| Part 1 The changes of spinal tumor necrosis factor-alpha in incisional pain in ratObjective To investigate the changes of behave, Hindpaw Withdrawal Mechanical Threshold (HWMT), Hindpaw Withdrawal Thermal Latency (HWTL) and spinal tumor necrosis factor-alpha in incisional pain in rat.Methods Sixty health adult male Sprague-Dawley rats weighing 200～250 gram were randomly divided into two groups: control group(group C) and etanercept group(group E), 30 in each group. A 1-cm longitudinal incision was made in left hindpaw after anesthesia with 1% sodium pentobarbital (40 mg/kg body weight, i.p.), then a 30-gauge needle, attached to a 25-μl microsyringe, was inserted through the skin and between the vertebrae into the subdural space of the L5-L6 spinal segments, and each injection was given within a period of 5 sec. The rats in group C received saline10μl and the rats in group E received etanercept 100μg(10μl)via intrathecal injection.The rats in each group were randomly subdivided into five groups (n=6 each) according to time points after surgery. Behavioral test was assessed using cumulative pain score(CPS), HWMT was assessed using a Dynamic Plantar Aesthesiometer (UGO BASILE,Italy) and HWTL was assessed using a Plantar test 7375(UGO BASILE,Italy), TNF-αmRNA was assessed using reverse transcript polymerase chain reaction(RT-PCR) and TNF-αprotein was assessed using Western blot assay. The tests were performed at -1, 4, 8, 16 and 24h after surgery. Results (1) The CPS in two groups were zero before surgery, after surgery, CPS increased significantly( P﹤0.01), but the CPS in group E were lower than those in group C( P﹤0.01). (2)The HWMT in two groups decreased sharply after surgery( P﹤0.01). The HWMT in group E were higher than those in group C at each time point after surgery(P﹤0.05 or P﹤0.01). (3) The HWTL in two groups decreased sharply after surgery( P﹤0.01). The HWTL in group E were higher than those in group C at each time point after surgery(P﹤0.05 or P﹤0.01). (4) After surgery, the expression of TNF-αmRNA increased significantly(P﹤0.01), whereas the level of expression of TNF-αmRNA in group E were lower than those in group C(P﹤0.05 or P﹤0.01). (5) After surgery, the expression of TNF-αprotein increased significantly(P﹤0.01), whereas the level of expression of TNF-αprotein in group E were lower than those in group C(P﹤0.05 or P﹤0.01).Conclusion Spinal tumor necrosis factor-alpha contributes to postoperative hyperalgesia in incisional rat.Part 2 Effect of intrathecal injection with tumor necrosis factor-alpha on pain threshold in ratObjective To investigate the effect of intrathecal different dose of recombinant rat tumor necrosis factor-alpha on Hindpaw Withdrawal Mechanical Threshold (HWMT) and Hindpaw Withdrawal Thermal Latency (HWTL).Methods Twenty-four health adult male Sprague-Dawley rats weighing 200～250 gram were randomly divided into four groups(n=6 each): control group received saline(10μl) via intrathecal injection; group T1, T5 and T10 received recombinant rat tumor necrosis factor-alpha 1ng(10μl), 5ng(10μl) and 10ng(10μl) via intrathecal injection respectively. After anesthesia with 1% sodium pentobarbital (40 mg/kg body weight, i.p.), a 30-gauge needle, attached to a 25-μl microsyringe, was inserted through the skin and between the vertebrae into the subdural space of the L5-L6 spinal segments, and each injection was given within a period of 5 sec. HWMT was assessed using a Dynamic Plantar Aesthesiometer (UGO BASILE,Italy) and HWTL was assessed using a Plantar test(UGO BASILE,Italy), The tests were performed at -1, 4, 8, 16 and 24h after intrathecal injection. Results (1)The HWMT in four groups have no difference before intrathecal injection(P﹥0.05). After intrathecal injection, HWMT in control group have no change(P﹥0.05).4～8 hours after intrathecal injection, HWMT decreased significantly(P﹤0.01) in three test groups; 16 hours after intrathecal injection, HWMT decreased significantly(P﹤0.05 or P﹤0.01) in group T5 and group T10, 24 hours after intrathecal injection, HWMT decreased significantly(P﹤0.01) in group T10. (2) The HWTL in four groups have no difference before intrathecal injection(P﹥0.05). After intrathecal injection, HWTL in control group have no change(P﹥0.05).4～8 hours after intrathecal injection, HWTL decreased significantly(P﹤0.01) in three test groups; 16 hours after intrathecal injection, HWTL decreased significantly(P﹤0.05 or P﹤0.01) in group T5 and group T10, 24 hours after intrathecal injection, HWTL decreased significantly(P﹤0.01) in group T10.Conclusion Intrathecal injection with recombinant rat tumor necrosis factor-alpha contributes to central hyperalgesia.Part 3 The changes of spinal phosphorylated p38 mitogen-activated protein kinase in incisional pain in ratObjective To investigate the changes of behave, Hindpaw Withdrawal Mechanical Threshold(HWMT), Hindpaw Withdrawal Thermal Latency(HWTL), spinal p38 mitogen-activated protein kinase(p38MAPK) and phosphorylated p38 mitogen-activated protein kinase(P- p38MAPK) in incisional pain in rat.Methods Ninty health adult male Sprague-Dawley rats weighing 200～250 gram were randomly divided into three groups: control group(group C), dimethylsulfoxide group (group D) and SB203580 group( group S), 30 in each group. A 1-cm longitudinal incision was made in left hindpaw after anesthesia with 1% sodium pentobarbital (40 mg/kg body weight, i.p.), then a 30-gauge needle, attached to a 25-μl microsyringe, was inserted through the skin and between the vertebrae into the subdural space of the L5-L6 spinal segments, and each injection was given within a period of 5 sec. The rats in group C received saline10μl and the rats in group D received dimethylsulfoxide(2%)10μl and the rats in group S received SB203580(SB203580 was dissolved in 2% dimethylsulfoxide) 10μg(10μl)via intrathecal injection. The rats in each group were randomly subdivided into five groups(n=6 each) according to time points after surgery. Behavioral test was assessed using cumulative pain score(CPS), HWMT was assessed using a Dynamic Plantar Aesthesiometer (UGO BASILE,Italy), HWTL was assessed using a Plantar test(UGO BASILE,Italy) , spinal p38MAPK and spinal P- p38MAPK were assessed using Western blot assay. The tests were performed at -1, 4, 8, 16 and 24h after surgery.Results (1) The CPS in three groups were zero before surgery, after surgery, CPS increased significantly( P﹤0.01), the CPS in group C and group D had no difference, but the CPS in group S were lower than those in group C and group D ( P﹤0.01). (2)The HWMT in three groups had no difference before surgery, but HWMT in three groups decreased sharply after surgery( P﹤0.01). The HWMT in group C and group D had no difference ,whereas the HWMT in group S were higher than those in group C and group D at each time point after surgery(P﹤0.05 or P﹤0.01). (3) The HWTL in three groups had no difference before surgery, HWTL in three groups decreased sharply after surgery( P﹤0.01). The HWTL in group C and group D had no difference ,whereas the HWMT in group S were higher than those in group C and group D at each time point after surgery(P﹤0.05 or P﹤0.01). (4)Compared with the level of expression of p38 before surgery, the level of expression of p38 had no change after surgery. The level of expression of P-p38 was zero almost before surgery, after surgery, the levels of expression of P-p38 in three groups increased sharply ( P﹤0.01), they had no difference between group C and group D, but the levels of expression of P-p38 in group S were lower than those in group C and group D at each time point after surgery(P﹤0.05 or P﹤0.01).Conclusion Spinal phosphorylated p38 mitogen-activated protein kinase contributes to postoperative hyperalgesia.Part 4 Effects of intrathecal injection with thalidomide and SB203580 on pain threshold, spinal tumor necrosis factor-alpha and phosphorylated p38 mitogen-activated protein kinase in incisional pain in ratObjective To investigate the effect of intrathecal injection with thalidomide and SB203580 on Hindpaw Withdrawal Mechanical Threshold(HWMT) , Hindpaw Withdrawal Thermal Latency(HWTL), spinal tumor necrosis factor-alpha spinal p38 mitogen-activated protein kinase(p38MAPK) and phosphorylated p38 mitogen-activated protein kinase(P- p38MAPK) in incisional pain in rat.Methods Ninty health adult male Sprague-Dawley rats weighing 200～250 gram were randomly divided into three groups(n=30 each): control group(group C) , thalidomide group(group T) and SB203580 group(group S). A 1-cm longitudinal incision was made in left hindpaw after anesthesia with 1% sodium pentobarbital (40 mg/kg body weight, i.p.), then a 30-gauge needle, attached to a 25-μl microsyringe, was inserted through the skin and between the vertebrae into the subdural space of the L5-L6 spinal segments, and each injection was given within a period of 5 sec. The rats in group C received saline10μl and the rats in group T received thalidomide100μg(10μl) and the rats in group S received SB203580(SB203580 was dissolved in 2% dimethylsulfoxide) 10μg(10μl)via intrathecal injection. The rats in each group were randomly subdivided into five groups(n=6 each) according to time points after surgery. Behavioral test was assessed using cumulative pain score(CPS), HWMT was assessed using a Dynamic Plantar Aesthesiometer (UGO BASILE,Italy), HWTL was assessed using a Plantar test(UGO BASILE,Italy) , spinal TNF-αmRNA was assessed using reverse transcript polymerase chain reaction(RT-PCR), spinal TNF-αprotein was assessed using Western blot assay, spinal p38MAPK and spinal P- p38MAPK were assessed using Western blot assay. The tests were performed at -1, 4, 8, 16 and 24h after surgery.Results (1) The CPS in three groups were zero before surgery, after surgery, CPS increased significantly( P﹤0.01), the CPS in group T and group S had no difference, but they were lower than those in group C at each time point after surgery ( P﹤0.01). (2)The HWMT in three groups had no difference before surgery, but HWMT in three groups decreased sharply after surgery( P﹤0.01). The HWMT in group T and group S had no difference ,while they were higher than those in group C at each time point after surgery(P﹤0.05 or P﹤0.01). (3) The HWTL in three groups had no difference before surgery, HWTL in three groups decreased sharply after surgery( P﹤0.01). The HWTL in group T and group S had no difference ,while they were higher than those in group C at each time point after surgery(P﹤0.05 or P﹤0.01). (4) After surgery, the expression of TNF-αmRNA increased significantly(P﹤0.01), whereas the level of expression of TNF-αmRNA in group T and group S had no difference, but they were lower than those in group C at each time point after surgery ( P﹤0.01). (5) After surgery, the expression of TNF-αprotein increased significantly(P﹤0.01), whereas the level of expression of TNF-αprotein in group T and group S had no difference, but they were lower than those in group C at each time point after surgery ( P﹤0.01). (6)Compared with the level of expression of p38 before surgery, the level of expression of p38 had no change after surgery. The level of expression of P-p38 was zero almost before surgery, after surgery, the levels of expression of P-p38 in three groups increased sharply ( P﹤0.01), the levels of expression of P-p38 had no difference between group T and group S, but they were lower than those in group C at each time point after surgery(P﹤0.05 or P﹤0.01). Conclusion The expression of Spinal TNF-alpha is increasing after surgery, and it promots the expression of Spinal P-p38; at the same time, the expression of Spinal P-p38 is increasing after surgery, and it promots the expression of Spinal TNF-alpha. Spinal TNF-alpha and P-p38 build a positive feedback, they both contribute to postoperative hyperalgesia.
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