double staining of GST-?/NG2 and BrdU displayed the promoted proliferation and differnetiation of OPCs and increased number of mature oligodendrocytes in Hv1-/-group compared to the wild-type group.The results of LFB staining and transmission eletromicroscopy revealed alleviated myelin damage,the behavior assessment of 8-arm maize test verified the improvement of neurological function in Hv1-/-mice.In vitro study showed that Hv1 deficiency reduced ROS,TNF-? and IL-1? production in microglia following OGD,thereby differentiation of OPCs co-cultured with microglia.PI3K/Akt signaling is involed in Hv1 deficiency mediated regulation of microglial ROS and inflammatory cytokines production.Conclusion: Chronic hypoperfusion leads to damage of myelin structure and cognitive dysfunction in mice,alongwith the activation of microglia,damage of oligodendrocyte lineage and inhibited proliferation and differentiation of OPCs.Hv1 plays an active role in the pathological process of white matter injury.Hv1 knockout can exert protective effects on myelin integraty and cognitive function,which might be mediated by reducing the ROS and proinflammatory cytokines generation in microglia and thereby promoting the survival and maturation of oligodendrocyte.Thus,we identify microglial Hv1 as a unique therapeutic target for white matter lesions in clinic practice.