The Effect Of Lhx8on Hippocampal Cholinergic Neural Regeneration

Lhx8(LIM homeobox8)is associated with the development of embryo basalcholinergic neurons, however the function of Lhx8on cholinergic neural regeneration incentral nervous system and hippocampal dentate gyrus is still unclear, this research willstudy the function of Lhx8on hippocampal cholinergic neural regeneration.Objective:Radial glia cells (RGCs) derived from rat hippocampus was cultured in vitro, weinvestigated and detected the effects of normal and transected hippocampal extracts in theproliferation and differentiation of RGCs, and then we studied the function of Lhx8onhippocampal cholinergic neural regeneration.Methods:(1) The rats' hippocampal right fimbria-fornix was transected and the normal andtransected hippocampal extracts were prepared successfully;(2) The RGCs was cultured in vitro, normal and transected hippocampal extractswas added into the culture mediums, then investigated the function of hippocampalextracts in the proliferation and differentiation of RGCs;(3) Using hippocapal extracts and NGF to promote the cholinergic differentiationof RGCs, then investigate the expression and location of Lhx8in ChAT positive cells;(4) Cloning of Lhx8cDNA sequence, the expression recombinant plasmidpLenti6.3-Lhx8-IRES2-EGFP was constructed successfully and packaged by lentivirus, thecollected lentivirus Lenti6.3-Lhx8were transfected into RGCs, then investigated thecholinergic differentiation of RGCs and the dynamic gene and protein expression of Lhx8and ChAT;(5) The lentivirus Lenti6.3-Lhx8were injected into the normal and fimbria-fornixtransected hippocampal dentate gyrus, then investigated the expression and location of theChAT positive cholinergic neurons in normal and transected hippocampal dentate gyrusand the dynamic gene and protein expression of Lhx8and ChAT. Results:(1)RGCs presented typical double processes morphological feature, they expressedastroglial markers BLBP, GFAP, Vimentin, and these astroglial markers co-expressed withstem cells marker Nestin, and these RGCs could differentiate into neurons, astrocytes andoligodendrocytes;(2) The transected hippocampal extracts could promote the proliferation andneuronal differentiation of RGCs; the transected hippocampal extracts combined with NGFcould promote the RGCs differentiation into cholinergic neurons, and these cholinergicneurons could co-express with Lhx8;(3) Lenti6.3-Lhx8was transfected into RGCs, the number of differentiated MAP2positive neurons was not increased, but the proportion of ChAT positive cells to MAP2cells was increased, and the gene and protein expression of Lhx8and ChAT were increasedsignificantly, it indicated that transfection of Lenti6.3-Lhx8could promote the cholinergicneurons differentiation;(4) The Lenti6.3-Lhx8were injected into normal hippocampal dentate gyrus, thenumber of ChAT/Lhx8positive cholinergic neurons were increased; the hippocampalfimbria-fornix transection combined with Lenti6.3-Lhx8injection of dentate gyrus, thenumber of ChAT/Lhx8positive cholinergic neurons were increased significantly, the geneand protein expression of Lhx8and ChAT were also increased, it indicated that thehippocampal fimbria-fornix transection combined with Lenti6.3-Lhx8injection couldpromote the level of cholinergic neural regeneration in hippocampus.Conclusions:(1) The RGCs presented typical double processes morphological feature in vitro,these cells co-expressed astroglial markers with stem cells marker. The fimbria-fornixtransected hippocampal extracts could mimic the hippocampal neural regeneration niche invitro. On the proliferation stage the transected hippocampal extracts could promote theproliferation of RGCs, on the differentiation stage the transected hippocampal extracts alsocould promote the RGCs differentiation into neurons;(2) Lentivirus Lenti6.3-Lhx8could promote the hippocampal RGCs differentiationinto cholinergic neurons, but had no effect on the total number of neurons. As a familymember of LIM homeobox genes, Lhx8was only associated with the establishment of cholinergic phenotype, but Lhx8could not promote hippocampal RGCs differentiation intomore neurons in vitro;(3) Lentivirus Lenti6.3-Lhx8were injected into the hippocampal dentate gyrus, theChAT positive cholinergic neurons was increased, Lenti6.3-Lhx8were injected into thefimbria-fornix transected hippocampal dentate gyrus, the number of ChAT positivecholinergic neurons was significantly increased, it indicated that LIM homeobox geneLhx8played an important role in the hippocampal cholinergic neural regeneration afterfimbria-fornix transection.