Effect Of GABA_B Receptor Antagonist On The Proliferation Of Hippocampal Neural Stem Cells In Acute Cerebral Ischemia Mice

Objective:To explore the effects of GABA_B receptor antagonist on adult hippocampal neurogenesis and cognitive function in actue cerebral ischemia(CI)mice.Methods:The bilateral common carotid arteries occlusion(BCCAO)was used for 20 minutes to establish a mouse cerebral ischemia model.C57BL/6J mice aged 6-8 weeks were randomly divided into 3 groups(21mice in each group):sham group,CI+vehicle group(vehicle group),CI+GABA_B receptor antagonist CGP52432 group(CGP group).GABA_B receptor antagonist CGP52432(10mg/kg)or an equivalent amount of vehicle solution were injected intraperitoneally 24 hours after BCCAO in mice.HE staining was performed on day 7 after CI to observe the pathological changes in the hippocampal CA1 region of mice.The Morris water maze test was performed to test spatial learning and memory at day28 after CI.Immunofluorescence staining of BrdU and DCX was used on day 14 after CI to detect neurogenesis in the DG region of hippocampal.Immunofluorescence staining BrdU/NeuN was used to observe the differentiation of newborn cells at day 28 after CI.Primary neural stem cells(NSCs)were cultured in vitro,and the purity of the cultured cells was identified by immunofluorescence staining,and the cell viability of NSCs cultured in vitro was detected by CCK-8 assay.The proliferation of NSCs cultured in vitro was detected by immunofluorescence staining.The expression of cAMP/CREB signaling pathway were detected by ELISA assay and Western blot.Results:The pyramidal neurons in the hippocampal CA1 region were damaged and the number of surviving neurons were significantly reduced in mice following CI(P<0.01).The spatial learning and memory abilities were significantly decreased in mice after CI(P<0.01).The administration of CGP52432 24 hours after BCCAO did not aggravate the ischemic neuronal damage on day 7 in CI mice,but significantly improved the cognitive dysfunction on day 28 caused by CI(P<0.05).Compared with the sham group,the number of BrdU~+and DCX~+cells in the DG region of hippocampus of the vehicle group increased significantly(P<0.01);compared with the vehicle group,the number of BrdU~+and DCX~+cells in the DG region of hippocampus of CGP group increased significantly(P<0.01).Compared with the sham group,the number of BrdU~+/NeuN~+cells in the vehicle group were significantly increased(P<0.01);compared with the vehicle group,the number of BrdU~+/NeuN~+cells in the CGP group were significantly increased(P<0.01).Immunofluorescence staining results showed that cell spheres and single cells cultured in vitro expressed Nestin,a maker of NSCs.CGP dose-dependently enhanced cell viability of NSCs(P<0.01).Immunofluorescence staining showed that CGP promoted the proliferation of NSCs(P<0.01).In addition,the expression of cAMP,PKA,and pCREB was increased in cultured NSCs treated with CGP.Conclusion:Inhibition of GABA_B receptor can promote hippocampal neurogenesis in acute CI mice and proliferation of NSCs cultured in vitro,and improve the spatial learning and memory abilities of mice with acute CI.