Pharmacological Regulation of Water Transport and its Effects on Physiological Optics in the Bovine Lens

Abstract

The reduced quality of life and financial burden associated with visual impairment and blindness will increase dramatically as world’s population ages. Although presbyopia and cataract are the two leading causes of age-related vision loss, the pathophysiological mechanisms responsible for these two lens pathologies is still not fully understood. Through a series of experiments, the Molecular Vision Laboratory (MVL) has shown that the optical properties of the bovine lens are actively maintained by circulating ionic and fluid fluxes that generate an internal microcirculation. In the mouse lens, it has been shown that this water circulation through gap junction channels generates a substantial intracellular hydrostatic pressure gradient that is subjected to a dual feedback regulation system. This discovery led to the hypothesis that the regulation of the lens water transport (pressure gradient) is directly linked to changes in the lens optics; it is this hypothesis I have endeavoured to test in this thesis. To achieve this, I have developed a pico-injector/microelectrode based whole lens measurement system and utilized it to study the regulation of water transport/pressure in the larger bovine lens, which is our chosen animal model to study lens optics. Using this system, I showed that like the mouse lens, the larger bovine lens has a hydrostatic pressure gradient that varies from 0 mmHg in the surface to 350 mmHg in the core of the lens, and that inhibition of microcirculation reduces the magnitude of this pressure gradient. Again like in the mouse lens, the surface pressure in the bovine lens was found to be regulated by a dual feedback system that was mediated by the reciprocal modulation of the transient receptor potential vanilloid channels, TRPV1 and TRPV4. These TRPV channels were confirmed in the bovine lens by both Western blotting and immunohistochemistry. Pharmacological activation of TRPV1 or TRPV4 induced a biphasic increase and decrease, respectively, in the hydrostatic pressure recorded at the lens surface. This biphasic response was abolished if lenses were pre-treated with either a TRPV1, or TRPV4 inhibitors, prior to activation of the channels. These results suggest that this TRPV1/4 mediated feedback control system is a conserved mechanism, in at least the mouse and bovine lenses, that serves to regulate lens hydrostatic pressure and thus maintaining a constant water transport and water content. To then test whether this TRPV1/4-mediated dual feedback system, which controls hydrostatic pressure, also modulates lens power, I utilised a laser ray tracing (LRT) system to measure the optical properties of the lens in the absence and presence of reagents that manipulate the microcirculation system and/or the pressure gradient. Using an LRT protocol that monitored the back focal length and therefore lens optical power, I showed that the biphasic decrease in lens surface pressure induced by TRPV4 activation had no significant effect on lens power. In contrast, the biphasic increase in lens surface pressure induced by the capsaicin-mediated activation of TRPV1 channels did produce a transient increase in the lens optical power. Because the results from these into the effects of biphasic changes in lens surface pressure on overall power using a simple measure of lens power were somewhat inconclusive, I utilised a slower LRT-GRIN scan protocol to determine the relative contributions to lens geometry and GRIN to overall power. Since this protocol necessitated longer scan times, I applied it in experiments where sustained changes in surface pressure were induced the pre-treated of lenses with either a TRPV1, or TRPV4 inhibitors, followed by activation of the channels. I found that the steady-state decrease in lens pressure induced by the activation of TRPV4, while blocking TRPV1 channels tended to cause a small decrease in the lens RI throughout the lens, which was associated with a small increase in anterior curvature of the lens. In contrast, the steadystate increase in lens pressure observed by the activation of TRPV1, in the presence of TRPV4 inhibition, tended to cause a small increase in the lens RI throughout the lens which was associated with a small decrease in anterior curvature of the lens. This pharmacological manipulation of TRPV1 and TRPV4 channel activities induced opposing effects on lens geometry and GRIN, showed a trend towards positive and negative shift in lens power, and had minimal effect on the lens longitudinal spherical aberration and overall vision quality. Taken together, my results show that the proposed link between lens pressure regulation and the lens optical properties is more complicated than the first envisaged and requires a modification to the lens microcirculation system. In my thesis, I present such a modified model that can be used to explain not only my current results but also provides deeper insights into the mechanisms responsible the onset of presbyopia and cataract that will inform the development of novel pharmacological therapies to maintain vision quality in the elderly. Testing this model will be the focus of ongoing work.