June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Steroid-Response in Primary Culture Scleral Cells, Implications for Distal Outflow Pathology.
Author Affiliations & Notes
  • Thania Bogarin
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
    University of California, Los Angeles, Los Angeles, California, United States
  • Sindhu Saraswathy
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
  • Elizabeth Tannous
    University of California, Los Angeles, Los Angeles, California, United States
  • Chi Zhang
    University of California, Los Angeles, Los Angeles, California, United States
  • Jose Gonzalez
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
  • James C Tan
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
    University of California, Los Angeles, Los Angeles, California, United States
  • David R Hinton
    Ophthalmology, University of Southern California, Los Angeles, California, United States
  • Robert N Weinreb
    Ophthalmology, University of California, San Diego, La Jolla, California, United States
    Shiley Eye Institute, San Diego, California, United States
  • Jie J Zheng
    University of California, Los Angeles, Los Angeles, California, United States
  • Alex S Huang
    Ophthalmology, Doheny Eye Institute, Los Angeles, California, United States
    University of California, Los Angeles, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Thania Bogarin, None; Sindhu Saraswathy, None; Elizabeth Tannous, None; Chi Zhang, None; Jose Gonzalez, None; James Tan, None; David Hinton, None; Robert Weinreb, Aerie Pharmaceutical (C), Alcon (C), Allergan (C), Bausch & Lomb (C), Carl Zeiss Meditec (F), Eyenovia (C), Forsight Vision V Sensimed (C), Genentech (F), Heidelberg Engineering (F), Optovue (F), Quark (F), Topcon (F), Unity (C); Jie Zheng, None; Alex Huang, Allergan (C), Heidelberg Engineering (F)
  • Footnotes
    Support  Supported by National Institutes of Health, Bethesda, Maryland (K08EY024674 [ASH]); Research to Prevent Blindness Career Development Award 2016 [ASH]; and an unrestricted grant from Research to Prevent Blindness (New York, NY).
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3480. doi:
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      Thania Bogarin, Sindhu Saraswathy, Elizabeth Tannous, Chi Zhang, Jose Gonzalez, James C Tan, David R Hinton, Robert N Weinreb, Jie J Zheng, Alex S Huang; Steroid-Response in Primary Culture Scleral Cells, Implications for Distal Outflow Pathology.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3480.

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      © ARVO (1962-2015); The Authors (2016-present)

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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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