Cystamine Effectively Promotes Functional Recovery After Ischemic Stroke In Mouse Model

Part ?:Experimental study of culture and induced differentiation of neural stem cells,and the effect of Cystamine on cellsObjective:The aim of this study was to investigate the isolation,culture and differentiation of neural stem cells(NSCs)from hippocampus in neonatal mice,and to identify the cultured cells.To investigate the effect of cystamine on the expression of brain-derived trophic factor(BDNF)in neurons.Material and methods:The hippocampus was isolated from the brain of newborn C57BL/6L mice.Single cell suspension was obtained by mechanical blowing.NSCs were induced by suspension culture and cell growth factor induction.The cultured NSCs were identified by cell morphology and immunofluorescence staining.The fourth generation of NSCs were cultured to induce neuronal differentiation,then cell morphology and immunofluorescence staining were used to identify neurons and astrocytes.The induced NSCs were treated with different concentrations of cystamine,then the concentration of BDNF in the supernatant and neurons was measured by ELISA and WB.Results:3-5 NSCs derived from hippocampus gathered into clusters after cultured in serum-free neural stem cells for 24 hours.After 5 days of culture,the cells proliferated rapidly and gathered into a typical neurospheres.Immunofluorescence staining of the fourth generation of NSCs showed high expression of NSCs specific marker Nestin.The positive rates of B-tubulin and GFAP were 5.2%,93.5%respectively after NSCs were induced differentiation.The increased level of BDNF induced by cystamine promoted the differentiation of neural stem cells into neuronal cells.Conclusion:NSCs were successfully isolated and cultured from the hippocampus of neonatal mice.The cultured cells were identified to be the neural stem cells.They could differentiate into neurons,astrocytes and oligodendrocytes.Cystamine could promote the expression of BDNF in neuronal cells.Part ?:Establishment of focal ischemic stroke model in mice by photochemical embolizationObjective:To establish a focal ischemic stroke mice model with photochemical embolization.Using magnetic resonance imaging to evaluate the stability and controllability of the model.And to evaluate the neurological function loss of the mice after model.Providing a reliable animal model for further study in stroke treatment with cystamine.Material and methods:20 male C57BL/6J mice were randomly divided into two groups:stroke model group(n = 10)and sham operation group(n = 10).10mg/kg Rose was injected intraperitoneally into the body.Then 4mm diameter cold light source was used to irradiate the cortex for 15min through the skull.The operation of the sham operation group was the same as the stroke group,only the roses were not given.T2WI was performed with 7.0T micro-MR at 24 hours and 7 days after stroke.Then the brain were stained with TTC and Nissl to evaluate the stability of the model.Results:Magnetic resonance imaging showed a stable lesion in motor function area at 24 hours.The infarct percentage of infract volume on T2WI and TTC were 17.1±3.7%.15.6±2.3%,respectively.Motor function showed contralateral limb movement dysfunction.The sham operation group showed normal MR signal,negative TTC staining and normal motor function.Conclusion:The focal ischemic stroke mice model was successfully established with photochemical embolization.The ischemic infarction was stable and controlled,and it could be used for the evaluation of motor function.Part ?:Cystamine improves functional recovery via axon remodeling and neuroprotection after stroke in miceObjective:Stroke is a leading cause of disability.However,there is no pharmacological therapy available for promoting recovery.Although treatment of stroke with cystamine has gained increasing interest,the detailed mechanisms underlying this process remain elusive.Thus,our aim is to examine the effect of cystamine on the function recovery after stroke and investigate further cystamine mechanisms.Material and methods:Adult male C57BL/6J mice were subjected to photothrombotic model of focal stroke or sham operation.Cystamine or saline was administered intraperitoneally at 24 hours after stroke.Functional recovery was analyzed using behavioral tests;axon remodeling was analyzed using magnetic resonance diffusion tensor imaging(DTI)and histological assessment.ANA-12,an antagonist of tropomyosin-related kinase B(TrkB),was administrated to examine the mechanisms underlying the neuroprotection mediated by cystamine.Results:Treatment with cystamine resulted in amelioration of impaired function with concomitant enhancement of axonal remodeling.Cystamine treatment significantly increased brain-derived neurotrophic factor(BDNF)levels and phosphorylation of TrkB in brain after stroke.Cystamine significantly enhanced neuronal progenitor cell proliferation,neuronal survival and plasticity through BDNF/TrkB pathway.Conclusions:These data provide evidence to investigate the promising utility of cystamine for therapy of stroke in a variety of ways,acting principally though BDNF/TrkB pathway.