| After spinal cord injury,myelin-forming cells die in large numbers so that axons will be demyelinated.This change of myelin sheath makes the electrical signal transmission of the axon blocked,which would eventually lead to individual motor and sensory dysfunction.How to promote the regeneration of myelin sheath rings and the recovery of body structure and function after demyelination,is currently the focus and difficulty worldwide.During the body's developmental period,the whole process of myelination including the migration,proliferation and differentiation of OPCs,maturation of oligodendrocytes,and myelination of axons.A complete myelin sheath structure is necessary for maintaining the normal conduction of axons.And studies have also shown that neuronal activity,as an axon signal,plays a crucial role in the developmental myelination process.In addition,some studies have found that after SCI,enhancing neuronal activity can effectively promote OPCs' proliferation and differentiation,OLs maturation,as well as improving the ability of axon remyelination.Therefore,studing how neuronal activity can affect the regeneration process of myelin sheath would provide us a theoretical basis for clinical application of diseases such as spinal cord injury and multiple sclerosis,and futher researches are needed.As a valuable scientific research tool,chemicogenetic technology can interfere or overexpress certain substances through small chemical molecules to achieve the regulation function of specific proteins in physiological processes following genetic principles.DREADDs(designer receptors executively activated by designer drugs)featured in precise control belongs to one of those chemicogenetic technologies.Through the combined use of N-clozapine(CNO),it can be a great manner for specific target neurons at specific times.The excitability of the neurons has attracted the attention of many neuroscience researchers.The anatomical and functional positioning of mice shows that pyramidal bundles controlling the motor function of mice hind limbs mainly distribute in the cortical spinal bundles of posterior cord of the tenth thoracic segment and below.These axons are mainly originated from the fifth or sixth pyramidal neurons of the somatic motor cortex.Therefore,in this study,after demyelination of the dorsal corticalspinal tract caused by a mild contusion spinal cord injury in the T10 segment of mice,we controlled neuronal activity of pyramidal neurons in the fifth layer of M1 through DREADDs strategy to explore how different levels of excitability of neuronal activity(Activation/Normal/Inhibition)can affect the process of d CST remyelination and motor function recovery.Methods:1.d CST demyelination verification of mild spinal cord contusion model: through Allen's impact device,the impact weight is 5g.Animals were divided into 4 groups,including sham group,2.5mm×5g group,5mm×5g group,7.5mm×5g group.The impact force caused spinal cord injury in mice T10 segment.Then the severity of spinal cord injury was evaluated by BMS scale;2 weeks after SCI,the demyelination of d CST in each group was examined by EC staining,immunofluorescence staining,and the transmission electron microscopy.2.First,the stereotaxic injection of the virus was performed in the primary motor cortex(M1)of adult wild-type mice at layer V(activated type: p AAV-h Syn-HA-h M3D(Gq)-IRES-m Citrine,inhibited type: p AAV8-h Syn-DIO-h M4D(Gi)-m Cherry).After 2 weeks of the injection,all animals were subjected to mild spinal cord injury.2 weeks later,daily intraperitoneal injection of mice was started and continued for 4 weeks,in which the experimental group was injected with N-clozapine(CNO),and the control group accepted the same amount of the normal saline.After the intraperitoneal injection was completed,all mice were perfused and sampled,and the changes of oligodendrocyte lineage cells(OPCs,oligodendrocytes)were observed by immunofluorescence staining;the motor function of hind limbs of mice after SCI was evaluated by BMS and irregular horizontal ladders.Results:1.Mouse BMS test: at every single time point detected within two weeks after SCI,the scores of mice in the 2.5mm×5g group were significantly higher than those in the 5mm× 5g group and 7.5mm×5g group(P<0.0001),and at SCI 2w point,there was no significance between 2.5mm×5g group and sham group.EC staining showed that two weeks after SCI,the staining of the sham group was normal within the white matter of the spinal cord;EC staining of the posterior cord tissue of the 2.5mm× 5g group including d CST was significantly lighter;EC staining of the 5mm×5g group became lighter and the range reached the pre-white matter union;in the 7.5mm×5g group,EC staining range also became lighter,and even reached the anterior cord.GFAP immunofluorescence staining: after SCI caused by 2.5mm×5g,the number of astrocytes increased,but the glial scar was very limited and not reached the d CST area;the glial scar seen in the 5mm×5g even extended to the gray matter around the posterior funiculus;in the 7.5mm×5g group,the limits of glial scar exceeded the anterior white gray matter joint,and most of the spinal cord tissue had been damaged severly.Transmission electron microscopy further showed that in the 2.5mm×5g group,most of the axons at d CST remained intact,and extensive myelination was also observed here.2.Immunofluorescence staining detected an increased expression of neuroproto-oncogene protein(c Fos)in the activated group that showed neuronal activity,while a decrease of c Fos expression in the inhibited group.After 4 weeks of neuronal activity regulation,the immunofluorescence staining detected that the MBP fluorescence intensity of the activated group was significantly higher than that of the other three groups(P<0.001).MBP fluorescence intensity of the inhibition group was apparently lower than the other three groups(P<0.001);for the proliferating oligodendrocyte precursor cells(Ki67~+/Pdgf R?~+/DAPI~+),the activation group was significantly higher than the other three groups(P<0.001),the inhibition group was lower than the control group(P<0.05);The mature group had more mature oligodendrocytes(APC/CC1~+Olig2~+/DAPI~+)than the other three groups(P<0.01),while the inhibition group was less than the control group(P<0.05).In addition,the BMS score decreased to the lowest one day after SCI,and then gradually returned to the preinjury level,but there was no significant difference among all groups;irregular horizontal ladders test was used to further detect the motor function of hind limbs in mice.Two weeks after the activation of neuronal activity,error rates of mice left hind limbs were lower than that of the other three groups(P<0.05).After four weeks of activation,error rates of left hind limbs of the stimulated mice were still significantly lower than that of the other three groups(P<0.01).Conclusions:1.Allen 's model of 2.5mm×5g can cause the mild spinal cord contusion injury of T10 in mice,which makes the d CST of this segment show extensive demyelination changes but the retained axons,which meets the needs of the experiment.2.Using chemical genetics strategies DREADDs by stereotaxic injection of brain AAVs combined with intraperitoneal injection of CNO,can successfully activate or inhibit cortical neuronal activity.3.Regulating neuronal activity bidirectionally can effectively control changes of the OL lineage cells and the axon remyelionation,mainly affecting the proliferation of OPCs,maturation of OLs and the axon remyelionation.4.Increasing the cortical neuronal activity can promote skilled motor function recovery after axon demyelination in mice with mild spinal cord contusion injury,however,decreasing neuronal activity has no obvious effect on the skilled motor function recovery. |
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