Purchase this article with an account.
Thania Bogarin, Sindhu Saraswathy, Ernesto Barron, Jie J Zheng, Alex Huang; Increased Actin Stress Fiber Formation in Scleral Fibroblasts Exposed to Steroids. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4719.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Increased intraocular pressures from steroid response has been seen after trabecular bypass implicating distal targets. Previously, we showed that steroid application to primary scleral fibroblasts from normal donors resulted in decreased cellular proliferation and increased cell size, similar to trabecular meshwork (TM) cell steroid response. Here we investigate actin changes in scleral fibroblasts exposed to steroids.
Methods: Scleral primary cultures were generated from published protocols using donor corneo-scleral rims after corneal transplantation via scleral mid-scleral depth excised explants. Scleral cells were grown until 70% confluent in chambered slides (n = 4 explants) under 3 treatment groups for 7 days, 100 nM dexamethasone (DEX), vehicle (VEH), and media (MED) (control; DMEM/F12 with 10% FBS). Cells were fixed (4% paraformaldehyde), blocked (5% FBS), and permeabilized (0.25% Triton-X-100). Fixed cells were incubated in Phalloidin (Alexa Fluor-568; Life Technologies) with 1% FBS overnight at 4°C. Cells were washed, stained/mounted with DAPI (Vector Labs), and viewed under BZ-X700 digital imaging microscope (Keyence). Cell size and fluorescence was quantified (ImageJ; n = 30 cells per condition). Data are expressed as mean +/- SD.
Results: DEX-treated scleral fibroblasts were larger (DEX = 43,616 +/- 9,897; MED = 37,793 +/- 9,401 VEH = 36,810 +/- 12,545 pixels2; DEX vs. MED p = 0.02, DEX vs. VEH p = 0.02, MED vs. VEH p = 0.73). Phalloidin staining showed the presence of stress fibers in all cells, but stress fibers were more packed with overall increased mean pixel intensity in DEX-treated cells (DEX = 42.8 +/- 13.9, MED = 35.4 +/- 14.4, VEH = 0 +/- 12.7 intensity units; DEX vs. MED p = 0.05, DEX vs. VEH p = 0.003, MED vs. VEH p = 0.34).
DEX treatment led to increased cell size that was both consistent with prior literature regarding TM cells and earlier observations by our group (ARVO 2017, 3480-A0103). DEX-treated scleral fibroblasts also showed greater stress fiber packing which is similar to, but not identical to TM observation where there is increased formation of cross-linked actin networks in response to steroids. Scleral fibroblast cellular change in response to steroids may explain maintained clinical steroid-response after trabecular bypass.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only