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1Department of Ophthalmology, Saint Louis University
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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.
Keywords: Infection, Issue 70, Immunology, Virology, Medicine, Infectious Diseases, Ophthalmology, Herpes, herpetic stromal keratitis, HSK, keratitis, pathogenesis, clinical evaluation, virus, eye, mouse, animal model
Morris, J., Stuart, P. M., Rogge, M., Potter, C., Gupta, N., Yin, X. T. Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK. J. Vis. Exp. (70), e4276, doi:10.3791/4276 (2012).
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.
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
2. Mouse Infection
3. Reactivation of Mice from Latency
4. Clinical Evaluation
5. Representative Results
The recurrent model which was described above was first published by Shimeld, et al. 1-3. They tested and our laboratory has been using a modified version of this model to publish many manuscripts over the years 4-15. The model was used to define the role that the cytokine interferon-γ plays in recurrent HSV-1 disease 10. As shown in Figure 3, mice lacking IFNγ displayed worse corneal disease than did wild-type mice 14. It was also used successfully to define the therapeutic value of several different vaccine constructs 6,10,11. Note that in one case the vaccine tested worked well prophylactically but did not prove to be therapeutically effective 6. However, when a replication-incompetent viral vaccine was tested, it was not only effective prophylactically, but was also very effective therapeutically 10,11, see Figure 5 in reference 10. More recently we have used this model to determine if gender differences were detectable in mice undergoing recurrent HSK. As Figure 1 demonstrates, male C57BL/6 mice display significantly less corneal opacity than do female C57BL/6 mice. Likewise, when corneal neovascularization was evaluated, male mice demonstrated significantly less new vessel growth in the cornea than did female mice (Figure 2). We are currently testing whether this phenotype is strain specific by performing similar analysis in other strains of mice. We will also determine whether the difference is due to the lack of testosterone or the presence of estrogen.
Figure 1. Corneal disease in male and female C57BL/6 mouse strains following UV-B induced reactivation. Latently infected mice were induced to reactivate with UV-B irradiation and mice were monitored for corneal opacity for 35 days. The numbers of mice used for these studies were as follows: males, n = 15; females, n = 15. Results indicate mean ± SEM. *There was significantly greater virus-induced disease in female C57BL/6 mice for days 14-35 (P = 0.001 to 0.01).
Figure 2. Corneal disease in male and female C57BL/6 mouse strains following UV-B induced reactivation. Latently infected mice were induced to reactivate with UV-B irradiation and mice were monitored for corneal neovascularization for 35 days. The numbers of mice used for these studies were as follows: males, n = 15; females, n = 15. Results indicate mean ± SEM. *There was significantly greater virus-induced disease in female C57BL/6 mice for days 14-28 (P = 0.001 to 0.01).
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, therapeutic intervention to prevent primary HSK will not always lead to amelioration of recurrent HSK, which we have shown in various publications when evaluating differences between primary and recurrent HSK 6,8,10-13,15. As indicated previously, this is particular the case when developing vaccines to prevent human HSK. Most vaccines will prevent the development of primary HSK but very few have been shown to be effective in reducing recurrent HSK 6,10,11.
The weaknesses of this model are that it takes much longer to perform an experiment. There is an almost 6 week period following primary infection during which the mice become latently infected and the administered human serum is metabolized. Furthermore, not all mice that are latently infected will shed detectable virus into the tear film of the infected eye. This does not mean that they did not reactivate, only that we cannot detect their reactivation by culturing virus. It is highly likely that most, if not all mice do reactivate but that they do not produce sufficient numbers of virus to be detected by plaque assay.
No conflicts of interest declared.
The authors would like to thank Dr. Jay Pepose for helping teaching the model to us. This work was supported by National Institutes of Health Grants EY11885 (PMS), EY21247 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.
|Fetal Bovine Serum||Atlanta Biologicals||S11150|
|Oak Ridge Tubes||Nalgene||Z 720410|
|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|
|10 ml Syringe||BD||309602|
|10 ml Syringe||BD||309604|
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