Experimental Study Of The Preventive Effect Of Parecoxib On Allodynia Of Spinal Cord

BackgroundParecoxib(Par) is a highly selective cyclooxygenase-2 inhibitor. Following IV injection, parecoxib is rapidly converted to the pharmacologically active substance (valdecoxib) by enzymatic hydrolysis in the liver. Parecoxib play a role in anti-inflammatory analgesic with COX -2 specific inhibition by blocking the synthesis of prostaglandins from arachidonic acid. In 2002 Parecoxib was approved on the market in Europe, and later was approved in China in 2008. As the first injectable COX-2 selective inhibitors in the world, Parecoxib is a milestone in the history of NSAIDs. Since put into use clinically, Parecoxib has been generally considered playing a role in analgesic peripherally, however, the role and mechanisms of inhibiting allodynia by Parecoxib are rarely reported.Cyclooxygenase, including two isoforms (COX-1 and COX-2), is the key enzymes of PG biosynthesis from arachidonic acid (AA). COX-1 known as structure type enzyme is the normal body composition, expressed in most tissues. The prostaglandins from COX-1 meet the ongoing needs of the physiological processes. COX-2 known as inducible enzymes is few in normal. But it can be induced by inflammation and cytokine. The prostaglandins from COX-2 related to pain, inflammation and so on. Prostaglandin E₂(PGE₂) is the metabolite of arachidonic acid under the action of the cyclooxygenase. It is an important medium of inflammation, and plays an important role in peripheral sensitization and central sensitization. In local injury area, PGE₂ can reduce the pain threshold, activate the peripheral pain receptors, and sensitize peripheral nociceptive afferent fibers. At the same time, PGE₂ can be synthesized and released from spinal cord, and increase excitability of dorsal root ganglion neurons, resulting in hyperalgesia. Studies have demonstrated that COX-1 and COX-2 exist in low level in normal spinal cord. Under noxious stimulation, PGE₂ can rapidly increased in the spinal cord. By using behavior, microdialysis, ELISA, immunohistochemistry methods and Brennan model of incisional pain, the effect of Parecoxib on inhibiting allodynia and the expression of PGE₂ and two subtypes of COX (COX-1 and COX-2) in the spinal cord was observed in the process of formation and the development of postoperative pain. Thus further explore the role and the mechanisms of Parecoxib on preventing allodynia.Part one The analgesic effects of Parecoxib in rat model of incisional painObjectiveTo investigate the analgesic effects and the preemptive analgesia of Parecoxib.MethodsWeighting 200²50g, forty male Wistar rats were randomly divided into 4 groups, as control group(C group), operation group(P₀ group), preemptive analgesia group(P₁ group), postoperative analgesia group(P₂ group). In the control group(C group), rats were given intravenous injection of 2ml 0.9% saline, and no Brennan's pain incision was performed under 2% isoflurane anesthesia. In operation group(P₀ group), rats were given intravenous injection of 2ml 0.9% saline, and Brennan's pain incision was performed under 2% isoflurane anesthesia. In preemptive analgesia group(P₁ group), rats were given intravenous injection of Parecoxib 5mg/kg, and Brennan's pain incision was performed under 2% isoflurane anesthesia. In postoperative analgesia group(P₂ group), Brennan's pain incision was performed under 2% isoflurane anesthesia, and rats were given intravenous injection of Parecoxib 5mg/kg at the end of incision. Using Von Frey and radiant heat tail flick test instrument, pain intensity was measured with mechanical paw withdrawal threshold (MWT) and thermal tail flick latency (TFL) at 30min before surgery and at 1h, 2h, 3h, 4h, 5h and 6h after surgery.Results1,Mechanical flinching reflex threshold (MWT) changesNo significant deviation of MWT baseline was shown (p>0.05) among all four groups. In group C, MWT showed no significant deviation at each checkpoint (p>0.05), and maintained at 14.52¹4.76 g. In group P₀, MWT dropped from (14.82±0.48)g to (4.93±0.48)g, and minimized at 2h after Brennan's pain incision (which was lower than the baseline by 74.49%), then recovered slightly, but still significantly lower than that of the control group C. In group P₁, MWT was slightly lower, fluctuated in 10.84¹1.96 g, and minimized at 2h after Brennan's pain incision(11.96±0.35g), which was slightly lower than the baseline by 18.92%. MWT of group P₁ and group P₂ was significantly higher than that of group P₀ in each time point after incision(P<0.05). Compared between group P₁ and group P₂, MWT was no significant difference at 1h after incision(P>0.05). MWT of group P₁ was significantly higher than that of group P₂ in 2⁶h after incision(P<0.05)。2,Tail flick latency (TFL) changesNo significant deviation of TFL baseline was shown (p>0.05) among all four groups. In group C, TFL maintained at 3.72³.84 s, and showed no significant deviation at each checkpoint(p>0.05). In group P₀, TFL dropped from (3.76±0.36)s to (1.89±0.17)s, and minimized at 2h after Brennan's pain incision(which was lower than the baseline by 73.40%), then recovered slightly, but still significantly lower than that of the control group C. In group P₁, TFL was slightly lower, fluctuated in 2.12³.03 s, and minimized at 2h after Brennan's pain incision(3.03±0.09s),which was slightly lower than the baseline by 20.26%. TFL of group P₁ and group P₂ was significantly higher than that of group P₀ at each time point after incision(P<0.05). Compared between group P₁ and group P₂, TFL was no significant difference at 1h after incision(P>0.05). TFL of group P₁ was significantly higher than that of group P₂ in 2⁶h after incision(P<0.05)。ConclusionsParecoxib has not only a good analgesic effect in rat model of incisional pain, but also has a clear effect of preemptive analgesia.Part two Efects of Parecoxib on Prostaglandin E₂ and Cyclooxygenase in the spinal cordObjectiveThe effect of Parecoxib on inhibiting allodynia and the expression of PGE₂ and two subtypes of COX (COX-1 and COX-2) in the spinal cord was observed in the process of formation and the development of postoperative pain. Thus further explore the role and the mechanisms of Parecoxib on preventing the induction of allodynia by postoperative pain.MethodsAfter successfully implanted microdialysis probe, weighting 200²50g, forty male Wistar rats were randomly divided into 4 groups, which were control group(C group), operation group(P₀ group), preemptive analgesia group(P₁ group), postoperative analgesia group(P₂ group). In the control group(C group), rats were given intravenous injection of 2ml 0.9% saline, and no Brennan's pain incision was performed. In operation group(P₀ group), rats were given intravenous injection of 2ml 0.9% saline, and Brennan's pain incision was performed. In preemptive analgesia group(P₁ group), rats were given intravenous injection of Parecoxib 5mg/kg, and Brennan's pain incision was performed. In postoperative analgesia group(P₂ group), Brennan's pain incision was performed, and rats were given intravenous injection of Parecoxib 5mg/kg at the end of incision. The Microdialysate was separately collected at 30min before surgery and in 6 hours at 30min intervals after incision. After collected, the Microdialysate, each tube 150μl, was stored at -80℃for subsequent ELISA analysis. The rats were fixed at 6 hours after incision, and then, lumbar spinal cords were taken and the spinal expression of cox-1 and cox-2 was measured with immunohistochemistry.Results1,PGE₂ concentration in the dorsal hornNo significant deviation of PGE₂ baseline was shown (p>0.05) agong all four groups,which were (153.08±2.07) pg/ml, (153.06±2.29) pg/ml, (154.17±1.78) pg/ml, and (154.38±1.61) pg/ml respectively. In group C, PGE₂ showed no significant deviation at each checkpoint(p>0.05), and maintained at 153.03¹54.66 pg/ml. In group P₀, PGE₂ sharply rose after Brennan's pain incision, and peaked at 120min after incision. PGE₂ of group P₀ was 82.47% higher than that of group C. Compared with group C, PGE₂ of group P₁ and group P₂ slightly rose. PGE₂ of group P₁ was lower than that of group C from 60min to 360min after incision, and minimized at 120min after incision. PGE₂ of group P₂ was lower than that of group C at 60min after incision, and was similar to group C at 240min after incision. Compared with group P₀, PGE₂ of group P₁ and group P₂ was significantly lower(P<0.05). PGE₂ of Group P₁ was similar to group P₂ at 90min after incision(P>0.05), but it was lower than group P₂ from 120min to 360min after incision(P<0.05).2,The expression of spinal COX-1 and COX-2 protein at 6 hours after incisionIn group C, COX-1 and COX-2 immunoreactive cells were constitutively expressed and lightly dyed in the lumber spinal cord, and the immunohistochemical composite scores of COX-1 and COX-2 were 3.29±1.86 and 2.78±2.05 respectively. Compared with group C, COX-1 and COX-2 immunoreactive cells of group P₀, group P₁ and group P₂ significantly increased and mainly distributed in the dorsal horn laminaⅠ^Ⅰ. In group P₀, group P₁ and group P₂, the expression of COX-1 and COX-2 was similar, but the COX-2 immunoreactive cells were more than the COX-1 immunoreactive cells. ConclusionsParecoxib does not inhibit the expression of cyclooxygenase in dorsal horn, but it can effectively inhibit the function of COX-2 and the release of PGE₂ in spinal cord which play a inhibitory role in central allodynia.