The Expression Change Of Spinal TRPV1 And P-JNK Was Involved In Resiniferatoxin-induced Neuropathic Pain

Background and objective:Postherpetic neuralgia( PHN) is a devastating disease, the most common complication of herpes zosters(HZ), which is considered as a neuropathic pain syndrome.In the area of North America, Europe, Africa and Asia, approximately 4 to 4.5 people diagnosed with HZ in 1’000 people in each year. In such a huge number of patients with chickenpox, approximately 10% to 20% of them will develop to PHN. The main clinical manifestation of PHN is characterized by dermatomal spontaneous pain, allodynia and dysesthesia, the symptoms of most patients will last about 3 mouths to 1 year, but minimal of them will persist over years. PHN has a severe effect on life qualities and work conditions to the patients, some serious cases will become depression and suicidal tendency, which will inevitably increase burden of finance and psychology of families and society. The principal methods in nowadays are using plenty of drugs to do symptomatic treatment. In order to make more effective and efficient methods and medication of treatment, hence, figuring out the exact etiology and pathogenesis of PHN is our priority.The aetiological agent behind PHN probably is the latent varicella-zoster virus(VZV) in cranial nerve ganglia, dorsal root ganglia and autonomic ganglia along the entire neuraxis is reactivated, which will leads to virus infection and cascade reaction. At 1990,some scholars described the first in vivo model of VZV latent infection in the adult ratperipheral nervous system and the latency was in long existence. And thus they successfully created the PHN animal model, which grew maturely and were recommended extensively. However, the model is difficult to build, low rate of success and usually ending in failure, including strict culture condition of VZV, difficulty in inducing thermal impairment, developing tissue inflammation, severe skin lesions and paralysis by the virus spreading into the central nervous system. Model building is hard to satisfy the standard levels, perhaps that is the reason why pathogenesis of PHN progress is slow. Scholar Hui-Lin Pan and his colleagues unexpectedly found that systemic injection of Resiniferatoxin(RTX) diminished the thermal sensitivity but increased sensitivity to tactile stimulation in adult rat at 2003, which is pretty similar to the clinical manifestation of PHN patients. Therefore, more and more scientists of pain area accepted the RTX model,the characteristics of such model is steady, convenient and more realistic to clinical manifestation of PHN patients, which has been used as a perfect non-viral PHN model to search for pathogenesis of PHN from symptomatology point.Transient receptor potential vanilloid receptor 1(TRPV1), a nonselective cation/sodium channel, expressed on primary afferent C-fibers, was first cloned by Caterina et al and described as a heat sensor and transducer of thermal nociception. RTX is an ultrapotent capsaicin analog, interacts and activates with TRPV1. Hence, we have reason to believe that TRPV1 is involved in regulation of pain sensation of PHN. Lots of work show that mitogen-activated protein kinases(MAPKs) play an important role in the induction and maintenance of neuropathic pain. Neuropathic pain models as spinal nerve ligation model and sciatic nerve ligation model, in which the expression of pJNK in astrocyte of the spinal cord is increased within a specific time. So we deduce that JNK may participate in control of pain sensation of PHN. To sum up, we have reason to believe that TRPV1 and JNK are involved in regulation of pain sensation of PHN. We can further testify the RTX model is a reliable and steady PHN model whereby the process of our study. Moreover, we may deliver some new theoretical foundation for studying pathogenesis and therapy strategy of PHN.Methods:In our study, firstly, we made PHN model through a single intraperitoneal injection of RTX to SD rats. After multiple time-points behavior test, we mark the patterns of TWL and PWT to assess the RTX model and to make sure the model has been successfully andsteadily built. According to the characteristics of patterns of TWL and PWT, we choose several appropriate time-points to determine the TRPV1 and JNK protein level in the spinal cord through Western Blot. Through patterns of TWL and PWT, furthermore, we choosing several special time-points to inject TRPV1 and JNK sensitive inhibitors intrathecally and observing changes of pattern of TWL and PWT via behavior test,meanwhile, determining TRPV1 and JNK protein levels in the spinal cord through Western Blot and clarify if there exist some changes of targeted protein levels.Results:Part one: the establishment and assessment of RTX induced PHN rat model(1) RTX was dissolved in a mixture of 10% Tween 80 and 10% ethanol in normal saline and the final concentration of RTX is 40ug/ml. In SD rats, RTX induced PHN model through a single intraperitoneal injection of RTX with 200ug/kg to the rats.Behavior test should be made before RTX treatment 2 days and every 3 days in 5 weeks after the RTX treatment. We used heat radiation device and von Frey filaments to determine TWL and PWT, respectively. And found that TWL was markedly increased after RTX treatment 1 day and reached the top(26.0±1.2s) after 2 days, which kept over 5weeks, while the TWL of sham group was not changed obviously(P<0.05). PWT was markedly decreased after RTX treatment 10 days and arrived the bottom(5.2±1.3g) after 2weeks, which also kept over 5 weeks, while the PWT of sham group was not changed obviously(P < 0.05). These data indicate that through RTX treatment to induce PHN model is basically succeed on behavioral point.(2) According to the characteristics of pattern of TWL and PWT, we chose several appropriate time-points to remove lumbar enlargement of spinal cord of rats on ice and determined the TRPV1 and JNK protein level through Western Blot. And Western Blot showed that, compared with sham group, TRPV1 protein level of spinal cord decreased obviously after RTX treatment 1 day and arrived the bottom after 2 days, kept over 5weeks(P<0.05); compared with sham group, JNK protein level of spinal cord increased obviously after RTX treatment 10 days and reached the top after 2 weeks, kept over 5weeks(P<0.05). These data suggest the level of TRPV1 and JNK protein levels may have some close relationship with patterns of TWL and PWT on several special time-points,respectively.Part two: intrathecal injection of inhibitors of TRPV1 and JNK respectively could affect pain sensation of RTX model and expression of TRPV1 and JNK in spinal(1) According to part one, we found that TWL reached its top after RTX management2 days, meanwhile, TRPV1 protein level of spinal cord decreased to its bottom. In the same way, PWT decrease to its bottom and JNK protein level reach its top after RTX management 2 weeks. The above indicates that dysesthesia(TWL increasement) has developed steadily after RTX management 2 days and allodynia(PWT decreasement) has developed steadily after RTX management 2 weeks.(2) Intrathecal injection of Capsazepine(50nmol, a TRPV1 inhibitor) on two time-points: 0.5 hour before RTX treatment and 2 days after RTX treatment. We found that the injection before RTX treatment 0.5 hour could obviously delay TWL increasement(dysesthesia)(P<0.05). However, the injection after RTX treatment 2 days showed little effect on TWL increasement. In the same way, intrathecal injection of SP600126(50nmol,a JNK inhibitor) on two time-points: 0.5 hour before RTX treatment and 2 weeks after RTX treatment. We found that the injection after RTX treatment 2 weeks could obviously alleviate PWT decreasement(allodynia)(P<0.05). However, the injection before RTX treatment 0.5 hour showed little effect on PWT decreasement. The above data on behavioral point manifests that TRPV1 may regulate induction phase of TWL increasement of RTX model, rather than maintenance phase; JNK may regulate maintenance phase of PWT decreasement of RTX model, rather than induction phase.(3) According to the time-points of intrathecal injection, we choosed several appropriate time-points to remove lumbar enlargement of spinal cord of rats on ice and determined the TRPV1 and JNK protein level through Western Blot. We found that TRPV1 protein level of spinal cord increased after intrathecal injection of Capsazepine(50nmol) before RTX treatment 0.5 hour. While, TRPV1 protein level was little changed after intrathecal injection of Capsazepine(50nmol) on RTX treatment 2days(P < 0.05). Similarly, JNK protein level of spinal cord decreased obviously after intrathecal injection of SP600125(50nmol) on RTX treatment 2 weeks. While, JNK protein level was little changed after intrathecal injection of SP600125(50nmol) before RTX treatment 0.5 hour(P < 0.05). The above data on point of protein level of spinal manifests again that TRPV1 may regulate induction phase of TWL increasement of RTX model, rather than maintenance phase; JNK may regulate maintenance phase of PWTdecreasement of RTX model, rather than induction phase.Conclusions:The major innovations of our experiment lie in that we confirm RTX can induce PHN animal model and it mimics the symptoms of PHN patients steadily. We further testify that RTX model is a reliable and steady PHN model. Meanwhile, through several special time-points to inject specific inhibitors intrathecally, we found that TRPV1 and JNK of spinal cord participates in the regulation of pain sensation of RTX model, which may suggest that TRPV1 and JNK are involved in the regulation of pain sensation of PHN patients.(1) The adult rats showed clear changes of TWL and PWT after RTX treatment for a while, and this diphase changes could be steady in a long time. The RTX model could precisely mimic clinical manifestation of PHN patients, which is considered as a reliable and steady PHN animal model. The above information partly reflects that we successfully built an non-viral PHN model induced by RTX, which laid a firm foundation for the subsequent experiment.(2) The expression change of TRPV1 and JNK in spinal were consistent in time window with TWL and PWT in RTX model, suggesting that TRPV1 and JNK in spinal may take part in the regulation of pain sensation of PHN patients: TRPV1 may regulate TWL, JNK may regulate PWT.(3) Through intrathecal injection of specific inhibitors on special time points, from behaviral and spinal protein expression angel, we found and verified that in RTX model TRPV1 may regulate induction phase of TWL increasement of RTX model, rather than maintenance phase; JNK may regulate maintenance phase of PWT decreasement of RTX model, rather than induction phase. These data remind us that on early stage intrathecal treatment of TRPV1 inhibitor without delay may have a positive effect on PHN patients with thermal impairment(TWL increasement / dysesthesia); in the same way, on maintenance stage, intrathecal treatment of JNK inhibitor may have a positive effect on PHN patients with tactile impairment(PWT decreasement / allodynia).