Effect of TGF-β2 on outflow facility and gene expression in anterior segments of calf eyes

Abstract

Purpose : Intraocular pressure (IOP) elevation in primary open angle glaucoma patients is believed to be due to an increase in outflow resistance through the trabecular meshwork (TM) and Schlemm’s canal. To understand long-term regulation of outflow resistance, anterior segment perfusion provides a valuable model. Here we report the development of an anterior segment perfusion assay that can evaluate 20 samples in parallel for up to 2 weeks.

Methods : Calf anterior segments (calfAS) were dissected and mounted onto a perfusion apparatus with a clamping ring. The apparatus was 3-D printed and optimized to fit calfAS. IOP was controlled by adjusting the reservoir height and flow rate was measured with flow sensors (Zhou et al. IOVS 2017). An effective antibiotic cocktail was identified to prevent bacterial contamination. Cell viability was assessed using a live-dead stain and lactate dehydrogenase (LDH) release. To evaluate the effects of TGF-β2, media containing vehicle or two doses of TGF-β2 (40 or 400 ng/ml TGF-β2 in 300 µl) was injected once daily for 5 days. Soluble fibronectin concentration was evaluated using ELISA. Expression of extracellular matrix genes was evaluated using real time qPCR in excised TM.

Results : After 2 weeks of perfusion, TM cells remained mostly viable. Minimal LDH release was detected in the perfusate. As a positive control, an injection of 30 µM Y-39983 (a ROCK inhibitor) increased outflow facility by 56 ± 24% (mean ± SD, n = 6-8, p < 0.01) after 8 hours of treatment. TGF-β2 treatment for 5 days at both doses reduced outflow facility by up to 30% relative to contralateral eyes receiving vehicle control. This reduction was accompanied by a 2-3 fold increase in soluble fibronectin concentration and increases in the transcript levels of fibronectin and alpha-smooth muscle actin (α-SMA).

Conclusions : We have extended our compact eye perfusion system to perform long-term perfusion of anterior segments and have demonstrated its utility by TGF-β2 and ROCK inhibition treatment. This assay enables the identification of molecules and pathways that chronically modify the outflow pathway to achieve a long lasting impact on IOP.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.