in situ rat eyes as a vehicle for evaluating conventional aqueous humor outflow facility in drug candidates and rat ocular hypertensive model

Abstract

Purpose : To develop a method for estimating conventional aqueous humor outflow facility in in situ rat eyes following euthanasia.

Methods : Following euthanasia, a 30G cannula connected to pressure transducers, a reservoir, and a minipump were used to cannulate the rat’s anterior chamber (AC). Stabilizing/standardizing the AC’s volume by the reservoir was performed prior to running the perfusion steps method. The AC was perfused by a fixed 4-steps constant flow rate method (0.1ul/min, 0.35 ul/min, 0.6ul/min, 0.8 ul/min). The plateau pressure (mmHg) of each perfusion step was plotted against the flow rate to determine the slope, whose inverse is the outflow facility, C. We determined C in naive Brown Norway (BN) and Lewis rat eyes, in rats treated with top-oc 0.02% Netarsudil (200ug/eye) and in a rat steroid-induced ocular hypertension (RSOHT) model eyes, where conscious intraocular pressure (IOP) was collected after subcutaneous prednisolone pellet (25mg x 60D slow released) implantation. Retinal ganglion cell (RGC) density (cells/mm²) was determined post-mortem. Unpaired two-tailed student t-test was used to determine statistical significance (p<0.05).

Results : No significant difference in C was observed between postmortem and live rat eyes. C (Mean ± SD) was 0.024 ± .003 ul/min/mmHg in Lewis rat eyes and 0.023±.010 ul/min/mmHg in BN rat eyes. C in 0.02% Netarsudil top-oc treated BN eyes was significantly increased (+40.6%, p =0.041) vs the untreated eyes (0.039 ± 0.023 ul/min/mmHg vs 0.023±0.010 ul/min/mmHg). In the RSOHT model using Lewis rats, average IOP (Mean± SEM) for steroid eyes after 8 weeks was 21.3% higher vs baseline (14.8 ± 1.1 vs 12.2 ± .7 mmHg) with no loss of RGCs was observed in steroid vs placebo retina. No significant change of C in the steroid vs placebo eyes (0.025±0.013 vs 0.028±0.015 ul/min/mmHg) was observed.

Conclusions : We are one of the first to report a method for estimating the conventional aqueous humor outflow facility in euthanized in situ rat eyes by a fixed 4-steps constant rate perfusion system. Importantly, our method can also be used to measure outflow facility of novel drugs and distinguish between normal and glaucoma model eye. When experimental conditions are properly handled, the method is a novel approach for assessing outflow facility.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.