Transcription Factor KLF7 Promotes Descending Propriospinal Neuron Axonal Plasticity After Spinal Cord Injury

Objective:Spinal cord injury(SCI)is a major source of major pain and financial burden for patients and their families.So to find an effective strategies to treat SCI has become an important social development issue to be solved urgently.Descending propriospinal neuron(DPSN)play an important role in the generation and regulation of spontaneous and voluntary movement.The corticospinal tracts(CST)can be reconnected for DPSN axons via axonal tracts following SCI.The plasticity of DPSN axons can promote it pass through and beyond the lesion site.The central command is effectively transmitted to the spinal cord structure below the lesion site through DPSN axonal functional mediation.This provides an alternative pathway for neural transmission,called "functional relay",to facilitate the reconstruction of spinal cord function.At present,the plasticity of DPSN axons are important therapeutic targets for functional reconstruction after spinal cord injuryKruppel Like Factor 7(KLF7)is widely present in the nervous system.Recent studies have shown that KLF7 plays an important role in axonal regeneration,glial cell differentiation and myelin sheath formation of the central nervous system.Knockout of KLF7 gene can cause axonal regeneration disorder after central nerve,retina and olfactory nerve damage.Overexpression of KLF7 can promote the regeneration of CST axons following SCI.The preliminary research findings of our research group:(1)AAV-KLF7 virus transfected acellular nerve ramus was constructed to bridge sciatic nerve defects and effectively promote peripheral nerve regeneration.(2)KLF7 can promote peripheral nerve injury,sensory and motor axon regeneration,myelin formation and functional recovery.(3)KLF7 transfected Schwann cells(SCs)have the ability to proliferate.However,the effect and mechanism of KLF7 on axonal plasticity of DPSN following SCI have not been reported.In this project,based on research of axon regeneration,synaptogenesis,axon plasticity and functional recovery of DPSN,to explore the mechanism of KLF7 on the morphology of spinal cord neurons and the axon regeneration of dorsal root ganglion(DRG)neurons in vitro,to elucidate the mechanism of KLF7 regulates axonal plasticity of DPSN following SCI.To solve these problems will further enrich the theoretical mechanism of spinal cord injury,provide new therapeutic strategies and targets for the treatment of SCI.Methods:1.In vitro experiments:? Spinal cord neurons were isolated and cultured,AAV-GFP virus was used to transfect spinal cord neurons to detect the transfection rate of AAV-NC virus,and AAV-KLF7?AAV were used to transfect spinal cord neurons.The cells were divided into AAV-KLF7 group,AAV-NC group and Control group.?Immunofluorescence staining was used to detect the transfection rate of AAV-GFP virus,KLF7 expression and morphological characteristics of spinal cord neurons.?Western blot and PCR used to detect the expressions of KLF7,target genes NGF and TrkA protein and mRNA in spinal cord neurons of each group.?IncuCyte ZOOM real-time analysis of AAV-KLF7 on the neurite growth of spinal cord neurons.?DRG cell culture,immunofluorescence staining and Sholl analysis were used to detect the regeneration of DRG axons after AAV-KLF7 and AAV-NC transfection2.In vivo experiment:?The model of Contusion injury of T10 segment of spinal cord in mice was established with the louisville injury system instrument(LISA).?Dynamic changes of KLF7 expression in spinal cord lesions were detected before and after SCI 1d,1w,2w,3 w and 4w by Western blot.?Injection of AAV-NC and AAV-KLF7 into the T7-9 spinal cord of mice,the experimental animals were divided into SCI+AAV-KLF7 group,SCI+AAV-NC group,and Sham group.?Expression of KLF7,NGF,TrkA GAP43 and PO were detected by Western blot.?Cresyl Violet-Eosin,Luxol Fast Blue and Neurolucida system were used to detect the lesion area,lesion volume and myelin sheath in each group after SCI 5w.?Combined FG retrograde tracking DP SN neuron with immunofluorescence were to detect AAV-KLF7 transfection DPSN,to verify that AAV-KLF7 transfected DPSN at T7-9 segment effectively.?T7-8 segment biotin glucan amine(BDA)was injected to trace the DPSN axon in the right direction,and immunofluorescence staining was performed to detect GFAP(labeled astrocyte)and BDA in T10 segment of the lesion,and KLF7's effect on the plasticity of DPSN axon in the lesion was detected.?CTB retrograde labeled lumbar motor neuron cell bodies and dendrites,DPSN axons were labeled by BDA injection and SYP immunofluorescence,the synapse formation of DPSN axon and spinal cord lumbar motor neuron was detected by laser confocal and immunofluorescence.?The formation of axon myelin was observed and detected by electron microscopy around the injury area.?Immunofluorescence staining of double-labeled KLF7 and CC1(labeled oligodendrocytes),NG2(labeled oligodendrocyte precursors),GFAP(labeled astrocytes)at T7-9 segments.To determine whether KLF7 is expressed in oligodendrocytes,oligodendrocytes precursor cells or astrocytes.3.Functional recovery:?Wet weight ratio of the tibialis anterior muscle(TA)and Roots-Karnovsky method;?BMS;?Grid walking test;?Electrophysiological test;?Hargreaves test.Results:1.The transfection rate of AAV-GFP transfected spinal cord neurons was 84.70±5.57%by immunofluorescence staining.Compared with the AAV-NC group,the expression of KLF7 protein in the AAV-KLF7 group was significantly increased(P<0.001)At the same time,AAV-KLF7 group significantly increased the average length of neurite protuberances compared with AAV-NC group(P<0.001)2.Western blotting and PCR results showed that compared with the AAV-NC group and the Control group,the expressions of KLF7 protein and mRNA in AAV-KLF7 group were significantly increased(P<0.05).NGF,TrkA and their mRNA expressions in the AAV-KLF7 group were more than that of the AAV-NC group and the Control group(P<0.05).However,there was no significant difference between the AAV-NC group and the Control group(P>0.05)3.IncuCyte ZOOM system show that compared to the AAV-NC group,the length of neurite and number of neurite branches in AAV-KLF7 group were significantly increased on 3d and 4d after virus transfection(P<0.05)4.Sholl analysis showed that the average length of DRG neuron axons in the AAV-KLF7 group was significantly higher than the average of the AAV-NC group(P<0.001)The AAV-KLF7 showed the greatest neurite length in 0-60°,180-240°,240-300° and 300-360° angle bins compared with AAV-NC(P<0.001)5.Western blot results showed that the baseline level of KLF7 protein in the thoracic spinal cord was low.KLF7 protein expression increased in the lesion 1 d after SCI,and the peak KLF7 expression appeared 1-2 w after SCI,and returned to the baseline level 3-4 w after SCI(P<0.001)6.Western blot results showed that KLF7 protein expression was significantly increased in SCI+AAV-KLF7 group compared with Sham group and SCI+AAV-NC group(P<0.001).Compared with Sham group,NGF,TrkA and GAP43 protein expressions were increased in SCI+AAV-KLF7 group and SCI+AAV-NC group,and NGF,TrkA and GAP43 protein expressions were significantly higher in SCI+AAV-KLF7 group than in SCI+AAV-NC group(P<0.05).Compared with Sham group,the expression of PO protein in SCI+AAV-KLF7 group and SCI+AAV-NC group was significantly decreased,and the expression of PO protein in SCI+AAV-KLF7 group was significantly higher than that in SCI+AAV-NC group(P<0.05)7.Results of Cresyl-echt violet,Luxol Fast Blue and Neurolucida system showed that AAV-KLF7 treatment had no effect on the lesion area nor the lesion volume compared to the AAV-NC group following SCI.However,Luxol Fast Blue staining showed that AAV-KLF7 treatment increased the percent of myelin sparing compared to the AAV-NC group(P<0.05)8.The results of FG retrograde tracking DPSN and immunofluorescence staining showed that there was no significant difference in the number of FG retrograde labeled neurons between the SCI+AAV-KLF7 group and SCI+AAV-NC group(P>0.05)However,KLF7 protein expression and the percentage of FG/KLF7 double labeled neurons in the population were significantly higher than that in the SCI+AAV-KLF7 group compared to the SCI+AAV-NC group(P<0.05)9.BDA forward tracking DPSN axon and immunofluorescence staining results showed that:at the T10 lesion level,GFAP protein expression of SCI+AAV-KLF7 group and SCI+AAV-NC group showed no significant difference(P>0.05).However,BDA protein expression of SCI+AAV-KLF7 group was significantly higher than that of SCI+AAV-NC group(P<0.05)10.CTB retrograde labeled lumbar motor neuron bodies and dendrites.BDA followed DPSN axon.Immunofluorescence staining results showed that at L4-5,there was no significant difference in the number of CTB retrograde labeled anterior angular motor neurons between SCI+AAV-KLF7 group,SCI+AAV-NC group and Sham group(P>0.05).Compared with Sham group,relative density of BDA and relative density of SYP/motor neurons in SCI+AAV-KLF7 group and SCI+AAV-NC group were significantly decreased,and BDA relative density and relative density of SYP/motor neurons in SCI+AAV-KLF7 group were significantly higher than those in SCI+AAVgroup(P<0.05)11.Electron microscopy showed myelinated ratio of T10 segment axons in the Sham group was significantly higher than the injured SCI+AAV-KLF7 and SCI+AAV-NC groups.The ratio of axonal myelin sheath around the lesion in SCI+AAV-KLF7 group were significantly higher than in the SCI+AAV-NC group(P<0.001)12.Immunofluorescence staining results showed that KLF7 was expressed in most NG 2-positive cells at T7-9,and the percentage of NG2/KLF7 co-labeled neurons was 84.22±6.95%.However,only a few CC1-positive cells expressed KLF7,and the percentage of CC1/KLF7 co-labeled neurons was 10.23±2.39%.No gfap-positive cells were found to express KLF713.After 5w of SCI,compared with that of Sham group,TA wet weight of SCI+AAV-KLF7 group and SCI+AAV-NC group decreased significantly.TA wet weight of SCI+AAV-KLF7 and SCI+AAV-NC group showed no significant difference(P>0.05)In addition,the dyeing of the sport end-plates showed that the number/fiber of the SCI+AAV-KLF7 group,the SCI+AAV-NC group and the Sham group had no significant difference(P>0.05)14.Behavioral test results:?BMS scores were significantly increased in the SCI+AAV-KLF7 group compared with the SCI+AAV-NC group 5 weeks after injury(P<0.05).?The experimental results of grid walking show that the number of foot drops during grid walking was significantly improved by AAV-KLF7 treatment compare to the AAV-NC group(P<0.05).? The electrophysiological results showed that the SCI+AAV-KLF7 and SCI+AAV-NC group displayed decreased wavelength amplitude compared to the uninjured sham group,however,the SCI+AAV-KLF7 group displayed a significant improvement over SCI+AAV(P<0.05).?Hargreaves experimental results show that compared with Sham group,the reflex latency of SCI+AAV-KLF7 group and SCI+AAV-NC group increased significantly,while there was no significant difference between two groups(P>0.05)Conclusion:1.AAV-KLF7 can effectively transfect spinal cord neurons and DRG neurons,promotes the expression of KLF7,NGF and TrkA in spinal cord neurons,and promote neurite outgrowth of spinal cord neurons and DRG axons in vitro2.KLF7 promotes DPSN axonal plasticity and synaptic formation with lumbar motor neurons by up-regulating the expression of target genes NGF,TrkA and GAP433.KLF7 can up-regulate the decrease of PO after SCI,and express in oligodendrocyte precursor cells,and promote the formation of myelin around the lesion.4.AAV-KLF7 promotes motor function recovery following SCI.