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

Emilie Courrier,Sylvie Pillet,Fabien Forest,Corantin Maurin,Zhiguo He,Victor Lambert,Thibaud Garcin,Chantal Perrache,Antoine Rousseau,Gilles Thuret,Philippe Gain,Thomas Bourlet,Didier Renault,Paul O Verhoeven,Marc Labetoulle,Nancy M Sawtell

doi:10.1371/journal.pone.0236183

Abstract

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. 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. 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). The ASM provides an innovative ex vivo model of HK in whole human cornea that reproduces typical epithelial lesions.

Highlights

Herpes simplex virus type 1 (HSV-1) has a particular tropism for human sensory neurons of the upper body
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)
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 organ culture (OC) method

Summary

Introduction

Herpes simplex virus type 1 (HSV-1) has a particular tropism for human sensory neurons of the upper body. The prevalence of ocular HSV infection was estimated in 1980s America at 149 cases per 100,000 individuals with an incidence of 8.4 new cases per 100,000 individuals per year [2]. These data are similar to the annual incidence of all types of new ocular HSV infection, estimated in a more recent study at 11.8 per 100,000 people in a similar population [3]. Herpetic keratitis (HK) models using whole human corneas are essential for studying virushost relationships, because of high species specificity and the role of interactions between corneal cell populations that cell culture cannot reproduce. 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

Objectives

Methods

Results

Conclusion