Study On The Mechanism Of Pain Caused By Nerve Compression Injury Aggravated By Contralateral Monoarthritis

Background:Osteoarthritis(OA)is a common disease in the elderly.The causes of OA are various.Secondary synovitis,progressive degeneration,destruction and even loss of articular cartilage are the main pathological manifestations.The imaging manifestations are the formation of articular cartilage and osteophyte at the edge of subchondral bone.The final results of clinical manifestations are pain,swelling,stiffness,deformity and dysfunction around the joint In many clinical manifestations,joint pain is the most prominent.It mainly affects knees,hips,hands and spine joints.As a painful disease,OA severely damages the body function and reduces the quality of life.Neuralgia caused by neuropathic or sensory impairment is also common in elderly patients,especially those with diabetes mellitus(diabetic neuropathy),herpes zoster(postherpetic neuralgia),low back pain(such as lumbar spinal stenosis),cancer and stroke.Both OA and neuropathic pain(NP)show hyperalgesia,and their comorbidity in elderly patients is not uncommon.When combined with OA and neuropathic pain,drugs for both diseases do not adequately alleviate symptoms.To improve the treatment of these two complications,it is necessary to clarify the pathophysiological relationship between these conditions.Chronic pain after arthritis is associated with chronic inflammation of the peripheral or central nervous system.When arthritis occurs,we can detect that many pro-inflammatory cytokines(IL-1β,TNF-a,IL-6,IL-17A)and chemokines(iNOS,eNOS,COX-2)are elevated in primary afferent neurons and dorsal horn neurons innervating joints.These elevated inflammatory mediators then enhance synaptic transmission in the spinal cord,known as central sensitization,and promote the development of neuropathic pain.Many studies on OA have focused on local or ipsilateral neuroinflammatory response,with the contralateral non-injured side as the control.There is a lack of relevant research on the inflammatory changes of the contralateral nerve and a lack of discussion on the mechanism.Part Ⅰ:Establishment of monoarthritis(MA),nerve chronic constriction injury(CCI)model and detection of pain behaviorObjective:In this study,complete Freund’s preparation(CFA)was injected into the right tibial-tarsal joint space by intra-articular injection to establish a model of uniarthritis and determine whether it affected the contralateral pain behavior.The animal model of sciatic nerve compression injury(CCI)with nerve pain was established by ligating the sciatic nerve to further observe whether the contralateral MA affected the nerve.Through pain behavior,the effect of MA on contralateral nerve compression injury was clarified.At the same time,in order to further determine whether the effect is related to the induction sequence,we changed the induction order to further observe its effect on pain behavior.Methods:1.1 Establishment of monoarthritis(MA)model and detection of pain behavior1.1.1 MA model was established by injecting 50uL CFA into the right tibial tarsal joint of rats under isoflurane inhalation anesthesia to induce MA.The rats in the blank control group were injected with 50μL sterile saline intra-articular.After injection of CFA,the right ankle of rats was markedly swollen,and the animals without or slightly swollen right ankle were removed.1.1.2 Effect of monoarthritis on contralateral pain:Pain was measured in rats of blank control group and MA group four days before induction,including mechanical stimulation foot-contraction threshold and thermal stimulation foot-contraction latency.The determination time was 4 days,2 days before induction,0 days,2 days,4 days,6 days,8 days,14 days and 21 days after induction of MA.1.1.3 Histological confirmation:After two weeks of injection,the right tibial tarsometatarsal joint of rats in control group and MA group was sliced at 5 μm and stained with HE.Microscopic observation showed pathological damage of the tibial-tarsal joint in MA rats,including deformations of the tibia and tarsus,enhanced mononuclear infiltration and synovial cell proliferation.It can be confirmed that we have successfully established a single arthritis model.1.2 Establishment of a model of nerve chronic constriction injury and detection of pain behavior1.2.1 Establishment of CCI model:Rats were anesthetized by intraperitoneal injection of pentobarbital sodium to expose and separate the left sciatic nerve and expose part of it.Four chromic gut ligatures were loosely tied around the sciatic nerveand knotted.The interval between the two adjacent knots was 1 mm.The blood flow outside the nerve was blocked but not blocked.CCI animal model was prepared.1.2.2 Pain behavior test after CCI:Three groups of experimental animals were selected,blank control group(saline injection into tibial tarsal joint),CCI group and CCI+MA group(CFA injection into tibial tarsal joint two days after CCI).Pain was detected 4 days before induction,including mechanical paw withdrawal threshold and Thermal withdrawal latency.The determination time was 4 days,2 days before induction,0 days,2 days,4 days,6 days,8 days,14 days and 21 days after induction of MA.The effects of CCI and CCI combined with contralateral MA on pain behavior were observed.1.3 Induction of monoarthritis before nerve compression injury1.3.1 To further determine whether the order of induction of the two models has an impact on pain behavior,change the order of induction and induce MA 2 days before CCI.The blank control group was injected with saline at the same time.1.3.2 Establishment of nerve compression injury model:CCI model was established by sciatic nerve ligation.1.3.3 Detection of pain behavior:According to the pre experimental scheme,the threshold value of mechanical stimulation and the thermal withdrawal latency were measured.The time was 4 days and 2 days before CCI induction,0 day,2 day,4 day,6 day,8 day,14 day and 21 day after CCI induction.Results:1.After inducing MA,we found that the tibial-tarsal joint of MA rats had pathological damage,including tibial and tarsal deformations,enhanced mononuclear infiltration,synovial cell proliferation and successful establishment of a monoarthritis model.There was no significant difference in mechanical stimulation foot contraction threshold and latency time of thermal stimulation foot contraction reaction before and within 3 weeks after operation between the contralateral MA group and the control group.2.After the establishment of CCI model,compared with the control group,the mechanical paw withdrawal threshold and thermal stimulation foot contraction latency were significantly reduced,lasting for 3 weeks,which confirmed the successful establishment of CCI-neuropathic pain model,showing hypersensitivity to mechanical stimulation and hyperalgesia.Compared with the CCI group,in the contralateral MA group induced 2 days after CCI,the CCI-related mechanical stimulation hypersensitivity increased at 2 days,while the thermal hyperalgesia aggravated at 4 days(the results were statistically significant).The worsening of the pain allergy lasted for 3 weeks.It was proved that the contralateral MA induced by CCI aggravated the mechanical stimulation hypersensitivity and thermal hyperalgesia induced by CCI.3.Similarly,in the contralateral MA group induced by CCI on the first two days,CCI-related mechanical stimulus hypersensitivity increased at 0 days(Fig.2A),while thermal hyperalgesia(Fig.2B)increased at 6 days and lasted for 3 weeks.It can be confirmed that the contralateral MA induced before CCI aggravates the mechanical stimulation hypersensitivity and thermal hyperalgesia induced by CCI.Conclusion:Through the above experiments,we have successfully established animal models of uniarthritis and nerve compression injury,and proved that the model has good repeatability.At the same time,we have confirmed that uniarthritis has no significant effect on contralateral hypersensitivity to mechanical nerve stimulation and hyperalgesia;but in the model of nerve compression injury,both before and after.Post-induced monoarthritis can significantly aggravate mechanical stimulation hypersensitivity and thermal hyperalgesia induced by CCI.Part II:Study on the transmission pathway of monoarthritis affecting neuropathic pain.Objective:In order to further study the mechanism of arthritis causing CCI pain aggravation and explore the pathway,we measured the levels of myelitis factor in the monoarthritis model.Methods:2.1 To prepare MA and control animal models:In group MA,50 L CFA was injected into the right tibial tarsal joint.In the sham group,50 L saline was injected into the right tibial tarsal joint.2.2 after 48 hours of injection,rats were killed,the left and right lumbar(L3-L6)spinal cord was isolated,and RNA was isolated.The integrity of RNA was confirmed in denatured agarose gel,and then reverse transcription polymerase chain reaction(RT-PCR)kit was used for RT-PCR.2.3 The samples were expanded to 25 μL reaction mixture and then detected by PCR(three copies for each sample):The levels of proinflammatory factors(IL-1β、INF-y,IL-17A and TNF-α)in the ipsilateral and contralateral spinal cord of the two groups were detected,and then the levels of chemokines(COX-2 and iNOS)were detected.To observe the effect of MA on inflammatory factors of ipsilateral and contralateral sides in two groups.Results:1.First of all,we found that the levels of IL-1β、INF-γ,IL-17A and TNF-a in the ipsilateral and contralateral sides of the blank control group were at a low level,and the values were basically close to each other.Compared with the uniarthritis group,the levels of IL-1β、INF-y,IL-17A and TNF-α in the ipsilateral side of the uniarthritis group were significantly higher(the difference was significant),and the increase of IL-lbeta was the most significant.To be significant,the increase of IL-17A was the lowest,but there was no significant difference in the contralateral side.2.Secondly,we measured the levels of chemokine COX-2 and iNOS.It was found that the levels of COX-2 and iNOS in the control group were similar to those in the control group,but there was no significant difference between the two groups.The levels of COX-2 and iNOS in the monoarthritis group were significantly higher than those in the control group,especially COX-2.Different from the previous factors,the level of iNOS in the contralateral side of the monoarthritis group also increased significantly,with statistical significance;compared with the same side,the level was slightly lower,but the difference was not significant.Conclusion:It can be concluded that monoarthritis can increase the levels of IL-1β,INF-γ,IL-17A,TNF-a and COX-2 in ipsilateral side,but not in contralateral side;monoarthritis can also elevate the levels of iNOS in ipsilateral side and contralateral side.Part Ⅲ:Inhibitory effects of iNOS inhibitors(1400W)on synaptic transmission and pain sensitization in the opposite side of MAObjective:Since elevated iNOS levels in the dorsal horn of the spinal cord have been reported to be associated with increased neuroexcitability and prominent transmission,we will next study whether the up-regulation of iNOS induced by MA affects synaptic transmission on the other side of the spinal cord,and whether blocking iNOS eliminates this effect and improves pain sensitization induced by MA.Methods:3.1 Upregulation of synaptic transmission by nitric oxide synthase and its inhibition3.1.1 To prepare animal models:MA group:50 μL CFA was injected into the right tibial tarsal joint of rats.Control group:rats were injected with 50 L saline into the right tibial tarsal joint.MA9+1400W group:rats were injected with 10 μg iNOS inhibitor(1400W)immediately after 50μL MA was injected into the right tibial tarsal joint.3.1.2 Patch clamp recording:48 hours after injection,rats were executed,lumbar spinal cord was taken out,sliced,rewarmed in artificial cerebrospinal fluid,immersed,and whole-cell patch clamp recording miniature excitatory postsynaptic currents(MEPSCs).When the stimulation effect reached the maximum of about 20 minutes,sucrose-based cerebrospinal fluid and cerebrospinal fluid containing Forskolin were lavaged,respectively.The frequencies and amplitudes of mEPSCs before and after treatment were compared.To observe the effect of MA on synaptic transmission of Forskolin activation and the changes after iNOS inhibitor application.3.2.Effect of 1400W on hypersensitivity to mechanical stimulation and hyperalgesia in uniarthritis in CCI model3.2.1 To prepare animal models:CCI group:As mentioned above,the left sciatic nerve was ligated with chromic gut ligatures to prepare CCI model.Control group:50μL saline was injected into the right tibial tarsal joint 2 days earlier.MA+CCI group:The contralateral MA model was made by injecting 50μL CFA into the right tibial tarsal joint two days before CCI.MA+CCI+1400W group:Right tibial tarsometatarsal joint was injected with 50μL CFA 2 days before CCI preparation to prepare contralateral MA model,and iNOS inhibitor 1400W 10 μg was injected at the same time.3.2.2 Pain behavior was measured 4 days before preparation of CCI,including mechanical paw withdrawal threshold and thermal withdrawal latency.The determination time was 4 days,2 days before induction of CCI,0 days,2 days,4 days,6 days,8 days,14 days and 21 days after induction.The effect of 1400W on pain behavior was observed.Results:1.First whole-cell patch clamp technique was used to record mEPSCs.The frequency and amplitude of mEPSCs in control and MA rats were the same(Fig.4A).After perfusion with Forskolin,the increase of mEPSCs in MA rats was stronger than that in control rats.In MA group,the forskolin-induced increase in frequency and amplitude of mEPSC in contralateral SG neurons was reduced to the level of control group after pretreatment with 1400 W.2.Secondly,from the aspect of pain behavior,mechanical stimulation hypersensitivity and thermal hyperalgesia were observed in CCI,MA+CCI,MA+CCI+1400W groups compared with the control group,and remained stable throughout the experiment.In addition,1400W pretreatment significantly reduced mechanical stimulation hypersensitivity and thermal hyperalgesia in MA+CCI group,reaching the same level as that in CCI group.These data suggest that inhibition of iNOS may reverse the deterioration of contralateral neuropathic pain induced by MA.Conclusion:It can be concluded that the up-regulation of iNOS in the contralateral spinal cord of MA rats contributes to the enhancement of forskolin-induced synaptic transmission,while 1400W can eliminate the enhancement of Forskolin on mEPSCs in MA rats.At the same time,1400W could reverse the deterioration of contralateral neuropathic pain induced by MA.Full text conclusion:In summary,our evidence suggests that initial localized arthritis can lead to increased iNOS levels in the contralateral spinal cord.This may in turn promote central sensitization(the activity of excitatory synaptic transmission)and exacerbate symptoms of neuropathic pain.This study suggests that patients with neuropathic pain may have more severe pain when combined with OA.We also provide evidence that iNOS inhibitors may have better therapeutic prospects in the treatment of neuropathic pain and arthritis.