Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Summary

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

Abstract

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity ¹.

Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability.

In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Protocol

All reagents and equipment were purchased sterile or were sterilized prior to the procedure. This article describes the steps of setting up the ex vivo model beginning with a pre-enucleated eye. In our experiments, we use fresh whole eyeballs from young (8-12 wks) albino rabbits (Pel-Freez Biologicals, Rogers, AR). In addition, we use human corneas obtained from the local eye bank. If performing enucleation yourself, take care to avoid damaging the cornea or the limbus during the procedure. Rabbit enucleation protocols can be found elsewhere ².

Part 1: Excision of the Corneoscleral Button

1. Wet a narrow ....

Discussion

In this video article we describe a method for studying acute corneal epithelial HSV-1 infection in an ex vivo model. This model is a useful stepping stone between the in vitro and the in vivo models, because it allows for physiologically-accurate validation of cell culture results and, thereby, limits the amount of animal experimentation that must be performed. In addition, the ex vivo model provides a unique and invaluable opportunity to study epithelial herpes infection in intact .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Minimum Essential Medium (MEM)	Mediatech, Inc.	10-010-CV
Non-Essential Amino Acids (100x solution)	Mediatech, Inc.	25-025-CI
L-Glutamine	Mediatech, Inc.	25-005-CI
Penicillin G Sodium Salt	Sigma-Aldrich	P3032
Streptomycin Sulfate Salt	Sigma-Aldrich	S9137
Agarose	Invitrogen	15510-027
Phosphate Buffered Saline (PBS)	Prepared in Lab	N/A
Trypsin EDTA (1x)	Mediatech, Inc.	25-053-CI
DNeasy Blood & Tissue Kit	Qiagen	69504
RNeasy Mini Kit	Qiagen	74104
Laemmli Buffer	Prepared in Lab	N/A
Optimal Cutting Temperature (OCT) Compound	Tissue-Tek	4583
Whole Rabbit Eyes (Young)	Pel-Freez Biologicals	41211-2
Human Corneas and Whole Eyes	Local Supplier	N/A
12 Well Porcelain Spot Plate	Avogadro’s Lab Supply	1877
35 mm Tissue Culture Dish	Falcon BD	353001
6 Well Cell Culture Plate	Greiner Bio-One	657160
Cryomold Intermediate	Tissue-Tek	4566