The Expression And Regulation Of Draxin In The Development Of Mice Spinal Cord And The Repair After Spinal Cord Injury

Objective: Draxin(dorsal repulsive axon guidance protein)is a novel inhibitory repulsive axon guidance protein that plays an important regulatory role in the development of the embryonic central nervous system.However,it is unclear whether the expression of Draxin in mice embryonic spinal cord and whether it plays a regulatory role in spinal cord injury in adult mice.In this study,C57BL/6 fetal mice,C57BL/6 mice and Draxin knockout mice were used as subjects.Double immunofluorescence staining,Western Blot and In vitro culture were used to observe the expression time and expression site of Draxin during embryonic development,the type of Draxin-expressing cells and the regulatory factors closely related to the dynamic changes of Draxin after adult mice spinal cord injury,to explore the role and mechanism of Draxin in spinal cord injury.Methods:1.Mice embryonic spinal cord at E9.5,E10.5,E11.5,E12.5,E13.5,E14.5,E15.5,E16.5 developmental stages,were used to make continuous coronal sections.The Immunohistochemical staining was used to observe the Draxin expression and its dynamic changes in the spinal cord at different embryonic stages.2.In vitro culture of embryonic spinal cord at E9.5 development stage and double immunofluorescence staining were used to check the type of Draxin expressing cells.Anti-Draxin antibody was used for double immunofluorescence staining respectively with Tuj-1,Nestin and GFAP.3.Preparation of Draxin knockout mice,detection of knockout efficiency of Draxin knockout mice by PCR and immunohistochemical staining.4.A half-clamp model of spinal cord thoracic 10 segments was performed in 2 months old wild-type C57BL/6 mice.The spinal cord tissues of the injured sites were taken at 1-10 days,14,21,28,42,49,56 days after spinal cord injury.The expression changes of Draxin,LRP6,Wnt5 a and RhoA were detected by Western Blot.5.Two-month-old wild-type C57BL/6 mice were fixed at 4 days after spinal cord half-clamp surgery.Continuous coronal sections were taken and immunostained by Draxin respectively with BLBP,GFAP and Nestin.Further explore the cell types that secrete Draxin after spinal cord injury.6.Two-month-old wild-type C57BL/6 mice were fixed at 4 days after spinal cord half-clamp surgery.Continuous coronal sections were taken and immunostained by Draxin respectively with LRP6,Fzd3,Wnt5 a and RhoA,to explore the relevant factors of Draxin's role after spinal cord injury.7.Spinal cord tissue at 4 days after spinal cord half-clamp surgery were taken out from sham operation group,wild type mice(WT)operation group,knockout mice(KO)operation group of two-month-old C57BL/6 mice.Western Blot was used to detect the correlation between the expression changes of LRP6,Fzd3,Wnt5 a and RhoA and the expression of Draxin.Results:1.Immunohistochemical staining showed that the expression of Draxin showed obvious dynamic changes in the inner and outer tissues of embryonic spinal cord at different developmental stages.2.The results of in vitro culture combined with immunohistochemical double staining showed that some Tuj-1 positive immature neurons,Nestin positive neural stem cells and GFAP positive astrocytes expressed Draxin.3.The PCR results showed that the PCR product of the wild type mice was a 634 bp fragment,and the PCR product of the Draxin knockout type mice was a 336 bp fragment.The results of immunohistochemical staining showed that the expression of Draxin in the injured spinal cord tissue of wild-type mice was obvious,but there was no obvious positive signal in the injured spinal cord tissue of Draxin knockout mice.4.At different time points after spinal cord injury in wild-type mice,Western Blot showed that the dynamic expression of Draxin protein increased,and LRP6,Wnt5 a and RhoA also showed similar expression changes after spinal cord injury.5.Four days after spinal cord injury in wild-type mice,immunohistochemical staining showed that some BLBP-positive,GFAPpositive glial cells and some Nestin-positive neural stem cells expressed Draxin.6.Four days after spinal cord injury in wild-type mice,immunohistochemical double staining showed that some LRP6-positive cells,some Fzd3-positive cells,some Wnt5a-positive cells and some RhoA-positive cells all expressed Draxin.7.Four days after operation in different operation groups,Western Blot showed that compared with WT sham operation group,Draxin expression was significantly increased in WT operation group,and no obvious Draxin protein expression was observed in KO operation group;LPR6 protein expression was significantly increased in WT operation group.The expression of LRP6 protein decreased after Draxin knockout;the expression of Fzd3,Wnt5 a and RhoA in WT group increased significantly,and the increase of Fzd3,Wnt5 a and RhoA decreased significantly after Draxin knockout(n=8,P<0.05).Conclusions:1.During the development of embryonic spinal cord,the expression of Draxin has a dynamic trend,and various types of neurons and astrocytes express Draxin.2.After spinal cord injury,the expression of Draxin in the basement membrane and in the surrounding area was obvious.Draxin has a distinct dynamic trend,and various types of neurons and glial cells express Draxin.3.Increased expression of Draxin after spinal cord injury may play a regulatory role through Wnt signaling pathway and Rho GTPase signaling pathway.