Experimental Research Of The Treatment Of Ischemic Stroke By Neural Stem Cell Transplantation

Chapter1. Isolation and identification neural stem cell from newborn mice hippocampus, olfactory bulb and cortexBACKGROUND: Neural stem cells can be used in the treatment of central nervous system diseases.OBJECTIVE: Establishment of newborn mice hippocampus, olfactory bulb, cortex neural stem cells isolated and cultured system;Observe the proliferation and differentiation of neural stem cells in vitro under conditions; Establishment of neural stem cell bank.METHODS:Neural stem cell were isolated from neuborn Kunming mice hippocampus, olfactory bulb, cortex tissue by mechanical separation and trypsin digestion. Using serum-free culture technology, mechanical pipetting and trypsin digestion extracted primary cultured neural stem cells and purified neural stem cells by passaged. Use 10% fetal bovine serum-induced differentiation of neural stem cells. Identification of neural stem cells and their differentiated products by immunofluorescent staining of Nestin、CD133、β-TubulinⅢ、 GFAP. Cryopreservation and recovery technology to build the neural stem cell bank.RESULTS AND CONCLUSION:The neural stem cell obtained from newborn mice hippocampus,olfactory bulb,cortex had the capacity of Self-renewal and differentiation which were positive for nestin and cd133. Neural stem cell can differentiation to β-Ⅲ-tubulin or glial fibrillary acidic protein positive cell after FBS induced. Description of laboratory have successfully established newborn mice hippocampus, olfactory bulb, cortex neural stem cell isolation and identification system.Chapter2. Effect of oxygen-glucose deprivation on the proliferation,differentiation and apoptosis of neural stem cellBACKGROUND: Hypoxia is one of the main pathological factors of ischemic stroke.Establishment of neural stem cells glucose oxygen deprivation model.In vitro oxygen glucose deprivation on neural stem cell to understand the proliferation, differentiation, and apoptosis is the fundamental for how neural stem cell survival in pathological conditions after transplantation.OBJECTIVE: Neural stem cell were isolated from neuborn Kunming mice olfactory bulb. Establishment of neural stem cells oxygen glucose deprivation model.Observation the influence of oxygen glucose deprivation on neural stem cell proliferation, differentiation and apoptosis.METHODS: Neural stem cell were isolated from neuborn Kunming mice olfactory bulb,establishment of neural stem cells oxygen model. The neural stem cells were divided into normoxic group and hypoxia group,observe the effect of oxygen glucose deprivation on neural stem cell proliferation by immunofluorescent staining. MTT assay detective effect of oxygen glucose deprivation for 24 hours and 48 hours on neural stem cell proliferation.Observe the influence of oxygen glucose on neural stem cell apoptsis by Hochest 33258.RESULTS AND CONCLUSION: Immunofluorescence show that CD133 positive when neural stem cell passage 4.Comapre with normoxic group,MTT show proliferation was significantly decline with oxygen glucose deprivation. Especially, the group of oxygen glucose deprivation 48 hours(P<0.05). GFAP, β-Tubulin Ⅲ immunofluorescence staining show that the number of GFAP andβ-Tubulin Ⅲ postive cell was significantaly decline(p<0.05)by oxygen glucose deprivation 48 hours. Apoptosis rate was significantly rise when oxygen glucose deprivation 48 hours(p<0.01) than 24 hours(p<0.01) compare with normoxic group. These results indicate that oxygen glucose deprivation have adversely on neural stem cell proliferation, differentiation, apoptosis, the impact depends on the concentration and time of hypoxia, and the ability of tolerance for hypoxia.Chapter3. Research of Photothrombosis animal model of ischemic strokeBACKGROUND:Experimental focal cerebral ischemia models is an indispensable tool in the stroke research Field.The photothrombosis model as good choice model cause have a lot of advantage such as easy to build;more stability;high repeatability;and easy to control.OBJECTIVE:Introduce how to bulid photothrombosis model. Analysis gender differences about in infarct volume and behaivioral manifestations after ischemic stroke.METHODS:Use photothrombosis establishe ischemic stroke model. Nissl staining detecting pathological changes and calculating infarct volume after ischemic stroke. Detective degeneration neuronal expression after ischemic stroke by Fluoro-Jade C staining. Fatigue rotarod test and forelimb grip strength test for the detection of ischemic stroke behavioral impact.RESULTS AND CONCLUSION: Nissl staining shown neuronal degeneration,necrosis,and vacuolar pathology in infarction central.Fluoro-Jade C staining shown neuronal degeneration change. Female group infarct volume was significantly smaller than the male group. Model group motor function was significantly greater than the sham group and female group which is less than the degree of impairment of motor function in male group. These results suggest that phtothrombosis method can be successfully established animal model of ischemic stroke. Male suffered more neurological damage from ischemic stroke than female on the same condition.Chapter4. Neural stem cell transplantation promotes behavioral recovery on photothrombosis stroke modelStem cell-based therapy provides a promising approach for treat stroke.Neural stem cells isolated from mice hippocampus possessing the capacity of differentiate into neurons and astrocytes both in vitro and vivo.Here,we investigated the capability of neural stem cell transplantation in photothrombosis stroke model.Nissl staining revealed that the cortical infarct significantly decreased by 16.32%(Vehicle:27.93±2.8mm3,n=6,NSC:23.37±2.25mm3,n=6,p<0.05) in the NSC group compared with the vehicle.More over transplantation of neural stem cells significantly(p<0.01) improved neurological performance compared with vehicle.These results indicate that transplantation of neural stem cell is a effective therapy in ischaemic stroke.