Induced Activation And Differentiate Into Cholinergic Neurons Of Hippocampal Radial Glial Cells In Vitro

Part I THE INDUCED ACTIVATION AND THE NEURAL STEM CELLS'CHARACTERISTICS OF HIPPOCAMPAL RADIAL GLIAL CELLS IN VITROObjective To explore the changes of hippocampal radial glial cells (RGCs) in the morphology, proliferation, embryogenicity and the neural stem cells (NSCs) identity towards to neurons and glial cells after the denervated fimbria-fornix transected hippocampal extracts induced.Methods On the postnatal 1 day, the hippocampi of SD rats were acquired. RGCs were separated and purified by differential velocity adherent method and shakingbed oscillation technique. The purified RGCs were plated into 24-well plates and then divided into transection group and normal group. The DMEM/F12 medium containing 5% (vol/vol) fimbria-fornix transected hippocampal extracts was added into the transection group, while the normal group added the the DMEM/F12 medium containing 5% (vol/vol) normal hippocampal extracts. At the same time, BrdU were added into both groups to label the proliferated cells, the cells of both groups were detected by BLBP immuno-fluorescence and marked by Hoechst on the 1st,3rd,7th and 14th d respectively. Double-labeling immunofluorescence of BLBP/BrdU,BLBP/nestin,BLBP/MAP-2,BLBP/GFAP,BLBP/CNP were used to investigate the characteristics of proliferation, embryogenicity and the cell differentiation into neurons, astrocytes and oligodendrocytes. The percentage of BrdU positive cells over the BLBP positive cells, the perimeter and area of BLBP positive cells and the percentage of BLBP/nestin, BLBP/MAP-2, BLBP/GFAP, BLBP/CNP Double-labeling cells over the BLBP positive cells in two groups were deteced. Comparing analysis between both groups were analyzed by Statal0.0 statistical software.Results Immunofluorescence assay of BLBP showed that we acquired nearly 100% RGCs by our purified method. BLBP expressed in the cytoplasm, nuclei and processes. The percentage of BrdU positive cells in the transection group was higher than the normal group (the transection group:56.86±8.52%; the normal group:31.11±4.28%; P< 0.01). On the 1 day after cell seeding, the cell bodies were small in both groups, the processes of both groups were also short and thin; On the 3rd day, compared to the normal group, the cell bodies of the transection group were slightly larger and the processes were also longer and thicker; On the 7th day, the cell bodies of RGCs in the transection group were significantly larger than the normal group, and the cell processes became much more longer and thicker, and interlaced into networks; on the 14th day, the cell bodies of both groups decreased slightly, and the processes also became shorter and thinner slightly. The statistical analysis of two groups'BLBP positive cells in the perimeter and area showed that the transection group is significant higher than the normal group (P<0.01) except the 1st day (P> 0.05). The percentage of nestin positive cells over the BLBP positive cells in the transection group was also higher than the normal group (the transection group:57.92±17.93%; the normal group: 23.26±9.85%; P< 0.01). And we observed more RGCs differentiated into MAP-2 positive neurons in the transection group (the transection group:46.13±14.92%; the normal group:29.13±10.07%; P< 0.01). Although there was no significant difference in the cell numbers between two groups when they differentiated into GFAP positive astrocytes and CNP positive oligodendrocyte, the cell bodies in the transection group were larger and the processes were also longer and thicker than the normal group.Conclusions The results indicate that the denervated fimbria-fornix transected hippocampal extracts can significantly promote the proliferation of RGCs, enlarge the cell bodies, thicken and elongate the processes of BLBP positive RGCs which presenting the state of "activation" and containing the embryogenicity, and also can make RGCs present the identity of neural stem cells and differentiate into neurons, astrocytes and oligodendrocytes.PartⅡTHE DIFFERENTION OF HIPPOCAMPAL RADIAL GLIAL CELLS INTO CHOLINERGIC NEURONS IN VITRO BY DENERVATED HIPPOCAMPAL EXTRACTSObjective Fimbria-fornix transected hippocampal extracts were added into the cell culture medium to simulate the hippocampal internal environment in vitro, and to observe the situation of the RGCs differentiation into cholinergic neurons.Methods Purified RGCs were divided into transection group and normal group. The DMEM/F12 medium supplemented with 5%(vol/vol) fimbria-fornix transected hippocampal extracts was added into the transection group, while the normal group the DMEM/F12 medium contains 5%(vol/vol) normal hippocampal extracts. BLBP/ChAT double-labeling immunofluorescence, Real-time PCR, and Western blot were used to observe the differentiation into cholinergic neurons, the levels of ChAT mRNA and protien in RGCs after 7 day inoculation.Results Immunofluorescence assay of ChAT showed that the percentage of ChAT positive cholinergic neurons over the BLBP positive cells in the transection group(41.62±9.97%) was higher than the normal group(16.08±7.31%)(P< 0.01), and the cholinergic neuron bodies of transection group were larger and the processes were also longer and thicker than the normal group. The level of ChAT mRNA in the transection group was 5 times higher than the normal group (P< 0.01). The relative amount of ChAT protein in the transection group(0.1141±0.0380)was significant higher than that in the normal group(0.0423±0.0106)(P< 0.05), although both groups had low expression.Conclusions The results indicate that the denervated hippocampal extracts after fimbria-fornix transection can significantly promote BLBP positive RGCs differentiate into cholinergic neurons.