IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries In Experimental Stroke Mice

BackgroundIschemic stroke involves a variety of pathophysiological mechanisms.Increasing evidence indicates that inflammatory response is one of the important pathogenesis of ischemic brain injury,and may provide a target for stroke treatment.Interleukin-1 receptor-associated kinase M(IRAK-M)negatively regulates Toll-like receptors(TLRs)signaling pathways and suppresses inflammatory responses by reprogramming negative feedback on innate immune responses.It is essential in the pathophysiology of a variety of infections and diseases,including ischemia-reperfusion injury processes.However,the role of IRAK-M in cerebral ischemia-reperfusion remains unclear.ObjectivesThis study aims to investigate whether IRAK-M deficiency could exacerbate neurovascular injuries during ischemic stroke.MethodsFocal cerebral ischemia was induced by middle cerebral artery occlusion in C57BL/6 mice,and laser Doppler was used to detect cerebral blood flow changes during and after surgery.1)qPCR was conducted to detectthe mRNA expression of IRAK-M at 1h,4h,12h,24h after cerebral ischemia-reperfusion;2)Male WT mice and IRAK-M KO mice were subjected to 45 min of middle cerebral artery occlusion and 4 or 24h of reperfusion.The effects of neurological function score and infarct volume were compared between the two groups after 24h of reperfusion;3)Dry-wet weight method measured the effect of IRAK-M deficiency on postoperative brain edema in mice;4)Evans Blue and immunohistochemically labeled IgG were used to detect postoperative blood-brain barrier damage in both groups;5)qPCR and western blot examined the mRNA and protein expression of inflammatory factors between the two groups of mice after cerebral ischemia-reperfusion.Results1.IRAK-M is significatively upregulated after cerebral ischemia-reperfusion.(1)IRAK-M is significatively upregulated after cerebral ischemia-reperfusion.The qPCR results showed that the mRNA expression of IRAK-M increased at 1h and peaked at 4 h after cerebral ischemia-reperfusion.(2)The upregulation of IRAK-M expression at lh after cerebral ischemia-reperfusion is associated with increased expression of HIF-la.2.IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries in Ex-perimental Stroke Mice(1)IRAK-M gene knockout aggravated the neurological function score of cerebral infarction.Compared with WT mice,the Bersdon score of IRAK-M mice increased,and the score of the grip test decreased,showing a worse neurological status.(2)Compared with WT mice,IRAK-M KO mice showed increased cerebral infarction volume and cerebral edema volume.The results of TTC staining 24h after cerebral ischemia-reperfusion showed that the infarct volume and edema percentage of IRAK-M KO mice were significantly increased compared with WT mice.(3)IRAK-M KO rats have higher water content and higher incidence of hemorrhagic transformation than WT mice.IRAK-M KO mice showed higher brain edema in both the naked eye and the data.IRAK-M deficiency remarkably increased the incidence of hemorrhagic transformation after cerebral ischemia-reperfusion.(4)There is more leakage of Evans blue in the brain tissue of IRAK-M KO mice.Frozen section IgG staining revealed a significant increase in the depth and area of IgG staining in brain tissue of KO mice.3.Increased expression of MMPs and inflammatory factors in the infarcted side of IRAK-M knockout mice.(1)The expression of matrix metalloproteinases involved in blood-brain barrier destruction was elevated in IRAK-M KO mice.The expression of MMP-2 and MMP-9 was upregulated at 4h and 24h after cerebral infarction in IRAK-M KO mice.(2)The expression of inflammatory factors was upregulated in the infarcted hemispheres of IRAK-M KO mice.QPCR and Western blot were used to detect the expression of inflammatory cytokines in the infarcted hemispheres of WT and KO mice,and IL-6,TNF-?,IL-1?,IL-10,iNOS,COX-2 and NLRP3 were involved.The expression of inflammatory factors in inflammatory response to cerebral ischemia-reperfusion is elevated.(3)The deficiency of IRAK-M increases activation of the NF-?B signaling pathway.The protein level of P65 in the nucleus of the infarct hemisphere of KO mice was found to be significantly higher than in the infarct side of WT mice.