title: Assessment of anti-scarring therapies in ex vivo organ cultured rabbit corneas.
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.
authors: Schepens Eye Research Institute, Harvard Medical School, University of Florida, USA. Electronic address: sriniwas_sriram@meei.harvard.edu.
authors: Sriniwas
authors: S
authors: Sriram
authors: Institute for Wound Research, Department of Obstetrics and Gynecology, University of Florida, USA.
authors: Daniel J
authors: DJ
authors: Gibson
authors: Department of Pediatrics, University of Florida, USA.
authors: Paulette
authors: P
authors: Robinson
authors: Department of Pediatrics, University of Florida, USA.
authors: Liya
authors: L
authors: Pi
authors: Department of Ophthalmology, University of Florida, USA.
authors: Sonal
authors: S
authors: Tuli
authors: Department of Molecular Genetics and Microbiology, University of Florida, USA.
authors: Alfred S
authors: AS
authors: Lewin
authors: Institute for Wound Research, Department of Obstetrics and Gynecology, University of Florida, USA.
authors: Gregory
authors: G
authors: Schultz
mesh_terms: D000199:Actins / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000627:therapeutic use; D002921:Cicatrix / Q000378:metabolism / Q000517:prevention & control*; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D003315:Cornea / Q000473:pathology / Q000601:surgery; D003316:Corneal Diseases / Q000209:etiology / Q000378:metabolism / Q000628:therapy*; D004195:Disease Models, Animal; D007150:Immunohistochemistry; D053685:Laser Therapy / Q000009:adverse effects*; D053758:Nanoparticles / Q000627:therapeutic use; D009924:Organ Culture Techniques; D011239:Prednisolone / Q000031:analogs & derivatives / Q000627:therapeutic use; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000627:therapeutic use*; D011817:Rabbits; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D000199:Actins; D000893:Anti-Inflammatory Agents; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; C009935:prednisolone acetate; D011239:Prednisolone
keywords: CTGF; TGFB1; corneal haze; corneal organ culture; siRNA