The Effect And Mechanism Of Chronic Sfn Intervention On White Matter Injury And Cognitive Impairment After Stroke In Mice

BackgroundExperts’ consensus on the management of post-stroke cognitive impairment(PSCI)in 2017 is the cognitive impairment within six months after stroke,including cognitive impairment from non-dementia of PSCI that meets the diagnostic criteria to dementia of post stroke.Due to the lack of targeted large-scale research,the treatment of PSCI mainly refers to vascular cognitive impairment,vascular dementia and Alzheimer’s disease and other related research and evidence,and no effective drug treatment has been determined.Therefore,the current research focus is to further study the pathogenesis of PSCI,develop new therapeutic drugs and programs,so as to achieve early intervention and improve the prognosis of PSCI.Ischemic stroke not only cause gray matter damage,but also lead to severe white matter injury(WMI).The severity of WMI can affect infarct volume,predict functional recovery and secondary cognitive dysfunction after stroke.Oxidative stress can directly lead to necrosis and apoptosis of ischemic tissue through many ways,which is the main factor of ischemic stroke,and the molecular mechanism of oxidative stress-induced cognitive impairment.Nuclear factor E2-related factor 2(Nrf2)is a transcription factor,which is the"main regulator" of antioxidant response and regulates the expression of phase II detoxification enzyme and antioxidant enzyme genes.Heme oxygenase-1(HO-1)is one of the antioxidant enzymes with neuroprotective effect.Sulforaphane(Sfn)is an effective natural Nrf2 activator,which widely exists in cruciferous vegetables.More and more studies have confirmed the protective effect of Nrf2 inducer in the early stage of cerebral ischemia or in the time window of 1-2 weeks after cerebral ischemia.However,there are few studies on the long-term protective effect of Nrf2 inducer on cerebral ischemia.First of all,we will explore whether chronic Sfn intervention can reduce the long-term sensorimotor and cognitive impairment,WMI caused by stroke,and promote axonal sprouting.On the basis of clarifying that chronic Sfn intervention can reduce white matter injury and cognitive impairment after stroke,we will take Nrf2 knockout mice as the research object to observe whether this effect of Sfn can be achieved by activating Nrf2/HO-1 pathway.This study was to investigate the protective effect and mechanism of chronic Sfn intervention on PSCI in mice.It can provide a theoretical basis and experimental basis for the development and clinical application of Sfn,and provides a potential diagnosis and treatment strategy for prevention and promotion of recovery of WMI and cognitive impairment in the chronic stage after stroke.Part I:The effect of Sfn on white matter injury and cognitive impairment after stroke in mice.ObjectiveOur study aimed to explore whether chronic Sfn intervention has protective effects on cognitive impairment,white matter injury,and neuroplasticity after stroke in mice.MethodsMale and OVX female C57BL/6J wildtype mice(WT)were divided randomly into three experimental groups using a random number table:sham group,vehicle group,and Sfn-treated group.The forelimb motor control function was evaluated by foot fault,and rotarod tests assessed the sensorimotor coordination function up to 35d post-stroke.The Morris water maze test estimated long-term spatial cognitive functions(spatial learning and memory)from day 29 to day 34 post-stroke.Brain tissue loss was measured by immunofluorescence staining of microtubule-associated protein 2(MAP-2).We examined myelin loss by Luxol fast blue staining,assessed myelin integrity and axonal damage by immunostaining for myelin basic protein(MBP)and non-phosphorylated pathological neurofilaments(SMI-32),evaluated the expression of HO-1 by western blot.The tract tracer biotinylated dextran amine(BDA)was injected at two sites in the contralesional motor cortex to access the plasticity of the corticobulbar and corticospinal tracts.Results1.Sfn improved long-term forelimb motor control deficits,the sensorimotor coordination deficits,spatial cognitive deficits and tissue loss after stroke in male and OVX female WT mice.2.Sfn attenuates myelin loss and white matter dysfunction after pMCAO in WT mice.3.Sfn attenuates axonal sprouting of the corticobulbar tracts after pMCAO in WT mice but improved axonal sprouting of the corticospinal tracts.Part II:The effect of Nrf2/HO-1 pathway on the improvement of cognitive impairment and white matter injury after stroke by Sfn in miceObjectiveOur study aimed to explore the protective mechanism of chronic Sfn intervention on white matter injury and cognitive impairment after stroke in mice,and to test the hypothesis that Sfn can reduce sensorimotor and cognitive impairment,white matter injury and axonal sprouting after stroke by activating Nrf2/HO-1 pathway.MethodsMale and OVX female C57BL/6J mice and Nrf2 knockout mice were divided randomly into three experimental groups using a random number table:sham group,vehicle group,and Sfn-treated group.The Nrf2 activation of wild-type mice and Nrf2 knockout mice was evaluated by HO-1 immunofluorescence staining and Western blot.The forelimb motor control function of Nrf2 knockout mice was evaluated by foot fault,and rotarod tests assessed the sensorimotor coordination function up to 35d post-stroke.The Morris water maze test estimated long-term spatial cognitive functions(spatial learning and memory)from day 29 to day 34 post-stroke.Brain tissue loss was measured by TTC staining and immunofluorescence staining of MAP-2.We examined myelin loss by Luxol fast blue staining,assessed myelin integrity and axonal damage by immunostaining for MBP and SMI-32,evaluated the expression of HO-1 by western blot.The tract tracer BDA was injected at two sites in the contralesional motor cortex to access the plasticity of the corticobulbar and corticospinal tracts.Results1.Sfn enhanced the HO-1 expression in both the acute and chronic phases after pMCAO.Nrf2 KO attenuates Nrf2 activation after stroke.2.Sfn failed to improve long-term forelimb motor control deficits,the sensorimotor coordination deficits,spatial cognitive deficits and tissue loss after stroke in male and OVX female Nrf2 KO mice.3.Sfn failed to attenuate myelin loss and white matter dysfunction after pMCAO in Nrf2 KO mice.4.Sfn failed to alter axonal sprouting of the corticobulbar and corticospinal tracts after pMCAO in Nrf2 KO mice.Conclusions1.The mouse model of permanent middle cerebral artery occlusion showed long-term motor sensory and cognitive impairment,as well as histopathological manifestations,which can be used as the mouse model of PSCI.2.Chronic Sfn intervention improved long-term sensorimotor and cognitive impairment of mice after stroke,protected the repair of white matter damage,and enhanced the neural plasticity of wild type mice after stroke.3.Sfn activated the Nrf2/HO-1 pathway after stroke in mice,while Nrf2 knockout weakened the effect of Sfn.4.Chronic Sfn intervention couldn’t improve the long-term sensorimotor and cognitive impairment,tissue loss,white matter injury and axonal sprouting of Nfr2 knockout mice after stroke.