Immunohistochemical detection of decorin in rat corneal transplants following treatment with immunomodulatory donor bone marrow derived dendritic cells

Abstract

Purpose: To investigate the immunohistochemical distribution of decorin in the corneal stroma as a potential marker for successful transplantation in a rat corneal transplant model.

Methods: A fully allogeneic rat corneal transplantation model (DA to LEW) was used for in vivo studies. BMDCs were propagated from Dark Agouti (DA) rat bone marrow precursor cells in complete medium supplemented with rat GMCSF (5ng/ml) and IL-4 (5ng/ml). For glucocorticoid treatment of BMDCs, dexamethasone (Dexa) (10uM) was added to the culture. Day 10 donor BMDCs +/- Dexa were harvested and 1x10⁶cells/ml injected intravenously into recipients 7 days prior to corneal transplant surgery. Graft survival and development of opacity, edema and neovascularisation were monitored throughout the therapy. On the average day of rejection (day 17) and on day 30 eyes from different treatment groups were collected, fixed and processed for embedding in paraffin. Five micron sections were cut and stained immunohistochemically by the anti-decorin antibody LF-113 and counter-stained by haematoxylin.

Results: At day 30, in the allografts which did not receive BMDC-treatment, i.e. in the rejected samples, decorin staining showed clear tendency to increase and accumulate in the corneal stroma. In contrast, in allografts at day 30 which received BMDC treatment, staining was much less evident. In the samples taken at the average rejection day, BMDC treated grafts showed higher decorin staining in the corneal stroma. However, in BMDC treated graft samples taken at the average rejection day in which the dendritic cells were treated with Dexamethasone, accumulation of decorin was less evident.

Conclusions: Although preliminary, these results suggest that extracellular matrix rearrangement occurs during corneal transplantation and that better graft integration (30d BMDC treated) displays little decorin accumulation. Therefore, increased decorin deposition could be considered as a novel biomarker in corneal rejection.

Keywords: 480 cornea: basic science • 554 immunohistochemistry • 741 transplantation