| Objective: To observe the proliferation, migration and neuronal differentiation of the endogenous neural stem cells(NSCs) in the bilateral hippocampus of adult rat with unilateral fimbria/fornix transection, and the relationship between the expression of Brn-4 and the neuronal differentiation, and to investigate the mechanism of neural regeneration and reparation in the hippocampus after fimbria/fornix transection.Methods: Section One: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, which served as right fimbria/fornix transected 3rd, 5th, 7th, 14th, 21st and 28th day group, respectively. Each rat was injected intraperitoneally with 5-bromodeoxyuridine (BrdU) at 8 hours intervals, three times together, in the day before killed. The cryostat sections of the brain were prepared. Then, BrdU immunofluorescence detection was employed, and the number of BrdU immunoreactive cells in the bilateral dentate gyrus was counted using a fluorescent microscope. Paired t test analysis was used in statistic analysis of the number of BrdU positive cells with Stata7.0 software. Section Two: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, which served as right fimbria/fornix transected 3rd, 5th, 7th, 14th, 28th and 42nd day group, respectively. On the 2nd day after transection, BrdU was injected intraperitoneally once a day for 5 days. Then the rats were perfused and fixed. The brains were taken, and the cryostat sections of hippocampus were prepared. The BrdU immunofluorescence test was used to investigate the migration and distribution pattern of newborn cells. To detect the number of BrdU-labeled cells differentiating into neurons or astrocytes, BrdU/NF-200 and BrdU/GFAP double-labeled immunofluorescence test were employed. The Leica Qwin software was used for the image processing. Paired t test analysis was used for statistic analysis of the number of BrdU/NF-200 double-labeled cells and BrdU/GFAP double-labeled cells in bilateral hippocampus. Data between every two time points were analyzed using the ANOVA and SNK test. Section Three: Thirty-six SD rats were randomly divided into 6 groups, 6 rats in each group, which served as right fimbria/fornix transected 3rd, 7th, 14th, 21st, 28th and 35th day group, respectively. On the 2nd day after operation, BrdU was injected intraperitoneally once daily for 5 consecutive days. Then the cryostat sections of the brain were prepared. BrdU/Brn-4/β-TubulinⅢimmunofluorescence triple-labeled method, and the Leica Qwin image processing software were used to observe the number of BrdU/Brn-4 double-labeled cells, the number of BrdU/Brn-4/β-TubulinⅢtrible-labeled cells, and the Brn-4 fluorescent intensity in hilus and subgranular zone(SGZ) of dentate gyrus. Paired t test analysis, and one-way analysis of variance and comparison were used for above data in bilateral hippocampus and different time points respectively with stata 7.0 software. Results: Section One: Comparing with non-transection side, BrdU positive cells increased slightly on day 3 after operation, thereafter, increased markedly on day 5 and maximally on day 7 after fimbria/fornix transection in transection side. The number of BrdU positive cells in transection side decreased on day 14 after operation, but was still greater than non-transection side. The number of BrdU positive cells appeared to return to control levels on day 28. Section Two: The location of BrdU-labeled cells in the dentate gyrus changed as the time went on. At 3rd day after fimbria/fornix transection, most labeled cells were located in the hilus of dentate gyrus. At 5th day some labeled cells migrated out into the SGZ. At 7th day the majority cells were located in the SGZ or the edge of the granule cell layer(GCL). At 14th day after fimbria/fornix transection, many of the cells were still in the SGZ, but some were found throughout the GCL. At 28th day more newborn cells were found throughout the GCL. At 5 days after fimbria/fornix transection, some labeled cells expressed astrocyte marker—GFAP, but not expressed neuronal marker—NF-200. However, BrdU/NF-200 double-labeled cells could be observed at 7 days, increased gradually at 14 days, reached the peak at 28 days, and did not increase significantly at 42 days. A similar increase was observed as the time went on in the number of BrdU/GFAP double-labeled cells. Among the BrdU-labeled population in the dentate gyrus, about 7% cells were BrdU/NF-200 double-labeled neurons, and about 70% cells were BrdU/GFAP double-labeled astrocytes. Several BrdU/GFAP double-labeled cells were observed only, but no BrdU/NF-200 double-labeled neurons at any time point in non-transection side's dentate gyrus. Section Three: In hilus and SGZ of dentate gyrus in transection side, BrdU/Brn-4 double-labeled cells could be observed on day 3, in which the intensity of Brn-4 labeling was less. The number of double-labeled cells and the fluorescent intensity of Brn-4 increased significantly on day 7, and reached peak on day 14. Then the double-labeled cells decreased slowly and the fluorescent intensity of Brn-4 was lost to leve1 of non-transection side on day 21. BrdU/Brn-4/β-TubulinⅢtriple-labeled cells appeared on day 14 after transection, increased on day 21 and reached peak on day 28.Conclusion: The endogenous NSCs in hippocampus proliferated significantly after fimbria/fornix transection. The intensity of proliferation changed as the time went on, that is, it increased at first and then decreased to normal level, and reached peak on day 7. The proliferating endogenous NSCs migrated and lined as a strip along the SGZ, and some migrated into the GCL. Approximately 7% of the proliferating cells could differentiate into neurons during migration. However, most endogenous NSCs differentiated into astrocytes. The increasing expression of Brn-4 might be related to the endogenous NSCs in the dentate gyrus differentiating into neurons after fimbria/fornix transection.
|
PDF Full Text Request