The use of amniotic membrane homogenate is improved by a retinoid pre-treatment in healing corneal alkali burns, in a mouse model.

Abstract

Purpose: Amniotic membrane and retinoids are known to promote corneal healing. The purpose of this study is to establish the effect of a human amniotic membrane homogenate (HAM) previously cultivated with all-trans retinoic acid (ATRA) on corneal alkali burns, in a mouse model.

Methods: This protocol was approved by the regional ethic committee (CEMEA Auvergne). Fifty male CD1 mice with alkali burn on the right eye were divided in five groups: no treatment, physiological serum (control group), amniotic membrane homogenate, DMSO cultivated amniotic membrane homogenate or ATRA cultivated amniotic membrane homogenate (10-6mol/L during 24 hours). Before instillation of homogenate, the HAM retinoid induction was assessed by PCR quantification of a well-known retinoid induced gene: RARβ. Wound healing was first evaluated through slit lamp photographs at days 1, 2 and 7 (for corneal re-epithelialization), then, histological analysis of ulcer depth was conducted and finally immuno-histochemical analysis of MMP9 and VEGF expression were done.

Results: We demonstrated first, that there was no difference at day 2 for our main criterion (corneal re-epithelialization). At day 7, we found an overall difference (ulcer surface) in the corneal healing between the groups (p<0,001). Significant differences were also found by histological analysis between the untreated group and the HAM+ATRA group, between physiological saline group and the HAM+ATRA group and between the HAM group and HAM+ATRA group. Finally, expressions of MMP9 and VEGF were visually weaker for the groups treated with HAM homogenate.

Conclusions: HAM induction by ATRA has a positive effect on corneal healing and on ulcer depth when used as a homogenate after alkali burns. Our results support the hypothesis that the effect of HAM seems to be more biological than mechanical. These first results need to be completed by further immuno-histochemical analysis using antibodies against TGFβ and thrombospondin but the use of HAM homogenate potentiated by ATRA already appears as a promising therapeutic alternative without the constraints of the more classical HAM graft.

Keywords: 480 cornea: basic science • 765 wound healing • 705 retinoids/retinoid binding proteins