| Part one: Establish and evaluation a rat model ofinflammation painObjective To establish and evaluate the rat model of inflammation pain induced byComplete Freund’s Adjuvant using behavioral tests.Methods Unmated male Sprague-Dawley rats (weight,180-220g) provided by theExperimental Animal Center of Soochow University were used in this study. IP wasinduced by intraplantar administration of CFA(50μl into the surface of the right hindpaw) suspended in an oil saline (1:1) emulsion. The rats assigned to the sham groupreceived50μl of normal saline. Pain-related parameters were collected prior tointraplantar injection,2、4、6、8、12h、1d、4d and14d after the intraplantar administrationof CFA or saline.Results After intraplantar injection of CFA2～3h, the right hind paw occurredinflammatory reactions, edema and red plaque. After4~5hours, significant behavioralhypersensitivity was observed on the rats in response to peripheral administration of CFA.Furthermore, the state of behavioral hypersensitivity lasts to14days after intraplantarinjection of CFA.Conclusion The present study successfully established a model of inflammationpain by CFA. Part two: The expression of HMGB1, TNF-α and IL-1β in spinalcord of the rat model of inflammation painObjective To observe the expression of HMGB1, TNF-α and IL-1β in spinal cord of the rat model of inflammation pain.Methods The expression of high-mobility group box1(HMGB1) in Spinal cord ofnomal, sham and inflammation pain group was detected by immunohistochemistry,western-blot, real-time-PCR after peripheral administration of CFA4、12hours and1、4、14days. In addition， the expression of tumor necrosis factor alpha (TNF-α) andinterleukin-1beta (IL-1β) was measured with real time-PCR on the same time and in thesame part.Results Compare with normal rats, the expression of HMGB1mRNA and Protein ofthe rat model of inflammation pain significantly increased after CFA injection12hours,and1,4,14days(P＜0.05), but not on the time of4hours (P＞0.05). The expression ofTNF-α and IL-1βmRNA increased on the all time (P＜0.05).Conclusion In spinal cord, HMGB1as a late proinflammatory cytokine plays apotent role in the inflammation pain. Part three: Pretreated administration of anti-HMGB1antibodyaffecting inflammatory pain in ratsObjective To evaluate the role of high mobility group box1(HMGB1) indevelopment of inflammatory pain in rats.Methods Forty adult male SD rats weighing180-220g in which intrathecal (IT)catheters were successfully implanted according to the method described by Yangwithout complication were used in this experiment. Inflammatory pain (IP) was inducedby subcutaneous (sc) injection of complete Freund′s adjuvant (CFA)50μl at the lateralside of ankle joint of the right hindpaw. Animals were randomly divided into5groups(n=8each): group Ⅰ sham operation-normal saline (NS)50μl was injected sc instead ofCFA; group Ⅱ（IP）; group Ⅲ control antibody IgG IT+IP（IgG+IP）; group Ⅳ anti-HMGB1antibody (Ab) IT+sham operation-Ab20μg/10μl was injected IT at30min before NS50μl sc injected and group Ⅴ （Ab IT+IP）. The behavioral testswere measured2d before induction of IP (T0,baseline), before and at4,8,12and24h (T1～5) after sc or CFA injection. All animals in each group were killed afterbehavioral tests. The lumber segment L4～6of the spinal cord was removed fordetermination of the expression of HMGB1by western-blot and the expression of TNF-αand IL-1βmRNA by real-time PCR.Results MWT and PWTL was significantly shortened after sc CFA in group ⅡandⅢ as compared with group Ⅰ. Mechanical and thermal hyperalgesia induced by sc CFAwas significantly suppressed by IT anti-HMGB1antibody in group Ⅴ but control IgGdid not. IP significantly increased HMGB1expressed in the spinal cord. IT anti-HMGB1antibody significantly attenuated IP-induced up-regulation of HMGB1, TNF-α andIL-1βmRNA in the spinal cord.Conclusion The activation of HMGB1in the spinal cord may be involved in thedevelopment of CFA-induced hyperalgesia in rats. Part four: The antinociceptive effect of intrathecaladministration of anti-HMGB1antibody in inflammatory ratsObjective To investigate the antinociceptive effect of intrathecally(i.t.)administration of anti-HMGB1antibody in inflammatory pain rats.Methods Adult male SD rats weighing180-220g in which intrathecal (IT) catheterswere successfully implanted were used in this experiment. Inflammatory pain (IP) wasinduced by subcutaneous (sc) injection of complete Freund′s adjuvant (CFA)50μl at thelateral side of ankle joint of the right hindpaw. The anti-HMGB1antibody or controlantibody IgG was intrafhecal injected at30min,1day and14day after the IP modelsestablished. The behavioral tests were measured after intrafhecal injection of administrations4hours. The lumber segment L4～6of the spinal cord was removed fordetermination of the expression of HMGB1by western-blot and the expression of TNF-αand IL-1βmRNA by real-time PCR.Results Anti-HMGB1iduced an antinociceptive effect on days4and14post-injection (P <0.05), but not4h (P>0.05) after the intraplantar administration ofCFA. Treatment with anti-HMGB1significantly decreased, at the protein and mRNAlevels, the expression of HMGB1in rats on days4and14(P <0.05), but no differencewas observed at4h (P>0.05). Intrathecal injection of anti-HMGB1significantlydown-regulated IL-1β and TNF-α expression on days4and14(P <0.05), but failed toreduce the expression at4h compared with vehicle treatment (P>0.05).Conclusion These results suggest that HMGB1plays an important role in thedevelopment of IP induced by intraplantar administration of complete Freund’s adjuvant.HMGB1blocking therapy holds potential in the treatment of IP. |
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