The Effects Of Glucocorticoid And It's Receptor On The Neuropathic Pain Of Rat In Spinal Level

Pain can be classified as either nociceptive or neuropathic according to pathologic characters.Neuropathic pain(NP) is defined as "pains resulting from disease or damage of the peripheral or central nervous systems,and from dysfunction of the nervous system"(IASP 1994).The neuropathic pain is often associated with the significant appearance of abnormal sensory signs of allodynia,hyperalgesia and spontaneous pain. It usually occurs without peripheral nociceptive stimulation,so is irrelated to the intensity and characters of the stimulation.Neropahtic pain is not only a tough disease in clinic,but also is a hot research topic in the field of neuroscience.Before,pain is generally considered as purely neural phenomenon,but now,it is recognized that pain and its modulation are also involved in neuroimmune interaction.Much more studies have revealed that astrocytes and microglia are also immunocompetent cells in spinal cord and thus can respond like immune cells within the central nervous system.Inhabited the activation of them may alleviate the hyperalgesia.Now,there have some agents which could modulate the glia cell' function those are propentofylline,pentoxifylline and fluorocitrate,et al.But now these agents are still in experimental researches,and have not been used in patients.In view of the clinical practice,glucocorticoids have been used frequently in pain management,and they have obvious pharmacological effects of anti-inflammation and immune inhibition.So we speculate that the glucocorticoids may modulate the neuropathic pain by depressing the activation of astrocytes in spinal cord. Which need to be tested by more experimentes.Moreover there have many specific issues about using glucocorticoids such as the pathway,dose,type, period treatment,et al.Recently Mao et al advanced that activation of the central glucocorticoids receptors will induce the hyperalgesia. Moreover as we know that the inappropriate use of glucocorticoids may cause severe adverse effects.So it will be beneficial for practical pain management to learn more about the mechanisms of glucocorticoids and its receptors in neuropathic pain.In the present study,first we establish the rat spared nerve injury(SNI) model,and then to observe the behavioral outcomes and the spinal cord morphological and biochemical results by means of behavioral parameters testing,immunohistochemical,Western blot and ELISA after given diprospan or RU486 in different ways on the rat's SNI model.Materials1,Animals:adult male SD rat weighting 250-350g,were provided by the experimental animal center of China Medical University.2,Major reagent:Diprospan,RU486,Anti-Rabbit Glial fibrillary acid protein,Anti-Rabbit Glucocorticoids Receptor antibody,Anti-Goat Nuclear transcription fator-κB p65,TNF-αand IL-1βELISA kits.3,Major instrument:Von Frey hair,BME-410 Full-automatic plantar analgesia tester, electrophoresis,semi-dry blotter,Spectrophotometer,MetaMorph/Olympus C-5050/BX41 photo analyse system,Fluochem V2.0 photo analyse systemMethods 1,intrathecal catheterization:Rats were anesthetized by intraperitoneal injection of pentobarbital sodium(40mg/kg),and the catheter was introduced into the subarachnoid space using a previously described modification of the method of Yaksh and Rudy:catheters were passed through a slit in the atlanto-occipital membrane and advanced 11 cm,to lie with the tip caudal to the conus medullaris.Animals were allowed to recover for 24 h before the study began. Rats that exhibited any evidence of sensory or motor dysfunction were excluded from the study2,Animal model of the spared nerve injury:After anesthesia the skin on the lateral surface of the thigh was incised and a section made directly through the biceps femoris muscle exposing the sciatic nerve and its three terminal branches:the sural, common peronea]and tibial nerves.The SNI procedure comprised an axotomy and ligation of the tibial and common peroneal nerves leaving the sural nerve intact.The common peroneal and the tibia]nerves were tight-ligated with 5.0 silk and sectioned distal to the ligation,removing 2±4 mm of the distal nerve stump.Great care was taken to avoid any contact with or stretching of the intact sural nerve.Muscle and skin were closed in two layers.3,The method of mechanical withdrawal threshold(MWT)testingAccording to Kingery's methods using Yon Frey hair to operate.4,The method of thermal withdrawal duration(TWD)testingAccording to Hargreaves's methods using BME-410 Full-automatic plantar analgesia tester to operate.5,Immunohistochemical method:To exame GFAP.6,Western blot method: To exame NF-κB and GR.7,ELISA:To exam TNF-αand IL-1β.8,Experimental paradigm:8.1 PartⅠ:Behavioral parameters testing48 rats were divided into six groups as:1 sham group(Sh group)2 SNI group(S group)3 SNI+diprospan intrathecal injection group(SD group)4 SNI+diprospan local injection(Sd group)5 SNI+RU486 intrathecal injection(SR group)6 SNI+RU486 intrathecal injection+diprospan local injection(SR-d group)MWT and TWD were detected at before operation,post-operative day 1,3,7,14,21,28 respectively.8.2 PartⅡ:Morphological and biochemical testing172 rats were divided into eight groups as below,in N group n=4,in other groups n=24.1 normal group(N group)2 sham group(Sh group)3 SNI group(S group)4 SNI+N.S intrathecal injection(SN group)5 SNI+ diprospan intrathecal injection group(SD group)6 SNI+ diprospan local injection(Sd group)7 SNI+ RU486 intrathecal injection(SR group)8 SNI+ RU486 intrathecal injection+diprospan local injection(SR-d group)Except the normal group the other groups rat's lumbar enlargement spinal cord were removed at post-operative day 1,3,7,14,21 28 respectively.9,Date analysis and statistics:Date were expressed as mean±SEM.The results were analyzed by analysis of variance(ANOVA)with Student-Newman-Keuls as the post hoc test.Values of P＜0.05 were considered as statistically significant.Results(一) Behavioral parameters results:1,Effects of spared nerve injury on the MWT and TWD:There were no significant differences on the values of MWT and TWD before ligation between Sham and SNI group.Compared with Sham group,the value of rats' MWT and TWD in SNI group had significant changes after ligation and maintained the state until 28 days afer ligation.2,Compared with SNI group the SD group and the SR group have no significant changes on the values of MWT and TWD.While the Sd group had a little significant changes and the SR-d group had the most significant difference on the values of MWT and TWD.(二) Rat's spinal GFAP results:The expression of GFAP in SNI group increased significantly,and which were depressed significantly in group Sh and group SD.There had no significant changes in group SR and SN.(三) Rat's spinal NF-κB P65 results:The relative density of NF-κB P65 significantly increased in group S,and which were significantly depressed in group SD and Sd.There had no significant changes in group SN and SR.(四) Rat's spinal TNF-αand IL-1βresults:The content of TNF-αand IL-1βsignificantly increased in group SNI,and which were significantly depressed in group SD and Sd.There had no significant changes in group SN and SR.(五) Rat's spinal GR resultsThe expression of GR increased after the spared nerve injury.There had no significant changes in group Sd.While the expression of GR increased significantly in group SD.Because of the intrathecal injection of RU486 in group SR and SR-d,there had significant decrease of GR's expression in spinal cord.Conclusion1,Ghcocorticoids can significantly depresse both of the activation of spinal astrocyte and the expression of NF-κB P65,also it can reduce the content of TNF-αand IL-1βin spinal cord.2,The activation of central glucocorticoid receptors could cooperate with the neuropathic pain of rat's SNI model.3,The local injection diprospan combined intrathecal injection of RU486 exerted not only the anti-inflammation function of ghcocorticoids but also avoided activating the central glucocorticoid receptors,so achieved excellent alleviation on the hyperalgesia.