The Role Of Activation Of ERK In Spinal Cord In Remifentanil Induced Postoperative Hyperalgesia

Opioids are the cornerstone therapy for alleviating moderate to severe pain. Opioids have long been used for alleviating acute and chronic pain and cancer pain. Common concerns regarding the use of opioids are the potential for detrimental side effects, such as tolerance, physical dependence,and addiction. However, recent research suggests that opioids may cause another problem, often referred to as opioid-induced hyperalgesia (OIH).Opioids were used to alleviated pain, somewhat paradoxically, may make patients become more sensitive to pain as a direct result of opioid therapy.From the classic opiate drugs morphine to the new opiate drugs remifentanil, regardless of long-term used (treatment of chronic pain) or a short application (in general anesthesia using a continuous infusion of opiates), may appear OIH.Remifentanil is a new type of short actingμ-opioid receptor agonist of its rapid onset, short half-life of its elimination.It is predominantly metabolized by nonspecific esterases in plasma and tissue,and little dependent on liver and kidney. It is ideal analgesic drugs for the patients with liver and kidney dysfunction. In recent years, it was found that continuous infusion of remifentanil leads to a rapid hyperalgesia when withdrawal.OIH reduced the analgesic effect, decreased sensitivity to noxious stimuli, and resulting in abnormal pain, affecting the treatment of postoperative pain, limiting the clinical application of opioids.The analgesic effects of remifentanil were quickly disappeared after withdrawal and induce a strong hyperalgesia. Patients were awake rapidly, but with strong hyperalgesia. It is difficulty to treat postoperative pain in early.Extracellular signal-regulated protein kinase (ERK) was belong to mitogen-activated protein kinase (MAPK) family, which is important for intracellular signal transduction and play critical roles in regulating neural plastictity and inflammatory responses.ERK activation in spinal cord neurons depends on nociceptive activity. Persistent noxious input such as imflammation pain and spinal injury and so on, could activate ERK in spinal cord.Activation of ERK may be the cytoplasm of a variety of target protein phosphorylation, and transfer to the nucleus to regulate the activity of transcription factors.This pathway is involved in pain mechanisms. Phosphorylated ERK can regulate ion channels, kinases and membrane excitability and synaptic plasticity, through direct or indirect phosphorylation of key structures such as receptors.In vitro studies showed that opioid receptor can activate ERK pathway by G protein-coupled receptor, and ERK pathway was involved in morphine tolerance and morphine induced hyperalgesia. Remifentanil is a short actingμ-opioid receptor agonists, by activating opioid receptors play its analgesic effect. Dose it induced hyperalgesia by activate the ERK in spinal cord?Postoperative hyperalgesia can occur either due to nervous system sensitization by surgical nociception (nociception-induced hyperalgesia) or as an effect of anestheticdrugs (opioids-induced hyperalgesia).The studies of remifentanil provide direct evidence for the existence of OIH in volunteers.But the results of whether remifentanil induced postoperative hyperalgesia are different. Is there same mechanism between remifentanil induced hyperalgesia and noxious stimulation caused allergic? The research of OIH and postoperative hyperalgesia mechanisms contribute to prevention of postoperative hyperalgesia, improvement analgesia effect of postoperative pain.The present study was designed to investigate whether activation (phosphorylation) of ERK in the spinal cord is involved in remifentanil induced hyperalgesia and plantar incision induced hyperalgesia in the rat hind paw. Using immunohistochemical and behavioral approaches, we examined the correlation between the remifentanil induced hyperalgesia and activation of ERK in the spinal cord.We also investigated the preventive effects of intrathecal injection of MEK inhibitor, U1026,before remifentanil administer and plantar incision on mechanical hyperalgesia. Using immunohistochemical,behavioral approaches and western blot, we also examined the relation between the OIH,incision-induced hyperalgesia and ERK activation in the spinal cord.Part 1 The role of activation of ERK in spinal cord in remifentanil induced hyperalgesia.ObjectivesTo evaluate the influence of remifentanil on nociceptive sensitivity when withdrawal in rats.The role of activation of ERK in spinal cord on opioid-induced pronociception was also investigated.MethodsThirty-two adult male Sprague-Dawley rats were randomly divided into 4 groups. Control group (C group).Normal saline 1.5ml·h-1 'was injected through ntravenous after intrathecal injection of PBS solution 20μl,and it was continued for 4h. Remifentanil and PBS solution (R+P group),intravenous injection with remifentanil, speed 1μg-mirn-1-kg-1,after intrathecal injection of PBS solution 20μl,and it was continued for 4h. Remifentanil and DMSO (R+D group), intravenous injection with remifentanil, speed 1μg·min-1·kg-1,after intrathecal injection of DMSO solution 10μl, and it was continued for 4h. Remifentanil and U1026 (R+U group), continuous intravenous injection with remifentanil, speed 1μg·mim-1·kg-1,after intrathecal injection of U1026 solution 10μl, and it was continued for 4h.Mechanical hyperalgesia was evaluated by the paw pressure test before venipuncture (TO), before administration(Tc),0.5h, 1h,2h and 24h after drug withdrawal (respectively T1-T4). Using immunohistochemical, p-ERK positive cells in spinal cord were examined after drug withdrawal 1h.Using SPSS 13.0 software for statistical treatment, measurement data were expressed as mean±standard deviation.Analysis of paired t test was used to evaluate pain threshold before and after catheterization.Comparisons among groups mechanical hyperalgesia were made by analysis of variance (ANOVA) and analysis of Repeated Measures. P-ERK positive cells in spinal cord were made by analysis of variance (ANOVA).Post Hoc multiple comparisons among each time spot in different groups and different time spots in each group were analyzed by using LSD test. P<0.05 was considered significant.ResultsIn 0.5 h,1 h,2h after drug withdrawal,the paw-pressure threshold of R+P group, R+D group and R+U group were significantly decreased Compared to C group and baseline.The mechanical pain threshold of R+P group, R+D group and R+U group were minimized in 1h after drug withdrawal.There were no significant different of the paw-pressure threshold in 24h after treatment between four groups.Intrathecal injection of U1026 and DMSO caused no effect on hyperalgesia.The numbers of p-ERK positive cells in rat spinal cord were no significant difference of between four groups.ConclusionsIntrathecal injection of U1026 does not affect the hyperalgesia caused by remifentanil.The number of p-ERK positive cells in spinal cord had no effect by intravenous infusion of remifentanil.Activation of ERK in spinal cord may be unrelated to remifentanil induced hyperalgesia.Part 2 The Study of Influence of Remifentanil on Mechanical Hyperalgesia of Incisional Pain in RatsObjectivesTo evaluated the influence of remifentanil used on nociceptive sensitivity of incisional pain in rats.MethodsTwenty-four adult male Sprague-Dawley rats were randomly divided into 3 groups. Control group, incisional and remifentanil group, incisional and NS group, each consisting of eight rats.A catheter for the drug infusion was implanted into the jugular vein.Rats underwent right hind paw surgery combined with administration of saline(1.5ml·h-1) or remifentani(1μg·min-1·kg-1)for 4h. Nociception sensitivity was evaluated daily for 3 days using paw-pressure.Using SPSS 13.0 software for statistical treatment, measurement data were expressed as mean±standard deviation. Analysis of paired t test was used to evaluate pain threshold before and after catheterization. Comparisons among groups mechanical hyperalgesia were made by analysis of variance (ANOVA) and analysis of Repeated Measures.Post Hoc multiple comparisons among each time spot in different groups and different time spots in each group were analyzed by using LSD test..P<0.05 was considered significant.ResultsThe comparison of the pressure threshold before and after catheter implanted had no significant analgesic effect(P>0.05).Remifentanil enhanced mechanical hyperalgesia induced by surgery. The pressure threshold in R group were significantly reduced compared to C group at T2 to T5.Remifentanil induced a decrease in nociceptive thresholds in the left paw after Remifentanil infusion.ConclusionsIntraoperative infusion of remifentanil significantly enhanced extent and duration of incisional pain in rats.Part 3 The role of activation of ERK in spinal cord in remifentanil induced postoperative hyperalgesiaObjectivesThe aim of the present study was to investigate the number of phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactive cells in the spinal dorsal horn after plantar incision, and the effects of intrathecal administration of MEK inhibitor U1026 on the behavior of pain in a rat model of incisional pain and remifentanil induced postoperative hyperalgesia.MethodsForty-eight adult male Sprague-Dawley rats were randomly divided into 6 groups. All rats were received a plantar incision surgery. (I+R+P group), intrathecal injection of PBS solution 20μl,intravenous injection with remifentanil.(I+N+P group) intrathecal injection of PBS solution 20μl, intravenous injection with normal saline. (I+R+D group), intrathecal injection of DMSO solution 10μl,intravenous injection with remifentanil.(I+N+D group), intrathecal injection of DMSO solution 10μl, intravenous injection with normal saline. (I+R+U group),intrathecal injection of U1026 solution 10μl, intravenous injection with remifentanil.(I+N+U group) intrathecal injection of U1026 solution 10μl,intravenous injection with normal saline. (and intrathecal DMSO group (D group).Mechanical hyperalgesia was evaluated by the pawpressure test before venipuncture (TO), before administration(Tc),0.5h,1h,2h and 24h after drug withdrawal (respectively T1-T4).Using immunohistochemical, p-ERK positive cells in spinal cord were examined after drug withdrawal 1h and 1d. Using western blot, protein of p-ERK were examined after drug withdrawal 1h.SPSS 13.0 software was used for statistical, measurement data were expressed as mean±standard deviation. Analysis of paired t test was used to evaluate pain threshold before and after catheterization. Comparisons among groups mechanical hyperalgesia were made by analysis of variance (ANOVA) and analysis of Repeated Measures.P-ERK positive cells and IOD of p-ERK protein in spinal cord were made by analysis of variance (ANOVA).Post Hoc multiple comparisons among each time spot in different groups and different time spots in each group were analyzed by using LSD test.P<0.05 was considered significant.ResultsThe paw-pressure threshold of I+R+P group in 1 h, ld,2d,3d after withdrawal were significantly decreased compared to I+R+U group and baseline of preoperative.I+P group was significantly lower than the I+U group in 1 h,1d,2d after withdrawal.I+R+P group and I+R+D group was no significant difference, I +P group and I+D group showed no significant difference.A significant increase in number of p-ERK immunoreactive cells was observed in the rat spinal cord form 1 h to Id in six groups. Intrathecal pretreatment of U1026 attenuated incision-induced mechanical hyperalgesia from 1 h to day 2 and significantly reduced activation of ERK in the spinal cord from 1h to 1 day after plantar incision. IOD of p-ERK protein in spinal cord in (I+R+P) group were significantly higher than the I+R+U group, and I+P group, I+P group than in I+U group.ConclusionsPlantar incision-induced mechanical hyperalgesia can be prevented by the U1026. ERK activation in spinal cord play a role in incision-induced mechanical hyperalgesia and remifentanil induced postoperative hyperalgesia in rats.