Study On The Role Of Spinal Cord α7nAChRs In Remifentanil Hyperalgesia And Its Mechanism

Part I The hyperalgesia of remifentanil and the effect of spinal a7nAChRs activation on remifentanil induced hyperalgesia behavioralFor the treatment of acute and chronic pain, opioids are still the most major drugs. Intravenous opioid receptor agonists, such as fentanyl, alfentanil, sufentanil, remifentanil are the most commonly used analgesic drugs in treating acute and chronic pain. And the use of these drugs shared the dominant position in the maintenance of anesthesia during the operation. During the use of these drugs in clinical, opioid induced hyperalgesia was found in the patients without these drugs medication anymore. This hyperalgesia not only reduces the analgesic effect of the drugs, but also increased postoperative discomfort and prolonged the recovery period after operation.Opioid-induced hyperalgesia OIH (opioid-induced hyperalgesia) refers to the abnormal phenomenons characterized by paresthesia with lower pain threshold and overreaction to normal stimuli in patients exposure to opioids. Remifentanil as an ultra short acting opioid receptor agonist characterized by many virtues, such as strong analgesic effect, rapid onset, short action time, rapid elimination, continuous infusion without accumulation, metabolism not dependent on the function of liver and kidney. Therefore, remifentanil was widely used in clinical anesthesia. But the study of the clinical application of remifentanil found that remifentanil provides satisfactory analgesia effect also induces hyperalgesia in these patients at the same time. The pharmacological properties of ultra short acting of remifentanil make the hyperalgesia more seriously, aggravating patients’discomfort, affecting postoperative recovery, limiting the use of remifentanil in clinical.The study of remifentanil induced hyperalgesia indicated that a variety of mechanisms and signal transduction pathways are involved in this process, but the exact mechanisms are unclear and remain to be elucidated. The immune responses of central and peripheral are mediated by alpha 7 nicotinic acetylcholine receptors (a7nAChRs) in glial cells. Research on central and peripheral glial cells found that a7nAChRs agonists could significantly reduce the generation of proinflammatory products from glial cells. In the study of inflammatory pain and neuropathic pain, activation a7nAChRs could decrease the hyperalgesia phenomena in these animal models. But the effect of a7nAChRs agonists in opioid induced hyperalgesia is still no relevant reports.In this experiment, we use the Brennan method to prepare the standard rat model of incision pain. During the process of model preparation, remifentanil was subcutaneous infused at the dose of 40μg/kg and the time of infusion was 30min. Using this method, we made the remifentanil induced hyperalgesia animal models. In order to investigate the effect of spinal a7nAChRs activation on remifentanil induced hyperalgesia, we intrathecal injected the a7nAChRs agonists and positive allosteric modulators.Assessments of pain behavior we used in this study were the mechanical withdrawal threshold (Paw Withdrawal Mechanical Threshold, PWMT) and thermal withdrawal latency (Paw Withdrawal Thermal Latency, PWTL). Evaluation of remifentanil induced hyperalgesia were recorded at 24h before the surgery and 2h,6h, 24h,48h after the operation respectively. The a7nAChRs agonists and positive allosteric modulators were injected at 24h after the operation in which the remifentanil induced hyperalgesia was relatively stable. The changes of behavior were measured at 1h before injection and 0.5h, 1h,2h,4h,6h after the injection. The results demonstrated:1. Intraoperative infusion of remifentanil induced postoperative hyperalgesia significantly; 2. The a7nAChRs agonists (PHA-543613) and type Ⅱ positive allosteric modulators (PNU-120596) could inhibition remifentanil induced hyperalgesia dose dependently, but the type I positive allosteric modulators (NS-1738) had no such effect; 3. The analgesic effect of PHA-543613 and PNU-120596 could be blocked by a7nAChRs antagonist ML A.4. The combined injection of sub-analgesic doses of a7nAChRs agonists and type Ⅱ positive allosteric modulators could inhibit remifentanil induced hyperalgesia.In conclusion, this experiment adopts the model of remifentanil induced hyperalgesia to imitate the clinical hyperalgesia by subcutaneously infusion of remifentanil during the process of making incision pain and studied the effect of spinal a7nAChRs activation on remifentanil induced hyperalgesia firstly. The a7nAChRs agonists and type II positive allosteric modulators and the combined use of these two drugs could inhibit remifentanil induced hyperalgesia, but type I positive allosteric modulators had no effect. This study provided new experimental evidence which will help to search and determine the drug targets of proper pathway for treating remifentanil induced hyperalgesia.Part Ⅱ The mechanism of activation spinal cord a7nAChRs inhibit remifentanil induced hyperalgesiaPain was due to plasticity changes in the passage from peripheral sense to the cerebral cortex. The central and outer peripheral immune cells play important roles in this process, involving the initiation and maintenance of pain. Spinal microglia and astrocytes are important immune-related cells in the central nervous. Actived glial cells and proinflammatory products release from these cells are thought to be important factors involved in maintained hyperalgesia in many pain models. Therefore, inhibition of glial activation and proinflammatory product reaction could be invoked as a target for the treatment of pain.The a7nAChRs were expressed on astrocytes and microglia in the spinal cord. Previously researchs showed that the activation of spinal a7nAChRs could produce significant antinociception effect in various pain models through downregulating the synthesis of proinflammatory cytokine. Combined with the role of proinflammatory cytokines in remifentanil induced hyperalgesia of our group previously study, suggesting that activation of spinal a7nAChRs could produce an analgesic effect by inhibiting microglia and astrocytes and proinflammatory cytokines derived from glial cell.In the occurrence and maintenance of hyperalgesia, the enhancement of excitatory pathway in synaptic transmission is one of the most essential parts. A large number of studies have shown that N-methyl-D-aspartate receptors (NMDA receptor) play a central role in the process of excitatory synaptic transmission pathway. The opioid induced hyperalgesia is closely related to the over activation of spinal NMDA receptors. Previous research of our team found that the expression of spinal cord tyrosine phosphorylation NR2B (functional NMDA receptor subunit) increased significantly in remifentanil induced hyperalgesia. Whether or not activation spinal α7nAChRs could attenuate remifentanil induced hyperalgesiapass by inhibiting the enhanced excitatory signal pathway through decreasing the overexpression of tyrosine phosphorylation NR2B is still remains unknown.When the excitatory pathway enhanced during the occurrence and maintenance of hyperalgesia, the decreased inhibiting signal pathway is also involved in this procession. Central neuronal synaptic inhibition is mediated through gamma aminobutyric acid (GABA) and glycine (Gly). GABAAR and GlyR could induce inhibitory effect through transport of anions, such as chloride ion (Cl-) and bicarbonate (HCO3-). The steady state of neurons chloride ion concentration is maintained by the transmembrane transport of K+-Cl- cotransporter 2 (KCC2). The KCC2 will downregulate the intracellular Cl- concentration by transporting the Cl- out of the cell along with the K+ gradient, thereby maintaining the intracellular concentration of lower Cl-. This was a precondition for the inhibition effect of GABAAR and GlyR. Brain derived neurotrophic factor (BDNF) released from activated spinal glial cells could downregulated the expression of neuronal KCC2 rapidly through the combination with neuronal TrkB receptors, thereby reducing extracellular transport of Cl- and attenuating the inhibittion of GABAAR and GlyR. These results suggested that activation of spinal α7nAChRs could inhibit remifentanil induced postoperative hyperalgesia probably by enhancing inhibitory pathway activity through downregulating BDNF-TrkB signal pathway.Therefore, combined with behavioral results in the first part, this part of the experiment study on possible mechanism by which α7nAChRs agonists and type Ⅱ PAMs inhibiting remifentanil induced hyperalgesia. This study carried through three aspects:1. Effect of a7nAChRs agonists and typeⅡPAMs on microglia, astrocytes and proinflammatory cytokines of the spinal cord in remifentanil induced hyperalgesia; 2. Effect of α7nAChRs agonists and type Ⅱ PAMs on tyrosine phosphorylation NR2B (p-NR2B) of the spinal cord in remifentanil induced hyperalgesia; 3. Effect of α7nAChRs agonists and type II PAMs on BDNF of the spinal cord in remifentanil induced hyperalgesia. Our experiments expounded the mechanism of activation spinal α7nAChRs attenuate remifentanil induced hyperalgesia through downregulating cholinergic anti-inflammatory pathway, decreasing the excitatory pathway and enhancing the inhibitory pathway. The results show that activation of spinal alpha 7nAChRs can significantly attenuate over-expressed the microglia, astrocytes, reduce the expressions of tumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), p-NR2B and BDNF.In summary, this is the first study and reported the possible mechanism of activation of spinal a7nAChRs inhibiting remifentanil induced hyperalgesia. Our results showed the weaken of glial cell and proinflammatory cytokines, the attenuation of the excitatory pathway and enhancement of inhibition transduction, are all involved in the mechanisms of analgesic effect of a7nAChRs on remifentanil induced hyperalgesia. This result provides a theoretical and experimental basis of looking for new rational control of remifentanil hyperalgesia.