The Mechanism Of Spinal Cord Stimulation In Neuropathic Pain By Low Intensity Focused Ultrasound

Objectives:Neuropathic pain(NP)affects 6.9%-10%of the world’s population,and there is still.a lack of effective treatments.Low intensity focus ultrasound(LIFU)can activate the central nervous system,but whether it can activate the spinal cord and regulate neuropathic pain is still unclear.The purposes of this study were to explore the efficacy and safety of spinal cord activation by LIFU,and to explore the efficacy and possible molecular mechanisms of spinal cord stimulation in neuropathic pain.Methods:This research included the following two parts:Part 1:The effectiveness and safety of LIFU stimulation of the spinal cord.1.1.Effectiveness of LIFU to stimulate the spinal cord:L4-L5 spinal cord level was stimulated by LIFU with different Acoustic pressure intensity(API)and Duty cycle(DC).Each API was stimulated for 20 s with an interval of 5 min between each API stimulation.The potential of muscle recruitment was recorded by Electromyogram(EMG).1.2.Safety of LIFU stimulation of the spinal cord.① Study on the safety of different APIs:Using ultrasound parameters of DC=20%and different APIs(API=0 MPa,API=0.5 MPa,API=1.5 MPa,API=3.0 MPa)to stimulate L4-L5 spinal cord of rats for 20min;②Study on the safety of different duty cycles,using ultrasound parameters as API=0MPa group,API=0.5MPa+DC(DC 1=20%,DC2=33%,DC3=50%),and API=1.5 MPa+DC(DC1=20%,DC2=33%,DC3=50%)LIFU stimulates the spinal cord for 20 min.Basso Beattie Bresnahan(BBB)was used to evaluate the motor function of rats.Electrophysiology of spinal cord was detected by Somatosensory evoked potential(SEP)and motor evoked potential(MEP).The gross morphology of the spinal cord tissue was observed with the naked eye,the cell structure was observed by Hematoxylin-Eosin staining(H&E)and Nissl staining,the apoptosis of cells was detected by Tunel staining,and the proteins such as Caspase3 and Bcl2 were detected by WB,and the expression of C-FOS and GAD65 in spinal cord tissue was detected by immunofluorescence(IF)staining.Part 2:The mechanism of LIFU stimulation of spinal cord in NP regulation.2.1.Effectiveness of LIFU stimulation on the L4-L5 level to regulate neuropathic pain.The peripheral nerve injurie(PNI)neuropathic pain rat model was established by selective ligation of sciatic nerve injury(SNI).Different ultrasound parameters(LIFU1+,API=0.5MPa,DC1=20%;LIFU2+,API=0.5MPa,DC2=33%;LIFU3+,API=1.5MPa,DC1=20%)were used to stimulate the spinal cord of NP model rats,the negative control group received negative ultrasound stimulation(LIFU-,API=0 MPa;),and the positive control group(Drug Group)received Gabapentin(1 mg/200 g)orally;Hind limb pain threshold of rats was evaluated by 50%mechanical paw withdrawal threshold(PWT50).2.2.The NP rats were randomly divided into 2 groups(LIFU+Group and LIFU-Group).According to scheme 2.1,effective ultrasound parameters(API=0.5mpa,DC=20%)were used to stimulate L4-L5 spinal cord in both groups for 4 weeks.PWT50 was used to evaluate the pain of the two groups;L4-L5 spinal cord was taken for transcriptomics testing following the last stimulation and PWT50 evaluation.2.3.NP rats were randomly divided into 4 groups:Normal group,Sham group,PNILIFU+group,PNI-LIFU-group.After 4 weeks of LIFU stimulation according to scheme 2.1,PWT50 was used to evaluate the pain of each group of rats;WB,IF,and other experimental techniques were used to test the expression of KCC2,pCREB,p-ERK,CaMK Ⅳ,IRF9,Caspase3,Bcl2,GFAP in the L4-L5 spinal cord;Tunel is used for detection of neuronal apoptosis in spinal cord tissue.2.4.After the successful establishment of NP rat model,WB and IF were used to detect the expression of IRF9,Caspase3,Bcl2 and GFAP at different time points,ie 1 day,3 days,5 days,7 days,and 2 weeks after modeling;Tunel is used for detection of neuronal apoptosis in spinal cord tissue.2.5.IRF9 knockdown/overexpression lentivirus(LV3-IRF9-/LV5-IRF9+)was injected into the spinal cord tissue of SD rats,while the control groups were injected with IRF9 lentiviral empty virus(LV3-NC/LV5-NC)and PBS in negative control group.The WB and IF were used to detect the expression of IRF9,Caspase3,Bcl2,GFAP in spinal cord tissue;Tunel is used for detection of neuronal apoptosis in spinal cord tissue.2.6.One week after IRF9 knockdown/overexpression lentivirus was injected into the spinal cord,an animal model of neuropathic pain was established by selective ligation of peripheral nerves.SD rats were randomly divided into 6 groups:Normal group,PBS-PNI group,LV3NC-PNI group,LV3IRF9--PNI group,LV5NC-PNI group,LV5IRF9+-PNI group.In the second week after modeling,WB and IF were used to evaluate the expression of IRF9,Caspase3,and Bcl2 in the spinal cord tissue of each group of rats.Tunel stain was used to detect apoptotic nerve cells in spinal cord tissue.Results:Part 1:The effectiveness and safety of low-intensity focused ultrasound stimulation of the spinal cord.1.1.When API≥0.5MPa,LIFU stimulation activated the spinal cord nerve circuit in rats and cause the electromyographic recruitment of the soleus muscle(Sol)of the hind limbs;under the same DC,with the increase of the API,the recruitment of Sol had increaseed with an increasing EMG amplitude value(P<0.05);under the same API,with the increase of the DC,the recruitment intensity of Sol also increases with an increasing EMG amplitude value(P<0.05);1.2.When the API=1.5MPa,DC=20%,or API=0.5MPa,DC=20%,33%or 50%,LIFU stimulation did not cause spinal cord damage;There were no significant differences in the morphology of spinal cord,H&E and Nissl staining,SEP and MEP detection,expression of Caspase3 and Bcl2,number of apoptotic cells in the ultrasonic stimulation group compared with the sham stimulation group(P>0.05).1.3.When API=3.0MPa,DC=20%or API=1.5MPa,DC=50%&33%,ultrasound intensity stimulation can cause spinal cord hemarrhage;H&E and Nissl staining showed significant changes in the structure of the spinal cord,and electrophysiological testing showed a decreasing of SEPs amplitude(P<0.05),the expression of Caspase3 and Bcl2 protein was significantly increased(P<0.05),the number of Tunel positive cells was increased(P<0.05),and the Immunohistochemical staining showed that the intensity of GFAP-positive cells(astrocytes)was increased(P<0.05);Part 2:Effectiveness and mechanism of LIFU spinal cord stimulation for neuropathic pain2.1.After establishing the NP model animal,the PWT50 had decreased significantly(P<0.05).After 3 weeks of LIFU stimulation,PWT50 increased in LIFU1+group compared with LIFU-group(P<0.05),but no statistical difference between LIFU1+group and LIFU-group(P>0.05).After 4 weeks of treatment,PWT50 increased in LIFU2+group compared with LIFU-group(P<0.05),but there was no statistical difference between LIFU3+group and LIFU-group(P>0.05).2.2.Transcriptomics results showed that there were 328 differential gene expressions between LIFU+group and LIFU-group,including 216 down-regulated genes and 112 up-regulated genes.Among them,the expression of the key genes of Slc12a5(KCC2;FC=1.851,Padjust=0.013)was up-regulated,Irf9(FC=0.404,Padjust=0.048)and Gfap(FC=0.483,Padjust<0.001)has been down-regulated.2.3.After 4 weeks of LIFU stimulation WB and IF tests showed that compared with the Normal group,the expression of KCC2 and Bcl2 in the LIFU-group was down-regulated(P<0.05),the expression of p-CREB,p-ERK,CaMK Ⅳ,IRF9,GFAP,and Caspase3 appeared up-regulation(P<0.05),and Tunel positive cells had also increased(P<0.05).Compared with LIFU-group,the expression of KCC2 and Bcl2 in the LIFU+group was up-regulated(P<0.05),and protein expressions such as p-CREB,p-ERK,CaMK Ⅳ,IRF9,GFAP,Caspase3 were down-regulated(P<0.05),and Tunel-positive cells were also down-regulated(P<0.05).2.4.3 days after the establishment of NP model,GFAP expression in spinal cord tissue began to increase(P<0.05);On the 5th day after modeling,the expression of IRF9 and Caspase3 began to increase(P<0.05),the Bcl2 decreased(P<0.05),and the Tunel positive cells in spinal cord tissue increased(P<0.05).2.5.After injecting with IRF9 knockdown/overexpressed lentivirus to the spinal cord,the expression of IRF9 was down-regulated/up-regulated compared with the PBS control or lentiviral empty injection group(P<0.05),while there is no significant statistical difference on the expression of Caspase3,Bcl2,GFAP,and Tunel positive cells among the three groups(P>0.05).2.6.Compared with PBS-PNI group and LV3NC-PNI group,the expression of Caspase3 of LV3IRF9--PNI had decreased(P<0.05),and the expression of Bcl2 increased(P<0.05),Tunel positive cells decreased(P<0.05);Compared with PBS-PNI group and LV5NC-PNI group,LV5IRF9+-PNI group showed a significant increase in Caspase3(P<0.05),Bcl2 decreased more significantly(P<0.05),and Tunel positive cells appeared more significant Increase(P<0.05).Conclusions:1.When API≥0.5 MPa,LIFU effectively activated the spinal nerve circulation and cause the recruitment of Sol muscles and recorded by EMG.2.When the API=1.5 MPa,DC=20%,or API=0.5 MPa,DC=20%,33%&50%,LIFU is a safe spinal cord stimulation method;when API=3.0 MPa,or API=1.5 MPa,DC=50%&33%,LIFU stimulation for 20 min can cause spinal cord injury.3.When API=0.5 MPa and DC=20%,LIFU stimulation of the spinal cord can significantly increase the PWT50 threshold of neuropathic pain rats;its molecular mechanism may be through inhibiting the expression of p-CREB and CAMK Ⅳprotein in the spinal cord and up-regulating the expression of KCC2.4.LIFU stimulation for spinal cord may inhibit the neuroinflammatory response and apoptosis in spinal cord of NP rats.LFIU inhibited the apoptosis of spinal neurons in NP rats by decreasing the expression of IRF9 in spinal neurons.5.IRF9 promoted the apoptosis of spinal cord neurons in rats with neuropathic pain.Knockdown IRF9 inhibited the apoptosis of spinal cord neurons,while overexpression of IRF9 promoted the apoptosis of spinal cord neurons in rats with neuropathic pain.