The Role Of Id2 In Axonal Regeneration And Functional Recovery After Sciatic Nerve Injury In The Mouse

Objective:To study the effect of Id2(inhibitor of DNA binding 2)on axonal regeneration and functional recovery after sciatic nerve injury,and to explore its underlying mechanism.Methods:1)Microinjection of AAV9 into spinal ventral horn and sciatic nerve crush injury:Under anesthesia,the T13 thoracic vertebrae of the mouse was immobilized and laminectomy was conducted to exposure the spinal cord.Microinjection of AAV9-GFP or AAV9-Id2-Flag-EGFP was performed stereotaxically into the ventral horn of the spinal cord.Two weeks after the AAV9 injection,the mice were anesthetized again,and the bilateral sciatic nerves were exposed and crushed.To confirm that all the axons are disrupted by this method,animals were perfused at 1hour after crush,and sciatic nerves were collected for histological analysis.Electromyography(EMG)was measured at 1 day after crush to further confirm the disruption of neuromuscular function.2)Assessing axonal regeneration after sciatic nerve injury:adult mice were injected with AAV9-GFP or AAV9-Id2,and bilateral sciatic nerves were crushed 2 weeks later.Animals were perfused 2 weeks after crush injury.The number and distance of GFP~+axonal regeneration in sciatic nerve,the number of GFP~+fibers and neuromuscular junctions(NMJs)within the gastrocnemius muscle were analyzed histologically.3)Evaluation of functional recovery:adult mice were injected with AAV9-GFP or AAV9-Id2,and bilateral sciatic nerves were crushed2 weeks later.Catwalk gait analysis were performed before and 30 days after injury,EMG were recorded 30 days after injury,and the number of NMJs in gastrocnemius muscle were counted.4)Neurite outgrowth assay:Primary cortical neurons were isolated from E18 mouse embryos.After infection with LV-shControl or LV-shNgn2lentivirus,neurons were plated and cultured for 2 days,followed by addition of Ad-Id2DBM or Ad-EGFP.Cells were cultured for additional 3 days before fixation.Neurite length was measured using Image J.Results:1)Verification of AAV9 expression and sciatic nerve crush.After AAV9recombinant virus injection,neurons and their neurites in the bilateral ventral horn of the lumbar spinal cord were labeled with GFP,including many ChAT~+motor neurons.GFP~+neurons in the Id2 group also expressed Flag,indicating the expression of Id2-Flag fusion protein.Many axons in the sciatic nerve were labeled with GFP,and all of the GFP~+axons were disrupted at 1 h after sciatic nerve crush.No activity was recorded in gastrocnemius muscle by EMG at 1 day after crush.2)Analyzing axonal regeneration after sciatic nerve crush.At 2 weeks after sciatic nerve injury,extensive axonal regeneration were observed in both GFP group and Id2 group.However,compare to GFP control group,Id2 group had greater numbers of GFP~+axons that regenerated over 3 mm.There were many GFP~+fibers re-growing into the gastrocnemius muscle of Id2 group,while only a few were found in that of GFP group.In addition,a few NMJs were formed in the gastrocnemius muscle at 2 weeks after sciatic nerve crush in the Id2 group,while no NMJ innervation was observed in GFP group.3)Functional recovery of animals 30 days after injury.Catwalk gait analysis showed,the print area of the mice in the GFP group after injury was significantly reduced compared with that before the injury,and the“base of support”of the left and right hind limbs was significantly wider than that before the injury.Compared with the GFP group,the print area in the Id2 group was increased,and the base of support was shortened.The gastrocnemius EMG showed that,compared with the GFP group,the amplitude of neuromuscular action potential was significantly increased in the Id2group,and the latency was significantly shortened.Histological staining showed that the number of NMJs in the gastrocnemius muscle of the Id2 group was significantly greater than that of the GFP group.4)Immunohistochemical staining showed that little Neurogenin2(Ngn2)expression was detected in the spinal cord neurons of GFP group.However,in Id2 group,Ngn2 expression was significantly upregulated in the GFP~+neurons expressing Id2.In vitro neurite outgrowth assay further showed that pre-knockdown of Ngn2 using shRNA significantly blocked the promoting effect of Id2 on axonal growth.Conclusion:1)Overexpression of Id2 in spinal motor neurons accelerates axonal regeneration after sciatic nerve injury.2)Id2 promotes the re-innervation of hindlimb muscle after sciatic nerve injury,promotes the recovery of motor nerve conduction velocity and neuromuscular action potential amplitude,and therefore promotes the recovery of hindlimb motor function.3)Id2 up-regulates the expression of Ngn2protein in adult neurons,which might be one potential mechanism for Id2 to promote axonal growth and regeneration.