Schecterson Leslayann C, Pazevic Alexander A, Yang Ruian, Matulef Kimberly, Gordon Sharona E
Department of Physiology & Biophysics, University of Washington School of Medicine, Seattle, WA.
Mol Vis. 2020 Aug 13;26:576-587. eCollection 2020.
The cornea is highly enriched in sensory neurons expressing the thermal TRP channels TRPV1, TRPA1, and TRPM8, and is an accessible tissue for study and experimental manipulation. The aim of this work was to provide a concise characterization of the expression patterns of various TRP channels and vesicular proteins in the mammalian cornea.
Immunohistochemistry (IHC) was performed using wholemount and cryostat tissue preparations of mouse and monkey corneas. The expression patterns of TRPV1 and TRPA1 were determined using specific antisera, and further colocalization was performed with antibodies directed against calcitonin-related gene protein (CGRP), neurofilament protein NF200, and the secretogranins ScgII and SCG3. The expression of TRPM8 was determined using corneas from mice expressing EGFP under the direction of a TRPM8 promoter ( mice). Laser scanning confocal microscopy and image analysis were performed.
In the mouse cornea, TRPV1 and TRPM8 were expressed in distinct populations of small diameter C fibers extending to the corneal surface and ending either as simple or ramifying terminals, or in the case of TRPM8, as complex terminals. TRPA1 was expressed in large-diameter NF200-positive Aδ axons. TRPV1 and TRPA1 appeared to localize to separate intracellular vesicular structures and were primarily found in axons containing components of large dense vesicles with TRPV1 colocalizing with CGRP and ScgII, and TRPA1 colocalizing with SCG3. Monkey corneas showed similar colocalization of CGRP and TRPV1 on small-diameter axons extending to the epithelial surface.
The mouse cornea is abundant in sensory neurons expressing TRPV1, TRPM8, and TRPA1, and provides an accessible tissue source for implementing a live tissue preparation useful for further exploration of the molecular mechanisms of hyperalgesia. This study showed that surprisingly, these TRP channels localize to separate neurons in the mouse cornea and likely have unique physiological functions. The similar TRPV1 expression pattern we observed in the mouse and monkey corneas suggests that mice provide a reasonable initial model for understanding the role of these ion channels in higher mammalian corneal physiology.
角膜富含表达热TRP通道TRPV1、TRPA1和TRPM8的感觉神经元，是一个便于进行研究和实验操作的组织。本研究的目的是简要描述各种TRP通道和囊泡蛋白在哺乳动物角膜中的表达模式。
使用小鼠和猴角膜的整装和冰冻切片组织制备进行免疫组织化学（IHC）。使用特异性抗血清确定TRPV1和TRPA1的表达模式，并进一步用针对降钙素相关基因蛋白（CGRP）、神经丝蛋白NF200以及分泌粒蛋白ScgII和SCG3的抗体进行共定位。使用在TRPM8启动子指导下表达增强绿色荧光蛋白（EGFP）的小鼠（TRPM8-EGFP小鼠）的角膜确定TRPM8的表达。进行激光扫描共聚焦显微镜检查和图像分析。
在小鼠角膜中，TRPV1和TRPM8在延伸至角膜表面的不同群体的小直径C纤维中表达，这些纤维以简单或分支末端结束，就TRPM8而言，则以复杂末端结束。TRPA1在大直径NF200阳性Aδ轴突中表达。TRPV1和TRPA1似乎定位于不同的细胞内囊泡结构，主要存在于含有大致密囊泡成分的轴突中，TRPV1与CGRP和ScgII共定位，TRPA1与SCG3共定位。猴角膜在延伸至上皮表面的小直径轴突上显示出CGRP和TRPV1的类似共定位。
小鼠角膜富含表达TRPV1、TRPM8和TRPA1的感觉神经元，为实施用于进一步探索痛觉过敏分子机制的活组织制备提供了一个便于获取的组织来源。本研究表明，令人惊讶的是，这些TRP通道定位于小鼠角膜中的不同神经元，可能具有独特的生理功能。我们在小鼠和猴角膜中观察到的相似TRPV1表达模式表明，小鼠为理解这些离子通道在高等哺乳动物角膜生理学中的作用提供了一个合理的初始模型。
