| Objective:To explore the effect of targeted knockdown of adenosine monophosphate-activated protein kinase（AMPK）expression in ventral horn motor neurons of the spinal cord on axon regeneration in the mouse model of acute motor axis neuropathy（AMAN）,and to provide ideas for therapeutic research in AMAN.Methods:Adeno-associated virus 2（AAV2）vector were constructed.The identified 5-7 weeks old GD3s-/-mice were divided into AMPK knockdown group and control AAV2 group.AMPK knockdown-AAV2was injected into the tibialis anterior muscle of the AMPK knockout group to target the knockdown of AMPK expression in ventral horn motor neurons,and Control-AAV2 was injected into the control AAV2group.After 2 weeks,the AMAN mouse model was established by crushing the sciatic nerve+intraperitoneal injection of anti-GM1 Ig G antibody.The successful knockdown of AMPK was verified by spinal AMPK immunofluorescence.Anti-GM1 Ig G antibody binding to nerve fibers was verified by sciatic nerve GM1 immunofluorescence.The motor function recovery of AMAN mice was evaluated by gait experiments.The recovery of nerve conduction function was assessed by the amplitude and latency of the sciatic nerve compound motor action potential（CMAP）.Regeneration of nerve fibers was assessed by neurofilament 200（NF200）and myelin basic protein（MBP）immunofluorescence staining.Results:1.AMPK immunofluorescence:Compared with the control AAV2 group,the expression of AMPK in the ventral horn motor neurons of the AMPK knockdown group was significantly knocked down（23.29±13.05 v.s.36.59±18.43,P=0.016）.2.Anti-GM1 Ig G antibody immunofluorescence:All AMAN mice in the two groups had obvious anti-GM1 Ig G antibody binding to the sciatic nerve.3.Gait experiment:The mean contact intensity of the AMPK knockdown group was greater than that of the control AAV2 group（156.20±8.36 v.s.146.40±12.31,P=0.023）,and the stance width was smaller than that of the control AAV2group（0.86±0.14 v.s.0.97±0.12,P=0.033）,the paw angle body axis was smaller than that of the control AAV2 group（24.52±6.82 v.s.29.47±5.70,P=0.040）,and the limb loading was greater than that of the control AAV2group（39.77±18.87 v.s.27.17±8.95,P=0.020）,and the remaining indicators were not statistically different.4.Electrophysiology:The amplitude of CMAP in the AMPK knockdown group was significantly greater than that in the control AAV2 group（21.61±3.62 v.s.16.58±4.02,P<0.0001）,and there was no statistical difference in the latency between the two groups（2.47±0.36 v.s.2.73±0.73,P=0.132）.5.The mean flourscence indensity of NF200 in the AMPK knockdown group was greater than that in the control AAV2 group[15.50（5.78）v.s.9.73（5.04）,P<0.0001],and the mean fluorescence intensity of MBP was also greater than that in the control AAV2 group[29.09（22.89）v.s.13.47（10.31）,P<0.0001].Conclusion:1.The AAV2 vector constructed in this study can effectively infect the ventral horn motor neurons of AMAN mice,and significantly knock down the expression of AMPK.2.Targeted knockdown of AMPK expression in ventral horn motor neurons promotes motor axon regeneration in AMAN mice,thus facilitating the recovery of motor function.Targeted knockdown of AMPK expression is a potential therapeutic strategy for AMAN. |
