Abstract
Purpose :
Increased IOP results in narrowing of Schlemm’s canal (SC), which increases the shear stress acting on SC cells. SC cells respond to shear stress by producing nitric oxide (NO), which increases outflow facility and lowers IOP. We hypothesize that increased IOP stimulates NO production by SC cells, comprising part of a homeostatic mechanism to regulate IOP. To measure in situ NO production within the outflow pathway of mice exposed to elevated IOP, we utilised a peptide-based biosensor that is sensitive to changes in NO. We also examined whether inhibition of NO synthase (NOS), which should block IOP-dependent NO production, exhibits a pressure-dependent effect on outflow facility (C).
Methods :
NO levels were measured using peptide-functionalized fluorescent particles that convert to 3-nitrotyrosine in the presence of nitroxidative stress. These NO biosensors were perfused into the TM of enucleated mouse eyes at 8 or 16 mmHg (C57BL/6J, n=8 mice). Anterior segments were labelled using anti-nitrotyrosine antibodies and imaged by confocal microscopy. Fluorescent signal from the particles was used to normalise the nitrotyrosine signal in order to account for local differences in particle concentration. To examine NOS activity at different pressures, 50µM L-NAME was perfused into enucleated mouse eyes while contralateral eyes were perfused with vehicle (C57BL/6J, n=7 mice). Eyes were perfused at 9 pressure steps ranging from 4 to 16 mmHg using iPerfusion. Power law fittings to flow-pressure data were used to estimate C at each pressure.
Results :
Eyes perfused at 16 mmHg exhibited higher nitrotyrosine levels than eyes perfused at 8 mmHg, relative pixel intensity was 1.15 times higher at 16 mmHg (p< 0.001). L-NAME decreased C, and its effect was pressure-dependent (p=0.02). At 20mmHg, L-NAME decreased C by roughly one-third (-32 [-50, -7]%; p=0.02), while at 8 mmHg L-NAME had a borderline significant effect (-17 [-34, 5]%; p=0.07).
Conclusions :
Elevated IOP stimulates NO production within the conventional outflow pathway of mice. L-NAME causes an IOP-dependent decrease in outflow facility, presumably by inhibiting NO production that would otherwise increase with IOP. Taken together, these data provide evidence for a rapid homeostatic mechanism involving IOP-dependent NO production that acts to increase outflow facility and oppose IOP elevation. Disruption of this homeostatic mechanism may contribute to ocular hypertension in glaucoma.
This is a 2021 ARVO Annual Meeting abstract.