| OBJECTIVESPersistent postoperative pain is an important clinical problem,which means it affects the quality of patients' life with operation.However the mechanism of persistent postoperative pain remains unclear.The purpose of this study is to clarify the role of TLR4,p38 and IL-1? signaling pathways which affects persistent postoperative pain in dorsal root ganglion.MEASUREMENTS1.Measuring the change of rats' behavior and the level of TLR4 in DRG.after SMIR1.1 SMIR surgery was performed on SD male rats.The ipsilateral and contralateral PWT were measured on days-1,1,5,10 and 20.1.2 Getting ipsilateral L3,L4 DRGs on days-1,1,5,10 and 20,and detecting the level of TLR4.1.3 The ipsilateral L3,L4 DRGs were obtained on the 10 th day after SMIR.We use immunofluorescence staining to find the cell types of TLR4 expression.2.Observing the change of MAPK in DRG after SMIR.2.1 Getting ipsilateral L3,L4 DRGs on days-1,1,5,10 and 20,and detecting the level of MAPK phosphorylation.2.2 Before SMIR 30 min to the 10 th day after SMIR we performed intrathecal injection of different doses p38 inhibitor SB203580(1?g,10?g,50?g)and vehicle in SMIR rats.Then we observed the change of mechanical pain in SMIR rats.Using the same methord,we performed intrathecal injection of SB203580 10?g in naive rats,and observed the change of mechanical pain in naive rats.3.Observing the relationship of between TLR4,PWT and p-p38 after SMIR.3.1 30 min before SMIR to the 10 th day after SMIR we performed intrathecal injection of different doses TLR4 antagonist LPS-RS(5?g,25?g,50?g)and vehicle in SMIR rats.Then we observed the change of mechanical pain in SMIR rats.Using the same methord,we performed intrathecal injection of LPS-RS 25?g in naive rats,and observed the change of mechanical pain in naive rats.3.2 Getting ipsilateral DRGs on day 10 after SMIR,we performed immunofluorescence staining of TLR4 and p-p38 to confirm the position relationship between them.3.3 30 min before SMIR to the 10 th day after SMIR we performed intrathecal injection TLR4 antagonist LPS-RS at the does of 25?g in SMIR rats.Then we got DRG on the 10 th day to detect the level of p-p38.4.After SMIR we observed the level of IL-1?.Then we researched the effect of TLR4 and p-p38 on IL-1?.4.1 Getting ipsilateral L3,L4 DRGs on days-1,1,5,10 and 20,and detecting the level of IL-1?.4.2 30 min before SMIR to the 10 th day after SMIR we performed intrathecal injection of different doses IL-1ra(10?g,50?g,100?g)and vehicle in SMIR rats.Then we observed the change of mechanical pain in SMIR rats.Using the same methord,we performed intrathecal injection of IL-1ra 50?g in naive rats,and observed the change of mechanical pain in naive rats.4.3 30 min before SMIR to the 10 th day after SMIR we performed intrathecal injection LPS-RS at the does of 25?g or SB203580 at the does of 10?g in SMIR rats.Then we got DRG on the 10 th day to detect the level of IL-1?.RESULTS1.The ipsilateral PWT of SMIR rats was decreased and the level of TLR4 was increased after SMIR.1.1 The ipsilateral PWT of SMIR rats was significantly declined on days 5,10,20 compared with pre-operation(n=12,p<0.05).1.2 The level of TLR4 in ipsilateral L3,L4 DRGs was significantly increased on days 5,10,20 compared with pre-operation(n=6,p<0.05).But it had no change on the first day.1.3 Immunofluorescence staining showed that TLR4 was co-expressed with IB4 and NF-200,but not GFAP.2.The level of p-p38 was SMIR increased after SMIR,and effected the PWT of SMIR rats.2.1 The level of p-p38 in ipsilateral L3,L4 DRGs was significantly increased on days 5,10,20 compared with pre-operation(n=6,p<0.05).But the levels of p-ERK,p-JNK were no significant change compared with pre-operation.2.2 The ipsilateral PWT significantly increased in intrathecal injection SB203580 at the does of 10?g and 50?g group compared with vehicle group on days 5,10 and 20 after SMIR(n=12,p<0.05).But the PWT of 1?g group was no significant change compared with vehicle group.And the PWT was no change in na?ve group that used 10?g SB203580.3.It can increase the PWT and decrease the level of p-p38 in SMIR rats to inhibit TLR4.3.1 The ipsilateral PWT significantly increased in intrathecal injection LPS-RS at the does of 25?g and 50?g group compared with vehicle group on days 5,10 and 20 after SMIR(n=12,p<0.05).But the PWT of 5?g group was no significant change compared with vehicle group.And the PWT was no change in na?ve group that used 25?g LPS-RS.3.2 Immunofluorescence staining showed that TLR4 was co-expressed with p-p38.3.3 The level of p-p38 significantly decreased after using LPS-RS at the dose of 25?g for 10 day compared with SMIR rats without using LPS-RS(n=6,p<0.05).4.The level of IL-1? was increased though TLR4 and p38 pathway.And it could affect the PWT of SMIR rats.4.1 The level of IL-1? in ipsilateral L3,L4 DRGs was significantly increased on days 5,10,20 compared with pre-operation(n=6,p<0.05).4.2 The ipsilateral PWT significantly increased in intrathecal injection IL-1ra at the does of 50?g and 100?g group compared with vehicle group on days 5,10 and 20 after SMIR(n=12,p<0.05).But the PWT of 10?g group was no significant change compared with vehicle group.And the PWT was no change in na?ve group that used 50?g IL-1ra.4.3 Intrathecal injection of 25?g LPS-RS or 10?g SB203580 can prevent IL-1? expression after SMIR.CONCLUSIONSThese findings suggest that the activation of p38 and IL-1? signaling pathway via TLR4 mediate mechanical allodynia after SMIR surgery. |
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