The Role Of Astrocytic TRPV1 In Epilepsy Induced By Neonatal Ischemia Hypoxia Brain Disease

Objective:Neonatal hypoxic-ischemic brain damage(HIBD),the main cause of neonatal death,which caused by perinatal hypoxia and decreased cerebral blood flow induced by a variety of factors.HIBD has complex etiology and severe illness,and survivors may have serious sequelae,such as cognitive dysfunction,cerebral palsy,epilepsy susceptibility increased in adulthood after neonatal HIBD,etc.Previous studies have shown that HIBD is closely related to the abnormal function of the central nervous system(CNS)ion channels.TRPV1 is a kind of non-selective cation channel widely distributed in the CNS and mainly leads to extracellular Ca2+ influx after activation.Recent studies have shown that TRPV1 can not only promote the occurrence of neuroinflammation,but also increase the excitability of neurons.Additionally,astrocytes,as the neuroglial cells with the largest number and the most extensive expression in the CNS,are also closely associated with neuroinflammation and neuronal excitability.Nevertheless,the mechanism between TRPV1 on astrocytes and epilepsy susceptibility in adulthood after neonatal HIBD is unclear.This study aimed to investigate whether TRPV1 knockout could reduce epilepsy susceptibility in adulthood after neonatal HIBD and the role of TRPV1 on astrocyte after OGD.Methods:(1)P9 WT and TRPV1 knockout C57BL/6 mice were taken to constructe HIBD model or sham model,respectively.Then,immunohistochemistry(IHC)was used to test the astrocyte activation in brain tissue slices of mice from different groups after 24 hours.The mice that didn't be treated with IHC continued to be reared to 7 weeks and electroencephalogram(EEG)were used to detect PTZ-induced seizures for behavioral scoring.(2)The brain tissue of P0 WT and TRPV1-/ C57BL/6 mice were used to culture primary astrocytes.Then,agarose gel electrophoresis,Western Blot and immunocytochemistry(ICC)were used to detect the expression and distribution of TRPV1 in astrocytes after oxygen and glucose deprivation(OGD).(3)The effect of TRPV1 on the migration of astrocytes after OGD was detected by scratch migration assay and intracellular calcium ion fluorescence intensity assay.(4)Western Blot,q PCR,ELISA and ICC were used to detect the effect of TRPV1 on the expression and secretion of inflammatory molecules(il-6,il-1,TNF,i NOS,Arginase-1)and the polarity change of astrocytes after OGD.(5)Whole-cell patch clamp was used to detect TRPV1 current on primary cultured astrocytes.Results:(1)Astrocytes activation and epilepsy susceptibility in adulthood after HIBD were reduced after TRPV1 knockdown at the animal level.(2)TRPV1 channel was expressed on astrocytes.,astrocytes were activated and the expression of TRPV1 was increased on the astrocyte membrane after OGD.In addition,the astrocytes turned hypertrophied and the distribution of TRPV1 was transferred from the astrocyte cytoplasm to the membrane.(3)TRPV1 regulates astrocytes migration by reducing the polymerization ability of actin via reducing intracellular calcium levels in astrocytes after OGD.(4)TRPV1 knockdown reduced the secretion and expression of inflammatory factors(IL-6,IL-1?,TNF,i NOS,Arginase-1)as well as the neurotoxic Gene expression of astrocytes after OGD.(5)Astrocytes have TRPV1 current which was dependented on the concentration of capsaicin.Conclusions:This study preliminarily explored the mechanism of how TRPV1 on astrocytes promoted epilepsy susceptibility increased in adulthood after HIBD.At the animal level,TRPV1-/-reduced astrocyte activation and epilepsy susceptibility in adulthood after HIBD.At the cellular level,TRPV1-/-suppressed the level of intracellular calcium ions and migration of astrocytes.In addition,TRPV1-/-also reduced the expression and secretion of pro-inflammatory factors of astrocytes after OGD.Therefore,TRPV1 on astrocytes may promote the increase of epilepsy susceptibility in adulthood after HIBD by promoting its migration as well as the expression and secretion of pro-inflammatory factors after HIBD.Therefore,targeting inhibition of TRPV1 may be an active therapeutic strategy to reduce epilepsy susceptibility in adulthood after HIBD.