Establishment Of Axonal Regeneration Model Of Motor Neurons In Anterior Horn Of Spinal Cord

Purpose:Nerve regeneration-especially central nerve system regeneration,has been a problem in clinical study.As the output channels of neurons,axons play an extremely important role in cell signaling transduction.Patient's sensory and motor function will be lost because axons can't transmit signals under the conditions that the spinal cord injured.Central axons can't regenerate due to its weak regenerative capacity and the inhibitory microenvironment around.Therefore,it is a critical step to solve the problem of axonal regeneration during the repairment of the nervous system.As the subordinate motoneurons of the corticospinal tract(CST)conduction pathway,the anterior horn motor neurons cooperate with CST to control the movement of muscles.But so far,we have not found a related study on the establishment a model of axon regeneration in anterior horn motor neurons.Therefore,we focus on establishing a model using Rosa-tdTomato +/-gene mouse and AAV-Cre virus in this study to provide an experimental method in axon regeneration research of spinal anterior horn motor neurons.Method:(1).We used the Rosa-tdTomato+/-gene mouse as the experimental object,and then injected the AAV-Cre virus into the anterior horn of the mouse spinal cord.The virus-infected cells expressed tdTomato fluorescent protein.(2).We obtained the spinal cord,DRG,sciatic nerve and muscle of the gene mouse,followed by frozen sectioning,specific immunofluorescence staining of the motoneurons and axons of the anterior horn of the spinal cord,and microscopic observation of the expression of tdTomato fluorescent protein in various tissues.(3).To measure the proportion of axons expressing tdTomato fluorescent protein in the sciatic nerve,we transected the sciatic nerve and immunofluorescently stained Tuj-1 antibody and ChAT antibody,and then imaged by fluorescence microscopy to count the number of axons.(4).AAV-Cre virus was injected into the anterior horn of spinal cord,and the EGFP plasmid was transfected with L3-L4 DRG electroporation.The sciatic nerve was injured 4 days later,then,the sciatic nerve was taken for compression on the 7th day,and the fluorescence microscope was used to image the sciatic nerve.Measurement of axon regeneration length of sensory and motor neurons in sciatic nerve(5).The Rosa-tdTomato+/-gene mouse was mated with the PTENflox/flox gene mouse to obtain Rosa-tdTomato+/-;PTENflox/flox gene mouse.Rosa-tdTomato+/-gene mouse and Rosa-tdTomato+/-;PTENflox/flox gene mouse underwent complete spinal cord injury surgery and anterior intraocular injection of AAV-Cre virus.After 4 weeks,the spinal cord was taken for frozen sectioning.The synaptic vesicles were detected by immunofluorescence staining with vGlut 1 antibody.The growth of tdTomato positive axons was observed under fluorescence microscope.BMS score was used to evaluate the effects of axonal regeneration of anterior horn motor neurons on motor function in mice with spinal cord injury.Results:(1).AAV-Cre virus was successfully injected into the anterior horn of the spinal cord,enabling tdTomato fluorescent protein to successfully label motoneurons in the anterior horn of the spinal cord.(2).The number of tdTomato positive axons accounted for 25.6%±2.42 of the total axons in the sciatic nerve,accounting for 72.6%±2.91 of the total motor neuron axons in the sciatic nerve(n=3).(3).The length of regeneration of tdTomato-positive axons in the sciatic nerve is significantly shorter than that of EGFP-positive axons.(4).After knockout of PTEN gene,the anterior horn motoneurons axon regeneration ability of Rosa-tdTomato+/-;PTENflox/flox gene mouse was enhanced.The axon passed through the spinal cord injury site and coincided with the vGlut 1 positive plaque,Remodeling synaptic structure.BMS score,relative to Rosa-tdTomato+/-gene mouse,Rosa-tdTomato+/-;PTENflox/flox gene mouse have relatively improved motor function,but not statistically significant.Conclusion:We have successfully established axonal regeneration model of spinal anterior horn motor neurons,and provide an experimental method for studying axonal regeneration of spinal anterior horn motor neurons.