The support of the Lions Eye Bank of Delaware Valley was invaluable for this work. Primary mouse monoclonal antibody against ICP8 was a kind gift from Dr. David Knipe (Harvard Medical School). We also thank Dr. Stephen Jennings (Drexel University College of Medicine) and Dr. Peter Laibson (Wills Eye Institute) for helpful discussions and expertise.

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 enucleati...

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	<li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+keratitis.">Herpes simplex keratitis.</a> Prog. Retin. Eye Res. 25, 355-380 (2006).</li><li>Holmberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+exotic+pets.">Enucleation of exotic pets.</a> Journal of Exotic Pet Medicine. 16, 88-94 (2007).</li><li>Remeijer, L., Osterhaus, A., Verjans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpes+simplex+virus+keratitis:+the+pathogenesis+revisited.">Human herpes simplex virus keratitis: the pathogenesis revisited.</a> Ocul. Immunol. Inflamm. 12, 255-285 (2004).</li><li>Foreman, D. M., Pancholi, S., Jarvis-Evans, J., McLeod, D., Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization.">A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.</a> Exp. Eye Res. 62, 555-564 (1996).</li><li>Xu, K. P., Li, X. F., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants.">Corneal organ culture model for assessing epithelial responses to surfactants.</a> Toxicol. Sci. 58, 306-314 (2000).</li><li>Klausner, E. A., Peer, D., Chapman, R. L., Multack, R. F., Andurkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy.">Corneal gene therapy.</a> J. Control Release. 124, 107-133 (2007).</li><li>Halford, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus:+implications+for+the+regulation+of+viral+latency.">ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency.</a> Virol. J. 3, 44 (2006).</li><li>Bhattacharjee, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model.">Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model.</a> Invest Ophthalmol Vis. Sci. 49, 4263-4268 (2008).</li><li>Novitskaya, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model.">Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.</a> Cornea. 28, 421-425 (2009).</li><li>Sharma, A., Shimeld, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice.">In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.</a> Br. J. Ophthalmol. 81, 785-788 (1997).</li></ol>

No conflicts of interest declared.

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 ...

<table border="1">
 <tbody>
 <tr>
 <td>Reagents/Supplies</td>
 <td>Company</td>
 <td>Catalogue 				#</td>
 </tr>
 <tr>
 <td>Minimum 				Essential Medium (MEM)</td>
 <td>Mediatech</td>
 <td>10-010-CV</td>
 </tr>
 <tr>
 <td>Non-Essential 				Amino Acids (100x solution)</td>
 <td>Mediatech</td>
 <td>25-025-CI</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Mediatech</td>
 <td>25-005-CI</td>
 </tr>
 <tr>
 <td>Penicillin G 				Sodium Salt</td>
 <td>Sigma</td>
 <td>P3032</td>
 </tr>
 <tr>
 <td>Streptomycin 				Sulfate Salt</td>
 <td>Sigma</td>
 <td>S9137</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Invitrogen</td>
 <td>15510-027</td>
 </tr>
 <tr>
 <td>Phosphate 				Buffered Saline (PBS)</td>
 <td>Prepared in 				lab</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Trypsin EDTA 				(1x)</td>
 <td>Mediatech</td>
 <td>25-053-CI</td>
 </tr>
 <tr>
 <td>DNeasy Blood 				&amp; Tissue Kit</td>
 <td>QIAGEN</td>
 <td>69504</td>
 </tr>
 <tr>
 <td>RNeasy Mini 				Kit</td>
 <td>QIAGEN</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>Laemmli 				Buffer</td>
 <td>Prepared in 				lab</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>Optimal 				Cutting Temperature (OCT) Compound</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 </tr>
 <tr>
 <td>Whole Rabbit 				Eyes (Young)</td>
 <td>Pel-Freez</td>
 <td>41211-2</td>
 </tr>
 <tr>
 <td>Human 				Corneas and Whole Eyes</td>
 <td>Local Eye 				Bank</td>
 <td>N/A</td>
 </tr>
 <tr>
 <td>12 Well 				Porcelain Spot Plate</td>
 <td>Avogadro's 				Lab Supply</td>
 <td>1877</td>
 </tr>
 <tr>
 <td>35 mm Tissue 				Culture Dish</td>
 <td>Falcon</td>
 <td>353001</td>
 </tr>
 <tr>
 <td>6 Well Cell 				Culture Plate</td>
 <td>Greiner 				Bio-One</td>
 <td>657160</td>
 </tr>
 <tr>
 <td>Cryomold 				Intermediate</td>
 <td>Tissue-Tek</td>
 <td>4566</td>
 </tr>
 </tbody>
</table>

ex vivo organotypic corneal model of acute epithelial herpes simplex virus type i infection

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.

Watch this Scientific Journal Video about Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection at JoVE.com

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

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

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

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 ...

ex-vivo-organotypic-corneal-model-acute-epithelial-herpes-simplex

Research

JoVE Journal

Neuroscience

12.6K Görüntüleme.  Drexel University College of Medicine. Bu video yazıda yeni bir kullanımı tarif Ex vivo modeli virüsü tip I kornea epitel enfeksiyon simpleks.