Imaging the activation of mechanosensitive signaling pathways in response in changes to zonular tension in different regions of the rat lens

Abstract

Purpose : To develop methods to image the activation of mechanosensitive signaling pathways in the different regions of the lens in response to changes to the tension applied to the lens via the zonules that attach the lens to the ciliary muscle.

Methods : The posterior surface of enucleated rat eyes was cut to produce four flaps that allowed the lens-ciliary body preparation to be pinned down, with its anterior surface facing up, to the bottom Sylgard-coated recording chamber that was mounted on a laser scanning confocal microscope (FV3000, Olympus). The iris and the cornea were then removed to facilitate imaging of the lens from its anterior surface using a water immersion objective. Ca²⁺-sensitive dyes were loaded into the lens anterior epithelial cells by incubating the lens in the esterified dye Cal520-AM (AAT Bioquest, Sunnyvale, USA) for 40 min at 33°C, while to study the underlying fiber cells, de-esterified dyes Fluo-8 and Fura Red dyes (Pentapotassium salt) were introduced by microinjection at various depths into the lens. The tension applied to the lens was changed by pharmacologically modulating the contractility of the ciliary muscle by the addition of either pilocarpine or tropicamide to decrease or increase, respectively, zonular tension in the absence or presence of reagents that modulate the activity of mechanosensitive ion channels.

Results : Using the optical sectioning ability of the confocal microscope to visualize the influx of Ca²⁺, we found distinct responses in the central and equatorial epithelium, along with differentiating fiber cells, upon exposure to Capsaicin and GSK1016790A, that activate the mechanosensitive channels TRPV1 and TRPV4, respectively. The application of tropicamide, leading to ciliary muscle relaxation, resulted in increased zonular tension, TRPV4 activation, and intracellular Ca²⁺ mobilization in the central anterior epithelium. Conversely, the pilocarpine-induced contraction of the ciliary muscle, accompanied by decreased tension and TRPV1 activation, induced a modest reduction in lens intracellular Ca²⁺ levels.

Conclusions : Our novel tissue preparation and imaging protocols allows us to investigate how changes in zonular tension are transduced via mechanosensitive channels into changes in Ca²⁺ that activate cell signaling pathways known to control lens water transport and therefore the optical properties of lens.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.