The Effect Of Lhx8 On Hippocampal Cholinergic Neural Regeneration

Lhx8, one member of the LIM homeobox gene family, is associated with the development of embryonic basal cholinergic neurons, while the effect of Lhx8 on hippocampal cholinergic neural regeneration is still unclear. The following four parts are included in this study. The first one is the change of Lhx8 protein expression and co-expression of Lhx8 and Ch AT in hippocampal neurons after fimbria-fornix transection. Then the differentiation of hippocampal neural stem cells into cholinergic neurons was detected while Lentivirus Lenti6.3-Lhx8 or Lhx8 RNAi was used to upregulate or downregulate the expression of Lhx8 respectively. And then the signaling path was studied while NGF upregulated Lhx8 to promote hippocampal neural stem cells to differentiate into cholinergic neurons.Objective:Dynamic change of Lhx8 protein expression and the co-expression of Lhx8 and Ch AT in hippocampus after fimbria-fornix transection were observed. And then we investigated the influence of upregulation and downregulation of Lhx8 on the differentiation of hippocampal neural stem cells to make clear that if there was connection between Lhx8 and hippocampal cholinergic neural regeneration. Then to study the possible signaling path participating in the process of NGF upregulating Lhx8 to promote hippocampal neural stem cells differentiated into cholinergic neurons.Methods:(1) Lhx8 protein expression was detected after the hippocampal fimbria-fornix of rats was transected for 1d, 3d, 7d, 14 d, 21 d and 28 d. And the co-expression of Lhx8 and Ch AT in hippocampus was detected on 7d after fimbria-fornix transection;(2) Hippocampal NSCs was cultured in vitro, and transected hippocampal extracts and lentivirus Lenti6.3-Lhx8 were added into the culture mediums. The differentiation of hippocampal NSCs into cholinergic neurons and the expression of Lhx8 and Ch AT gene and protein were detected;(3) The lentivirus Lenti6.3-Lhx8 was injected into the normal and fimbria-fornix transected hippocampal dentate gyrus, then the expression and location of the Ch AT positive cholinergic neurons were investigated together with the gene and protein expression of Lhx8 and Ch AT in hippocampal dentate gyrus;(4) Hippocampal NSCs was cultured with transected hippocampal extracts and NGF. Then Lhx8 gene and protein were detected. Lhx8 RNAi was added into the culture mediums to investigated the differentiation of hippocampal NSCs into cholinergic neurons and the expression of Lhx8 and Ch AT m RNA and protein;(5) Using lateral ventricle infusion of NGF to promote the hippocampal cholinergic activity, Lhx8 RNAi was injected into the normal and fimbria-fornix transected hippocampal dentate gyrus, then the expression and location of the Ch AT positive cholinergic newborn neurons were investigated together with the gene and protein expression of Lhx8 and Ch AT in hippocampal dentate gyrus;(6) We used NGF to promote the cholinergic differentiation of hippocampal NSCs in vitro, then added inhibitors of different signaling paths into the culture mediums. Then relevant protein phosphorylation level, gene and protein of Lhx8 were detected and the differentiation of hippocampal NSCs into cholinergic neurons was observed after inhibitions being used.Results:(1) The protein expression of Lhx8 was increased gradually and it reached the peak on 7d after transection. There were Lhx8 positive cells in the hippocampal subgranular zone and hilus on 7d. The number and average optical value of Lhx8 positive cells in the transected side were obviously higher than those in the normal side and there were also Lhx8/Ch AT positive cells;(2) Lenti6.3-Lhx8 was transfected into hippocampal NSCs, the number of differentiated MAP2 positive neurons was not increased, but the proportion of Ch AT positive cells to MAP2 cells was increased. The gene and protein expression of Lhx8 and Ch AT were increased significantly.(3) The number of Ch AT/Lhx8 positive cholinergic neurons was increased after Lenti6.3-Lhx8 being injected into normal hippocampal dentate gyrus. After the hippocampal fimbria-fornix transection and Lenti6.3-Lhx8 being injected into dentate gyrus, the number of Ch AT/Lhx8 positive cholinergic neurons was increased significantly, and the gene and protein expression of Lhx8 and Ch AT were also increased. The results showed that the hippocampal fimbria-fornix transection combined with upregulation of Lhx8 could raise the level of cholinergic neural regeneration in hippocampus.(4) Lhx8 RNAi was transfected into hippocampal NSCs, the proportion of Ch AT positive cells to MAP2 cells was decreased. And the gene and protein expression of Lhx8 and Ch AT were decreased significantly. The results indicated that downregulation of Lhx8 could inhibit the cholinergic neurons differentiation of cultured hippocampal NSCs;(5) After the hippocampal fimbria-fornix transection and lateral ventricle infusion of NGF, Lhx8 RNAi was injected into hippocampal dentate gyrus. The number of Ch AT/Lhx8 positive cholinergic neurons was decreased and the gene and protein expression of Lhx8 and Ch AT were decreased significantly either. The results indicated that downregulation of Lhx8 could inhibit the cholinergic neural regeneration in hippocampus;(6) The phosphorylation levels of AKT,IKB,NFκB,p38 MAPK were increased in cultured hippocampal NSCs with NGF added. The gene and protein expression of Lhx8 were decreased when corresponding inhibitors of signaling paths were added and it was the most significant in NGF+BAY117082 group, but the inhibitors had no synergistic effect. The differentiation of cultured hippocampal NSCs into cholinergic neurons could be influenced significantly when NFκB signaling path was blocked.Conclusions:(1) The protein expression of Lhx8 were increased and reached the peak on 7d after fimbria-fornix being transected and there were Lhx8 positive cells in the hippocampal subgranular zone and hilus. The results showed that high expression of Lhx8 may be relevant to the hippocampal cholinergic neural regeneration;(2) Overexpression of Lhx8 in cultured hippocampal NSCs could increase the proportion of Ch AT positive cells to MAP2 cells while the number of differentiated MAP2 positive neurons was not effected. Upregulation of Lhx8 in hippocampus could promote the cholinergic neural regeneration after fimbria-fornix being transected;(3) Differentiation of cultured hippocampal NSCs into cholinergic neurons could be influenced by Lhx8 RNAi used. NGF was used to raise the cholinergic activity, and the Lhx8 RNAi could influence hippocampal cholinergic neural regeneration after fimbria-fornix transected;(4) NGF might regulate Lhx8 expression through NFκB signaling path and then promote hippocampal NSCs to differentiate into cholinergic neurons.