Mechanism Of Allodynia Signal Regulation Induced By Tumor Necrosis Factor-alpha In The Red Nucleus

Objective:Tumor necrosis factor-alpha (TNF-α) in the red nucleus (RN) playsfacilitated roles in the development of abnormal pain. Here, the roles of nuclearfactor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), p38mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) inTNF-α evoked mechanical allodynia were investigated.Methods:1Establishment of the pain model in ratsRepeated microinjection of recombinant rat TNF-α (20ng daily for3days) into theunilateral RN of normal rats induced a significant mechanical allodynia in thecontralateral but not ipsilateral hind paw at the fifth day and disappeared24h later.Re-injection of a single bolus of20ng TNF-α into the same RN reproduced thismechanical allodynia within30min, which was used as a pain model for furtherexperiments.2Detection of Behavioral changes and abnormal painAll rats were habituated in the experimental arena for20min daily during the3daysbefore testing. Briefly, the rat was placed under a transparent plastic chamberpositioned on a metal wire mesh floor, and ten von Frey filaments were applied tostimulate the hind paw.Make von Frey nylon bent into a form of S or C shape, for6to8second. Observe reactions,the rats raise the paws was positive reaction. Andusing the same method to stimulate normal rats, the above reaction.3Drug application and ImmunohistochemistryThrough the red nucleus microinjection NF-κB, ERK,JNK and p38MAPK inhibitorto model rats. Then observe the changes of starting phase and maintaining phase, at the same time through immunohistochemistry to observe the expression changes.4Data analysisStatistical analyses were performed using SigmaStat2.03and all data werepresented as mean±SEM. Differences in drug effect among groups were testedstatistically by two-way repeated measures of analysis of variance (two-way RMANOVA) with a multiple comparison for analysis of the differences in entireobservation time or at each time point among different groups. P<0.05wasconsidered statistically significant.Results: Immunohistochemistry demonstrated that NF-κB, phospho-ERK(p-ERK) and p-p38MAPK in the RN were significantly up-regulated at1h afterTNF-α microinjection, the up-regulations of NF-κB and p-ERK but not p-p38MAPK remained at high levels till4h later. A significant up-regulation of p-JNKoccurred at4h (but not1h) after TNF-α microinjection, which was later than thoseof NF-κB, p-ERK and p-p38MAPK. Pre-treatment with NF-κB inhibitor PDTC,ERK inhibitor PD98059or p38MAPK inhibitor SB203580at30min before TNF-αmicroinjected into the RN completely prevented TNF-α-evoked mechanicalallodynia. Pre-treatment with JNK inhibitor SP600125did not prevent but reversedTNF-α-evoked mechanical allodynia during the subsequent detection time.Post-treatment with PDTC, PD98059or SP600125(but not SB203580) at4h afterTNF-α microinjected into the RN significantly reversed TNF-α-evoked mechanicalallodynia.Conclusion: These results further prove that TNF-α in the RN plays a crucialrole in the development of abnormal pain, and the algesic effect of TNF-α isinitiated through activating NF-κB, ERK and p38MAPK. The later maintenance ofTNF-α-evoked mechanical allodynia mainly relies on the activation of NF-κB, ERKand JNK, but not p38MAPK.