| Spinal cord injury is one of the most cause of disability around the world,but there’s no unequivocal and viable treatment strategy at show.Our previous clinical trials confirmed that the increased excitability of motor cortex is significantly related to the functional prognosis of patients with spinal cord injury.However,it is not clear which cell types in the motor cortex lead to the later functional recovery and the neural mechanism of recovery.Here,we use optogenetic techniques to target the activation of glutamate neurons in the primary motor cortex(region M1)and investigate whether activation of glutamate neurons in the primary motor cortex promotes functional recovery after spinal cord injury in rats and the preliminary neural mechanisms involved.The results showed that the targeted activation of glutamate neurons in the motor cortex could significantly improve the motor function scores of rats.It can shorten the latency period of motor evoked potential and improve the amplitude of motor potential effectively.Here,we use optogenetics technology to selectively activate glutamate neurons in the primary motor cortex,and we will research if activation of glutamate neurons in the primary motor cortex can promote functional recovery after spinal cord injury in rats and the preliminary neural mechanisms related.The results prove that the activation of glutamate neurons in the motor cortex is able to meaningfully further the score of motor function in rats,and efficiently weaken the latency period of motor evoked potentials and enhance the amplitude of motor potentials.On the side,HE staining and nerve fiber staining at the injured site showed that accurate activation of primary motor cortex could effectively promote tissue recovery and neurofilament growth(GAP-43,NF)at the injured site of spinal cord,and the content of some growth-related proteins(BDNF,NGF)at the injured site increased.These results suggest that selective activation of glutamate neurons in primary motor cortex can promote functional recovery after spinal cord injury,and may be of great significance for comprehending the neural cell mechanism of functional recovery induced by motor cortex stimulation.Objective: To observe the effect of optogenetic targeted activation of glutamate neurons in M1 region on motor function recovery in rats with spinal cord injury(SCI).Methods:1.90 female SD rats were haphazardly separated into 4 bunches: light incitement bunch(SCI+Ch R2)and pretense incitement bunch(SCI+m Cherry)and SCI alone bunch(SCI)and pretense operation bunch(Sham).The SCI rodent show was set up by clamping strategy.SCI modeling was just implemented in the simple SCI group,whereas,in the sham operation group,the skin of the corresponding segment only was cut and the lamina with bone-biting forceps without any intervention was opened.The pretense incitement bunch(SCI+m Cherry)injected viruses AAV-Ca MK Ⅱ alpha m Cherry,the virus contains Ca MK Ⅱ alpha gene promoter sequences,can be precisely infection can glutamate neurons,but do not contain light channel protein gene,so light can’t activate the M1 glutamate neurons.light incitement bunch(SCI+Ch R2)injected viruses AAV-Ca MK Ⅱ alpha Ch R2-m Cherry,containing Ca MK Ⅱ alpha channel protein gene,gene promoter sequences and light can infect neurons and glutamic acid can be precisely corresponding blu-ray activation.After the successful injection of the virus,the virus was expressed in the injected M1 region(about 3 weeks after the injection of the virus).Used immunofluorescence method to stain the sections of the injected brain region with DAPI and c-Fos staining by immunofluorescence method,and observed with VS200.At the same time,the rats were subjected to light stimulation by electrophysiological method to see whether the area where the virus was injected could produce electrical response.This is to determine where the virus is injected and whether the injection volume is sufficient to elicit a response.2.After the expression of the virus,the lamina of T10 was peeled off,and 50 g aneurysm clip was used to compress the spinal cord of T9 for 60 s.During this period,there was tail wagging and hind limb reflex,indicating the success of SCI model.To prevent infection,the rats were injected subcutaneously for 7 consecutive days.In addition,in order to detect the success of modeling,motor evoked potential was detected one day after modeling to see whether the amplitude and latency were consistent with the waveform characteristics of paraplegia.3.Rats in the light stimulation group and the sham stimulation group received 473 nm blue light stimulation twice a day for 2 weeks on the 3rd to 14 th day after successful modeling.Routine manual urination gave the SCI group,while the sham group was fed in the cage without any other operation until the end of the experiment.4.After the completion of light stimulation(2 weeks after SCI surgery),cardiac perfusion was enforced within 90 minutes after the last light stimulation,and the spinal cord was dissected.1cm before and after the injury was cut and frozen in a refrigerator at-80℃.We used WB to detect the protein expressions of GAP-43,BDNF and NGF in the spinal cord injury of rats in each group.5.From 1 to 6 weeks after modeling,we used BBB score and slope test to evaluate the motor function of SCI rats weekly.6.At the 6th week after SCI,we perfused spinal cord tissue of rats in each group for HE staining and NF immunofluorescence staining,and observed the repair of spinal cord in the injured area.Furthermore,we detected motor evoked potential(MEP)again,and observed the amplitude and latency of MEP.Results:1.Activation of glutamate neurons in M1 regionThree weeks after the rats were injected with AAV,DAPI staining showed significant and stable expression of AAV in bilateral M1 region,and c-Fos staining of the brain also showed activation of glutamate neurons in M1 region.We through the installation of rat fiber porcelain head,using the 473 nm blue light on rat brain M1 area light stimulation,discharge situation of cells stimulation test,found that M1 area were stimulated cell of rats obviously increase the discharge,and to stimulate the camera to observe in rats,rats found in light stimulate strong hind legs appear when not voluntary movement,the light stops and involuntary movement when stop,in the whole process,the physiological characteristics of normal rats,epilepsy,and other mental symptoms.These results demonstrate that our virus can activate glutamate neurons in the rat M1 region and control the movement of the hind limbs of rats by light exposure.2.Activation of glutamate neurons in M1 region by light stimulation can promote the recovery of motor function in rats with spinal cord injuryDuring 1-6 weeks after SCI,BBB score and slope test were used to test the weekly functional changes of SCI rats’ hind limbs,and it was found that BBB score and slope stability Angle could be improved in the injured groups with the enhancement of time.It is crucial to take notice that we have found that light stimulation group(group SCI + Ch R2)weekly score were higher than other two groups,to the end of the experiment of 6 weeks,BBB score and the stability of the slope angles were significantly better than the other two groups,test results and SCI + m Cherry and SCL group was no remarkable deviations,suggests that light stimulates the M1 area can really promote the movement functions of SCI in rats,and in the long-term functional recovery has obvious advantages.3.Glutamate neurons in the M1 region of rats after spinal cord injury can excite the descending conduction bundle by light stimulationIn order to confirm whether light stimulation of the M1 region in rats can activate the descending conduction bundle,thereby triggering the movement of the hind limbs of SCI rats,we first detected the motor evoked potentials on the first day after SCI,and confirmed that almost no motor evoked potentials were produced in the rats after SCI.When the M1 region of the rat brain was stimulated by 473 nm blue light,the same strong involuntary movement of the hind limbs was observed in the stimulated rats by video camera.6 weeks after SCI,once again we test each rat motion evoked potential,found that each group rats the amplitude of motor evoked potentials were improved,compared with the Sham group,the extension of incubation period between groups of spinal cord injury to different extent,the good news is,M1 area receives the light stimulation rats group(SCI + Ch R2)of motor evoked potential amplitude is higher than other injury group,and the incubation period was closer to normal rats,moreover SCI + m Cherry group and SCL group motion evoked potential test results there were no significant differences.These results indicate that the precise activation of M1 region after SCI can stimulate the descending conduction bundle,thereby triggering the movement of the hind limbs of SCI rats,and at long last advancing the recuperation of motor function of SCI rats.4.Light stimulation of glutamate neurons in the M1 region of rats with spinal cord injury can promote tissue repair at the injured siteFor purpose of searching after the reasons for the obvious improvement in the functional recovery of rats after reiterant stimulation of M1,we used HE staining at the 6th week to observe the postoperative histological changes of SCI injury in the three groups.It was found that the rats receiving light stimulation in M1 region(SCI + Ch R2 group)had more neat and clear tissue and smaller cavity area.We calculated the cavity area of the three groups for statistics,and found that the cavity area of the light-stimulated group was indeed significantly smaller than that of the other two groups,proving that the precise activation of M1 region can promote the repair of SCI rats’ damaged tissues.5.Light stimulation activated glutamate neurons in the M1 region of rats with spinal cord injury and increased the expression of neurotrophic protein at the injury siteBecause the recovery of injured nerves was not able to be gained without the expression of neurotrophic factors,Western blot was used to detect the expression of neurotrophic factors(BDNF and NGF)at the spinal cord injury site after the end of light stimulation(the 2nd week after SCI).The comes about appeared that,compared with the ordinary rats(Sham),the expression levels of BDNF,NGF and BDNF were expanded in all harm bunches,but the expression level of SCI+ Ch R2 gather was more essentially expanded,while the level of neurotrophic factor in SCI+ m Cherry group and SCI group was similar without significant difference.These results indicate that the precise activation of M1 region can promote the increase of neurotrophic factor content at the injured site,hence advancing the repair of the harmed tissue,and at last accomplishing the reason of advancing the recuperation of motor function in SCI rats.6.Light stimulation activates glutamate neurons in M1 region of rats with spinal cord injury and promotes the regeneration of nerve filaments at the injured site of spinal cord injuryThe expression levels of nerve growth-related molecules were also examined,such as GAP-43,which plays a significant role in axial germination and synaptic plasticity.The results also examined that the expression of GAP-43 was enhanced in rats with spinal cord injury after iterative stimulation in the M1 region.In addition,immunofluorescence staining was used to detect the generation of nerve fibers at the injury site of rats in each group at the6 th week,and the results showed that the fluorescence expression of NF at the injury site of rats with spinal cord injury after repeated stimulation in M1 zone(SCI + Ch R2 group)was slightly higher than that in other injury groups.We suppose that the precise activation of glutamate neurons in the M1 region of the brain after SCI in rats can improve the regeneration of nerve filaments at the site of spinal cord injury,therefore advancing the repair of the harmed tissue,and at last advancing the recuperation of motor function in SCI rats.Conclusion:These results suggest that the activation of glutamate neurons in the M1 region of the brain after spinal cord injury can promote the recovery of motor function,and the cause of this promotion may be related to the increased expression of neurotrophic factors at the injury site. |
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