The present study showed that after laparotomy, OXA levels in the hypothalamus, PAG and spinal cord decreased significantly. Bilateral EA at ST36 and SP6 elevated OXA levels in the above regions. Rats displayed a local and systematic mechanical allodynia state after laparotomy. The allodynia could be relieved by fentanyl in a dose-dependent manner with a limited time course of 2 h. OXA at 0.3 nmol and EA at 2/15 Hz both showed analgesic effects in the rat model. SB-334867 30 nmol blocked OXA-induced analgesia. Pre-surgical, but not post-surgical, administration of SB-334867 treatment significantly reduced EA analgesia. These results suggested the involvement of OXA in acupuncture analgesia via OX1R. As we focused on laparotomy induced pain, indexes such as blood pressure, heart rate and breath during surgery were not included in the present study.
Relevance and validity of the model and pain assessment
In this study, we used the mechanical measurement of ACT in addition to PWT to evaluate the pain state of the animal. PWT decreased significantly after laparotomy; this effect lasted for over 24 h, which is in agreement with previous reports [41, 42]. Because paw withdrawal is a complex reflex that requires spinal and supraspinal reflexes and includes cerebral involvement, this reflex can be used to represent the systemic pain state. The hind paw test is commonly used to evaluate mechanical pain, including hind paw pain, patellar osteoarthritis, abdominal incisional pain and tibial cancer pain, among others. In abdominal pain following surgery, nerves innervating the hind paw (superficial and deep branches of the peroneal nerve as well as medial and lateral plantar nerves) terminate in the lumbar-sacral segments L6–S2. The surgical site is served by the lumbar spinal cord segments L1–L4, with some input from T13. Assessments of the pain state of the abdomen, not the hind paw, could be more consistent with typical clinical scenarios. The idea of using the ACT came from the “abdominal constriction score” or “abdominal withdrawal response” when assessing visceral inflammatory pain. We introduced the ACT to evaluate abdominal mechanical allodynia and compared it with PWT. As shown in Figure 1A and B, the same tendency of ACT and PWT was observed in the model group and fentanyl groups, but ACT remained at a relatively steady level, whereas PWT gradually increased towards baseline levels. Thus, the ACT could be more suitable than other tests in assessing abdominal pain.
Acupoints and EA frequency selection
In the present study, we selected the bilateral acupoints of ST36 and SP6 to relieve laparotomy pain. In Chinese medicine, pain is believed to be caused by either Qi flow stagnation or Qi-blood insufficiency. In the laparotomy case, abdominal pain is caused by incision, which induces Qi and blood stagnation. ST36 and ST6 have been reported to alleviate many kinds of abdominal pain by regulating and activating Qi and blood movements. When Qi flows smoothly, the pain is relieved. Some studies have found that after EA at ST36, high IL-1 Receptor-I mRNA expression of PAG due to peripheral inflammation was inhibited and the high pain threshold significantly increased . This indicates the analgesia of EA at ST36 may be a consolidation of signals at the PAG level. In the present study, we have found that EA at the bilateral ST36 and SP6 increased the mechanical threshold of rats after laparotomy, as compared with the model group.
EA analgesia is achieved by integration of the wave, pulse width, frequency and intensity of stimulation on acupoints. Among these factors, frequency is a key factor affecting the analgesic effect of EA. We selected 2/15 Hz and 2/100 Hz for EA administration and compared the analgesic effects of these two frequencies. Our results show that 2/15 Hz produced a better analgesic effect in the rat model. Experimental studies have shown that different frequencies can cause the brain and spinal cord to release different types of opioid peptides, which may have different effects . EA stimulation at 2 Hz accelerates the release of endorphins in the brain and large amounts of enkephalin in the spinal cord [45–47], while 15 Hz EA stimulation causes the release of endorphins, beta-endorphin, endomorphin and dynorphin, and 100 Hz EA stimulation causes the spinal cord to release dynorphin [10, 48–51]. Thus, 2/15 Hz EA stimulation may accelerate the release of many different endogenous opioid peptides , each of which strengthens the effect of the others. Therefore, this frequency of EA can produce a strong analgesic effect.
OXA and EA analgesia
Recent studies show that orexins, especially OXA, may play an important role in pain modulation. Previous studies showed that OXA injection into the posterior hypothalamus of rats reduced the afference of facial A- and C-fibers to electrical and heat stimulation, while OXB increased the afferent response to heat stimuli . In addition, prepro-orexin (PPO) knockout mice displayed significant hyperalgesia and reduced stress-induced analgesia compared with wild-type mice, suggesting that pain and stress stimulate the orexin system to modulate the pain transmission process . Orexin participates in the nociceptive process both at the spinal cord and supraspinal cord level and may also be involved in the descending inhibitory system of pain regulation [26–28].
The present study showed that intrathecal injection of OXA can relieve the pain state induced by laparotomy, and that this effect was antagonized by the selective OX1R antagonist SB-334867. The OXA analgesia effect of 0.3 nmol and 1.0 nmol was not dose-dependent. Similar observations were made by Bingham et al. . This result may be because OX1R was already saturated for OXA at the dose of 0.3 nmol. In the present study, pre-surgery treatment with SB-334867, instead of post-surgical SB-334867, blocked EA analgesia. We can infer it is because EA activated OXA release to achieve the analgesic effect, which was proven by the ELISA. OXA has a much higher affinity for OX1R than OXB. The above results strongly indicate the involvement of orexin, particularly OXA in EA analgesia. However we can’t completely exclude the possibility that OXB is also involved. In another experiment, the analgesic efficacy of intrathecal injection of OXA was superior to OXB in the paw incision induced pain model . Nevertheless we can infer from the present results that OXA is a more potent factor in participating in EA analgesia than OXB.
Because EA mainly achieves its analgesic effect by stimulating the release of opioid peptides, whether SB-334867 blocked EA in an opioid-dependent way was also studied. We conducted experiments using intrathecal injections of naloxone, which has an extremely high affinity for μ-opioid receptors and a lower affinity at κ- and δ-opioid receptors in the CNS. Therefore, naloxone mainly antagonizes the analgesic effect of endorphins, as well as part of the effects of dynorphin and enkephalin. There is strong evidence that naloxone can block EA analgesia; however, inhibition is not complete. Therefore, we can’t exclude the role of opioids in acupuncture analgesia. Unfortunately, we did not repeat the naloxone + EA experiment because our study focused on OXA involvement in acupuncture analgesia.
We found that naloxone (28 nmol), at a dose that blocked dynorphin-induced analgesia, was unable to block OXA-induced analgesia. This result is in accordance with previous studies [21, 29]. Also, the OX1R receptor antagonist SB-334867, at a dose that blocked EA analgesia, did not block fentanyl-induced analgesia.
In addition, OXA levels in the hypothalamus, PAG and spinal cord also decreased significantly after laparotomy and were reversed by EA. The above areas are included in the descending inhibitory system. This result has shown that EA can regulate the descending inhibitory system to achieve an analgesic effect. We can only infer that the possible reason for an OXA decrease in the model group is that the laparotomy is a traumatic injury to rats and induces a stress response, which causes exhaustion of OXA in the CNS. OXA is produced through hydrolysis of its precursor PPO by the proteolytic enzyme during the process of axonal transport . The mechanism why EA increased OXA levels remains to be explored. One possible mechanism is that EA stimulation promotes the axon transmission of OXA and accelerates proteolysis, which in turn increases OXA production and release . Whether EA initiates the rapid response of cells to achieve the subsequent effect remains to be clarified. In the present study, only 2/15 Hz increased OXA levels in the above areas. This may provide further evidence that different frequencies of EA stimulate the release of different peptides.