Study On Exercise-induced Hypoalgesia In Mice With Neuropathic Pain

Neuropathic pain is a chronic pain state with a complex etiology and currently lacks effective treatments in clinical practice.Nonpharmacological treatment methods such as treadmill training have been shown to alleviate neuropathic pain nociceptive hyperalgesia,but the exact mechanism has not been elucidated to date.Therefore,exploring the mechanism of exercise-induced hypoalgesia(EIH)will provide a theoretical basis for the application of exercise therapy to improve chronic pain and provide a new direction for the treatment of chronic pain.In this study,we will use a neuropathic pain spared nerve injury(SNI)mouse model to investigate the effect of exercise on hyperalgesia in mice with neuropathic pain through treadmill exercise training,and explore the specific mechanisms involved.PART1.Effects of different intensity running exercise on pain behavior in miceObjective:To establish a mouse SNI model of neuropathic pain,to observe the effects of different intensity treadmill training on mouse pain behavior,and to verify the stability of mechanical nociception induced by the mouse SNI model and the effect of exercise on hyperalgesia in mice with neuropathic pain.Methods:Forty C57BL/6 mice,randomly divided into five groups(n=8):sham group(Sham group),SNI model group(SNI group),SNI model+7m/min exercise group(SNT-7 group),SNI model+10m/min exercise group(SNT-10 group)and SNI model+15m/min exercise group(SNT-15 group).The SNI mouse model was used,the Sham group exposed the sciatic nerve and its branches but did not ligate and cut the nerve,and the SNI group did not participate in treadmill training after modeling.The rest of the mice in the SNI exercise group were trained for 60 minutes per day starting from day 3 after modeling and for 5 days per week until 21 days after surgery.Body weight and mechanical paw withdrawal threshold(PWT)were measured preoperatively,3,7,14 and 21 days postoperatively,respectively.Results:Significant mechanical hyperalgesia was observed in the lateral plantar of mice after SNI.Compared with the Sham group,PWT was significantly lower in the remaining four groups of mice starting from 3 days postoperatively and continued until 21 days postoperatively.Compared with the SNI group,PWT gradually increased in the SNT-7,SNT-10,and SNT-15 groups from the 7th day postoperatively and was significantly higher than that in the SNI group at 14 and 21 days postoperatively(P<0.05).There was no significant difference between the analgesic effects of the three different velocity trainings performed in the SNI model mice.PART2.Proteomic study of spinal cord tissue in mice with neuropathic painObjective:It uses Tandem Mass Tag(TMT)proteomics technology to detect neuropathic pain exercise and non-exercise mice spinal cord tissue proteomics to explore the key factors,related signaling networks,and signaling pathway of EIH.Methods:Six mice were randomly divided into 2 groups(n=3),namely SNI model group(SNI group)and the SNI model+exercise group(SNT group).SNI model were established in both groups,and the SNT group was trained by treadmill running at a speed of 10 m/min for 60 min every day from the 3rd day after modeling for 5 days per week until 14 days postoperatively when L4-6 spinal cord tissue were taken from both groups for TMT proteomics detection.Combined with IPA analysis and other bioinformatics analysis methods to screen the differentially expressed proteins,to explore the biological functions and interaction networks of the differentially expressed proteins,and to screen the core factors regulating critical signaling pathways.Results:270 differentially expressed proteins were screened.392 biological processes were enriched by GO analysis,mainly including neurotransmitter transport,signal release from synapse,regulation of membrane potential,etc.137 cellular components were enriched by GO analysis,mainly including postsynaptic membrane,neuron to neuron synapse,proteasome complex,etc.37 molecular functions were enriched by GO analysis,primarily including ubiquitin protein ligase binding,ion channel binding,glutamate receptor binding,etc.Among the top 20 enriched pathways in KEGG pathway analysis,regulation of neurotransmitter levels,autophagy,and regulation of membrane potential are closely related to neuropathic pain.IPA analysis revealed significant enrichment in EIF2 pathway,synaptogenesisrelated signaling pathway,mTOR pathway,cAMP-mediated signaling pathway,autophagy,and neuropathic pain signaling in dorsal horn neurons.Autophagy was the classical signaling pathway located in top 1 of the differential protein significant enrichment analysis in the IPA analysis of proteomics data in this study,suggesting that this pathway may play an important role in EIH.The differential proteins involved in this pathway include ATF2,AKT3,GNAI3,GSK3B,PIK3C3,PIK3CB,PPP2CA,and SQSTM1.PART3.Treadmill training regulates microglial polarization by promoting autophagyObjective:To investigate the effect of autophagy on treadmill training induced microglia polarization in the dorsal horn of the mouse spinal cord by establishing a mouse SNI model,and to further explore the specific molecular mechanisms of autophagy in EIH.Methods:Seventy-eight C57 mice were randomly divided into 5 groups,shamoperated group(S group,n=18),SNI model+non-exercise group(SD group,n=18),SNI model+exercise group(SNT group,n=18),SNI model+exercise+rapamycin treatment group(SNR group,n=12),SNI model+exercise+3-MA treatment group(SNM group,n=12).The S group isolated and exposed the sciatic nerve and its branches but did not damage the nerve,and the rest of the groups established SNI mouse models.The mice in the exercise groups were trained by running at a speed of 10 m/min for 60 min every day from day 3 after modeling,and the training was performed 5 days a week until 14 days after surgery.10 mg/kg of rapamycin and 15 mg/kg of 3-MA were injected intraperitoneally every day from 30 minutes after SNI in the SNR and SNM groups,respectively.PWT was measured in mice before surgery and 3,7 and 14 days after surgery,respectively.The expression of autophagyrelated proteins LC3,Beclinl and p62,as well as CD86 and CD206 proteins were detected by protein blotting and double immunofluorescence in the L4-6 segment of the spinal cord of mice on 14 days postoperatively.The formation of autophagosomes in the spinal dorsal horn was observed by electron microscope in each group of mice.Results:PWT levels were significantly higher in the SNT group compared with the SD group at 7 and 14 days postoperatively.Double immunofluorescence and western blot showed that both CD86 and CD206 protein expression in the spinal dorsal horn were significantly increased in the SD group of mice after SNI compared with the S group.CD206 protein expression was further increased after treadmill exercise training(P<0.05),while CD86 expression was decreased(P<0.01).CD206 expression was significantly decreased in the SNM group compared with the SNT group,while CD86 protein expression was increased.The expression of CD206 protein in the SNR group was significantly increased,while the protein expression of CD86 was significantly decreased.Spinal autophagy-related proteins LC3-II/LC3-I and Beclinl expression was significantly increased in the SD group compared with the S group,and further increased after treadmill training(P<0.05).Compared with the SNT group,LC3-II/LC3-I expression was significantly increased in the SNR group,while LC3-II/LC3-I expression was decreased and p62 expression was increased in the SNM group instead.Transmission electron microscopy showed that swollen and vacuolated mitochondria in the SD group 14 days after SNI,and a small number of autophagosomes were observed in microglia,whereas mitochondria swelling was reduced and the number of autophagosomes was significantly increased after treadmill training.PART4.BDNF/Akt/mTOR pathway is involved in treadmill training to induce microglia autophagyObjective:To observe the role of BDNF/mTOR pathway in EIH and its relationship with microglia autophagy through treadmill training in SNI mouse model.Methods:Forty-eight C57 mice were randomly divided into four groups(n=12):sham operation group(S group),SNI model+non-exercise group(SD group),SNI model+exercise group(SNT group),SNI model+Exercise+BDNF group(SNB group).In the S group,the sciatic nerve and its branches were isolated and exposed without damaging the nerve;in the SD group,the SNI model was established without exercise;in the SNT and SNB groups,the SNI model was established and trained for 60 minutes every day from day 3 until 14 days postoperatively;in the SNB group,BDNF 50 ng/kg was injected intrathecally every 2 days starting 30 minutes after SNI.PWT was measured in mice before surgery and 3,7 and 14 days after surgery,respectively.The expression of autophagy-related proteins LC3 and p62,as well as BDNF,p-TrkB,CD86,CD206,Akt and were detected by western blot and immunofluorescence in L4-6 spinal cord tissues from mice 14 days postoperatively.Results:Spinal cord BDNF and TrkB phosphorylation levels were significantly increased in mice 14 days after SNI,while treadmill training reduced the expression of BDNF(P<0.05)and p-TrkB(P<0.01).Compared with the SNT group,the expression of autophagy-related proteins LC3-II/LC3-I was decreased in the SNB group,while the expression of p62 was significantly increased(P<0.001).Compared with the SNT group,CD86 protein expression was significantly increased and CD206 protein expression was decreased after intrathecal BDNF injection.Spinal cord pAKT and p-mTOR levels were significantly increased in the SD group compared with the S group after SNI(P<0.001),whereas both Akt and mTOR phosphorylation levels were decreased in the SNT group compared with the SD group,and mTOR phosphorylation levels in the spinal cord of mice increased after intrathecal BDNF injection instead.Conclusions:Treadmill training can significantly improve hyperalgesia in mice with neuropathic pain,and there was no significant difference in the analgesic effects of different intensities of training.Autophagy is involved in the generation and maintenance of neuropathic pain,and treadmill training alleviates hyperalgesia symptoms by promoting autophagic flux to regulate microglia polarization toward the M2 phenotype.The BDNF/Akt/mTOR pathway is involved in the process of exercise training-induced microglia autophagy,and the activation of microglial autophagy stimulates their polarization toward the M2 phenotype,thereby attenuating BDNFinduced microglial neurotoxicity and alleviating neuropathic pain hyperalgesia.