Steroid-Response in Primary Culture Scleral Cells, Implications for Distal Outflow Pathology.

Abstract

Purpose : Steroid-response glaucoma is thought to be due to trabecular meshwork (TM) changes in response to steroid exposure either after topical administration in intact eyes or after drug exposure in situ using cell culture models. Continued steroid-response can be observed with steroid delivery even after trabecular bypass surgeries suggesting distal aqueous humor outflow (AHO) pathway steroid-response mechanisms as well. This project proposes to study steroid-response in primary scleral cultures to understand distal outflow pathway pathology.

Methods : Scleral primary cultures were generated from published protocols using donor corneo-scleral rims after corneal transplantation via scleral mid-scleral depth excised explants. Preliminarily, scleral cells were grown until 70% confluent in chambered slides (n = 2) under 3 treatment groups for 7 days, 100 nM dexamethasone (DEX), DMSO (vehicle), and MEDIA (control; DMEM/F12 with 10% FBS). Cells were prepared for western blot for myocilin expression; or were fixed with 4% paraformaldehyde, blocked with 5% fetal bovine serum, and permeabilized with 0.25% Triton-X-100. Fixed cell were incubated in Phalloidin (Alexa Fluor-568; Life Technologies) with 1% fetal bovine serum overnight at 4°C. Cells were washed, stained/mounted with DAPI (Vector Labs), and viewed under BZ-X700 digital imaging microscope (Keyence). Western blot was processed by standard procedure with GAPDH as a control. Band pixel intensities were quantified (ImageJ).

Results : DEX treated scleral cells exhibited the development of cross-linked actin networks (CLANS). By western blot, myocilin expression was increased (DEX = 2.05, DMSO = 0.95, and MEDIA = 0.78; arbitrary intensity units normalized to GAPDH). Alternatively, DMSO and MEDIA treatment did not show increased myocilin protein or CLAN formation but instead typical uniform and parallel F-actin fiber organization.

Conclusions : Increased CLANS and myocilin expression are known hallmarks of steroid-response as described in TM cell-culture. DEX treatment of primary scleral cultures in a small sample size can lead to a steroid-response like phenotype with CLAN and increased myocilin protein formation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

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