Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Summary

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Abstract

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Protocol

Animal Care Note: Analgesics are not used in this model as they are anti-inflammatory and thus would invalidate the model.

1. Preparation of Virus

1. Grow HSV-1 on Vero cells (80% confluent) using a starting dilution of 0.01 multiplicity of infection (in T-150 flask, about 5 x 10⁵ plaque forming units) for 3-4 days until all of cells have rounded-up and are easily dislodged by hitting the flask.
2. Remove media to sterile centrifuge tubes and centrifuge in Sorvall Legend RT at 4 °C and 3,500 rpm for 15 min. After removing supernatant (see 1.3), resuspend large pellet from each 50 ml tube with 5 ml of media total. Son....

Discussion

The recurrent model of herpetic stromal keratitis (HSK) described here enables the investigator to study human HSK in a model that is more consistent with that which is observed during human disease. Thus, the model's strengths are that disease occurs in the context of an immune system that has already by stimulated during primary disease. Since these mice already have an immune response to HSV-1, the factors that reactivate that immune response are likely different from those that originally generated it. Therefore, the.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Sigma	D5796
Fetal Bovine Serum	Atlanta Biologicals	S11150
Human Serum	Sigma	S7023
L-Glutamine	Cellgro	25-005-C1
Pen/ Strep	Sigma	P4333
Fungizone	Invitrogen	15290018
Centrifuge	Sorvall	Legend RT
Centrifuge	Beckman	52-21
Transilluminatior	UVP	95-0452-02
Sonicatior	Branson	Sonifier 450
Oak Ridge Tubes	Nalgene	Z 720410
Flasks- 7150	Corning	430823
Plates- 12 well	Corning	CLS 3513
Plates- 48 well	Corning	CLS 3548
Sterile Conical Tubes- 30 ml	Corning	CLS 430828
Sterile Cotton-tipped Applicators	Fisher	23-400-125
30G Needles	Becton-Dickinson	305106
25G Needles	BD	305122
10 ml Syringe	BD	309602
10 ml Syringe	BD	309604