Closed-loop Electrical Stimulation Promotes Reconstruction Of Spinal Sensory-motor Neural Circuits After Spinal Cord Injury In Mice

Objective:Through the changes in the number of the proprioceptive axon terminals and synaptic connections between of the spinal cord premotor neurons and motor neurons.Exploring changes in spinal cord sensory-motor neural circuits in spinal cord injury(SCI)mice with closed-loop electrical stimulation training at different frequencies Observation of changes in spinal cord evoked potentials by intrathecal injection of neurotransmitter antagonists.Exploring the types of synaptic connections between neurons in the sensory-motor neural circuits of the mice after a specific frequency closed-loop electrical stimulation training.It provides important clues for the mechanism of closed-loop electrical stimulation training to promote reconstruction of spinal cord sensory-motor neural circuits after spinal cord injury in miceMethods:(1)The motor neurons that dominate the anterior tibial muscle(TA)are labeled by the retrograde marker of fluorescent gold(FG),and the implant position of the spinal cord electrode is established by the spinal cord segment where the motor neurons are located.(2)The spinal cord evoked potentials were recorded in normal mice TA,and the waveforms formed by electrical signals through different neural circuits pathways were determined by intrathecal injection of tetrodotoxin(TTX)(3)Intrathecal injection of different types of neurotransmitter antagonists(AP5 NMDA receptor antagonist;PTZ:GABA-A receptor antagonist;CNQX:AMPA/Kainate receptor antagonist)was used to analyze the changes of spinal cord evoked potentials after spinal cord injury in mice,and to determine the types of synaptic connections between neurons in the spinal cord local neural circuits associated with TA motor function.(4)Immunohistochemistry and Imaris software 3D modeling were used to quantify the changes in the number of synaptic connections between the proprioceptive axon terminals and premotor neurons and motor neurons associated with TA motor function before and after spinal cord injury(5)Immunohistochemistry and Imaris software 3D modeling were used to observe the proprioceptive nerve fibers associated with TA motor function,and the synaptic connections between premotor neurons and motor neurons related to TA motor function,and to determine the effective closed-loop electrical stimulation frequency of spinal cord sensory-motor neural circuits reconstruction after spinal cord injury.(6)Using the intrathecal injection of the AMPA/Kainate receptor antagonist CNQX,the types of synaptic connections between neurons in the spinal cord sensory-motor neural circuits that may be reconstructed after an effective frequency closed-loop electrical stimulation training was analyzed.Results:(1)The results of FG retrograde labeling showed that motor neurons that innervated TA were distributed in the L2?L4 segment of the spinal cord,and the location of spinal epidural electrode implantation was determined.(2)The results of intrathecal injection of TTX showed that the waveform of the spinal cord evoked potentials recorded by TA included early response(electrical signals through the efferent nerve to TA),middle response,and late response(electrical signals through multiple synaptic transmission to TA).(3)The results of intrathecal injection of neurotransmitter antagonists show that spinal cord injury disrupts the connections of glutamatergic(Glu)synapses between neurons in the sensory-motor neural circuits of the spinal cord,blocking the binding of glutamate to AMPA/Kainate receptors.(4)Immunohistochemical method combined with Imaris software 3D modeling,quantitative statistical analysis showed that in spinal cord injury mice,the number of proprioceptive axon terminals associated with TA motor function and the number of Glu synaptic connections between premotor neurons and motor neurons were significantly reduced.(5)Immunohistochemical methods combined with Imaris software 3D modeling,quantitative statistical analysis results show that compared to other stimulation frequencies,the 10 Hz?20 Hz closed-loop electrical stimulation can significantly increase the number of proprioceptive axon terminals associated with TA motor function and the number of Glu synapses between premotor and motor neurons.(6)The results of intrathecal injection of the AMPA/Kainate receptor antagonist CNQX showed that the 10 Hz?20 Hz closed-loop electrical stimulation promoted the formation of Glu synaptic connections between neurons in the spinal sensory-motor neural circuits.Conclusion:Spinal cord transection injury disrupts the transmission of multiple synapses between neurons in the sensory-motor nerve circuit of the mice spinal cord.The 10 Hz?20 Hz closed-loop electrical stimulation training can increase the connections of Glu synapses between premotor neurons and motor neurons in the spinal cord sensory-motor neural circuits of the SCI mice and the number of proprioceptive nerve fibers.This is an important guiding significance for further study of closed-loop electrical stimulation at specific frequencies for the reconstruction mechanism of spinal cord sensory-motor neural circuits after spinal cord injury.