Mild Hypothermia Enhanced The Potent Therapeutic Effect Of Neural Precursor Cell Transplantation In A Neonatal Mouse Model Of Cerebral Hypoxia-ischemia

1 Objectives1.1 To observe electrophysiological characteristics of neuron-like cells differentiated from murine neural stem cells(NSCs) in vitro. 1.2 To analysis electrophysiological function of neuron-like cells differentiated from transplanted murine NSCs in HIE mice to explore the therapeutic mechanism of NSCs transplantation, and to evaluate its safety. 1.3 To evaluate the efficacy of NSCs transplantation combined with mild hypothermia for HIE,and to explore the mechanism of synergistic effect of mild hypothermia for NSCs transplantation.2 Methods2.1 Culturing NSCs in vitro, study the differentiation potential of NSCs in vitro by immunofluorecence, finally analysing the electrophysiological properties of the neuron-like cells differentiated from NSCs in vitro with patch clamp. 2.2 mice were divided into three groups: SHAM group; HIE+PBS group(placebo group); HIE+ NSCs group(treatment group). Tracking the GFP labeled transplanted cells with fluorescence microscope,observing behavior of mice from each group with rotarod test,cylinder test and morris water maze evaluating safety of NSC transplantation with pathological examination 2.3 mice were divided into five groups: Normal control group; HIE+hypothermia group; HIE group; HIE+NSCs + hypothermia group; HIE+ NSCs group. Evaluate the efficacy of NSCs transplantation with mild hypothermia by Tunnel assay, survival test, differentiation test, and behavior test.3 Results3.1 NSCs from C57/BL6 fetal mice was adherent monocells, expressing pluripotent markers such as Nestin and Sox-2. The original single form of adherent cultured cells changed in differentiation medium. Differentiated cells expressing Neu N, GFAP, CNPase as neurons, astrocytes and oligodendrocytes glial markers respectively. The resting membrane potential and whole-cell capacitance values of undifferentiated NSCs was significantly different from differentiated neuron-like cells(p <0.05). A rapid activation of the Na+ channel which can be blocked by TTX and transient outward-fast loss activity of potassium current was recorded in differentiated neuron-like cells. 3.2 There is a statistically significant difference(P <O.05) between the placebo and treatment group on infarct volume. No significant difference between week 1 and week 4 of treatment group. Transplanted cells are visible with GFP marker, expressing Neu N, GFAP, and CNPase, which account for implanted cell ratio of 11 to 14%, about 63 to 77% and 2% to 6%, respectively. GFP labeled Cells reduced by about 60% at the 4th week compared with those at the 1st week. Resting membrane potential and membrane input impedance of Neuron-like cells did not show any significantly difference from those cultured in vitro.The Na+ action potentials and spontaneous excitatory postsynaptic current can be recorded. The rotarod Test: there were significant differences(P <0.05) between NSC treatment group mice and the PBS treatment group at 21 th day after HIE; Cylinder Test: there were significant differences(P <0.05) between NSC treatment group mice and the PBS treatment group at 21 th day after HIE; Morris water maze: The NSC group of mice have a shortened escape latency compared with the PBS group at 2month and 6month(P <0.05). 3.3 The mild hypothermia provide neuroprotection for transplanted cells, Number of positive TUNEL staining cells in NSCs + hypothermia group is less than HIE group and NSCs group, but similar to Hypothermia group. The protein expression level of NF-κ B and the active form of caspase3 in hypothermia group and NSCs + hypothermia group were significantly lower than HIE and NSCs groups. The infarct volume of NSCs+hypothermia group is significantly smaller than NSCs group(p <0.05), also smaller than the hypothermia group(p <0.05). Rotarod test: NSCs + hypothermia group improve significantly compare with NSCs and hypothermia groups on behavior(p <0.05). Cylinder test: the behavior of NSCs, Hypothermia and NSCs + Hypothermia groups improved immediately after treatment without significant difference. Morris water maze: there is a significant improvement in NSCs + Hypothermia group compare with NSCs and Hypothermia groups on behavior after treatment(p <0.05). The survival rate of transplanted cells in NSCs + hypothermia group was significantly higher than NSCs group(p <0.05).NSCs of NSCs + hypothermia group is easier to differentiate into Neu N-positive neuron-like cells than the ones of NSCs group(p <0.05).4 ConclusionNSCs can differentiate into neuron-like cells with electrophysiological properties of mature neurons in vitro. NSCs implanted into brain of HIE mice prefer to migrate into ischemic area,then differentiate into functional neuron-like cells which could recover HIE mice behavior as well as cognitive function. Mild hypothermia can provide neuroprotection for transplanted cells, the mechanism is probably through inhibition of inflammation and apoptosis.