GABA_B Receptor Promotes The Recovery Of Spinal Cord Injury Through The Activation Of Pkd2l1~+ CSF-cNs Neural Stem Cells Properties

Objective:To investigate the potential of promoting spinal cord injury repair in mice by modulating GABA_Breceptor activation and enhancing the neural stem cell properties of Pkd2l1~+cerebrospinal fluid-contacting neurons.Methods:This study comprises both animal and cellular experiments.In the animal experiment section,C57BL/6 mice aged 6-8 weeks were subjected to spinal cord clamping at the T10 level.Western blotting,real-time quantitative polymerase chain reaction(qRT-PCR),and frozen section immunofluorescence were employed to detect GABA_Breceptor expression following spinal cord injury.Immunofluorescence was used to analyze the co-expression of Pkd2l1~+cerebrospinal fluid-contacting neurons and GABA_Breceptors in the mouse spinal cord.The mice were then randomized into five groups:(1)Sham Group:T9-T11 lamina was opened only,without spinal cord clamping;(2)SCI Group:T9-T11 lamina was opened,and a vascular clamp was applied to the T10 spinal cord segment for 10 seconds;(3)SCI+PBS Group:1X PBS solution was intraperitoneally injected daily for 7 or 35days,starting 24 hours after injury;(4)SCI+Baclofen Group:Baclofen solution was intraperitoneally injected daily for 7 or 35 days,starting 24 hours post-injury;(5)SCI+CGP52432 Group:CGP52432 solution was intraperitoneally injected daily for7 or 35 days,starting 24 hours post-injury;with n=6 mice/group.The proliferation of Pkd2l1~+cerebrospinal fluid-contacting neurons with upregulated GABA_Breceptors following spinal cord injury was detected using 5-ethynyl-2’-deoxyuridine(Ed U)labeling and immunofluorescence.The expression of Pkd2l1~+cerebrospinal fluid-contacting neurons in each group was observed via immunofluorescence.Hematoxylin-eosin(HE)and Nissl(tar violet)staining were used to examine the morphology and neuronal injury post-injury.Luxol Fast Blue(LFB)staining and NF-200 immunofluorescence staining were utilized to observe the loss of myelin sheath and axons after spinal cord injury.Lastly,the Basso Mouse Scale(BMS)score assessed the recovery of motor function in injured mice.In the cellular experiment section,Pkd2l1~+ cerebrospinal fluid-contacting neurons were extracted and cultured from C57BL/6 pups within 24 hours of birth.Immunofluorescence was employed to detect the co-expression of Pkd2l1~+cerebrospinal fluid-contacting neurons and GABA_Breceptors in vitro.The third-generation Pkd2l1~+cerebrospinal fluid-contacting neurons were randomly divided into four groups:(1)Control Group:fresh serum-free neural culture medium;(2)PBS Group:fresh serum-free neural culture medium+PBS solution;(3)Baclofen Group:fresh serum-free neural culture medium+Baclofen solution;(4)CGP52432Group:fresh serum-free neural culture medium+CGP52432 solution.The proliferation and apoptosis of Pkd2l1~+cells were observed using Ed U staining and terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL).Results:Our data demonstrate that the upregulation of GABA_B receptors activates the neural stem cell potential of Pkd2l1~+cerebrospinal fluid-contacting neurons,thus promoting the repair of spinal cord injury in mice.Initially,we observed that the expression levels of GABA_Breceptors in the spinal cord’s central canal and surrounding tissues exhibited an increasing trend followed by a decrease after spinal cord injury.On the 7th day post-injury,the expression levels significantly diminished as assessed by protein immunoblotting,quantitative reverse transcription-polymerase chain reaction,and immunofluorescence in frozen sections compared to sham-operated mice.Concurrently,we discovered that Pkd2l1~+cerebrospinal fluid-contacting neurons expressed GABA_Breceptors both in vitro and in vivo.Moreover,compared to the control group,Pkd2l1~+cerebrospinal fluid-contacting neurons treated with Baclofen were activated,exhibiting stem cell potential,robust proliferation capacity in vitro and in vivo,and partial resistance to cell apoptosis.We also confirmed that,compared to the SCI group and SCI+PBS group,the SCI+Baclofen group displayed better morphological recovery of the central spinal canal and surrounding tissues,reduced neuronal damage,and decreased axonal myelin sheath and axon loss.Lastly,we found that the BMS score in the SCI+Baclofen group was higher than in the SCI and SCI+PBS groups at the same time points,but lower than in the Sham group.Conclusions:GABA_B receptors were expressed in Pkd2l1~+CSF-cNs,with the lowest expression on day 7 post-SCI.The proliferative capacity of Pkd2l1~+CSF-c Ns was activated by GABABR.Activation of GABABR improved locomotor recovery in SCI mice.