| Background and objective:Chronic pain include inflammatory pain and neuropathic pain, it is a major challenge in the clinical treatment,20%of adults suffer more affliction from chronic pain. Chronic pain involves complex regulatory mechanism and neural plasticity, because there are various receptors, neurotransmitter, ion channels and second messenger. So the treatments are insufficient and ineffective, or there are serious side effects, and it is difficult to find a specific and effective treatment. But ion channels as drug targets, which are in cell membrane surface, are easy to control. So it is meaningful to clinical ion channels disease, such as human single gene pain syndromes. In recent researches, ion channels which expressed by immune system cells (e.g. P2X7) played an important role in the occurrence and development of chronic pain. At same time, ion channels involved in sensory conduction, the regulation of neuronal excitability, action potential propagation and neurotransmitter release, and ion channels have also all been proved to be specific and selective targets of the analgesics in some pain models of animal. Also, some chronic pains have a close relation with ion channels monogenic mutations. Therefore, it is provide specific and potentially selective targets of treat chronic pain in the clinical treatment for us by understanding channelopathies deeply.According to genetic research over the past few years, Nav1.7of voltage-gated sodium channel dysfunction is closely related to human three single gene pain diseases caused by the genetic defect in SCN9A. That is the primary erythema erythromelalgia and paroxysmal extreme pain disorder, in which the disorders result from gain-of-function mutations of Nav1.7, and congenital indifference to pain, in which the disorder result from loss-of-function mutation of Navl.7. Thus, Nav1.7is also a key factor in the occurrence and development of the pain. In this study, we observed the expression changes of voltage-gated sodium channel Navl.7in L5spinal dorsal root ganglion (DRG) in the rat model of chronic constriction injury of the sciatic nerve (CCI, ligation of right sciatic nerve)-induced neuropathic pain and right plantar pedis of the rats received the injection of complete Freund’s adjuvant-induced inflammatory pain model by immunohistostaining, westernblot and reverse transcription-PCR, and we observed the changes of hyperalgesia threshold during chronic pain by behavioral tests, we revealed the relationship between expression of Navl.7of voltage-gated sodium channel in spinal dorsal root ganglion and pain in chronic pain.The research found that Navl.7expression in the dorsal root ganglion of rats had changed in inflammation pain and neuropathic pain. We observed the distribution of Nav1.7-positive neurons in L5dorsal root ganglion and explored how the nociceptive stimulus play a crucial role in large or small neurons by Immunofluorescence double-labeled staining.Methods:1. Establishment of Chronic constriction injury of the sciatic nerve model and inflammatory pain model:114SD male rats were randomly divided into5groups:CCI model group (n=30), inflammatory pain model group (n=30), CCI sham operation group (CCI Sham group, n=22), inflammatory pain sham operation group (inflammatory pain sham group, n=22) and control group (n=10). CCI model group received ligation of right sciatic nerve; The CCI sham surgery exposed only the right sciatic nerve. Inflammatory pain model group received the injection of complete Freund’s adjuvant (100μl), The inflammatory pain sham received the injection of9%physiological saline (100μl). Control groups did not receive any surgery and injection.2. Behavioral Observations and Tests Mechanical withdrawal (MW) and paw withdrawal latency (PWL) to evaluate mechanical hyperalgesia and thermal hyperalgesia. Measurements were carried out between8:00～10:00am,5rats from each group were used for behavioral testing at1,3,7,14,28days after surgery and1day before surgery.3. RT-PCR for Nav1.7mRNA expression in the right L5DRG in neuropathic pain and inflammatory pain ratsThree rats from each group were used for RNA extraction of the right side of L5dorsal root ganglion at1,7,28days after surgery, and a two-step method was used for mRNA extraction, then we detect mRNA relative expression of Nav1.7by RT-PCR and agarose gel electrophoresis.4. Immunohistochemistry for Nav1.7expression in the right L5DRG in neuropathic pain and inflammatory pain ratsThe right L5dorsal root ganglions of three rats from each group were used for paraffin-embedded biopsy at1,7,28days after surgery. Sections were cut using slicing machine, immunohistochemical staining labeled Nav1.7, then we photographed by optical microscope, and image pro plus6.0analyzed positive expression of Nav1.7, we analyzed the changes of Nav1.7expression.5. Western blot for Nav1.7expression in the right L5DRG in neuropathic pain and inflammatory pain ratsThree rats from each group were used for protein extraction of the right side of L5dorsal root ganglion at1,7,28days after surgery, and we observed Nav1.7relative expression by Western blot6. Immunofluorescence double-labeled staining for Subcellular localizationRespectively, we take positive tissue sections of the right L5dorsal root ganglion of rats at1,7day after surgery and control group, and the tissue sections were labeled Nav1.7and NF200(the marker of large neuron), and we observed the distribution of Nav1.7in dorsal root ganglion neurons.Results:1. Changes of behavior in neuropathic pain and inflammation pain ratMTW and PWTL were decreased in CCI group and inflammatory pain group after surgery. Compared to sham group and control group, CCI group and inflammatory pain group occur significant threshold of thermal and mechanical hyperalgesia from the3day to28day after surgery (P<0.05), following gradual recovery. Compared to the control group, there were no significant changes in paw withdrawal latency threshold and mechanical threshold in the sham group (P>0.05). These results indicate that the CCI model and inflammatory pain model was established successfully.2. RT-PCR for Nav1.7expression in the right L5DRG in neuropathic pain ratThe expressions of Nav1.7-mRNA were increased in CCI group and inflammatory pain group after surgery. Compared to sham group and control group, expressions of Nav1.7-mRNA were significantly increased in the injured dorsal root ganglion of CCI group and inflammatory pain group at1,7,28days (P<0.05).3. Immunohistochemistry for Nav1.7expression in the right L5DRG in neuropathic pain and inflammation pain ratsThe expressions of Nav1.7were increased in CCI group and inflammatory pain group after surgery. There was a significant increase (p<0.05) in the expression of Nav1.7in the right L5DRG specimens of CCI group and inflammatory pain group at1,7,28days after surgery, compared to the sham groups and control groups.4. Western blot for Nav1.7expression in the right L5DRG in neuropathic pain and inflammation pain ratsCompared to the control groups and sham groups, Nav1.7levels were significantly increased in the model group (P<0.05), These results indicate that Nav1.7expression level changed with the behavioral alteration of rat right foot pain model in CCI group and inflammatory pain group.5. Subcellular localization of Nav1.7in the right L5DRG of CCI group and inflammatory pain groupImmunofluorescence double-label indicated that, compared to the control groups, Nav1.7expression were significantly increased, and small neurons were significantly increased, whereas Nav1.7expression enhanced in large neurons。 In control group, Nav1.7expression less and weak in large neurons and small neurons.Conclusions:1.In neuropathic pain and inflammation pain rats, voltage-gated sodium channels a subunit——Navl.7, in which are activated in dorsal root ganglion, and Navl.7expression were significantly increased in neuropathic pain and inflammation pain. It is indicated that algesthesia change concerned with Navl.7activation in neuropathic pain and inflammation pain rats.2.In neuropathic pain and inflammation pain, voltage-gated sodium channels play a key role in algesthesia signal transduction, It is indicated that Navl.7play a key role by large neurons and small neurons, but small neurons may be more important. Nociceptive stimulus may propagate themselves by small neurons. |
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