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Exp Eye Res. 2014 Aug;125:173-82. doi: 10.1016/j.exer.2014.06.014. Epub 2014 Jun 24.

Assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas.

Author information

1
Schepens Eye Research Institute, Harvard Medical School, University of Florida, USA. Electronic address: sriniwas_sriram@meei.harvard.edu.
2
Institute for Wound Research, Department of Obstetrics and Gynecology, University of Florida, USA.
3
Department of Pediatrics, University of Florida, USA.
4
Department of Ophthalmology, University of Florida, USA.
5
Department of Molecular Genetics and Microbiology, University of Florida, USA.

Abstract

The effects of a triple combination of siRNAs targeting key scarring genes were assessed using an ex vivo organ culture model of excimer ablated rabbit corneas. The central 6 mm diameter region of fresh rabbit globes was ablated to a depth of 155 microns with an excimer laser. Corneas were excised, cultured at the air-liquid interface in defined culture medium supplemented with transforming growth factor beta 1 (TGFB1), and treated with either 1% prednisolone acetate or with 22.5 μM cationic nanoparticles complexed with a triple combination of siRNAs (NP-siRNA) targeting TGFB1, TGFB Receptor (TGFBR2) and connective tissue growth factor (CTGF). Scar formation was measured using image analysis of digital images and levels of smooth muscle actin (SMA) were assessed in ablated region of corneas using qRT-PCR and immunostaining. Ex vivo cultured corneas developed intense haze-like scar in the wounded areas and levels of mRNAs for pro-fibrotic genes were significantly elevated 3-8 fold in wounded tissue compared to unablated corneas. Treatment with NP-siRNA or steroid significantly reduced quantitative haze levels by 55% and 68%, respectively, and reduced SMA mRNA and immunohistostaining. This ex vivo corneal culture system reproduced key molecular patterns of corneal scarring and haze formation generated in rabbits. Treatment with NP-siRNAs targeting key scarring genes or an anti-inflammatory steroid reduced corneal haze and SMA mRNA and protein.

KEYWORDS:

CTGF; TGFB1; corneal haze; corneal organ culture; siRNA

PMID:
24971495
PMCID:
PMC4496961
DOI:
10.1016/j.exer.2014.06.014
[Indexed for MEDLINE]
Free PMC Article

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