Closed-loop Electrical Stimulation Promoted The Recovery Of Motor Function In Mice After Spinal Cord Injury

Spinal cord injury(SCI)is one kind of severe central nervous systemic disease.After spinal cord injury,information transmission between the brain and spinal cord is impaired,so different degree and the parts of the spinal cord injury can lead to upper or lower limb paralysis and seriously affected the daily life of the patients.As a result,the limb functional recovery after spinal cord injury becomes the primary evaluation standard of clinical treatment.There are many treatment methods for spinal cord injury,including electric stimulation therapy,stem cell transplantation,drug intervention,etc.But only a few of them can achieve effective functional recovery,while most of them cannot restore the motor function of paralyzed limbs.In recent years,the application of electrical stimulation in the treatment of spinal cord injury has made substantial progress.Research shows that this method can enhance the electrical activity of spinal cord neurons,and can make the limbs functional recovery after spinal cord injury.However,the challenge is how to choose the stimulus parameters and explore the possible influencing mechanism.Accordingly,this study focuses on the selection of stimulation parameters under the closed-loop mode of electrical stimulation and the influence of this mode of electrical stimulation on the neural circuits of the spinal cord.Part 1: Establishment of closed-loop electrical stimulation system and selection of effective stimulation frequencyExperiment 1: characteristics of spinal cord closed-loop electrical signals in normal miceObjective: Electrodes were implanted in normal mice under deep anesthesia to detect the characteristics of the electrical signals emitted by the spinal cord closed-loop,so as to determine the parameters of the closed-loop electrical stimulation.Methods: 1.Using fluorogold(FG)retrograde tracing method,the distribution range of motor neurons related to the anterior tibial muscle(TA)in the spinal cord was determined.2.C57 BL/6J male mice weighing 22-25 g were implanted with surface electrodes of tibial anterior muscle(TA)and epidural electrodes of spinal cord L2-L4 segments(electrodes can stimulate or receive signals).3.Electrical stimulation of different intensity was given to the spinal epidural and tibial anterior muscles of mice respectively to detect the characteristics of the release of electrical signals of the spinal cord closed-loop in mice,and the detected electrical signal components were identified by intrathecal injection of tetrodotoxin(TTX).4.The received electrical signals were statistically analyzed and the closed-loop electrical stimulation parameters were determined.Results: 1.The distribution range of TA-related motor neurons was L2-L4 segments of the spinal cord.2.The epidural electrode and the muscle surface electrode of the tibia anterior muscle were successfully implanted.3.The threshold value of epidural electrical stimulation was 300 m V.4.After 400 m V,1Hz and 0.2 ms of suprathreshold stimulation of the epidural in mice,the detected waveform components included the latency period and spinal cord reflex(early,middle and late response;ER,MR,LR).5.Give various intensity of epidural stimulus(300 m V,400 m V,500 m V,600 m V,700 m V,800 m V,900 and 1000 m V),in the spinal reflex,middle-late waveform can be detected since 400 m V.When the stimulus intensity reached 600 m V,middle-late waveform peaked.With the strength of the stimulus continuously increasing,waveform amplitude decreased.The whole conduction time after electrical stimulation was no more than 30 ms.6.After intrathecal injection of tetrodotoxin(TTX),the attenuation and disappearance of waveforms in the middle and late stages of spinal cord reflex can be observed.7.The threshold value of electrical stimulation on the surface of the tibial anterior muscle was 900 m V,and the conduction time was no more than 20 ms.Conclusion: Spinal cord epidural stimulation in mice can induce different spinal cord reflexes under different intensities.The early spinal cord reflexes may be related to the direct stimulation of efferent nerves,while the middle and late spinal cord reflexes may be formed by the involvement of multiple neurons in the spinal cord.Therefore,we not only successfully established the closed-loop stimulation system but also obtained the specific closed-loop stimulation parameters.Namely,the intensity of epidural stimulation was 400-600 m V,and after 50 ms,the tibial anterior muscle surface stimulation was started,and the intensity was 1V.Experiment 2: Selection of effective stimulation frequency of closed loop electrical stimulation in mice after spinal cord injuryObjective: A closed-loop electrical stimulation system was established in mice with spinal cord injury to explore the most effective stimulation frequency in mice with spinal cord injury.Methods: 1.T9 transection model of spinal cord was performed in C57 BL/6J male mice weighing 22-25 g.2.Electrodes were implanted in the spinal epidural segment L2-L4 and the tibial anterior muscle,respectively,to connect the closed-loop stimulation system.3.Closed-loop electrical stimulation was given to the mice 1 week after spinal cord injury at a frequency of 1 Hz,5 Hz,10 Hz,20 Hz,30 Hz and 40 Hz for 3 weeks.4.Biopac was used to detect the EMG of the tibial anterior muscle in mice 1 week before and 1 and 3 weeks after stimulation.5.Matlab was used to conduct quantitative analysis of data collected by Biopac.Results: 1.We successfully established the spinal cord T9 complete transection injury model.2.The possibility of long-term implantation of mouse spinal cord epidural electrode and muscle surface electrode was realized.3.After 1 week of complete transection injury of the spinal cord,myoelectric signals of mice in each group showed the disappearance of the middle and late spinal cord reflex waveforms,and only the early waveforms existed.4.In mice without closed-loop electrical stimulation,the middle and late spinal cord reflex waveforms were not induced after 4 weeks of injury.5.After 3 weeks of closed-loop electrical stimulation at 1 Hz,5 Hz,30 Hz and 40 Hz,the results of myoelectric signal detection in mice were consistent with those in the non-closed-loop electrical stimulation group,that is,no late spinal cord reflex was induced.6.After 10 Hz and 20 Hz closed-loop electrical stimulation in mice,the detection results of EMG signal after 3 weeks showed that,in the presence of early spinal cord reflex,the middle and late spinal cord reflex also recovered to a certain extent.Conclusion: The effective frequency of closed-loop electrical stimulation was 10 Hz and 20 Hz in mice after spinal cord injury.Part 2: The effect of closed-loop electrical stimulation at a specific frequency on functional remodeling of motor nerve circuits in mice with spinal cord injuryObjective: In view of the specific closed-loop electrical stimulation frequency identified in the first part,the second part aims to explore the functional remodeling of the mouse spinal cord motor loop under the action of specific frequency closed-loop electrical stimulation.Methods: 1.r AAV-Retro-EGFP was used to retrograde label TA related motor neurons,and the number and distribution of these neurons in the lumbar segment of the spinal cord were observed.2.Changes in TA related motor neurons and their synaptic density and volume were analyzed by Imaris software in each group.3.CUBIC 3D transparent technique was used to analyze the overall distribution of TA related motor neurons in the spinal cord of each group of mice.4.Flexible pressure sensor device was used to detect the activity of the tibial anterior muscle of the lower limbs of mice stimulated by a specific frequency.Results: 1.After 3 weeks of retrograde labeling with r AAV-Retro-EGFP,immunofluorescence re-staining showed that TA related motor neurons were only distributed in the middle and lateral parts of the gray matter of the spinal cord.2.Observation of the staining results of mice in each group showed that the number of neurons in the group without closed-loop electrical stimulation was significantly lower than that in the control group,both from the coronal plane and the sagittal plane.However,the number of motor neurons in the closed-loop stimulation group with specific frequencies(10 Hz and 20 Hz)was significantly higher than that in the other frequency stimulation groups(1 Hz,5 Hz,30 Hz and 40 Hz).3.At a certain frequency(10 Hz,20 Hz),the Intensity mean of the total density of neurons in the closed-loop electric stimulation group was far lower than that in the injured group,while the results were no difference from those in the control group.Similarly,in the densities of dendrites around neurons,Intensity mean was also significantly lower in the closed-loop electrical stimulation group(10 Hz,20 Hz)than that in the injured group,while there was no significant difference from the control group.4.The overall volume of neurons and surrounding dendrites was quantitatively compared.The results showed that after specific frequency(10 Hz,20 Hz)closed-loop electrical stimulation,the overall volume of neurons was much larger than that of the injured group,and there was no significant difference between the two groups.The quantitative results of the volume of dendrites around neurons showed that after specific frequency(10 Hz,20 Hz)closed-loop electrical stimulation,the volume of motor neuron dendrites was significantly larger than that of the injured group,but there was no significant difference from the control group.5.CUBIC 3D transparent result showed that the number of TA motor neurons in L2-L4 segment of the whole spinal cord in the 10 Hz closed-loop electrical stimulation group was far higher than that in the injured group,indicating increased connections between neurons and full shape of neurons,but the number did not reach the level of the control group.6.The flexible pressure sensor device detected that after 10 Hz closed-loop electrical stimulation intervention,mice lower limbs tibial anterior muscle has a certain recovery of contraction function compared with the control group mice,while the mice without closed-loop stimulation did not see any recovery of contraction function.Conclusion: The specific frequency of the closed-loop electrical stimulation may promote the changes of the local nerve loop in the SCI mice,increase the activity of related motor neurons,and lead to the recovery of specific motor function.Part 3: Analysis of neurotransmitter metabolism in the gray matter of spinal cord before and after total spinal cord transection injury and a preliminary study of premotor neurons in local spinal cord circuitsObjective: In view of the influence of specific frequency of closed-loop electrical stimulation on spinal motor circuits,the possible mechanism of the influence and whether premotor neurons are involved in it remain to be explored,this part aims to investigate the substance metabolism in the gray matter of the spinal cord and the distribution of TA related premotor neurons,laying a foundation for future studies.Methods: 1.Magnetic resonance spectroscopy(MRS)was used to analyze the substance metabolism in the spinal gray matter of mice before and after spinal cord transection injury.2.Monosynaptic rabies virus(RV)revealed the distribution of TA-related premotor neurons in the spinal cord.Results: 1.Multi-directional and high-resolution T2 WI imaging of the mouse spinal cord was performed,and the L2 segment of the mouse spinal cord could be clearly seen in the results.The components of gray matter and white matter in the spinal cord were clearly divided.2.STEAM was used on the selected location.The distribution characteristics of neurotransmitter metabolism in the spinal cord of mice were as following: the highest peak was Cho,including phosphatidylcholine(PCho)and glycerine choline phosphate(GPC).There were other metabolites,including glutamic acid(Glu),glutamine(Gln),gamma-aminobutyric acid(GABA)and Gly(Gly).3.After T9 spinal cord transection injury,the glutamate content in the gray matter of L2 segments below the injury showed an increasing trend compared with the metabolite rate of creatine in the control group.4.The rabies virus(RV)label premotor neurons that were related to the TA and position are mainly distributed in the ipsilateral Rexed lamellar Ⅵ,Ⅶ,Ⅹ layer.And the contralateral premotor neurons focused on Ⅶ Rexed plate layer and the ventromedial Ⅷ layer.Conclusion: 1.After spinal cord injury,the neurons below the site of injury may lead to the increase of glutamate release,thus resulting in the enhancement of extensor excitability and the occurrence of spasm.2.Spinal cord premotor neurons can be the focus of follow-up studies,including the loop connections between them and motor neurons.