| Postoperative pain remains a major health problem affecting up to19-56%patients undergone surgical procedures, according to a statistic data from Euroanaesthesia (ESA). The occurrence of postoperative pain is closely associated with the way of anesthesia. Therefore, selecting a safe and effective anaesthesia way with little physiological interference is a main solution to prevent from postoperative pain.Combined acupuncture and medicine anaesthesia has shown its clinical effectiveness in keeping patient’s homeostasis during surgical operation, reducing side effects of anesthetics, as well as relieving postoperative pain. However, when referred to mechanism research, there are few animal models that can mostly comply with clinical surgical procedures. Thus it’s necessary to establish a postoperative pain model for further study of acupuncture mechanism on acute traumatic pain.In the last decades, global studies have focused on the CNS (central nervous system) mechanism of acupuncture analgesia and partially revealed that the primary mechanism of acupuncture in the anti-nociceptive effect involved release of opioid peptides in the CNS in response to the long-lasting activation of ascending sensory tracks during the intermittent stimulation. Besides, naloxone, an opioid receptor antagonist, could partly block analgesic effect of acupuncture. Moreover, analgesic effect of OXA couldn’t be blocked by naloxone at a dose that blocked dynorphine analgesia. Another accepted theory is acupuncture activates descending inhibitory system by regulating neurotransmitters, neuromodulators and neuropeptides.Orexins (Orexin A or OXA and Orexin B or OXB) are peptides that are exclusively expressed in the lateral and perifornical areas of the posterior hypothalamus. Usually, orexinergic neurons are considered to localize within the hypothalamus and project extensively and widely to many regions of the nervous system, where orexins have been shown to regulate a number of physiological and homeostatic functions. Orexins act at two different G-protein coupled receptors:the Orexin-1receptor (OX1R) which is selective for OXA and the Orexin-2receptor (OX2R) which is nonselective for both OXA and OXB. Growing evidences of morphology and behaviors have shown the nociceptive role of orexins, especially OXA.Identification of its projection in the spinal cord provides basis of its role in primary nociception. Researches on the constriction/diabetic neuropathic pain, carrageenan/formalin test, inflammatory pain, trigeminovascular pain etc., identified the anti-nociceptive role of OXA. Besides, in the Brennan’s post-operative pain model, in which the rat hindpaw was made an incision resulting in an incisional pain, intrathecal OXA was revealed an anti-allodynic effect. Moreover, orexins have been attributed to the pain-inhibited pathway and it is also reported that analgesic effect of OXA couldn’t be blocked by naloxone at a dose that blocked dynorphine analgesia.From the above evidence we could postulate that, acupuncture analgesia includes not only opioids but also other mechanisms, and OXA is independent from opioids analgesia. Moreover, OXA and acupuncture both attribute to descending inhibitory system in their anti-nociception roles. Our hypothesis is that, OXA might participate in acupuncture analgesia process in a way independent of opioids.Therefore the present study is designed to testify the hypothesis that OXA participates in acupuncture analgesia, using an acute pain model induced by operative trauma.We used von Frey filaments to test mechanical threshold, Hargrave’s test and hot water to measure the thermal threshold. Also, Immunohistochemistry, RT-PCR, ELISA and laser confocal microsopy were used for detecting the expression of OXA and OX1R in levels of mRNA and protein.The results are following:1. Influence of acute trauma on mechanical and thermal thresholds of rats1.1Influence of diethyl ether on mechanical and thermal thresholds To exclude the influence of diethyl ether on basic mechanical and thermal thresholds, rats were randomly divided into Anaesthesia group and Nonanaesthesia group,8rats in each group. Rats in Anaesthesia group received15min systematic anaesthesia. Time0was defined as the end of the anaesthesia. Time points of pain measurement were0.5h,1h,2h. Results showed that diethyl ether had no obvious effect on PWT, ACT, PWL or TFL (P>0.05). As PWT and ACT exhibited more steady thresholds than thermal ones, the following experiments adopted PWT and ACT as the indicators of pain threshold.1.2Influence of surgery trauma on the abdominal and paw allodynia of ratsRats were randomly divided into Normal group, Sham Model group and Model group,8rats in each group. After being anaesthized, rats received laparotomy which induced a6-cm long incision on the abdominal wall. Time0was defined as the end of the surgery. Mechanical thresholds of paw and abdomen were measured respectively at0.5h,1h,2h,4h,6h and24h. Results showed that0.5h post-surgery, rats in Model group displayed abdominal and paw allodynia and maintained for over24h, with significant differences with Sham Model group and Normal group (P<0.01).1.3Analgesic effect of fentanyl on traumatic ratsTo further testify the validity of the acute traumatic postoperative pain model, Saline or opioid μ,-receptor angonist fentanyl (5,10and20μg/kg) was intramuscular injected immediately after surgery. No statistic difference was found among baselines. Mechanical thresholds of paw and abdomen were measured respectively at0.5h,1h,2h,4h,6h and24h. Results showed that10and20pμg/kg fentanyl dose dependently increased mechanical threshold of both paw and abdomen, and the effect could last for2hours, with significant differences with Saline group (ACT:P<0.01,PWT:P<0.05). The ACT and PWT in Fentanyl5μg/kg group had no difference with Saline group. There was no MPE difference between ACT and PWT.Brief summary:Laparotomy in rats induced both paw and abdominal allodynia, which could last for over24h.10and20μg/kg fentanyl dose dependently relieved allodynia state. In conclusion, the rat pain model induced by acute trauma is steady and valid for further study. 2. Acupuncture analgesic effect on the rat model of postoperative pain2.1Effects of EA at2/15Hz and2/100Hz on postoperative painRats were randomly divided into Model group, Sham EA group, EA2/15Hz group and EA2/100Hz group,8-11rats in each group. After being anaesthized, rats received bilateral EA at ST36and SP6, with intensity of1-3mA in30min. Laparotomy was undergone within the middle10min of EA application. Mechanical thresholds of paw and abdomen were measured respectively at0.5h,1h,2h,4h,6h and24h post EA application. ACT results showed that,0.5h and1h after surgery, both frequencies of EA could increase ACT with statistical differences compared with Model group (P＜0.05). At0.5h, EA2/15Hz showed statistical difference with Sham EA group (P<0.05). PWT results showed that,0.5h after surgery, both frequencies of EA could increase PWT with statistical differences with Model group (P＜0.05). At0.5h, EA2/15Hz showed statistical difference with Sham EA group (P＜0.05).2.2Influence of EA combined with low dose fentanyl on postoperative painBased on the better immediate analgesic effect of EA2/15Hz,2/15Hz was chosen as the EA parameter for the following studies. Rats were randomly divided into Saline group, Fentanyl5μg/kg group, EA2/15Hz+Saline group, EA2/15Hz+Fentanyl5μg/kg group,7-8rats in each group. Results showed that there is no difference between the Fentanyl5μg/kg group and the Saline group. Compared with Fentanyl5μg/kg group, EA2/15Hz+Fentanyl5μg/kg group showed increased ACT at0.5h,1h and24h (P＜0.05), and increased PWT at0.5h,1h, and4h to24h (P＜0.05). Compared with EA2/15Hz+Saline group, EA2/15Hz+Fentanyl5μg/kg group showed increasing tendency of ACT at24h and increased PWT at0.5h,6h and24h (P＜0.05).Brief summary:2/15Hz and2/100Hz both have immediate analgesic effect on postoperative pain. Combined EA with low dose fentanyl showed enhanced and prolonged analgesic effect.3. Intrathecal OXA, naloxone or OX1R antagonist SB-334867on postoperative pain3.1Effect of intrathecal OXA on postoperative pain Rats were randomly divided into Saline group, OXA0.1nmol group, OXA0.3nmol group and OXA1nmol group,6-8rats in each group. Drugs were injected after surgery at time0. ACT was measured at0.5h,1h,2h,4h,6h and24h post-surgery. Results showed that compared with Saline group, OXA0.1nmol group had no effect on ACT, while OXA0.3nmol group and OXA lnmol group significantly increased ACT at0.5h (P<0.05), but there was no difference between OXA0.3nmol group and OXA1nmol group.3.2Influence of intrathecal OXA, naloxone and selective OX1R antagonist SB-334867on postoperative painRats were randomly divided into Saline group, OXA0.3nmol group, SB-33486730nmol group, SB-33486730nmol+OXA0.3nmol group and naloxone28nmol+OXA0.3nmol group,7-8rats in each group. SB-334867and naloxone were inthrathecally injected before surgery, while saline and OXA were injected after surgery at time0. ACT was measured at0.5h,1h,2h. Results showed that, compared with Saline group, SB-33486730nmol showed no significant effect (P>0.05) at the above time pionts. Compared with OXA0.3nmol group, ACT in SB-33486730nmol+OXA0.3nmol group was significantly decreased (P<0.05) at0.5h post-surgery. ACT in naloxone28nmol+OXA0.3nmol group showed no difference with OXA0.3nmol group (P>0.05).3.3Influence of intrathecal OXA on acupuncture analgesiaRats were randomly divided into Saline group, OXA0.3nmol group, EA group and EA+OXA0.3nmol group,8-11rats in each group. OXA was injected after EA application. Compared with OXA0.3nmol group, ACT in EA+OXA0.3nmol group increased significantly (P＜0.05). Compared with EA group, there was an increasing tendency of ACT in OXA0.3nmol group.3.4Influence of intrathecal selective OX1R antagonist SB-334867on acupuncture analgesiaRats were randomly divided into DMSO group, EA group, SB-33486730nmol group, SB-334867100nmol group, SB-33486730nmol+EA group and SB-334867100nmol+EA group,7-11rats in each group. Results showed that compared with DMSO group, SB-33486730nmol and SB-334867100nmol had no influence on the model (P>0.05). Compared with EA group, ACT in SB-334867100nmol+EA group decreased significantly (P＜0.05), There was no difference between EA group and SB-33486730nmol group (P>0.05).3.5Influence of intrathecal selective OX1R receptor antagonist on acupuncture analgesiaRats were randomly divided into Saline group, fentanyl10μg/kg group, SB-334867100nmol group and SB-334867100nmol+fentanyl10μg/kg group,7-8rats in each group. SB-334867was inthrathecally injected before surgery, and fentanyl was intramuscular injected after surgery. Results showed that, compared with Saline group, there was no obvious change in ACT in SB-334867100nmol (P>0.05). At0.5h and1h, both of fentanyl10μg/kg group and SB-334867100nmol+fentanyl10μg/kg group showed statistical difference with Saline group (P<0.05). There was no difference between fentanyl10μg/kg group and SB-334867100nmol+fentanyl10μg/kg group (P>0.05).Brief summary:(1) Intrathecal injection of OXA relieved postoperative pain in rat model and, its analgesic effect was enhanced when combined with EA.(2) Intrathecal injection of SB-334867blocked OXA analgesia, and when the dose was up to100nmol, SB-334867blocked EA analgesia.(3) Naloxone28nmol didn’t block OXA analgesia, and SB-334867100nmol didn’t block fentanyl analgesia.4. Influence of surgery and EA on the expressions of central OXA and OX1R4.1Influence of surgery and EA on hypothalamus, PAG and spinal cord OXA expressionWe used ELISA to detect the OXA expression in hypothalamus, PAG and spinal cord among Normal group, Model group, EA2/15Hz group and EA2/100Hz group. Results showed that, compared with Normal group at1h, hypothalamus OXA in Model group decreased significantly (P＜0.05); EA2/15Hz group significantly up-regulated the OXA expression compared with Model group in hypothalamus (P<0.05). Compared with Normal group at1h, PAG and spinal cord OXA in Model group decreased significantly (P＜0.05); EA2/15Hz group significantly up-regulated PAG and spinal cord OXA expression compared with Model group (P<0.05). There were no difference between EA2/100Hz group and Model group in hypothalamus (P>0.05).4.2Influence of surgery and EA on hypathalamus OXA mRNA expressionUsing RT-PCR, we detected hypothalamus OXA mRNA expression among Normal group, Model group, EA2/15Hz group and EA2/100Hz group at2h and4h post-surgery. Results showed that, compared with Normal group, OXA mRNA expression in hypothalamus was down-regulated at2h (P＜0.05); EA2/15Hz significantly up-regulated OXA mRNA compared with Model group (P＜0.05). There was no difference at4h among groups.(P>0.05)4.3Influence of surgery and EA on Orexinergic neurons in the hypothalamusWe used immunohistochemistry to detect Orexinergic neurons among Normal group, Model group, EA2/15Hz group and EA2/100Hz group in the hypothalamus. Results showed that, compared with Normal group, there were no obvious difference of Orexinergic neuron expressions among groups.4.4OX1R expression in the hypothalamus, PAG, medulla and spinal cord of ratsWe used immunohistochemistry to detect OX1R expression in the hypothalamus, PAG, medulla and spinal cord of rats. Results showed that,OX1R-LI expressions and distributions were distributed in ventro-hypothalamus, PAG, RVM and dorsal horn.4.5Influence of surgery and EA on hypothalamus, PAG and spinal cord OX1R mRNA expressionWe used RT-PCR to detect OX1R mRNA expressions in the hypothalamus, PAG and spinal cord among Normal group, Model group, EA2/15Hz group and EA2/100Hz group at2h post-surgery. Results showed that compared with Normal group, OX1R mRNA expression in the hypothalamus was up-regulated at2h (P＜0.05); EA2/15Hz significantly up-regulated OXA mRNA expression compared with Model group (P＜0.05). There was no difference at4h among groups in the hypothalumas (P>0.05). Compared with Normal group, OX1R mRNA expressions in the PAG and spinal cord were up-regulated at2h (P＜0.05); EA2/15Hz significantly up-regulated OX1R mRNA expression compared with Model group (P<0.05).4.6Orexinergic neuron projections in the hypothalamus in Model ratsWe used double labeling immunohistochemistry to detect the cell location of OXA and results showed that the OXA-LI signals were mainly located within cytoplasm of NeuN positive neurons. We had also recorded thicker projections of OXA-LI in posterior hypothalamus of rat after surgery.Brief summary:2h post surgey in Model rats, OXA protein expressions in hypothalamus, PAG and spinal cord decreased significantly; in the hypothalamus, OXA mRNA was down-regulated and could be up-regulated by EA2/15Hz; OX1R mRNA of Model rats was up-regulated in the hypothalamus and down-regulated in PAG and spinal cord, while EA2/15Hz up-regulated OX1R mRNA in the three areas.The present study suggested:(1) Perioperative acupuncture produced immediate analgesia in the postoperative pain rats; Combined treatment of EA and low dose fentanyl could efficiently enhance and prolong EA analgesia;(2) Intrathecal injection of OXA produced analgesic effect on postoperative pain, which could be blocked by OX1R antagonist but not by naloxone; Intrathecal OX1R antagonist could also block EA analgesia but not fentanyl analgesia. These results indicate that OXA analgesia is independent of opioid system.(3) There were significant changes of endogenous OXA and OX1R in traumatic rats. EA could up-regulate hypothalamus OXA, as well as OXA mRNA and OX1R mRNA of hypothalamus, PAG and spinal cord, which indicates a participation of OXA through OX1R in descending inhibitory system in EA analgesia. |
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