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A new organ culture system to study the regenration of the human ocular surface

Poster Details

First Author: H.Elbadawy ITALY

Co Author(s):    G. Salvalaio   C. Cagini   D. Ponzin   S. Ferrari     

Abstract Details

Purpose:

To design and test a new corneal organ culture system, namely, the multipurpose artificial chamber (MAC). The system was designed to mimic natural eye conditions including temperature, tear flow and ocular pressure. Regeneration of the ocular surface after injuries was tested and the recovery rate was determined. Additionally, the corneal response to injury was characterized at a molecular level, providing an immune response map to detect minor inflammatory changes at to increase sensitivity and offer an alternative to conventional in vitro and in vivo test of substances on the ocular surface.

Setting:

The Veneto Eye Bank foundation, Venice, Italy

Methods:

The system is composed of a sterile chamber with 3 inlets connected to a multichannel peristaltic pump to organise the artificial tears dripping. Four types of wounds were tested; scratch to the surface using Algerbrush II, removal of the whole epithelial layer, deep cut resembling penetrating keratolpasty and horizontal cut as in lamellar keratoplasty. Superficial wounds healed in 7 days while deep wounds did not heal (3 weeks), however, the epithelium migrated to fill the gaps. Superficial wounds were found to be more reproducible. Using this method, we show the profile of expression of proliferation markers (P63 and Ki67) during 7 days; more expression in the wounded area and recovering after 5 days. The expression of cytokeratin 12 as a marker for epithelialisation was normal while the expression of markers of inflammatory cells (MPO and F4/80) and their migration towards the injured area was evident after 6 hours, 1 day and 3 days. Corneas recovered after 5 days. Finally, signals for neovascularisation of corneas were studied, showing the expression of VEGF (vascular endothelial growth factor) higher in the stroma underneath the injured area on day 1. The expression was restored to normal on day 3 and thereafter.

Results:

A novel ex vivo corneal habitat was developed and tested as a model for ocular surface regeneration. Human corneas regularly irrigated with artificial tears and maintained in a sterile condition were used for testing healing of the ocular surface after injury. The expression of proliferation, epithelialization and inflammatory markers were shown at different time points, from 6 hours to 7 days post injury to form the basis of this new eye test using the MAC. Unlike other animal eye tests, the system provides analysis of the inflammatory response at molecular level by detecting inflammatory cells using immunohistochemistry. Additionally, the model can serve as a testing platform for new pharmaceutical compounds and to replace or reduce animal testing.

Conclusions:

-

Financial Disclosure:

NONE

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