Ex vivo model of herpes simplex virus type I dendritic and geographic keratitis using a corneal active storage machine

BACKGROUND: Herpetic keratitis (HK) models using whole human corneas are essential for studying virus-host relationships, because of high species specificity and the role of interactions between corneal cell populations that cell culture cannot reproduce. Nevertheless, the two current corneal storage methods (hypothermia and organ culture (OC)) do not preserve corneas in good physiological condition, as they are characterized by epithelial abrasion, stromal oedema, and excessive endothelial mortality. METHODS: To rehabilitate human corneas intended for scientific use, we used an active storage machine (ASM) that restores two physiological parameters that are essential for corneal homeostasis: intraocular pressure and storage medium renewal (21mmHg and 2.6 μL/min, respectively). ASM storage regenerates a normal multilayer epithelium in 2 weeks. We infected six pairs of corneas unsuitable for graft by inoculating the epithelium with herpes simplex virus type 1 (HSV-1), and compared each ASM-stored cornea with the other cornea stored in the same medium using the conventional OC method. RESULTS: Only corneas in the ASM developed a dendritic (n = 3) or geographic (n = 2) epithelial ulcer reproducing typical HSV-1-induced clinical lesions. Corneas in OC showed only extensive desquamations. None of the uninfected controls showed epithelial damage. Histology, immunohistochemistry, transmission electron microscopy and polymerase chain reaction on corneal tissue confirmed infection in all cases (excluding negative controls). CONCLUSIONS: The ASM provides an innovative ex vivo model of HK in whole human cornea that reproduces typical epithelial lesions.