Name: Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection (Video) | JoVE Journal
Uploaded: 2012-11-03T13:00:00.000+00:00
Duration: 7 min 55 s
Description: Learn ex vivo corneal culture for studying herpes simplex virus type I infection using rabbit and human donor corneas. This model offers improved physiological relevance compared to traditional cell cultures.

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5 minutes

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 enucleation protocols can be found elsewhere 2.
Part 1: Excision of the Corneoscleral Button
<ol>
 <li> Wet a narrow strip of gauze with PBS and wrap it around the eyeball to facilitate holding it during the procedure. </li>
 <li> Use a sharp scalpel to make a tangential incision in the sclera, about 0.5 cm away from the limbus. </li>
 <li> Use sharp scissors to extend the incision and completely excise the corneoscleral button. </li>
 <li> Once the corneoscleral button is isolated, tease away the iris while holding the scleral rim with forceps. Avoid damaging the endothelium or exerting excessive stress on the cornea. </li>
</ol>
Part 2: Introduction of Cornea into Organotypic Culture
<ol>
 <li> Explanted corneas are cultured in Minimum Essential Medium (MEM) supplemented with Non-Essential Amino Acids (1X), L-Glutamine (2 mM), Penicillin (200 U/ml), and Streptomycin (200 μg/ml). An antifungal agent, such as amphotericin, may also be added if necessary. </li>
 <li> Prepare a 1% agarose solution in culture medium and keep it ready at 55 °C. </li>
 <li> Thoroughly rinse the cornea in sterile PBS containing Penicillin (200 U/ml) and Streptomycin (200 μg/ml).</li>
 <li> Place the cornea epithelial side down into a well of a sterile spot plate. </li>
 <li> Add the agarose-containing medium to the endothelial concavity of the cornea just enough to fill it completely. Allow ~1 min for the agarose to solidify. </li>
 <li> Place the cornea with the supporting gel scaffold epithelial side up into a 35 mm tissue culture dish and add culture medium to cover the epithelial surface. </li>
 <li> The cornea may be cultured in this way for over a week, with medium changes every 48 hr. </li>
</ol>
Part 3: Infection with HSV-1 and Treatment with Experimental Drugs
<ol>
 <li> Add 1 ml of medium containing the desired amount of virus into a 35 mm tissue culture dish. We usually infect with 1x104 PFU of strain KOS HSV-1 per cornea. Our stock titers are determined by performing plaque assays on CV-1 monolayers. </li>
 <li> Place the cornea together with the agarose scaffold epithelial side down into the virus-containing medium. </li>
 <li> Place the cornea into a tissue culture incubator to infect for 1 hr with intermittent rocking every 10-15 min. </li>
 <li> After 1 hr, aspirate the virus-containing medium and rinse 2-3 times with PBS to remove any residual viral particles. </li>
 <li> Place the cornea back into its previous position and add fresh culture medium to cover the epithelial surface. </li>
 <li> Experimental treatments may be started before or after infection, depending on the nature of the experiment. </li>
 <li> Culture medium is changed every 48 hr. </li>
</ol>
Part 4: Sample Collection
<ol class="lalpha">
 <li> Medium for plaque assay analysis. Mix the medium well and use it immediately for a plaque assay or store at -80 °C for later use if collecting samples at different time points.</li>
 <li> DNA, RNA, protein, or cells from corneal epithelium 
 <ol>
 <li> Remove the agarose scaffold and rinse the cornea well with PBS. </li>
 <li> Excise the ring of scleral tissue from the cornea to ensure that only corneal epithelial material is collected. </li>
 <li> Using a scalpel, scrape off the corneal epithelial cells into the appropriate buffer, depending on the type of material to be isolated. For isolation of DNA and RNA, we use the DNeasy Blood &amp; Tissue Kit and the RNeasy Mini Kit, respectively (QIAGEN, Hilden, Germany). For collection of protein lysates, we use Laemmli buffer. For flow cytometry analysis of corneal epithelial cells, we pre-incubate the corneas in a small amount of trypsin to loosen the cells, and then dislodge them into trypsin with a scalpel. Since scraping the corneal epithelium does not yield equal amounts of tissue, it is important to include appropriate controls when analyzing the samples. We use 18S rRNA as a reference transcript in qRT-PCRs, GAPDH as a reference gene in qPCRs, and nucleolin as a loading control for Western blots. </li>
 </ol>
 </li>
 <li>Tissue samples for immunohistochemistry or immunofluorescence
 
 <ol>
 <li> Remove the agarose scaffold and rinse the cornea well with PBS. </li>
 <li> Depending on your experimental goal, use the cornea to make a paraffin tissue block or a frozen tissue block, as shown in the video. We follow standard procedures when preparing corneal tissue sections.</li>
 </ol>
 </li>
</ol>
Representative Results
Most of the methods available for studying cells in tissue culture can be easily adapted for use in corneas infected with HSV-1 ex vivo. Shown here are representative results for a few of the most common techniques applicable to studying HSV-1 infection - qPCR (Figure 1D), qRT-PCR (Figure 1B), Western blot (Figure 1F), plaque assay (Figure 1C), and tissue immunofluorescence (Figure 1E). 
<img src="/files/ftp_upload/3631/3631fig1.jpg" alt="Herpes simplex virus analysis: cornea infection diagram, viral transcript graph, plaque assay, ICP8 staining." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Analysis of corneas infected with HSV-1 ex vivo. (A) Schematic representation of the ex vivo model of acute corneal epithelial HSV-1 infection. (B-F) Analysis of corneas infected using the method outlined in this video article. Explanted rabbit corneas were infected with 1x104 PFU/cornea of strain KOS HSV-1 and cultured in the presence (PAA) or absence (Mock) of phosphonoacetic acid (400 μg/ml), a known inhibitor of HSV replication. (B) Total RNA was collected at the indicated time points, and viral transcription was assessed by qRT-PCR with primers for HSV-1 polymerase. Primers against 18S rRNA were used as a reference. n=3. Error bars indicate ± SEM. (C) Culture medium was collected at 48 hpi, and infectious particle production was assessed by plaque assay on CV-1 monolayers. (D) Total DNA was collected at 48 hpi, and viral genome replication was assessed in a qPCR assay with primers for the HSV-1 genome. Primers against GAPDH were used as a reference. n=3. Error bars indicate ± SEM. (E) Mock-treated corneas were flash-frozen at 48 hpi, sectioned, and analyzed by indirect immunofluorescence for the presence of a viral early protein (ICP8) within the infected epithelium. Nuclei are counterstained with Höchst 33258. (F) Protein lysates were collected at 48 hpi, and expression of a viral protein (ICP0) was assessed by Western blot. BCA assay was used to ensure equal loading of samples. hpi = hours post infection.

<ol><li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Herpes+simplex+keratitis%5BTitle%5D)+AND+%22Prog.+Retin.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Enucleation+of+exotic+pets%5BTitle%5D)+AND+%22Journal+of+Exotic+Pet+Medicine%22%5BJournal%5D)">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/pubmed?term=((Human+herpes+simplex+virus+keratitis%3A+the+pathogenesis+revisited%5BTitle%5D)+AND+%22Ocul.+Immunol.+Inflamm%22%5BJournal%5D)">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/pubmed?term=((A+simple+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization%5BTitle%5D)+AND+%22Exp.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants%5BTitle%5D)+AND+%22Toxicol.+Sci%22%5BJournal%5D)">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/pubmed?term=((Corneal+gene+therapy%5BTitle%5D)+AND+%22J.+Control+Release%22%5BJournal%5D)">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/pubmed?term=((ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus%3A+implications+for+the+regulation+of+viral+latency%5BTitle%5D)+AND+%22Virol.+J%22%5BJournal%5D)">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/pubmed?term=((Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis.+Sci%22%5BJournal%5D)">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/pubmed?term=((Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model%5BTitle%5D)+AND+%22Cornea%22%5BJournal%5D)">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/pubmed?term=((In+vivo+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice%5BTitle%5D)+AND+%22Br.+J.+Ophthalmol%22%5BJournal%5D)">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><tbody><tr><td>Minimum Essential Medium (MEM)</td><td>Mediatech, Inc.</td><td>10-010-CV</td><td></td></tr><tr><td>Non-Essential Amino Acids (100x solution)</td><td>Mediatech, Inc.</td><td>25-025-CI</td><td></td></tr><tr><td>L-Glutamine</td><td>Mediatech, Inc.</td><td>25-005-CI</td><td></td></tr><tr><td>Penicillin G Sodium Salt</td><td>Sigma-Aldrich</td><td>P3032</td><td></td></tr><tr><td>Streptomycin Sulfate Salt</td><td>Sigma-Aldrich</td><td>S9137</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Prepared in Lab</td><td>N/A</td><td></td></tr><tr><td>Trypsin EDTA (1x)</td><td>Mediatech, Inc.</td><td>25-053-CI</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit</td><td>Qiagen</td><td>69504</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Laemmli Buffer</td><td>Prepared in Lab</td><td>N/A</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Whole Rabbit Eyes (Young)</td><td>Pel-Freez Biologicals</td><td>41211-2</td><td></td></tr><tr><td>Human Corneas and Whole Eyes</td><td>Local Supplier</td><td>N/A</td><td></td></tr><tr><td>12 Well Porcelain Spot Plate</td><td>Avogadro&amp;rsquo;s Lab Supply</td><td>1877</td><td></td></tr><tr><td>35 mm Tissue Culture Dish</td><td>Falcon BD</td><td>353001</td><td></td></tr><tr><td>6 Well Cell Culture Plate</td><td>Greiner Bio-One</td><td>657160</td><td></td></tr><tr><td>Cryomold Intermediate</td><td>Tissue-Tek</td><td>4566</td><td></td></tr></tbody></table>

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

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

I'm Jane Clifford is econ. I'm professor and chair of biochemistry and and molecular biology at the Drexel University College of Medicine, And my name is Ola iv and I'm an MD PhD student in Dr.Clifford's lab. The video that Oli and I are prepared for you demonstrates the ex vivo culture of corneas obtained from rabbits and human don donors.We have utilized these corneas for studying herpes virus infection and they are certainly applicable for other diseases of the corn for study of other diseases of the cornea, and it's a particularly relevant ex vivo model as compared with Cells and mono culture. Herpes keratitis is one of the most severe Pathologies associated with a herpes simplex virus type one. It is currently the leading cause of cornea derived and infection associated blindness in the developed world.Herpes keratitis is traditionally studied in two types of experimental models. The in vitro model in which cultured mono layers of corneal epithelial cells are infected in a Petri dish offers simplicity, a high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model of herpes keratitis 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 acute corneal epithelial HSV one infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organ typically maintained in culture and infected with HSV. The use of the ex vivo model allows for highly physiologically based conclusions, yet it is rather inexpensive and requires STEM commitment comparable to that of the in vitro Model.Prepare the following before Starting the procedure. Petri dish phosphate buffered saline, corneal culture and scaffold media spot plate strip of gauze, fresh eyeball, scalpel, Forceps, and sharp scissors. Wet a narrow Strip of gauze with PBS and wrap it around the eyeball to securely hold it.During the procedure, use a sharp scalpel to make a tangential incision in the sclera about half a centimeter away from theus. Use scissors to completely excise the corneal scleral button. As the incision is extended, the pressure within the eyeball will decrease, making it difficult to hold and manipulate the tissue.For this reason, we recommend using a sharpened reliable pair of scissors at this step. Once the corneal scleral button is isolated, tease away the iris while holding the scleral rim with forceps. Avoid damaging the endothelium or exerting excessive stress On the cornea.Prepare A 1%agros in medium solution and keep it ready. At 55 degrees Celsius, thoroughly rinse the cornea in sterile PBS containing penicillin and streptomycin. Place the cornea epithelial side down into the well of a sterile Spotlight.Add agros containing Medium to the endothelial concavity of the cornea. Just enough to fill it completely. Allow about one minute for the agros to solidify.Place the cornea with the supporting agros scaffold epithelial side up and to a tissue culture dish and add supplemented MEM medium to cover the epithelial surface. The cornea may be cultured in this way for over a week with medium changes every 48 hours. Add one milliliter Of medium containing the desired amount of virus into a tissue culture dish.Place the Cornea with the agro scaffold epithelial side down into the virus containing medium. Place the cornea in the tissue culture incubator to infect for one hour with intermittent rocking every 10 to 15 minutes after one hour. Aspirate the virus containing medium and rinse two to three times with PBS to remove any residual viral particles.When rinsing with PBS, be careful not to dislodge the cornea from the agro scaffold. Add fresh medium to cover the epithelial surface. Experimental treatments may be started before or after infection depending on the nature of the experiment.Culture medium Is changed every 48 hours. Mix the medium well and use it immediately for a plaque assay Alternatively stored at minus 80 degrees Celsius for later use. If collecting samples at different time points, Rinse the cornea well with PBS.Remove the agro scaffold, Excites the ring of scleral tissue from the cornea to ensure that only corneal epithelial material is collected. Using a scalpel Scrape off the corneal epithelial cells into the appropriate buffer, depending on the type of material to be isolated. Removal of the scleral rim allows for the corneal str to rapidly swell by absorbing water.Since the swelling may complicate the scraping, we recommend performing the step quickly rinse the Petri dish to ensure that the Entire sample has been collected. Remove the agro scaffold and rinse The cornea. Well with PBS.Use the cornea to make a paraffin tissue block or a frozen tissue block as demonstrated here depending on your experimental goal. Embed the cornea in optimal cutting Temperature compound within a specimen mold Flash freeze the cornea in an ethanol dry ice bath. What we've shown here In this video is the preparation of corneas, the dissection of the cornea, placing it in exvivo culture and infecting corneas with herpes virus.This has provided the technique for a model that has been very useful to us in studying herpes virus infection, ex vivo, and we hope it will be useful to others for their experiments with corneal cells.

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JoVE Journal

Neuroscience

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The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

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Learn ex vivo corneal culture for studying herpes simplex virus type I infection using rabbit and human donor corneas. This model offers improved physiological relevance compared to traditional cell cultures.

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

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