Embryonic MGE Procursor Cells Grafted Into Cortex And Hippocampus Ameliorated Autism-like Behavior In TRIM32^-/- Mouse

PurposeExplore the way to separate and extract the MGE precursor cells from the area of mouse brain MGE while detect the specific markers of GABA neural precursor cells and the ability of neuron-oriented differentiation.Investigate the survival rate,migration and differentiation ability of MGE precursor cells after transplantation.Probe the effects of MGE precursor cells transplantation on the MGE precursor cells transplantation on behavioral defects and electroencephalogram epileptic discharges in TRIM32-/-mice.MethodsSeparate MGE precursor cells from mouse brain,then the expression of Nestin,Lhx6,Nkx2.1 and GABA,GAD67 and PV were detected by immunofluorescence staining.The co-expression with GFP of GAD67,PV,CR,NPY and NeuN were detected by immunofluorescence staining.The behavior improvement of TRIM32-/-mice after transplantation was evaluated by behavioral test.And epileptic discharge change was assessed by electroencephalography.Content and Process1.Part of the cells suspension continue to culture,dry maintenance and directional differentiation culture,respectively,followed by the immunofluorescence staining of Nestin,Nkx2.1 and LHX6 and GABA,GAD67,PV or other protein markers.2.Orientate hippocampus(±1.8,-2.0,-2.0)and cortical(± 1.8,0,-1.0)as the injection point,and 2ul cells suspension(1 × 107 cells/mL)was injected with the rate of 200nl/min and needle stayed for 5min after injection.2ml(2ml/g;5ug/g)Cyclosporine was applied before and after operation.The injection continues 14 days3.GABA,GAD67,PV,CR,NPY and NeuN were stained by immunofluorescence after four weeks of MGE precursor cells transplantation.The number of positive cells in the cortex and hippocampus and hippocampus were counted.4.3 rooms social preference assay,Social interaction experiment,Nesting experiment Y maze spontaneous choice experiment and Self rub fur experiment were performed to evaluate the behavior of TRIM32-/-mice with MGE precursor cells transplantation group,TRIM32-/-mice with invalid MGE precursor cells transplantation group and TRIM32-/-mice group after 4 weeks.5.The difference of electroencephalographic epileptic discharges in the transplanted areas of each experimental group was detected at 4 weeks after MGE precursor cells transplantation.Results1.Isolated MGE precursor cells assessed with trypan blue stain 80%-90%were viable.2.The expressions of Nestin,NK2 homologous box 1(NKX2.1),type 6 LIM homologous framework protein(LHX6)and MGE-specific proteins were positive and the positive expression of GABA,GAD67 were(0.718 ± 0.091),(0.694 ± 0.058)and(0.433 ± 0.08),respectively.3.Frozen brain tissue immunofluorescence microscopy showed that MGE precursor cells migrated and differentiated MGE precursor cells in hippocampus,cortex and striatum after transplantation were more than in specific migration areas such as corpus callosum and hippocampus.4.Immunohistochemical staining showed that NeuN(0.764 ± 0.098),GABA(0.704±0.124),GABA synthase GAD(0.752±0.062),PV(0.255 ± 0.055),CR(superscript 2+)were significantly higher than those in the control group(0.193 ± 0.062),NPY(0.163 ± 0.047).Similarly,GABA(0.600 ± 0.107),GAD67(0.546 ± 0.125),PV(0.171± 0.053),CR(0.100 ±0.030),NPY(0.130 ± 0.041)and NeuN(0.631 ± 0.064)were obtained.And in the hippocampus region GABA,GAD67,PV,CR,NPY,NeuN,etc.were negative.5.Immunohistochemical staining showed that myelin basic protein(MBP)and expression of synaptic vesicle protein(SYP)were positive in some of the transplanted cells,and the expression of glial fibrillary acidic protein(GFAP))And microglia(Ibal)were negative expression.6.Behavioral results of TRIM32-/-mice were compared with TRIM32-/-mice in the control group.The TRIM32-/-mice in the control group were compared with the control group.There was a significant difference between the two groups(P<0.05).7.The cortical and hippocampal electroencephalographic records showed that the TRIM32-/-mice in the TRIM32-/-mice had a significant decrease in the rate of epileptic discharges compared with the control group,and the difference was statistically significant with the control group.(P<0.05).