Mechanoreceptive TG Nerve Ending in the Anterior Chamber Structures of Rat Eye

Program Number: 3253 Poster Board Number: A0173 Presentation Time: 11:00 AM–12:45 PM Mechanoreceptive TG Nerve Ending in the Anterior Chamber Structures of Rat Eye Haixia Liu2, Qingli Meng2, Peng Fang1, Zhuangli Hu1, Yun Ling2, Hong zhang2, Xiongwu Zhou2, Bo Chen2. 1Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Purpose: Although studies have shown that the primary afferent nerve in the intraocular pressure (IOP) neural regulation exist in the trigeminal ganglion (TG) nerve, it is still not known whether the nerve endings of the afferent neurons in TG sense IOP through a mechanotransduction mechanism as the other mechanoreceptors that have been documented. This study was designed to determine whether the TG nerve endings distributing in the anterior chamber structures had the function of mechanotransduction. Methods: Rat TG neurons innervating the anterior chamber structures were labeled by anterior chamber injection of 1,1’-Dilinoleyl-3,3,3’,3’-Tetramethylindocarbocyanine, 4-Chlorobenzenesulfona (FAST DiI). The neuronal cell bodies were voltage clamped using whole-cell patch-clamp techniques, while it was deformed by ejection of bath solution to verify mechanotransduction. Immunofluorescence staining of transient receptor potential (TRP) A1, TRPV4, acid-sensing ion channel (ASIC) 3, ASIC2, Piezo1, and Piezo2 were performed on the sections of TG ganglia to determine the specific mechanosensitive (MS) channel proteins in the membrane of FAST DiI-labeled TG neurons. Results: Mechanical stimulation induced an inward current in 55 out of 96 DiI-labeled TG neurons. The magnitude of the current was related to the intensity of stimulation. The blockage effect of lanthanide gadolinium, a reported blocker of MS channels on the inward currents indicated that the inward currents were evoked via MS channel activation. Mechanical stimulation further enhanced the membrane potential and increased the frequency of action potentials.The mechanically evoked inward current was inhibited by ruthenium red and amiloride.TRPA1, TRPV4, ASIC2, and ACIC3 channel proteins were expressed in FAST DiI-labeled TG neurons. Allylisothiocyanate, a specific analog of TRPA1, evoked an inward current in FAST DiI-labeled TG neurons, while the mechanically evoked current was inhibited by HC-030031, a specific inhibitor of TRPA1.These results indicated that TRPA1 might be an essential mechanotransduction channel protein in the membrane of DiI-labeled TG neurons. Conclusions: We proved the mechanotransduction by TG neurons innervating the anterior chamber structures in vitro. Therefore, we propose Mechanoreceptive TG nerve endings are present in the anterior chamber structures of the rat eye. These mechanosensitive terminals may act as baroreceptor of IOP. Commercial Relationships: Haixia Liu, None; Qingli Meng, None; Peng Fang, None; Zhuangli Hu, None; Yun Ling, None; Hong zhang, None; Xiongwu Zhou, None; Bo Chen, None Support: This work was supported by a grant from the National Natural Science Foundation of China (No. 81070727). Program Number: 3254 Poster Board Number: A0174 Presentation Time: 11:00 AM–12:45 PM Direct interaction of cochlin with mechanosensing channel TREK-1 in intraocular pressure regulation Teresia Carreon1, Carmen Piqueras1, Aida Castellanos2, Xavier Gasull2, Sanjoy K. Bhattacharya1. 1Ophthalmology, University of Miami Bascom Palmer Eye Institute, Miami, FL; 22. Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain. Purpose: To determine the direct interaction between the extracellular matrix protein cochlin and the mechanotransducing channel TREK-1 in intraocular pressure regulation Methods: TREK-1 mRNA was silenced in the DBA/2J mouse model in an effort to determine its effect on intraocular pressure. A sodium fluorescein dye assay along with a gel expansion assay was implemented to mimic the effect of the cochlin/TREK-1 interaction on trabecular meshwork cell conformation. The interaction was further characterized by a co-immunoprecipitation and the utilization of a yeast – two hybrid system. Patch – clamp whole cell recording was also performed in the presence or absence of cochlin in order to measure TREK-1 channel activity. Results: TREK-1 mRNA silencing prevented a cochlin-induced increase in intraocular pressure (IOP). The presence of cochlin and TREK-1 together causes a spatial change in the cells of the trabecular meshwork as demonstrated by the increase of the fluorescein dye and the increase in collagen length in the gel expansion assay. A coimmunoprecipitation further demonstrated the increase of cochlin and TREK-1 specifically under shear stress and ionic stress conditions. The yeast-two hybrid system using a T7 promoter sequence demonstrated direct interaction between the bait (hCochlin) and TREK-1. In addition, cochlin interaction reduced TREK-1 channel currents as demonstrated by whole cell patch clamp recordings. Conclusions: The results support the potential interaction occurring between cochlin and TREK-1 under stressful conditions, which in turn may result in extracellular matrix remodeling and an increase in IOP. Commercial Relationships: Teresia Carreon, None; Carmen Piqueras, None; Aida Castellanos, None; Xavier Gasull, None; Sanjoy K. Bhattacharya, None Support: National Institute of Health Grants R01 EY016112, EY015266, EY021012, EY016775, and EY014801 and an unrestricted grant to the University of Miami’s Bascom Palmer Eye Institute from Research to Prevent Blindness. Funding to XG was provided by Instituto de Salud Carlos III, Spain (FIS PI11/01601 and RETIC RD12/0034/0003) and Generalitat de Catalunya (2009SGR869).