Evaluation of Trigeminal Ganglia Sensory Neuronal Population/s Mediating MIF-Induced Anti-Nociception in a Model of Apical Periodontitis.

摘要 尽管根管治疗在治疗根尖周炎等疾病方面取得了成功，但仍有180万患者 可能在治疗六个月后出现持续性疼痛。根尖周炎是由感染根尖的 牙髓会导致机械性超敏和炎症。持续的牙痛增加了负担成本 以及服用止痛药。此外，非类固醇抗炎药(NSAID)和阿片类药物提供 长期服用不完全缓解疼痛和不良反应，突出了对新治疗方法的需要 这可以为数百万经历持续性牙痛的患者提供缓解。干细胞移植的研究进展 为了开发一类新的止痛药，止痛药的性能一直在快速增长。药效 间充质干细胞已在神经病理性疼痛的临床前模型和患者中得到证实 被诊断为偏头痛和骨性关节炎。初步数据显示，静脉注射人 根尖乳头干细胞(HSCAP)完全逆转与根尖周炎相关的过敏反应 老鼠。HSCAP的RNA测序显示，感染牙齿的hSCAP表达增加了133倍 细胞因子巨噬细胞迁移抑制因子(MIF)。此外，MIF受体CD74和CXCR4 共定位于三叉神经节内的TRPV1+神经元。此外，局部注射重组MIF可逆转 与根尖周炎相关的过敏反应。最后，小鼠共培养的条件培养液 根尖周围肉芽肿(受感染的牙齿)和hSCAP减弱辣椒素引起的钙反应 MIF抗体对三叉神经节神经元有抑制作用。这一数据支持了一种新的 干细胞抗伤害性作用的机制可能是通过MIF介导的干细胞的直接作用。 然而，尽管初步数据表明MIF直接抑制三叉神经节神经元活动，但神经元 MIF诱导的抗伤害性反应的亚型尚不清楚。由根尖周炎引起的疼痛通常是 据报道，由于中枢敏化，提示非伤害性纤维参与其中。 了解这些亚群中的哪些参与了MIF诱导的抗伤害感受，将使我们能够开发出 与根尖周炎相关的持续性牙痛的靶向治疗。我们将测试中央 假设MIF对三叉神经节神经元活动的抑制是由伤害性和非伤害性介导的 伤害性神经元。为了验证这一假设，我们将1)进行单细胞RT-PCR和免疫组织化学 检测MIF受体CD74和CXCR4与不同神经元标志物的共表达；2)用Cre- 重组酶条件性敲除MIF受体CD74和CXCR4 并评价其在根尖周炎模型中对MIF抗伤害性的作用。这些研究将 不仅为介导MIF诱导的抗伤害性感受的神经元群体提供了新的见解，而且还 为我的临床科学家职业生涯提供了极好的培训工具。

Abstract Despite the success of root canal treatments at treating diseases such as apical periodontitis, 1.8 million patients may experience persistent pain six months after treatment. Apical periodontitis is caused by an infection of the dental pulp leading to mechanical allodynia and inflammation. Persistent dental pain increases cost of burden and intake of analgesic drugs. Furthermore, non-steroidal anti-inflammatory drugs (NSAIDs) and opioids provide incomplete pain relief and adverse effects when taken chronically, highlighting the need for a novel treatment that can provide relief to millions of patients experiencing persistent dental pain. The study of stem cells for their analgesic properties has been rapidly growing in hopes of developing a novel class of analgesics. The efficacy of mesenchymal stem cells has been demonstrated in pre-clinical models of neuropathic pain and in patients diagnosed with migraines and osteoarthritis. Preliminary data demonstrates that intravenous injections of human stem cells of the apical papilla (hSCAP) fully reverses hypersensitivity associated with apical periodontitis in mice. RNA sequencing of hSCAP homed to the infected tooth shows a 133-fold increase in the expression of the cytokine Macrophage Migratory Inhibitory Factor (MIF). Additionally, MIF receptors CD74 and CXCR4 colocalize on TRPV1+ neurons in the trigeminal ganglia. Moreover, a local injection of recombinant MIF reverses hypersensitivity associated with apical periodontitis. Lastly, conditioned media from co-cultures of mouse periapical granulomas (the infected tooth) and hSCAP attenuates capsaicin evoked Ca2+ response from trigeminal ganglia neurons and is inhibited with pre-treatment of a MIF antibody. This data supports a novel mechanism for stem cell anti-nociception through the direct effect of stem cells that is possibly mediated by MIF. However, despite primary data suggesting MIF directly inhibits trigeminal ganglia neuronal activity, the neuronal subtypes that MIF-induced anti-nociception are unknown. The pain induced by apical periodontitis is often reported as referred, suggesting the involvement of non-nociceptive fibers due to central sensitization. Understanding which of these subpopulations mediate MIF-induced anti-nociception will allow us to develop targeted treatments for persistent dental pain associated with apical periodontitis. We will test the central hypothesis that MIF-induced inhibition of trigeminal ganglia neuronal activity is mediated by nociceptive and non- nociceptive neurons. To test this hypothesis, we will 1) conduct single cell RT-PCR and immunohistochemistry to determine co-expression of MIF receptors CD74 and CXCR4 with different neuronal markers and 2) use Cre- recombinase to conditionally knockout MIF receptors CD74 and CXCR4 from specific neuronal populations in vivo and evaluate their contributions to MIF anti-nociception in a model of apical periodontitis. These studies will not only provide novel insight into the neuronal populations that mediate MIF-induced anti-nociception but also serve as an excellent training vehicle for my career as a clinician-scientist.