术中应用瑞芬太尼对术后慢性疼痛的影响及脊髓小胶质细胞TLR4/PGE2信号通路在其中的作用

Chronic post-surgical pain (CPSP) can result in a decrease of quality of life, physical incapacitation, and an economic burden, therefore needs to be prevented and managed actively. In previous studies, we found that acute fentanyl induced hyperalgesia and increased acute postoperative pain in rats mediated through activating of toll-like receptor 4 (TLR4) on spinal microglia. However, the role of intraoperative remifentanil, a short acting opioid, on CPSP is still unclear. Intraoperative remifentanil may increase the production of Prostaglandin E2 (PGE2) through increasing the activation the TLR4 on spinal microglia induced by peripheral nervous injury. The increase of PGE2 results in a blockade of microglial migration in the spinal cord. The retaining microglia may engulf both injured and uninjured myelinated axons and induce injuries to spinal cord neurons. Activation of both TLR4 and PGE2 may also induce the apoptosis of neurons in spinal cord and dorsal root ganglion (DRG). Thus, excessive microglia activation induced by intraoperative remifentanil may introduce central pain components to peripheral nervous injury and increase the incidence of CPSP. In the present study, we try to understand if intraoperative remifentanil increase the risk of CPSP by investigating multi-dose intraoperative remifentanil on the incidence of chronic post-thoracotomy pain in mice with enough sample size. Then the possible mechanism underlying remifentanil on risk of CPSP will be investigated. The mice will be pre-administrated with opioid antagonist naloxone or intercostal nerve block, or to block the activation of TLR4 and PGE2 by injecting with microglial inhibitor, TLR4 antagonist and PGE2 inhibitor before surgery or using TLR4 and microsomal PGE synthase (mPGES)-1 knockout mice. After these interventions, mice will be conducted a surgery of thoracotomy or/and intraoperative remifentanil infusion. 14 days after operation, the spinal cord and DRG will be removed. The differences of activation of microglia and TLR4 inflammatory cascade, the neuron apoptosis and nervous injury will be investigated by molecular immunological tests. The data from all of these experiments will clarify the role of spinal TLR4/PGE2 signaling pathway on mechanism of remifentanil inducing increase of CPSP, thereby, providing novel strategy for clinical treatment of CPSP.

防治术后慢性疼痛是重要的临床课题。前期研究发现急性芬太尼可激活脊髓小胶质细胞和Toll样受体4（TLR4）炎症通路，引起痛觉过敏并加重大鼠术后急性疼痛，但术中瑞芬太尼对术后慢性疼痛的影响还不明确。瑞芬太尼将加重外周神经损伤引起的脊髓小胶质细胞和TLR4炎症通路激活，加重神经炎症和神经损伤，可能增加术后慢性疼痛发生。本研究拟探究术中多剂量瑞芬太尼对足够样本量的小鼠开胸术后慢性疼痛模型成功率的影响，明确其是否增加术后慢性疼痛；预先给予阿片受体拮抗剂纳洛酮或肋间神经阻滞等干预措施，以及行药理学或基因敲除阻断加重神经损伤的关键因子TLR4和前列腺素E2活性后，进行开胸术或瑞芬太尼干预，取材脊髓和背根神经节，以分子免疫学方法明确不同干预下脊髓和背根神经节小胶质细胞和TLR4炎症通路激活、神经元凋亡和神经损伤的差异，明确其影响术后慢性疼痛发生的机制，为临床防治术后慢性痛提供实验依据。

临床上开胸手术可30-50%的术后慢性疼痛发生，防治术后慢性疼痛是重要的临床课题。作为术中主要镇痛用药，瑞芬太尼对术后慢性疼痛的影响尚未明确。本课题采用开胸后牵拉60分钟建立了大鼠的术后慢性痛模型；给予不同剂量的瑞芬太尼，观察瑞芬太尼对开胸术后慢性痛大鼠模型的行为学及分子学影响，初步探索其发生机制。研究发现：术中使用160、320、480μg/kg的瑞芬太尼可显著加剧开胸术后大鼠的早期疼痛（2小时-3天），320、480μg/kg的瑞芬太尼可显著增加开胸术后大鼠在后期（7-40天）的疼痛发生率，480μg/kg的瑞芬太尼作用更加明显。开胸牵拉并术中盐水或瑞芬太尼（160-480μg/kg）可引起大鼠脊髓早期（1-7天）NF-κB表达上调；开胸牵拉和术中低剂量瑞芬太尼（0-160μg/kg）脊髓ATF3表达没有改变，术中高剂量瑞芬太尼（320-480μg/kg）可引起大鼠脊髓晚期（14-40天）表达上调；开胸牵拉并术中盐水或瑞芬太尼（160-480μg/kg）可引起大鼠脊髓Iba1（小胶质细胞标记物）表达上调（1-40天）；开胸牵拉并术中盐水或瑞芬太尼（160-480μg/kg）可引起大鼠脊髓GFAP表达（星形胶质细胞标记物）表达晚期（14-40天）上调；开胸牵拉可引起大鼠前扣带皮层及脊髓中肾脑表达蛋白（KIBRA）表达上调，术后14天痛敏阳性的大鼠KIBRA上调更加明显。上述结果说明：大剂量瑞芬太尼可引起开胸术后慢性疼痛的发生率增加，其机制可能与胶质细胞的活化以及脊髓中相关通路的激活有关，该研究结果将为临床防治术后慢性疼痛的提供新的思路。。