The Dynamic Expression Of Brn-4and Lhx8in NGF-induced Hippocampal Neuroregeneration

Objective:To observe the dynamic expression of POU homeobox gene Brn-4and LIM homeobox gene Lhx8in NGF-induced hippocampal neuroregeneration, neuronal differentiation and cholinergic neuronal differentiation, and the effect of overexpression of Lhx8in cholinergic differentiation of hippocampal newborn neurons.Methods:Experiment1:1. SD rats were dividied into control group, transected group, NGF group, NGF combined with transected group randomly,18rats in each group; rats in transected group were processed by fimbria-fornix transection, rats in NGF group were processed by intracerebroventricular NGF injection, rats in NGF combined with transected group were processed by fimbria-fornix transection and intracerebroventricular NGF injection;2. Hippocampi of6rats in each group were isolated and total RNA was extracted, the first strand cDNA was synthesized, Real-time PCR was applied to detect the gene expression of Brn-4;3. Hippocampi of6rats in each group were isolated and the total protein was acquired, Western blot was applied to detect the protein expression of Brn-4;4.6rats in each group were perfused and fixed, then freezing sections were processed by immunohistochemistry of Brn-4/BrdU/NeuN and the nuclei were stained with Hoechst;5. Hippocampal neural stem cells were cultured in vitro and divideded into control group, normal group (contains5%normal hippocampal extracts), transected group (contains5%transected hippocampal extracts), NGF group (contains20ng/ml NGF), NGF+normal group (contains20ng/ml NGF and5%normal hippocampal extracts), NGF+transected group (contains20ng/ml NGF and5%transected hippocampal extracts), then cultured for7d;6. The total RNA was extracted and the first strand cDNA was synthesized, Real-time PCR was applied to detect the gene expression of Brn-4;7. The total protein in each group was acquired, Western blot was applied to detect the protein expression of Brn-4;8. The number of DCX/Brn-4double-labeled cells were detected by immunofluorescence;9. Image processing and statistical analysis.Experiment2:1. SD rats were dividied into control group, transected group, NGF group, NGF combined with transected group randomly,18rats in each group; rats in transected group were processed by fimbria-fornix transection, rats in NGF group were processed by intracerebroventricular NGF injection, rats in NGF combined with transected group were processed by fimbria-fornix transection and intracerebroventricular NGF injection;2. Hippocampi of6rats in each group were isolated and total RNA was extracted, the first strand cDNA was synthesized, Real-time PCR was applied to detect the gene expression of Lhx8;3. Hippocampi of6rats in each group were isolated the total protein was acquired, Western blot was applied to detect the protein expression of Lhx8;4.6rats were perfused and fixed, then freezing sections were processed by immunohistochemistry of ChAT/Lhx8and nuclei were stained with Hoechst;5. Hippocampal neural stem cells were cultured in vitro and divided into control group, normal group (contains5%normal hippocampal extracts), transected group (contains5%transected hippocampal extracts), NGF group (contains50ng/ml NGF), NGF+normal group (contains50ng/ml NGF and5%normal hippocampal extracts), NGF+transected group (contains50ng/ml NGF and5%transected hippocampal extracts), then cultured for14d;6. The total RNA in each group was extracted and the first strand cDNA was synthesized, Real-time PCR was applied to detect the gene expression of Lhx8;7. The total protein in each group was acquired, Western blot was applied to detect the protein expression of Lhx8;8. The number of ChAT/Lhx8double-labeled cells were detected by immunofluorescence;9. Image processing and statistical analysis.Experiment3:1. The newborn hippocampal neurons were acquired and divided into control group and transfected group respectively after treatment of negative control lentivirus and Lhx8overexpressed lentivires, then cultured for5d in vitro;2. The total RNA was extracted and the first strand cDNA was synthesized, Real-time PCR was applied to detect the gene expression of Lhx8;3. The total protein in each group was acquired, Western blot was applied to detect the protein expression of Lhx8;4. The number of EGFP/ChAT/VAChT tri-labeled cells were detected by immunofluorescence;5. Image processing and statistical analysis.Results:Experiment1:In vivo studies:1. The result of Real-time PCR in vivo indicated the gene expression of Brn-4in NGF combined with trnasection group was higher than other groups, the difference were statistically significant (P<0.05);2. Western blot indicated the protein expression of Brn-4in NGF group was higher than control group and trnasection group (P<0.05), the difference were statistically significant (P<0.05); Brn-4expression in NGF combined with transection group was higher than other groups, the difference were statistically significant (P<0.05or P<0.01);3. There was only a few Brn-4/BrdU/NeuN positive cells in hippocampal subgranular zone (SGZ) and hilus of control group, the number of Brn-4/BrdU/NeuN positive cells in transection group and NGF group were more than the control group, the difference were statistically significant between control group and transection group (P<0.05), also between control group and NGF group (P<0.01); the positive cells number in NGF combined with transection group was obviously more than the three others, the difference were statistically significant (P<0.05. P<0.01or P<0.001). In vitro studies:1. The result of Real-time PCR in vitro indicated that the Brn-4gene expression in transected group, NGF group, NGF+normal group, NGF+transected group was higher than the control group and normal group (P<0.05);2. The result of Western blot indicated that the protein expression of Brn-4in transected group, NGF group, NGF+normal group, NGF+transected group was higher than the control group and normal group (P<0.01);3. The Brn-4protein expression in NGF group, NGF+normal group, NGF+transected group was higher than the transectd group (P<0.05), no statistically significant existed in three groups; the number of DCX/Brn-4positive cells in transected group, NGF group, NGF+normal group, NGF+transected group was more than the control group and normal group (P <0.01or P<0.001); The number of DCX/Brn-4positive cells in NGF group、NGF+normal group、NGF+transected group was more than the transected group (P<0.05or P<0.01), no statistically significant existed in three groups.Experiment2:In vivo studies:1. The result of Real-time PCR indicated that the Lhx8gene expression in NGF group was higher than control and transected group (P<0.01) and the Lhx8gene expression in NGF combined with transected group was obviously higher than other groups (P<0.001);2. The result of Western blot indicated that the Lhx8protein expression in transected group, NGF group, NGF combined with transected group was obviously higher than the control group (P<0.05or P<0.01);3. The Lhx8protein expression in NGF group and NGF combined with transected group was higher than the transected group (P<0.05); there was no ChAT/Lhx8positive cells in hippocampal SGZ and hilus of control group, and just a few ChAT/Lhx8positive cells could be detected in transected group; there was some ChAT/Lhx8positive cells in NGF groups, the number was obviously more than the control group and trnasected group (P<0.001); there was a lot ChAT/Lhx8positive cells in NGF combined with transected group, and the cells number was obviously more than other groups (P<0.05, P<0.01or P<0.001). In vivo studies:1. The result of Real-time PCR indicated that the Lhx8gene expression in transected group, NGF group, NGF+normal group, NGF+transected group was obviously higher than control goup and normal group (P<0.05or P<0.01), the Lhx8gene expression in NGF+transected group was obviously higher than NGF group (P<0.05);2. The result of Western blot indicated that the Lhx8protein expression in transected group, NGF group, NGF+normal group, NGF+transected group was obviously higher than the control group and normal group (P<0.05or P<0.01); the Lhx8protein expression in NGF+transected group was higher than the transected group (P<0.05);6. The number of ChAT/Lhx8positive cells in normal group, transected group, NGF group, NGF+normal group, NGF+transected group was obviously higher than the control group (P<0.05, P<0.001or P<0.001); the number of ChAT/Lhx8double-labeled cells in NGF+transected group was more than the NGF group (P<0.05), but not NGF+normal group.Experiment3:1. The result of Real-time PCR in transfection experiments indicated that the Lhx8gene expression in transfected group was higher than the control group, the difference showed statistically significant (P<0.05);2. The result of Western blot indicated that the Lhx8protein expression in transfected group was higher than the control group, the difference showed statistically significant (P<0.05);3. Cells in control group and transfected group co-expressed EGFP with cholinergic neuronal markers ChAT and VAChT; the choinergic cells number in transfected group was obviously more than the control group, the results showed significant difference between two groups (P<0.001).Conclusion:1. Intracerebroventricular NGF injection can promote the gene and protein expression of Brn-4and Lhx8in hippocampal neuronal regeneration;2. The expression of Brn-4was increased in NGF-induced neuronal differentiation of hippocampal NSCs in vitro, and the Lhx8was also increased in NGF-induced cholinergic neuronal differentiation of hippocampal NSCs in vitro.3. Lentivirus transfection and overexpression of Lhx8gene in hippocampal newborn neurons can promote their cholinergic neuronal differentiation in vitro;4. The higher expression of Brn-4and Lhx8was associated with the hippocampal neuroregeneration and cholinergic neuroregeneration.