Role Of Spinal Cyclooxygenase In Allodynia Induced By Incisional Pain In Rats

Role of Spinal Cyclooxygenase in Allodynia Induced by Incisional Pain in RatsObjectivePostoperative pain is a common form of acute pain. Inadequate controlled postoperative pain may be one of main reasons that acute pain develops chronic pain after all kinds of surgery. AlthOugh basic and clinical research has increased our understanding of the pathophysiology of pain mechanisms, postoperative pain control continues to be a problem. After tissue damage, both sensitization of dorsal horn neurons in the spinal cord (central sensitization) and sensitization of peripheral nociceptors (peripheral sensitization) are thought to play an important role in the development and the maintenance of mechanical hypersensitivity.Previous studies have shown that prostaglandins (PGs), especially PGE₂, which are potent inflammatory and pronociceptive mediators, are thought to play an important role in peripheral and central sensitization at peripheral sites and in the spinal cord. PGs directly stimulate WDR neurons in the rat dorsal horn, sensitize these same neurons to noxious mechanical stimulation, enhance glutamate and substance P released from primary afferent terminals in the spinal cord, thereby leading to sensitization of pain processing, resulting in hyperalgesia and allodynia. PGs are synthesized in tissue by cyclooxygenase (COX), which is the rate-limiting enzyme that catalyzes the conversion of arachidonic acid to generate PGs. There are two isoforms of COX, namely, COX-1 and COX-2. COX inhibitor exerts antinociceptive action partly by inhibition of PGs synthesis in the central nerve system, and particularly in the spinal cord. Therefore, further studies of spinal two COX enzymes expression in the process of incision-induced mechanical allodynia will be important to find appropriate treatment of acute postoperative pain.To identify the role of spinal two COX isoforms in the developmerit and maintenance of postoperative pain, we examined the changes of COX-1, COX-2 and their catalytic products PGE₂ expression in lumbar spinal cord by immunohistochemistry and western blot technique in rat plantar incision model at different time points. The mechanical allodynia was evaluated by using paw withdrawal threshold (PWT) response to mechanical stimulation. We also studied the anti-allodynic effects of the COX inhibitors by intrathecal administration of selective COX-1 (SC-560) or COX-2 inhibitor (NS-398) after incision and we evaluated the anti-allodynic effect of SC-560 injected intrathecally at different time points before or after surgery to explore the best timing of intrathecal selective COX-1 inhibition administration which is likely to benefit its clinical effectiveness.Several studies have reported the extracellular signal-regulated kinase (ERK) phosphorylation in spinal dorsal horn neurons after acute noxious stimulation. We also examined changes of spinal p-ERK protein expression in the process of mechanical allodynia and effect of intrathecal injection of selective COX-1 inhibition SC-560 on its expression to explore the signal transduction mechanism of anti-allodynic effect of selective COX-1 inhibitor on rat postoperative pain model.Part One Expression of Spinal Cyclooxygenase and Prostaglandin E₂ in Rat Postoperative Pain Model Materials and Methods1,Animals76 adult male Wistar rats weighing 220-260g were used in this study. The rats were supplied by the Laboratory Animal Center of Chinese Medical University. 2,postoperative pain modelIsoflurane anesthesia was used for the skin incision surgery. As describe by Brennan, a 1cm longitudinal incision was made with a number 11 blade through skin and fascia of the plantar aspect of the left foot, starting 0.5cm from the proximal edge of the heel and extending toward the toes. The plantaris muscle was elevated and incised longitudinally, and the muscle origin and insertion remained intact. After hemostasis with gentle pressure, the skin was closed with two mattress sutures of 5-0 nylon threads.3,Experimental paradigmSurgical and non-surgical Paw withdrawal threshold (PWT) in response to mechanical stimulation was measured to assess mechanical allodynia by applying a von Frey filament at pre-incision, 2, 4, 6h and 1, 2, 3, 5, 7d after surgery (n=8). Expression of spinal COX-1 and COX-2 were detected at pre-incision, 2, 4, 6, 12 and 24h after surgery using immunohistochemical assay (n=4) and Western blot technique (n=4). Content of spinal PGE₂ protein was examined by ELISA at pre-incision, 2, 4, 6 and 24h after surgery (n=4).4,Data analysisData are presented as mean±SD and are analyzed by SPSS 11.5 statistical software. To compare the mean values between groups, one-way ANOVA method was used, while multiple comparisons versus pre-surgery group were assessed using the Student-Newman-Keuls test. Statistical significance between pre-and post-incision groups was analyzed with a student's t test. The significance level was set at P＜0.05.Results1,The changes of MWT in the process of postoperative painThere were no significant differences on the value of MWT before incision between surgical and non-surgical hind-paw of the rats. All rats showed allodynia in response to innocuous mechanical stimulation. Compared with pre-incision group and non-surgical paw, the MWT decreased significantly and peaked at 2h after surgery (P＜0.01), inreased thereafter and returned to normal by 5d. There were no changes of MWT of non-surgical paw at all time points.2,Expression of spinal COX-1 and COX-2 protein in rat postoperative painCOX-1 and COX-2 protein were constitutively expressed in the lumbar spinal cord in normal rats. COX immunoreactive (IR) cells mainly focus in the superficial laminae of spinal dorsal horn. The number of spinal COX-1 IR cells and protein expression all increased dramatically 2h after incision and peaked at 4h (P＜0.01). There are no significant differences in COX-1 expression 24h after surgery compared with pre-incision group. Although the number of spinal COX-2 IR cells and protein expression increased 2h after incision, they were less than those of COX-1 and there were no significant differences 12h after surgery compared with pre-incision group.3,Effects of incision pain on the content of PGE₂ in spinal cord of ratsThe content of PGE₂ protein in spinal cord of rat postoperative pain model increased dramatically at different time points and peaked 4h after surgery (P＜0.01). The level of spinal PGE₂ protein decreased 24h after incision, but there were also significant differences compared with pre-incision group (P＜0.05).Part Two Effects of Intrathecal Administration of Two Selective COX Inhibitors on Mechanical Allodynia and Spinal PGE₂ in Rats Postoperative Pain Model Materials and Methods1,Animal The same as that in Part One2,Postoperative pain model The starve as that in Part One 3,Intrathecal administrationAs described by Mestre et al., the rats were anesthetized by 2ï¼…isoflurane and were firmly held by one hand, a microsyringe (25μl) was inserted to L₅-L₆ intervertebral column perpendicularly. When the needle entered the subarachnoidal space, a sudden lateral movement of the tail was observed. The rats showing neurological deficits and/or inflammation postoperatively were discarded. All intrathecal drugs were administered in a 10μl volume and SC-560 and NS-398 were dissolved in 100ï¼…dimethyl sulfoxide (DMSO) 10μl.4,Experimental paradigm(1) Behavioral test one After paw incision, 40 rats developed allodynia were divided five groups (n=8): DMSO group, SC5 group, SC50 group, SC100 group, which were intrathecally injected with 10μl DMSO, 5μg, 50μg and 100μg SC-560 and 50μg NS-398 respectively. MWT were recorded before treatment, 30min, 1h, 2h, 4h, 6h and 24h after drug administration respectively and MWT of SC-560 treatment group were calculated by percentage of the maximum possible effect (ï¼…MPE).(2) Behavioral test two 48 rats randomly divided into three groups (n=16): BI group, AI 2 group and AI 24 group, which were intrathecally injection with SC-560 100μg (n=8) or DMSO 10μl (n=8) respectively at 15min before incision, 2h or 24h after incision. MWT were recorded 30min after treatment and were calculated by percentage of anti-aliodynia (ï¼…antiallodynia).(3) ELISA test 12 rats divided randomly into three groups (n=4): DMSO group, SC group and NS group, which were intrathecal injection with DMSO 10μl, SC-560 100μg or NS-398 50μg respectively 15min before incision. Lumbar enlargement of spinal cord in rats was removed 4h after incision and detected the protein content of PGE₂ by ELISA technique respectively.5,Data analysis The same as that in Part One. Results1,Effect of intrathecal injection of SC-560 and NS-398 after surgery on mechanical allodynia induced by paw incision in rats andï¼…antiallodynia of SC-560 treatment at different time pointThere were no significant differences on the value of MWT of all group before surgery and treatment. Compared with pre-incision and DMSO group, MWT of SC50 and SC100 group increased significantly, lasting for 2h and 6h, respectively (P＜0.01).ï¼…MPE of intrathecal treatment of SC-560 increased in a dose-dependent manner. There were no significant differences at all time points on MWT of SC5 and NS group compared with those of pre-incision and DMSO group.ï¼…antiallodynia of SC-560 treatment before and 2h after incision was significant higher than that 24h after surgery (P＜0.01).2,Effect of SC-560 and NS-398 treatment before surgery on the protein content of spinal PGE₂ in rat postoperative pain modelCompared with DMSO group, the protein content of spinal PGE₂ 4h after incision remarkably decreased in SC group (P＜0.01). There were no significant differences on PGE₂ preotein content between NS and DMSO group (P＞0.05).Part Three Effects of Selective COX-1 Inhibitor SC-560 on Spinal ERK Expression in Postoperative Pain of Rats Material and Methods1,Animal The same as that in Part One2,Postoperative pain model The same as that in Part One3,Intrathecal administration The same as that in Part Two 4,Experimental paradigm32 rats randomly divided into four groups (n=8): Control group, normal rats; PP group, no treatment; SC group, intrathecal injection of SC-560 100μg 15min before incision; DMSO group, intrathecal injection of DMSO 10μl 15min before incision. Lumbar spinal cord of rats in all groups were removed 1h after incision and examined p-ERK IR cells and protein expression using by immunohistochemical assay (n=4) and Western blot technique (n=4).5,Data analysis The same as that in Part One.Results1,Effects of intrathecal treatment of SC-560 on spinal p-ERK IR cells in postoperative pain of rats.p-ERK positive neurons in the rat superficial spinal dorsal horn increased significantly 1h after incision compared with that of non-incision group (P＜0.01). Intrathecal administration of SC-560 preoperatively can significantly reduce the number of p-ERK IR cells compared with PP group (P＜0.01). DMSO has no effects on reducing the number of ERK IR cells in spinal cord.2,Effects of intrathecal treatment of SC-560 on spinal p-ERK protein expression in postoperative pain of rats.Expression of p-ERK1/2 protein in the lumbar spinal cord increased significantly 1h after incision and decreased by intrathecal injection of SC-560 (P＜0.01). DMSO has little effect on it.Conclusions1,Spinal COX, mainly COX-1 and PGE₂ involved in the development and early maintenance of mechanical allodynia followed by paw incision in rats.2,Intrathecal treatment of selective COX-1 inhibitor SC-560 can reduce incision-induced mechanical allodynia in rats. Maybe it benefits the treatment of incisional pain by early interrupt synthesis of spinal COX-1-mediated PGE₂.3,Selective COX-1 inhibitor SC-560 administrated intrathecally can alleviate mechanical hypersensitivity induced by postoperative pain in rats and this anti-allodynic process may mediated by spinal ERK.