Brn-4 MRNA Expression In Rat Hippocampus And The Effects Of NGF On The Proliferation And Differentiation Of Endogenous NSCs In The Hippocampus Into Neurons After Fimbria Fornix Transection

PartⅠUsing RT-PCR/Southern Blot to Detect the Change of Brn-4 mRNA Expression in Rat Hippocampus after Fimbria Fornix TransectionObjective: Using RT-PCR/Southern blot to observe the diference of Brn-4 mRNA expression between the fimbria fornix transected rats'hippocampus and the normal ones, to investigate the molecular mechanisms of nerve regeneration and repaire in hippocampus after fimbria fornix transection. Methods: Forty-two SD rats were randomly divided into 7 groups, 6 rats in each group. One group served as normal control and the others served as the 1st, 3rd, 7th, 14th, 21st and 28th day group after fimbria fornix transection, respectively. Then hippocampi were isolated and total RNA was extracted. RT-PCR/Southern blot was used to detect the change of Brn-4 mRNA expression in hippocampus after fimbria fornix transection. The relative expression level of Brn-4 mRNA was indicated by the ratio of the optical density value of Brn-4 hybridization bands to that of reference gene GAPDH. Single factor analysis of variance and multiple comparison was performed using Stata7.0 statistical software. Results: The relative expression level of Brn-4 mRNA in normal group was 0.254±0.018, and in the 1st, 3rd, 7th, 14th, 21st and 28th day group after transection was 0.277±0.019, 0.516±0.014, 1.497±0.081, 2.357±0.133, 1.140±0.054 and 0.264±0.020, respectively. Statistical analysis showed that there were no statistically significant differences between the normal control group and the 1st, 28th day group after transection(P>0.05), there were statistically significant differences between the normal control group and the 3rd, 7th, 14th, 21st day group after transection(P<0.01); there were statistically significant difference between each two groups (P<0.01 or 0.05) except between 1d group and 28d group(P>0.05), compared different time groups after transection. Conclusion: The expression level of Brn-4 mRNA started to increase on the 3rd days after fimbria fornix transection, the peak appeared on the 14th days, then decreased slowly to pre-transection level on the 28th days. Combined with our groups's previous research data, the expression of Brn-4 mRNA increased after fimbria fornix transection, perhaps, might be related to the neural stem cells diferentiating into neurons in hippocampus.PartⅡThe Effects of NGF on the Proliferation and Differentiation of Endogenous NSCs in the Hippocampus into Neurons after Fimbria Fornix TransectionObjective: To study the effects of fimbria fornix transection and intraventricular injection of nerve growth factor (NGF) on the proliferation and differentiation of endogenous neural stem cells (NSCs) in the hippocampus into neurons. Methods: Twelve SD rats were divided into the treatment group and control group randomly, six rats in each group. The right fimbria fornix of rats were transected. NGF and artificial cerebrospinal fluid was injected into the lateral ventricle of rats in the treatment group and control group respectively immediately and on the 2nd and 4th day after transection. Bromodeoxyuridin (BrdU) was injected intraperitoneally twice per day from 3 to 7 days after operation. On the 28th day after operation, the brains were perfused for fixation and frozen section. BrdU/NF-200 double-label immunofluorescence was used to detect the proliferatiation and differentiatiation of NSCs into neurons. The pictures of BrdU positive cells and NF-200 positive cells in the same visual field in dentate gyrus were taken, respectively. Then the Spot 4.6 image processing software was used to make pictures overlap, and the numbers of BrdU positive cells and BrdU/NF-200 double labeled cells in dentate gyrus, subgranular zone and granular layer were counted, respectively. Using Stata7.0 statistical software, the data were analyzed with the 2-factors variance analysis(2×2 factorial design), and the q test (SNK) was used for comparison among groups. Results: In dentate gyrus, the number of BrdU positive cells in the transection side were much more than those of normal side both in the treatment group and the control group, and the BrdU positive cells in treatment group were more than those of control group both in the transection side and normal side; The number of BrdU/NF-200 double labeled neurons was the most in the transection side of the treatment group, less in normal side of treatment group and transection side of the control group, but absent in the normal side of the control group. Conclusion: Fimbria fornix transection could cause endogenous neural stem cells in the hippocampus proliferate and differentiate into neurons, and intraventricular injection of NGF could facilitate its proliferation and differentiation of endogenous NSCs in the hippocampus into neurons.