| Object:Ischemic stroke is a leading cause of disability and death in humans.With the improvement of Thrombolysis and Embolectomy treatments,the mortality decreased significantly.However,thrombolysis is still limited to a narrow therapeutic window(embolism is no more than 6 hours).Most recent studies have shown that vascular delayed recanalization beyond the therapeutic window also can significantly improve neurobehavioral function in patients without causing serious complications.Apoptosis is one of the pathways that lead to neuronal death after ischemic stroke.Penumbra is the neural tissue that has not died around the core area of infarction.Studies have shown that the enlargement of apoptosis and penumbra plays a crucial role in the treatment of ischemic stroke.Hepatocyte growth factor(HGF)is a cytokine that regulates angiogenesis,neurogenesis,organogenesis,and tissue reconstruction as well as mediates antiapoptotic signals through AKT.The tyrosine kinase receptor c-Met is a specific receptor of HGF.The HGF/c-Met signaling pathway can be activated by ischemia.However,the role of HGF/c-Met activation has not been clarified in delayed recanalization after middle cerebral artery occlusion(MCAO).The aim of this study was to investigate the anti-apoptotic effect of c-Met activation by HGF and the underlying mechanism in rats model of delayed recanalization after MCAO.Methods:First,the permanent MCAO(pMCAO)was performed by filament through internal carotid artery(CCA)to middle cerebral artery(MCA)to stop the blood flow.The delayed recanalization after MCAO(rMCAO)was performed by withdrawing the filament from the middle cerebral artery in 3 days after the initial MCAO.The endogenous expression of HGF and c-Met were measured at 4 d,6 d,10 d in pMCAO and rMCAO respectively by Western Blot.The double immunofluorescence staining of HGF and c-Met with neuronal nuclei(NeuN),Microglia(Iba-1)and Astrocytes(GFAP)were performed for the cellular co-localization in rats.Secondly,the HGF level in blood was checked in pMCAO 2 d,pMCAO 6 d and rMCAO 6 d by enzyme linked immunosorbent assay(ELISA).Thirdly,the neurobehavioral function changing of rats was evaluated by Modified Garcia scale and Beam Balance test at day 6 after pMCAO(day 3 after rMCAO),the infarction volume was measured by 2,3,5 triphenyltetrazolium choride(TTC)and Nissl staining,the neuronal apoptosis and degeneration were checked by terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling(TUNEL)staining and Fluoro-Jade C staining respectively.The limb movement and brain coordination of rats were evaluated by Rotarod test at continue 3 weeks after pMCAO and rMCAO,the spatial memory and potential learning functions of rats were measured by Morris Water Maze test,the brain tissue loss of rats was measured by Nissl stainin.For mechanism research,the small interfering ribonucleic acid(siRNA)for HGF were administered intracerebroventricularly 24 h before rMCAO(2 day after MCAO),or the inhibition of c-Met(SU11274)was administered intranasally immediately after recanalization.The infarction volume and neuronal apoptosis were measured by TTC and TUNEL respectively.The protein level of HGF;p-STAT3;STAT3;Bcl-2;Bax and Cleaved Caspase-3(CC-3)in ischemic hemisphere were detected by western blot.Results:(1)The expression of endogenous HGF in ischemic hemisphere was increased in both pMCAO group and rMCAO group,peaked at day 6 in rMCAO group,the HGF level increased significantly in rMCAO at day 6 when compared with sham group and pMCAO group.The expression of c-Met also increased after MCAO,but there are no significant difference between pMCAO and rMCAO.(2)HGF and c-Met were mainly expressed in neurons,but also expressed in astrocytes or microglia.(3)The serum HGF concentration increased significantly at 2 day after MCAO,but decreased after recanalization.(4)Delayed recanalization significantly reduced the infarct volume and improved the short-term neurobehavioral function as well as decreased neuronal apoptosis and degeneration in penumbra.(5)Delayed recanalization improved the limb movement and brain coordination ability of rats as well as spatial memory and potential learning functions.(6)Delayed recanalization ameliorated the brain tissue loss after MCAO.(7)Activation of c-Met with HGF activated STAT3 and then up-regulated the expression of Bcl-2 and down-regulated the expression of Bax and CC-3.(8)HGF siRNA or the inhibition of c-Met SU11274 both reversed the anti-apoptosis effects of HGF/c-Met axis and its effects on the expression of downstream pathway proteins.Conclusions:(1)The serum HGF level increased after MCAO.(2)Delayed recanalization increased the expression of HGF in brain,then combined with c-Met to activate STAT3 signaling pathway,up-regulated the expression of Bcl-2 and down-regulated the expression of Bax and CC-3.(3)Delayed recanalization after MCAO attenuated neuronal apoptosis,reduced infarct volume,and proved neurological function to save the penumbra brain tissue. |
PDF Full Text Request