Microglial Hydrogen Sulfide Synthase CBS Mediates Endogenous Hematoma Resolution After Intracerebral Hemorrhage

Research Purposes:After intracerebral hemorrhage(1CH),the hematoma squeezes the brain parenchyma and the rapid entry of blood into the brain parenchyma leads to the increase in local pressure in the brain parenchyma,resulting in primary injury.Moreover,once hematoma contents enter the brain parenchyma,they also induce secondary brain injury.In the present study,hematoma can be endogcnlusly cleared fbllowing ICH and the prognosis of patients is positively correlated with the rate of hematoma absorption.Therefore,increasing the speed of hematoma is the potential therapeutic strategy of ICH.Microgliti can be activated during hematoma resolution.Recent rcsearches show that hydrogen sulfide(H2S)is the third gaseous signal molecule,playing an important role in the treatment of various central nervous system.But no research studied the function and mechanism of hydrogen sulfide for hematoma resolution after ICH.Here we investigated whether endogenously H2S synthesis is involved in hematoma clearance after cerebral hemorrhage.Research Methods:(1)ICH was induced in mice by injecting autologous blood or collagense into the left striatum,termed as the bICH or cICH model respectively.The protein expression levels of H2S synthase cystathionine ?-synthase(CBS)was assessed at 1-14 days after ICH.The H2S synthesizing activity in the hemorrhagic striatum was assayed in the cICH model.(2)In the cellular model,primary microglia were treated with lysate of red blood cells(RBC Lysate).After treatment,the expression of CBS was assessed by western blot,and microglial phagocytosis of red blood cells was also assessed.(3)To explore whether RBC lysate promoted microglial phagocytosis of red blood cells via endogenous H2S,we used ZnCl2 to block the effects of free H2S,We examined whether ZnCl2 blocked the promoting effects of RBC lysate on microglial phagocytosis.(4)Primary microglia with cbs deletion(Cx3crl-cre:cbsfl/fl)were used to explore whether cbs knockout blocked the promoting effects of RBC lysate on microglial phagocytosis.(5)To explore whether CBS in microglia/macrophages contributed to endogenous hematoma clearance in vivo,we injectied collagenase into the striatum of the mice with specific deletition of cbs in microglial/macrophages(Cx3crl-cre:cbsfl/fl)or control mice(cbsfl/fl).After ICH,neurologic deficits were determined with neurologic deficit scores,right paw placement and corner test.Haematoma volumes and haemoglobin content in the striatum were also assessed to indicate hematoma clearance.(6)To further investigate whether the role of CBS in endogenous hematoma clearance was specifically mediated by microglia,we used genetically engineered mice with conditional knockout of cbs in microglia(Cx3crl-creER:cbsfl/fl).First,tamoxifen was injected into Cx3crl-creER:cbsfl/fl mice to induce cbs deletion only in microglia.Then ICH was induced by collagense injection in these mice or Cxicrl-creER:cbsfl/fl mice without tamoxifen injection.Hemoglobin content in the striatum and neurological deficits were assessed at 5 and 14 days after ICH.(7)To further investigate whether CBS promoted endogenous hematoma clearance via H2S,we examined whether supplementation of exogenous H2S promoted endogenous hematoma clearance in mice with cbs deletion following ICH.In the end,exogenous H2S donor ADT was injected into Cx3crl-cre:cbsfl/fl mice and Cx3crl-creER:cbsfl/fl mice with tamoxifen injection or control mice(cbsfl/fl mice,or Cx3crl-creER:cbsfl/fl mice without tamoxifen injection)following ICH.Hemoglobin content in the striatum and neurological deficits were determined at 5 and 14 days following ICH.Results:(1)The protein level of CBS and endogenous H2S synthesizing activity in brain tissues were enhanced after cerebral hemorrhage in mice.In the cICH model and bICH model,the protein level of CBS significantly increased at 3-5 days following ICH.In cICH model,H2S synthesizing capacity increased significantly 3-5 days following cerebral hemorrhage.(2)The protein level of CBS increased in primary microglia at 2-12 hours following the treatment with RBC lysate.RBC lysate also promoted microglia to engulf red blood cells.(3)ZnCl2 block the promoting effects of RBC lysate on microglial phagocytosis of red blood cells.(4)Compared with control microglia(cbsfl/fl),deletion of cbs in microglia blocked the promoting effects of RBC lysate on microglial phagocytosis of red blood cells.(5)Compared with control mice(cbsfl/fl),Cx3crl-cre:cbsfl/fl mice displayed enhanced hemoglobin content in the hemorrhagic striatum,enlarged hematoma volumes and aggravated neurological deficits at 5 days and 14 days following cerebral hemorrhage.(6)Compared with the control Cx3 crl-creER:cbsfl/fl mice without tamoxifen injection,the tamoxifen-injected Cx3crl-creER:cbsfl/fl mice displayed enhanced hemoglobin content in the hemorrhagic striatum,enlarged hematoma volumes and aggravated neurological deficits at 5 days and 14 days following ICH.(7)Supplementation of H2S donor ADT promoted endogenous hematoma clearance and improved neurological deficits in mice with cbs deletion in microglia/macrophages.Conclusion:Our results suggest that microglial CBS mediates endogenous hematoma clearance possibly via endogenously produced H2S after intracerebral hemorrhage.