TNF-αMediates Hyperalgesia And Allodynia By Upregulating P-NR1 Expression In Spinal Dorsal Horn In Rats

Recent decades, many researches suggest that after activation of microglia, someproinflammatory cytokines, such as TNF-α, will be upregulated, and subsequently, induce thedevelopment of central sensitization ---- hyperalgesia. However, more evidences are requiredand the mechanisms underlying remind undetermined. In consequence, the present study wasundertaken to determine whether interaction between TNF-αand its receptor TNFR1 couldincrease the phosphoralation of NR-1 on lumbar spinal cord, thereby hyperalgesia developed inrats. The experiment included two parts: 1) to observe the effect of intrathecal application ofanti-rTNF-αin the models of CFA-induced peripheral inflammation hyperalgesia; 2) to conformthat TNF-αand TNFRI are involved in the hyperalgesia by mediating the spinal NR-1phosphorylation.Part IAdult male Sprague—Dawley rats weighing 250-270 g were divided into the following 4groups randomly:< Saline+vehicle > as control, 24h and 2h prior to saline peripheral injection and 23h aftersaline injection, PBS (0.01M, pH 7.4, 5μl) was intrathecally injected into the lumbarenlargement of the spinal cord;< CFA+vehicle > 24h and 2h prior to CFA (0.08 ml, 40μg Mycobacterium tuberculosis)peripheral injection and 23h after CFA injection, PBS was intrathecally injected into the lumbarenlargement of the spinal cord.< Saline+anti-rTNF-α>, 24h and 2h prior to saline peripheral injection and 23h aftersaline injection, anti-rTNF-α(70 fmol / 5μl ) was intrathecally injected into the lumbarenlargement of the spinal cord;< CFA+anti-rTNF-α> 24h and 2h prior to CFA peripheral injection and 23h after CFAinjection, anti-rTNF-αwas intrathecally injected into the lumbar enlargement of the spinal cord.All the 4 groups were implemented: 1) paw withdrawal latency (PWL) in response tothermal stimuli was tested 2h and 24h after CFA/saline peripheral injection; 2) Western Blot wasperformed to observe the level of phosphorylated NR-1 expression (p-NR1) in ipsilateral lumbarspinal cord at 24h after CFA/saline peripheral injection. Pain behavioral tests revealed that compared to < Saline+Vehicle > group hyperalgesia in< CFA+Vehicle > group was observed at 2h after CFA peripheral injection and lasted in 24h,which displayed the significant increase of PWL (P < 0.05). However, in < CFA+anti-rTNF-α>group, no matter higher or lower dose of inhibitors of TNFR1 intrathecally administration, PWLin hindpaw increased significantly (P < 0.05).It was determined with Western Blot assay that compared to < Saline+Vehicle > group,CFA peripheral injection induced the significant increase of p-NR-1 expression bilaterally (P<0.01), however, which could be reversed, when TNF-αwas inhibited by intrathecal injection ofanti-rTNF-α. (P< 0.01).These results suggested that: 1) peripheral administration of CFA induced thermalhyperalgesia, which could be attenuated by inhibiting the combination of TNF-αand its receptorTNFR1; 2) peripheral administration of CFA induced thermal hyperalgesia upregulated thep-NR1 expression, which also could be reversed by anti-rTNF-α, an antagonist of TNF-α.Part IIAdult male Sprague—Dawley rats weighing 250-270 g were divided into the following 4groups randomly:< Vehicle+Vehicle > as control, PBS (0.01M, pH 7.4, 5μl ) was intrathecally injected intothe lumbar enlargement of spinal cord 1h prior to vehicle contained 0.1% BSA in PBS (5μl)intrathecal administration;< Vehicle+rTNF-α> PBS was intrathecally injected into the lumbar enlargement of spinalcord 1h prior to rTNF-α(120fmol/5μl)intrathecal injection;< anti-rTNF-α+Vehicle > anti-rTNF-α(70fmol,5μl)was intrathecally injected into thelumbar enlargement of spinal cord 1h prior to vehicle contained 0.1% BSA in PBS intrathecaladministration;< anti-rTNF-α+rTNF-α> anti-rTNF-αwas intrathecally injected into the lumbarenlargement of spinal cord 1h prior to rTNF-αintrathecal injection.All the 4 groups were implemented: 1) paw withdrawal latency (PWL) in response tothermal stimuli was tested 0.5h and 2h after rTNF-α/PBS intrathecal injection; 2) Western Blotwas performed to examine the level of phosphorylated NR1 expression in ipsilateral lumbarspinal cord at 2h after rTNF-α/ PBS intrathecal injection.Pain behavioral responses were observed at 0.5h after rTNF-αtreatment in , which displayed the significant decrease of PWL compare to group (P<0.05) ; and PWL returned to the baseline but still were less than thebaseline 2h after rTNF-αtreatment in < Vehicle+rTNF-α>. Alone anti-rTNF-αintrathecal injection cannot increase or decrease PWL in < anti-rTNF-α+Vehicle >, nevertheless,anti-rTNF-αintrathecal injection significantly increased PWL to baseline level.Western Blot results showed that compared to < Vehicle+Vehicle > group, TNF-αinducedthe significant and rapid increase of p-NR1 expression (P < 0.01) at 2h after intrathecal injection.However, when less TNFR1 were activated with anti-rTNF-αinjection into the lumbarenlargement of the spinal cord, the increasing p-NR1 expression were reversed (P < 0.01) at 2hafter inthrathecal injection. Besides there were no significantly difference in p-NR1 expressionbetween bilateral spinal dorsal horn. Results showed that rTNF-αinduced hyperalgesia rapidly.Thereby, the increasing p-NR1 expression induced by TNF-αin lumbar enlargement of spinalcord could be reversed by anti-rTNF-α.The present results from part II suggested that : 1) intrathecal rTNF-αinjection inducedrapid hind paw withdrawal behavior and thermal hyperalgesia, which could be attenuated byanti-rTNF-αintrathecal administration; 2) intrathecal rTNF-αinjection upregulated the p-NR1expression in lumbar spinal cord rapidly, which also could be reversed by intrathecaladministration of anti-rTNF-α.Conclusion:1) TNF-αand TNFR1 are involved in the development of CFA-induced hyperalgesia byupregulating the p-NR1 expression in lumbar spinal cord; 2) TNF-αand TNFR1 can inducecentral hyperalgesia rapidly by mediating the level of p-NR1 expression in lumbar spinal cord.