The Repair Effect Of Netrin-1/DCC In The Subchronic Stage Of Spinal Cord Injury Research On Mechanism

Objective(s):The spinal cord(SCI)injury can be divided into four stages according to the course and prognosis time: in the acute,subacute,subchronic,and chronic stages of spinal cord injury,this study explores the repair function and mechanism of Netrin-1after combination with the receptor Deleted in colorectal cancer(DCC)gene in the subchronic stage of spinal cord injury(the 28 th day after injury).The regulatory relationship between this neural repair mechanism and the signaling pathway NgR1-RhoA-ROCK was also attempted to be verified,opening a new idea for clinicians to treat SCI with drugs,hoping to achieve the ideal therapeutic effect and improve the later life of patients.Methods:A total of 210 SD rats with body weight ranging from 200 to 250 g were selected.They were randomly divided into 14 groups: Sham operation group(Sham),spinal cord transection group(SCT),Netrin-1 expression increased group(Netrin-1-ORF),Netrin-1 expression increased control group(Netrin-1-ORF-NC),Netrin-1 expression silence group(Netrin-1-si),Netrin-1 expression silence control group(Netrin-1-si-NC),DCC expression increased group(DCC-ORF),DCC expression increased control group(DCC-ORF-NC),DCC expression silence group(DCC-si),DCC expression silence control group(DCC-si-NC),Netrin-1 and DCC co-expression increased group(Netrin-1/DCC-ORF),Netrin-1 and DCC co-expression increased control group(Netrin-1/ DCC-ORF-NC),Netrin-1 and DCC co-expression silence group(Netrin-1/ DCC-si),Netrin-1 and DCC co-expression silence control group(Netrin-1/DCC-si-NC),15 rats in each group.Specific experiments are as follows:1.The animal injury model of complete spinal cord transection in rats was constructed.According to the conditions of each group,sham operation,simple complete spinal cord transection and three points above and below the severed end were injected with lentivirus as required.After surgery,determine whether the animal injury model was established successfully,and then select 6 time points(day 1,day 3,day 7,day 14,day 21,day 28)and adopt BBB rating scale(Basso Beattie&Bresnahan locomotor rating scale,BBB scale)to evaluate the recovery of hind limb joint movement,somatic coordination and tail placement in each group.2.On day 28 after the complete spinal cord transection,the rats died from overanesthesia,and the spinal cord was removed and stored in-80℃ or 4%Paraformaldehyde(PFA)solution to observe the continuity of the broken point and measure the length of the glial scar.3.Hematoxylin-eosin staining(HE)was used to observe the pathological changes and structure restoration.The morphological structure and survival of neurons around the broken end of the spinal cord were observed with Nishi in tar-purple stained nerve tissue.The expressions of nucleoproteins(Neu N),synaptophysins(SYP),nuclear markers(DAPI)and Growth associated protein-43(GAP-43)in the severed cranial and caudal neurons were detected by immunofluorescence(IF)method.4.Reverse Transcription Quantitative Real-time Polymerase Chain Reaction,(RT-qPCR)was used to detect the relative m RNA expression levels of Netrin-1,DCC and NgR1-RhoA-ROCK pathway related factors Nogo receptor 1(NgR1),RhoA,ROCK1,ROCK2 in spinal cord neurons.5.Western blot(WB)was used to detect the protein expressions of Netrin-1 and DCC in the neurons at the severed end of the spinal cord and the four factorsnamely NgR1,RhoA,ROCK1 and ROCK2 in the signaling pathway NGR1-Rhoa-Rock that inhibit the regeneration of neurons’ axons.Results:1.The BBB behavioral score of rats in all groups except Sham group was 0 one day after total spinal cord transection,indicating that the rat model of total spinal cord transection injury was successfully constructed.2.BBB behavioral scores showed that with postoperative recovery,hind limb motor ability of rats in all groups was improved,and behavioral scores of Netrin-1overexpression group,DCC overexpression group and Netrin-1 and DCC co-overexpression group were significantly higher than those of control group.The behavioral scores of Netrin-1 low expression group,DCC low expression group and Netrin-1 and DCC low expression group were significantly lower than those of the control group.3.The whole spinal cord was removed and the severed ends were observed.It was found that the cranial and caudal sides of the severed ends of each group grew back together,and the continuity of the severed ends of the Netrin-1 overexpression group and the DCC overexpression group were better and no obvious tissue was observed absent and relatively short length of glial scar;The results of Netrin-1 low expression group and DCC low expression group were opposite.4.The HE staining results showed that compared with the control group,the Netrin-1 overexpression group,the DCC overexpression group and the Netrin-1 and DCC co-overexpression group had regular cell morphology,orderly arrangement,and fewer nerve tissue defects;The conclusion of Netrin-1 low expression group,DCC low expression group and Netrin-1 and DCC co-low expression group is contrary.5.The observation of Nishi staining showed that more neurons in the Netrin-1overexpression group,the DCC overexpression group and the Netrin-1 and DCC co-overexpression group were positive for Nishi,and more neurons survived around the broken end of the spinal cord.In the low expression group of Netrin-1,low expression group of DCC,and low expression group of Netrin-1 and DCC,only fewer neurons were nistite-positive,and fewer neurons survived around the broken end of the spinal cord.6.By immunofluorescence detection of Neu N,SYP and GAP43 around the broken end,it was found that the Neu N positive rate around the broken end became higher in the Netrin-1 overexpression group and the DCC overexpression group.The number of GAP43 positive cells increased,and the length of new axons increased significantly.SYP positive cells increased and fluorescence intensity increased.The results of Netrin-1 low expression group and DCC low expression group were opposite.7.Through the analysis of RT-qPCR results,it was found that the expression of Netrin-1 and DCC in neurons was successfully increased and/or decreased in accordance with the grouping requirements.The m RNA relative expression levels of NgR1,RhoA,ROCK1 and ROCK2 decreased in Netrin-1 and DCC co-overexpression group.The m RNA relative expression levels of NgR1,RhoA,ROCK1 and ROCK2 were increased in the low expression group of Netrin-1 and DCC.8.The protein expression levels of Netrin-1,DCC,NgR1,RhoA,ROCK1 and ROCK2 in the co-overexpression and co-underexpression groups of Netrin-1 and DCC were detected by WB,and the results showed the same trend as the results of RT-qPCR.Conclusion(s):In the subchronic stage of complete spinal cord transection of rats(day 28),the expression level of Netrin-1 and DCC in the Netrin-1 overexpression group and the DCC overexpression group was still increased,which could promote the recovery of motor function of the hind limbs of rats,make more neurons survive and promote the regeneration of axons after injury.When fully combined with the increased co-expression of Netrin-1 and its receptor DCC,it can better promote axon regeneration after nerve cell injury,increase the number of nerve cells that survive,improve cell morphology,promote the recovery of motor function of posterior limb in rats with spinal cord injury,and facilitate spinal cord repair,and this repair mechanism is related to the inhibition of NgR1-RhoA-ROCK signaling pathway.Therefore,in clinical practice,Netrin-1 and its receptor DCC can be used to promote spinal cord repair with innovative ideas for patients with spinal cord injury,so as to reduce secondary injuries and complications and improve patients’ self-care ability.