Inflammatory Cell and Fibroblast Model to Investigate Candidate Anti-Scarring Therapies for Ocular Disease

Abstract

Purpose: : Failure of glaucoma and retinal surgery is often associated with scar tissue formation mediated by an inflammatory response. Various cytokines and growth factors are believed to be involved. Microglia are a major source of inflammatory cytokines in the retina, as such they provide an ideal model of inflammation. Here we describe a preliminary model of inflammation to assess both anti-inflammatory and anti-scaring medicines and their delivery systems.

Methods: : Microglia were activated with 1 µg ml^-1 lipopolysacharide (LPS) for 17 hours to create inflammatory conditioned media (CM). RT-PCR was used to confirm microglial activation and change in mRNA expression of inflammatory mediators. Gel contraction assays using Human Tenon Fibroblasts (HTFs) were used as a model of wound healing to investigate the effect of inflammatory cytokine rich CM on the degree of gel contraction over 72 hours.

Results: : Pretreatment of microglia with LPS resulted in significant increased mRNA expression of TNF (56%), MMP-9 (93 %), IL-6 (not expressed in control) and IL-1β (60 %), with a decrease in TGFβ₂ (48%), confirming the activation of microglia. Preliminary data using gel contraction assays with HTFs and CM showed significantly more contraction (P<0.002, 47 % greater than control) in the first 24 hours of contraction, the difference in contraction after 72 hours was insignificant.

Conclusions: : Microglia conditioned media provides a means to investigate the effect of simulated inflammation on a variety of cell lines and to gain a clearer understanding of inflammatory processes in the eye. This model will continue to be developed with the aim of testing candidate anti-scarring molecules.

Keywords: inflammation • wound healing • microglia