Ghovanloo Mohammad-Reza, Effraim Philip R, Tyagi Sidharth, Aldrich Alecia M, Cheng Xiaoyang, Yuan Jun-Hui, Schulman Betsy R, Jacobs Deborah S, Dib-Hajj Sulayman D, Waxman Stephen G
From the Department of Neurology (M.-R.G., S.T., A.M.A., X.C., J.-H.Y., B.R.S., S.D.D.-H., S.G.W.), Yale University School of Medicine, New Haven; Center for Neuroscience and Regeneration Research (M.-R.G., P.R.E., S.T., A.M.A., X.C., J.-H.Y., B.R.S., S.D.D.-H., S.G.W.), Yale University, New Haven; Neuro-Rehabilitation Research Center (M.-R.G., P.R.E., S.T., A.M.A., X.C., J.-H.Y., B.R.S., S.D.D.-H., S.G.W.), Veterans Affairs Connecticut Healthcare System, West Haven; Department of Anesthesiology (P.R.E.), Yale University School of Medicine, New Haven, CT; and Department of Ophthalmology (D.S.J.), Massachusetts Eye and Ear, Harvard Medical School, Boston.
Neurol Genet. 2024 Nov 14;10(6):e200206. doi: 10.1212/NXG.0000000000200206. eCollection 2024 Dec.
Despite extensive efforts, the mechanisms underlying pain after axonal injury remain incompletely understood. Pain following corneal refractive surgery offers a valuable human model for investigating trigeminal axonal injury because laser-assisted in situ keratomileusis (LASIK) severs axons of trigeminal ganglion neurons innervating the cornea. While the majority of patients are pain-free shortly after surgery, a minority endure persistent postoperative ocular pain. Through genomic analysis of patients experiencing persistent postoperative ocular pain, we identified rare variants in genes encoding ion channels and receptors, including TRPM8, which codes for the menthol-sensitive and cold-sensing transient receptor potential cation channel.
We conducted a profiling of 2 TRPM8 mutant variants, D665N and V915M, which were identified in patients suffering from persistent pain after LASIK surgery. We used patch-clamp and multielectrode array (MEA) recordings to investigate the biophysical and pharmacologic properties of mutant vs wild-type (WT) channels.
Patch-clamp analysis shows that these mutations shift the activation curves of TRPM8 in a hyperpolarized direction, with this effect being significantly different between WT and D665N channels. In addition, both mutations significantly increase channel sensitivity to the canonical ligand, menthol. MEA recordings from transfected rat trigeminal ganglion neurons indicate that expression of D665N and V915M mutant channels increases spontaneous activity compared with WT channels. Consistent with patch-clamp results, neuronal activity in MEA recordings was increased on exposure to menthol.
Collectively, our findings suggest that proexcitatory mutations of TRPM8, in the context of axonal injury within the cornea, can produce trigeminal ganglion neuron hyperexcitability that contributes to persistent postoperative ocular pain. In addition to providing additional evidence for a role of TRPM8 in human pain, our results suggest that inhibitors of this channel merit future study.
尽管进行了大量研究，但轴突损伤后疼痛的潜在机制仍未完全明确。角膜屈光手术后的疼痛为研究三叉神经轴突损伤提供了一个有价值的人体模型，因为准分子原位角膜磨镶术（LASIK）会切断支配角膜的三叉神经节神经元的轴突。虽然大多数患者术后不久就没有疼痛，但少数患者会忍受持续性的术后眼痛。通过对经历持续性术后眼痛的患者进行基因组分析，我们在编码离子通道和受体的基因中发现了罕见变异，包括TRPM8，它编码对薄荷醇敏感且能感知寒冷的瞬时受体电位阳离子通道。
我们对在LASIK手术后遭受持续性疼痛的患者中鉴定出的两种TRPM8突变变体D665N和V915M进行了分析。我们使用膜片钳和多电极阵列（MEA）记录来研究突变型与野生型（WT）通道的生物物理和药理学特性。
膜片钳分析表明，这些突变使TRPM8的激活曲线向超极化方向移动，WT和D665N通道之间的这种效应存在显著差异。此外，两种突变均显著增加了通道对典型配体薄荷醇的敏感性。转染大鼠三叉神经节神经元的MEA记录表明，与WT通道相比，D665N和V915M突变通道的表达增加了自发活动。与膜片钳结果一致，MEA记录中的神经元活动在暴露于薄荷醇时增加。
总体而言，我们的研究结果表明，在角膜内轴突损伤的情况下，TRPM8的促兴奋性突变可导致三叉神经节神经元兴奋性过高，从而导致持续性术后眼痛。除了为TRPM8在人类疼痛中的作用提供更多证据外，我们的结果还表明该通道的抑制剂值得未来研究。
