Neuroprotective Role Of Novel Sigma-1 Receptor Agonist In The Acute Ischemic Stroke Model

Part 1 The distribution characteristics of Sigma-1 receptor in the mouse brain of acute ischemic stroke modelObjective: The research was aimed to investigate the distribution characteristics of Sigma-1 receptor in the brain of acute ischemic stroke model mice.Methods: In the experiment,healthy male C57BL/6J mice aged about 6 to 8 weeks were used to perform the acute ischemic stroke model(permanent middle cerebral artery occlusion,p MCAO)by using light embolization method.Mouse brain tissue samples were harvested after peritoneal lavage with PBS at the time 24 hours after operation.The brain tissue was distinguished as ischemic core,peri-infarct area and contralateral zone.The protein level of Sigma-1 receptor in the mouse brain between p MCAO and sham operation in ischemic core and peri-infarct tissue was test by western blot assay.The distribution characteristics of Sigma-1 receptor in the mouse brain after acute stroke were observed by immunohistochemistry staining and immunofluorescence stanning.Multiple sequences(T2WI,DWI,PWI)of magnetic resonance imaging(MRI)were used to determine the penumbra area in the mouse brain after embolization.And the relationship between penumbra and distribution characteristics of the Sigma-1 receptor was further detected and analysed by immunofluorescence staining.Results: To evaluate the protein level of Sigma-1 receptor after acute ischemic stroke,we tested the protein level in ischemic core and peri-infarct area between experimental group and sham operation group.The protein level of Sigma-1 receptor was significantly increased in periinfarct area at 24 hours after acute ischemic stroke compared with sham operation group and was decreased in ischemic core.This finding was further confirmed by immunostaining,Sigma-1 receptor was increased in peri-infarct area compared with contralateral zone at 24 hours after acute ischemic stroke.The area where Sigma-1 receptor was increased was matched with the penumbra defined by MRI.The protein level of Sigma-1 receptor at 1 hour after acute ischemic stroke in the penumbra was significantly increased compared with the sham group and the ischemic core.Conclusion:The expression of Sigma-1 receptor was increased in peri-infarct tissue after acute ischemic stroke compared with that in the ischemic core or contralateral zone.The expression pattern of Sigma-1 receptor in the peri-infarct area matched with MRI evaluated penumbra at 1 hour after ischemic stroke,indicating that new drugs will be designed for the diagnosis and treatment of acute ischemic stroke by targeting the increased Sigma-1 receptor in penumbra.Part 2 Neuroprotective role of novel Sigma-1 receptor agonist in the acute ischemic stroke modelObjective: The research was to demonstrate the neuroprotective effects of the new self-synthesized Sigma-1 receptor agonist-M4 in the mouse model of acute ischemic stroke,and reveal the underlying mechanismsMethods: In the experiment,healthy male C57BL/6J mice aged about 6 to 8 weeks were used to perform the acute ischemic stroke(transient middle cerebral artery occlusion,t MCAO)model,and Laser doppler flow imager was used to evaluate whether the model was successful.Different concentrations(0.15 mg/kg,0.5 mg/kg,1.5 mg/kg)of Sigma-1 receptor agonists(M4,SA4503)were immediately administrated according to the experimental design when the models were successfully performed.Infarct volume was detected by magnetic resonance imaging(MRI)and 2,3,5-triphenyltetrazolium chloride(TTC)staining,and were quantified by software Image J.Modified neurological severity scores(m Nss)was used to evaluate the neurological deficits.Western blot assay was used to examine the expression of astrocyte marker(GFAP),apoptotic proteins(Bax,Bcl-xl,Caspase-3,Cleaved Caspase-3)and autophagy protein(LC3B-II)in the ischemic core.For the in vitro study,primary astrocytes and human astrocyte cell line-A172 cells were subjected the oxygen glucose deprivation/reperfusion(OGD/R).Cells were treated with OGD for 3 h and reperfusion for 6 h.The attached cells were collected and detected the viability by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-htetrazolium bromide(MTT)assay after the OGD/R treatment.Astrocyte marker protein,apoptotic proteins and autophagy protein were examined the protein level by Western blot assay.Mice were trained with grid-walking task and cylinder task three days before the operation,and those with abnormal behavior(hyperactive or negative)were excluded.Mouse was tested gridwalking task and cylinder task at 2d,7d,14 d,28d and 56 d after operation.Mice were sacrificed at 56 d after behavioral tests,and mouse brain tissue samples were harvested for Western blot assay to detect the protein level of post-synaptic density protein 95(PSD-95)and synaptic proteins(Synapsin).Results: Our study demonstrated that compound M4 decreased the ischemia outcomes of the acute ischemic stroke.M4 significantly decreased the cerebral infarction volume and the scores of modified neurological severity scores.M4 significantly inhibited the activation of astrocytes,apoptosis and autophagy in vivo.Pretreating with M4 significantly restored the decline of cell viability(primary astrocyte and human astrocyte cell line-A172)induced by OGD/R.After a long-term administration of compound M4,the sensorimotor function deficits were ameliorated,and the neuroplasticity were recovered in mouse of acute ischemic stroke.Conclusions: Our novel Sigma-1 receptor agonist-M4 reduced the scores of modified neurological severity scores and decreased the volume of cerebral infarction in the mouse of acute ischemic stroke.This effect may be achieved by the inhibition of astrocyte activation,apoptosis and autophagy.Additionally,long-term administration of Sigma-1 receptor agonist M4 the enhanced the neuroplasticity and improved sensorimotor function in mouse of acute ischemic stroke.These findings indicated that the novel Sigma-1 receptor agonist M4 was a promising therapeutic strategy for acute ischemic stroke.