Name: porcine corneal tissue explant to study the efficacy of herpes simplex virus-1 antivirals
Uploaded: 2021-09-20T15:00:00.000+00:00
Duration: 8 min 31 s
Description: Watch this Scientific Journal Video about Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals at JoVE.com

This study was supported by NIH grants (R01 EY024710, RO1 AI139768, and RO1 EY029426) to D.S. A.A. was supported by an F30EY025981 grant from the National Eye Institute, NIH.Study was conducted using the porcine corneas obtained from Park Packing company, 4107 Ashland Avenue, New City, Chicago, IL-60609

All the porcine tissue used in this study was provided by a third-party private organization and none of the animal handling was performed by University of Illinois at Chicago personnel.
1. Materials
<ol>
	<li>Reagents
	<ol>
		<li>Use following reagents for Plaque assay: powder methylcellulose, Dulbecco&#39;s modified eagle&#39;s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin (P/S) for Plaque assay.</li>
		<li>Use crystal violet tablets and ethanol (molecular biology grade) for preparing crystal violet solution for plaque assay.</li>
	</ol>
	</li>
	<li>Vero cell growth media - DMEM whole media
	<ol>
		<li>Open the DMEM media bottle inside the tissue culture hood. Remove 55 mL of media from the bottle using a serological pipette and discard it. Add 50 mL of FBS of P/S to DMEM. Refrigerate at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Plaque assay media - Stock of 5% methylcellulose media
	<ol>
		<li>Measure 2.5 g of methylcellulose powder with a stirring magnet inside a 500 mL glass bottle and autoclave it. After the bottle cools to room temperature, take 500 mL of DMEM whole media containing 50 mL of FBS and 5 mL of P/S and add it to glass bottle containing methylcellulose.</li>
		<li>Stir this at 4 &#176;C for 2 days using a magnetic stirrer. Refrigerate at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Media preparation for excised porcine cornea
	<ol>
		<li>Open a minimum essential media (MEM) bottle inside the tissue culture hood. Discard 10 mL of MEM from the bottle using a serological pipette.</li>
		<li>Add 5 mL of insulin-transferrin-selenium (ITS) and 5 mL of antimycotic-antibiotic (AA) to the media and refrigerate at 4&#176;C.</li>
	</ol>
	</li>
	<li>Master and working stock of crystal violet:
	<ol>
		<li>To make the crystal violet stock solution, add 1 g of crystal violet powder to 100 mL of 20% ethanol in water. This stock (1% w/v crystal violet) can be stored and used for up to year when stored in a place that is dark.</li>
		<li>To make a working stock from this, add 50 mL of original stock solution to 350 mL of water. Add 100mL of ethanol to this solution to make a 500 mL working crystal violet solution (0.1% w/v crystal violet). 
		&#8203;NOTE: Both these solutions need to be stored in the dark.</li>
	</ol>
	</li>
</ol>
2. Procedure
<ol>
	<li>Isolation of Porcine corneas from whole eyes17
	<ol>
		<li>Upon receiving the porcine eyes from a suitable vendor, store on ice if there is a delay with tissue processing as pictured in Figure 1.</li>
		<li>Ensure that personal protection equipment is used and worn during this procedure to avoid contamination as well as accidents from spillage of vitreous humor.</li>
		<li>Spray the working area with 70% ethanol to clean and disinfect. To ensure the working space is stable, spread a bench cover and tape down the sides securely as pictured in Figure 2.</li>
		<li>Place porcine eyes on gauze (Figure 3). Using 50 mm gauze, hold posterior section (Figure 4A) of porcine eyes as shown in Figure 4B with one hand.</li>
		<li>With a 30 G needle, gently make a single poke at approximately the center of epithelial surface of the eye carefully and ensure that there no damage to stroma (Figure 5). The poke should be limited to the epithelium (~100-200 &#181;m) to avoid stromal involvement.</li>
		<li>Using a sharp sterilized blade, make a small incision on the sclera at 1 mm distance from the cornea. Cut the edge of the cornea ensuring that the vitreous humor does not leak using a swift and smooth rotating action of the hand (Figure 6A). By holding the cornea at the cornea-sclera edge with a flat tweezer, cut off the remaining tethering membranes of eye using the blade (Figure 6B).</li>
		<li>Take the cornea and place it in a 12-well plate overlaid with 2 mL of cornea medium. The cornea should be placed facing up, demonstrated in a series of steps in Figures 7A-D, Figure 8).</li>
		<li>Add 5 &#181;L of the virus solution containing 5 x 105 plaque forming units (PFU) of 17 GFP to the debrided site on the corneal surface. Place the 12-well plate containing infected porcine corneas in an incubator with 5% CO2 for 72 h.</li>
		<li>Spray any additional eyes not used in experiment with 10% bleach and securely disposed in biohazard bags.</li>
	</ol>
	</li>
	<li>Visualization 
	NOTE: The virus should be visualized every day prior to addition of drugs.
	<ol>
		<li>Turn on the stereo microscope and LED light source and allow lamp of machine to warm up before imaging the corneas. Carefully carry the plate of corneas to instrument without disturbing the solution. Change the filter so that GFP (380-460 nm) is used to look at specimens. Set the exposure time to 500 ms to capture images.</li>
		<li>First, place the cornea plate under the stereoscope and capture the images at the lowest magnification of 7.5x. Follow this up with a series of increasing magnification images (e.g., 15x, 25x, 35x) such that all the viral spread and dendrite formation is visualized clearly</li>
		<li>Make sure to return the infected corneas back into the tissue culture incubator and save all the images that were taken.</li>
	</ol>
	</li>
	<li>Virus infection quantification 
	NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.
	<ol>
		<li>To quantify virus, seed Vero cells at a density of 5 x 105 of cells per well if using a 6-well plate as done in this experiment. Do this a day prior to the infection. Incubate the plated cells overnight to ensure they are confluent for virus quantification.</li>
		<li>Aliquot 500 &#181;L of serum free media into multiple microcentrifuge tubes. Insert sterile cotton tipped swab dipped into the serum free media filled tubes. The cotton swabs need to be dipped and wetted for at least 5 min prior to the use.</li>
		<li>Without disturbing the underlying media, transport the infected porcine cornea plate into a biosafety cabinet. Using the wet cotton swabs, make 3 revolutions clockwise and 3 revolutions anti-clockwise at a diameter of 5 mm from the center of the infected porcine cornea without applying excessive pressure.</li>
		<li>Insert back the cotton swab into the serum free media filled microcentrifuge tube and rotate it clockwise and anti-clockwise 5 times. The metal tip of the cotton swab should be cut short so that it fits entirely into microcentrifuge tube and the lid can be closed.</li>
		<li>Place the microcentrifuge tube containing the swabbed cotton tip on a vortex machine and vortex at high speed for 1 min.</li>
		<li>Perform virus quantification via a plaque assay on these samples. 
		NOTE: This quantification step needs to be performed on days 2, 4 and optionally on 6 days post infection.</li>
	</ol>
	</li>
	<li>Virus quantification by Plaque assay 
	NOTE: To perform a plaque assay, grow and plate Vero cells into a 6 well plate and ensure 90% confluency of cells before start of assay. Use a confluent 75 cm2 flask of these cells. All the steps below need to be performed inside a biosafety cabinet.
	<ol>
		<li>Wash the confluent monolayer of Vero cells in the flask with 10 mL of fresh phosphate buffer saline (PBS) after aspirating the culture medium. Repeat the wash step once again with PBS after aspirating the first set of wash solution.</li>
		<li>Add 1 mL of 0.05% Trypsin to the cell monolayer. Incubate the flask at 37 &#176;C for 5 min. With the naked eye, ensure that the cells are detached from the inside surface of the flask. If not, wait for another 5 min before examining the flask again. When cells appear to be detached, add 9 mL of whole media to the monolayer to ensure that the cells dislodge completely from the flask surface.</li>
		<li>Collect all the cells from the flask along with the whole media and place them in a 15 mL centrifuge tube.</li>
		<li>For every well of the 6 well plates that are used for plaque assay, use 300 &#181;L from the centrifuge tube. Top up each well of the 6 well plate with 2 mL of whole media. Leave the plates in the incubator overnight to allow them to grow and form a confluent monolayer in each well.</li>
		<li>In order to perform a plaque assay, perform a serial dilution of samples needs to be conducted before the quantification of virus.</li>
		<li>Perform a log101 fold dilution of the virus in micro-centrifuge tubes using serum free media until a dilution of 10-8 is reached. When at a 10-3 dilution, transfer 1 mL of the dilution to the monolayer of plated cells after aspirating the growth media on the cells from the 6 well plate, this will be the infection step. Incubate the infected plate at 37 &#176;C incubator for 2 h.</li>
		<li>Aspirate the existing infection media, wash with PBS twice gently to coat cells and add 2 mL of methylcellulose laden media per well to all 6 wells. Incubate for 72 h or until formation of plaques can be seen. Plaques can be identified by the formation of small gaps between cells in the cell monolayer.</li>
		<li>Add 1 mL of methanol slowly to the corner of each well, using the wall as guiding tip. Incubate the 6 well plate at room temperature for 15 min. Slowly aspirate the contents of each well from the plate without disturbing or agitating the cell monolayer.</li>
		<li>Add 1 mL of crystal violet working solution to each well of 6 well plate, ensuring all cells are covered. Incubate the 6 well plate in the dark for 30 min. Discard the crystal violet solution by aspirating it and dry the wells on a sheet of absorbent paper.</li>
		<li>Count the number of plaques at the highest dilution well to quantify the total virus content in the starting solution. Repeat the process twice to confirm the viral titer.</li>
	</ol>
	</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antiviral+drugs+of+herpes+simplex+virus+isolated+from+a+patient+treated+with+Acyclovir.">Resistance to antiviral drugs of herpes simplex virus isolated from a patient treated with Acyclovir.</a> New England Journal of Medicine. 306 (6), 343-346 (2010).</li><li>Yildiz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+due+to+acyclovir.">Acute kidney injury due to acyclovir.</a> CEN Case Report. 2 (1), 38-40 (2013).</li><li>Fleischer, R., Johnson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyclovir+nephrotoxicity:+a+case+report+highlighting+the+importance+of+prevention,+detection,+and+treatment+of+acyclovir-induced+nephropathy.">Acyclovir nephrotoxicity: a case report highlighting the importance of prevention, detection, and treatment of acyclovir-induced nephropathy.</a> Case Rep Med. 2010, 1-3 (2010).</li><li>Thakkar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+corneas+show+dendritic+spread+and+restrict+herpes+simplex+virus+infection+that+is+not+observed+with+cultured+corneal+cells.">Cultured corneas show dendritic spread and restrict herpes simplex virus infection that is not observed with cultured corneal cells.</a> Science Report. 7, 42559 (2017).</li><li>Pescina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al+Development+of+a+convenient+ex+vivo+model+for+the+study+of+the+transcorneal+permeation+of+drugs:+Histological+and+permeability+evaluation.">et al Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: Histological and permeability evaluation.</a> Journal of Pharmaceutical Sciences. 104, 63-71 (2015).</li><li>Jaishankar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+off-target+effect+of+BX795+blocks+herpes+simplex+virus+type+1+infection+of+the+eye.">An off-target effect of BX795 blocks herpes simplex virus type 1 infection of the eye.</a> Science Translational Medicine. 10, 5861 (2018).</li><li>Duggal, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+oxide+tetrapods+inhibit+herpes+simplex+virus+infection+of+cultured+corneas.">Zinc oxide tetrapods inhibit herpes simplex virus infection of cultured corneas.</a> Molecular Vision. 23, 26-38 (2017).</li></ol>

The authors declare no conflict of interest and no competing financial interests.

Prior research has shown BX795 to have a promising role as an antiviral agent against HSV-1 infection; by inhibiting the TANK-binding kinase 1 (TBK1)16. Both TBK1 and autophagy have played a role in helping inhibit HSV-1 infection as demonstrated on human corneal epithelial cells. BX795 was shown to be maximally effective with antiviral activity at a concentration of 10&#181;M and using both western blot analysis and viral plaque analysis of key viral proteins, BX795 was shown to inhibit HSV-1 infection comparable to the activity of TFT16. The study on porcine corneas followed the same analysis and obtained similar results as demonstrated above; BX795 is shown to be just as effective as TFT in inhibiting infection - images taken of porcine corneas at day 4 and 6 of infection show comparable results in both TFT and BX795. Plaque assays performed to quantify secreted virions also reinforce these findings16. BX795 has also shown to have antiviral effects in vitro and its use topically in mouse models in vivo has also shown suppression of corneal HSV-1 infection16.
The study contributes to establishing BX795 as an effective and leading compound for broad-spectrum antiviral application against HSV-1 infection. By showing its efficacy in porcine models as comparable to TFT, BX795 stands to be successful in multiple infection models16. Additionally, BX795 is important as it has been shown to be effective in multiple virus strains, even HSV-1 (KOS)tk12 which is resistant to another widely used drug Acyclovir (ACV)16. BX975 treated cells show very little expression of HSV-1 viral protein gB which certify its inhibition efficacy of HSV-1 virus. BX795 is also demonstrates better anti-viral efficacy at lower doses compared to other treatments and anti-herpesvirus therapies. Furthermore, therapeutic concentrations of BX795 (even proposed concentration of 10 &#181;M) show no adverse cytotoxicity towards cells - no apoptosis inducement and cell death, which is again comparable to the TFT control16.
Critical steps within the protocol section include isolation of porcine cornea from the whole eye. This involves the debridement of the cornea at the center of the eye using a needle and requires very little force to ensure no stromal involvement followed by excising the cornea from the ocular surface gently without disturbing the iris. Another set of critical steps include parts involving pre-wetting cotton tips and swabbing the corneal surface. The motion should be gentle to ensure corneal epithelium is not being dislodged from the ocular surface.
Modification can be done to the epithelial debridement step. Instead of using a 30 G needle to make a single poke at the center of the cornea, the experimenter can use a sterile blade or 30 G needle to gently make grid shaped scratches to the corneal epithelium. This ensures robust infection to the epithelium.
Porcine corneas should be used on the same day of procurement and should not be held for longer than 24 h. Porcine corneas kept in ice for longer than 4 h will result in the iris sticking to cornea. This makes it harder to separate the cornea from the rest of the eye during excision. Not all porcine corneas will get infected alike. The experimenter should infect a minimum of 5 eyes per group and then pick equally infected corneas on day 2 post infection to proceed with the experiment.
The significance of the current technique involves the cost effectiveness of using porcine over human corneas. The technique is also significant because of the relative freshness of the corneas being used when compared to human corneas.
Future applications of the current technique include but not limited to its use in testing drug permeability studies, ex vivo pharmacokinetic and pharmacodynamic studies. Porcine corneas can also be used to test antibacterial and antifungal drugs in addition to antiviral drugs. The corneas can also be used for ex vivo wound healing assays to study diabetic wounds.

People suffering from ocular infections often incur vision loss1. With a high seroprevalence worldwide, HSV infected individuals suffer from recurring eye infections which lead to corneal scarring, stromal keratitis and neovascularization2,3,4,5. HSV infections have also shown to cause less frequently, a range of serious conditions among immunocompromised, untreated patients like encephalitis and systemic morbidity6,7,8. Drugs like Acyclovir (ACV) and its nucleoside analogs have shown consistent success in curbing HSV-1 infection and even control reactivation yet the prolonged use of these drugs is associated with renal failure, fetal abnormalities and failure to restrict the emergence of drug-resistance to evolving viral strains9,10,11,12,13. Complexities associated with HSV-1 ocular infections, have been previously studied in vitro using monolayers and 3D cultures of human corneal cells and in vivo using murine or rabbit ocular infections. While these in vitro models provide significant data on the cellular biological components of HSV-1 infections, they, however, fail to mimic the intricate complexity of corneal tissue and do little to illuminate the dendritic spread of the virus14. In contrast, although in vivo systems are more insightful in showing infection spread in corneas and immune activation responses during HSV-1 infection, they do come with the caveat that they require trained investigators and large facilities for animal care to overlook the experiments.
Here we use porcine corneas as an ex vivo model to examine the HSV-1 infection induced wound system. Both the potential pharmacology of certain drugs as well as the cell and molecular biology of the wound system caused by the infection can be studied through tissue explant cultures. This model may also be amended for the use for other viral and bacterial infections as well. In this study, porcine corneas were used to test the antiviral efficacy of a preclinical small molecule, BX795. The use of porcine corneas was preferred due to ease of access and cost effectiveness. Additionally, porcine corneal models are good models of human eyes with the corneas being easy to isolate, adequately sized for infection and visualization and robust to handle15. Porcine corneas are also comparable to the complexity of human corneal models in both trans corneal permeability and systemic absorption15. By using this model for the study, we were able to elucidate how BX795 is worthy of further investigation as a competent inhibitor of HSV-1 virus infection and adds to the literature of classifying it as a potential small-molecule antiviral compound16.

<table><tbody><tr><td>30 G hypodermic needles.</td><td>BD</td><td>305128</td><td></td></tr><tr><td>500 mL glass bottle.</td><td>Thomas Scientific</td><td>844027</td><td></td></tr><tr><td>Antimycotic and Antibiotic (AA)</td><td>GIBCO</td><td>15240096</td><td>Aliquot into 5 mL tubes and keep frozen until use</td></tr><tr><td>Benchtop vortexer.</td><td>BioDot</td><td>BDVM-3200</td><td></td></tr><tr><td>Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification.</td><td>Thermofisher Scientific</td><td>Herasafe 2030i</td><td></td></tr><tr><td>Calgiswab 6&quot; Sterile Calcium Alginate Standard Swabs.</td><td>Puritan</td><td>22029501</td><td></td></tr><tr><td>Cell scraper - 25 cm</td><td>Biologix BE</td><td>70-1180 70-1250</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma Aldrich</td><td>C6158</td><td>Store the powder in a dark place</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium - DMEM</td><td>GIBCO</td><td>41966029</td><td>Store at 4 &amp;deg;C until use</td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E7023</td><td></td></tr><tr><td>Fetal bovine serum -FBS</td><td>Sigma Aldrich</td><td>F2442</td><td>Aliquot into 50 mL tubes and keep frozen until use</td></tr><tr><td>Flat edged tweezers &amp;ndash; 2.</td><td>Harward Instruments</td><td>72-8595</td><td></td></tr><tr><td>Freezers --80 &amp;deg;C. -</td><td>Thermofisher Scientific</td><td>13 100 790</td><td></td></tr><tr><td>Fresh box of blades.</td><td>Thomas Scientific</td><td>TE05091</td><td></td></tr><tr><td>Guaze</td><td>Johnson &amp;amp; Johnson</td><td></td><td>108 square inch folder 12 ply</td></tr><tr><td>HSV-1 17GFP</td><td>grown in house</td><td>-</td><td>Original strain from Dr. Patricia Spears, Northwestern University. GFP expressing HSV-1 strain 17</td></tr><tr><td>Insulin, Transferrin, Selenium - ITS</td><td>GIBCO</td><td>41400045</td><td>Aliquot into 5 mL tubes and keep frozen until use</td></tr><tr><td>Magnetic stirrer.</td><td>Thomas Scientific</td><td>H3710-HS</td><td></td></tr><tr><td>Metallic Scissors.</td><td>Harward Instruments</td><td>72-8400</td><td></td></tr><tr><td>Micropipettes 1 to 1000 &amp;micro;L.</td><td>Thomas Scientific</td><td>1159M37</td><td></td></tr><tr><td>Minimum Essential Medium - MEM</td><td>GIBCO</td><td>11095080</td><td>Store at 4 &amp;deg;C until use</td></tr><tr><td>OptiMEM&amp;nbsp;</td><td>GIBCO</td><td>31985047</td><td>Store at 4 &amp;deg;C until use</td></tr><tr><td>Penicillin/streptomycin.</td><td>GIBCO</td><td>15140148</td><td>Aliquot into 5 mL tubes and keep frozen until use</td></tr><tr><td>Phosphate Buffer Saline -PBS</td><td>GIBCO</td><td>10010072</td><td>Store at room temperature</td></tr><tr><td>Porcine Corneas</td><td>Park Packaging Co., Chicago, IL</td><td></td><td>Special order by request</td></tr><tr><td>Procedure bench covers - as needed.</td><td>Thermofisher Scientific</td><td>S42400</td><td></td></tr><tr><td>Serological Pipettes</td><td>Thomas Scientific</td><td>P7132, P7127, P7128, P7129, P7137</td><td></td></tr><tr><td>Serological Pipetting equipment.</td><td>Thomas Scientific</td><td>Ezpette Pro</td><td></td></tr><tr><td>Stereoscope</td><td>Carl Zeiss</td><td>SteREO Discovery V20</td><td></td></tr><tr><td>Stirring magnet.</td><td>Thomas Scientific</td><td>F37120</td><td></td></tr><tr><td>Tissue culture flasks, T175 cm2.</td><td>Thomas Scientific</td><td>T1275</td><td></td></tr><tr><td>Tissue culture incubators which can maintain 5% CO2 and 37 &amp;deg;C temperature.</td><td>Thermofisher Scientific</td><td>Forma 50145523</td><td></td></tr><tr><td>Tissue culture treated plates (6-well).</td><td>Thomas Scientific</td><td>T1006</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>GIBCO</td><td>25-300-062</td><td>Aliquot into 10 mL tubes and keep frozen until use</td></tr><tr><td>Vero cells</td><td>American Type Culture Collection ATCC</td><td>CRL-1586</td><td></td></tr></tbody></table>

porcine corneal tissue explant to study the efficacy of herpes simplex virus-1 antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

To understand the efficacy of the experimental antivirals, they need to be tested extensively before they are sent for in vivo human clinical trials. In this regard, positive control, negative control and test groups have to be identified. Trifluorothymidine (TFT) has long been used as the preferred treatment to treat herpes keratitis topically16. Used as a positive control, the TFT treated corneal groups show lower infection spread. As a negative control, we used DMSO or vehicle control dissolved in PBS. BX795, the experimental preclinical drug was the test group. A total of 4 corneas were assigned to each group and the drugs were added 3-times every day to the porcine corneas. Our results using stereoscopic fluorescence imaging show that the antiviral efficacy of BX795 is similar to TFT in controlling viral spread. Viral spread in our studies can be visualized by imaging the green fluorescence channel in the stereoscope. We observed that vehicle only treated negative control group corneas showed spread of the virus from the central infection zone to its periphery by 6 days post initial viral inoculation, while both drugs (BX795 and TFT) clear the infection in day 4 - 6 images (Figure 9A). Similarly, the ocular swabs taken on days 2 and 4 post infection show a complete inhibition of virus in the positive control and BX795 treated samples while a sharp increase in infectious virus titer is observed in the negative control group (Figure 9B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62195/62195fig01.jpg" /> 
Figure 1: Porcine eyes kept on ice until tissue is processed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62195/62195fig02.jpg" /> 
Figure 2: Work bench setup. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62195/62195fig03.jpg" /> 
Figure 3: Porcine eyes placed on gauze. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62195/62195fig04.jpg" /> 
Figure 4: Porcine eye with 30G needle pictured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62195/62195fig05.jpg" /> 
Figure 5: 30 G needle used; hole made at center of epithelial surface. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62195/62195fig06.jpg" /> 
Figure 6: Rotating action used to cut around corneal edge using sharp sterilized blade (A,B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62195/62195fig07.jpg" /> 
Figure 7: Cornea images. (A,B) The cornea finally cut using sharp scissors (C) Isolated cornea is held by tweezers (D) Completely isolated cornea, ready for use <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62195/62195fig08.jpg" /> 
Figure 8: Corneas placed in 12-well plate and incubated for 72 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62195/62195fig09.jpg" /> 
Figure 9: Progression of viral spread taken during course of infection. Freshly excised porcine corneas were infected with HSV-1 17-GFP and (A) imaged using microscope on days 2, 4 and 6 post infection. Topical treatment with DMSO, TFT or BX795 was started on day 2 post infection. (B) Plaque assays were performed using swabs taken from the porcine corneas on Vero Cells. Two-way ANOVA test was performed to understand significant differences between the treatment groups. n=4, ****p=0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62195/62195fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals at JoVE.com

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

porcine-corneal-tissue-explant-to-study-efficacy-herpes-simplex-virus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.9K Views.  University of Illinois at Chicago. We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

Video: Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

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

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.

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.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

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.

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.

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 ...

Neuroscience

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

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.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

Plaquing of Herpes Simplex Viruses

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can ...

Ex Vivo Porcine Corneal Culture System for Toxicity Testing and Wound Healing

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 &#176;C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

Author Spotlight: Exploring Corneal Innate Immunity and Delayed Wound Healing in Diabetic Patients

Porcine corneal ex vivo organ culture and epithelial wound healing provide an economical, ethical, reproducible, and quantitative means for testing the ocular toxicity of chemicals. They also aid in elucidating mechanisms underlying the regulation of epithelialization and tissue repair, and in evaluating therapeutics for treating diabetic keratopathy and delayed wound healing.

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular ...

Medicine

Porcine Ex vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Source: Okurowska, K., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61156">Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.</a> J. Vis. Exp (2020).
This video describes a protocol to set up an ex vivo model for bacterial keratitis using a corneoscleral button dissected from the porcine eye. Inoculated bacteria traverse the corneal epithelial incisions to invade the underlying stroma and establish an infection within the corneal tissues that mimic the in vivo infection.

Model rogówki ex vivo świńskiego bakteryjnego zapalenia rogówki: technika ustalania infekcji bakteryjnej w komórkach nabłonka rogówki

Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp (2020).
...

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

Source: Holzhey, A. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60542">Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss.</a> J. Vis. Exp. (2020)
This video demonstrates a non-invasive laser-assisted procedure to selectively damage the corneal endothelial cells in the porcine eye. The developed ex vivo model of endothelial cell damage can be used to study diseases of the corneal endothelium.

Selektywna ablacja wspomagana laserowo komórek śródbłonka rogówki świń: technika wywoływania skoncentrowanego uszkodzenia śródbłonka rogówki świni do badań ex vivo

Source: Holzhey, A. et al., Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss. J. Vis. Exp. (2020)
This ...

Determining Antiviral Efficacy of Experimental Drugs Through a Plaque Assay

Source: Yadavalli, T., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62195">Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals.</a> J. Vis. Exp. (2021)
This video demonstrates the method to perform a plaque assay using a virus obtained from infected porcine cornea. The porcine cornea is used to test the antiviral efficacy of experimental drugs.

Określanie skuteczności przeciwwirusowej leków eksperymentalnych za pomocą testu płytki nazębnej

1. Virus infection quantification
NOTE: Virus titers from porcine corneas should be evaluated every day to analyze the effect of drug treatment.

	To ...

Immunology

Immunodiagnostics

Specialized Assays

Porcine Ex Vivo Cornea Model of Bacterial Keratitis: A Technique to Establish Bacterial Infection in Corneal Epithelial Cells

Remove the media from the Petri dish, and rinse the corneas twice with one milliliter of sterile PBS. Hold the cornea with forceps and squeeze gently. Use a 10A scalpel to make four cuts - two vertical, two horizontal - in the central section of the corneoscleral button through the epithelial layer to the underlying stroma.
Place a sterile glass mold in a six-well plate with the wide part up. Then, place the cornea in the middle of the glass mold, with the epithelium side facing down, and the wounded part of the cornea centered in the glass mold.
Fill the glass mold completely by adding one milliliter of agar solution. After allowing the agar to set, invert the glass mold so that the corneal epithelium is facing upwards.
The glass mold has to be sealed with agar up to the brim to prevent leaking of the inoculum or drug solution.
Pipette 200 microliters of the bacterial culture directly into a cut area. Also, for each experiment, set up a control by adding 200 microliters of sterile PBS to one cornea, instead of adding the bacterial culture.
Next, add 185 microliters of PBS to the top of each cornea to keep the epithelium moist. Then, add one milliliter of DMEM without antibiotics to the bottom of each well. Incubate the six-welled plate at 37 degrees Celsius with humidity and 5% carbon dioxide, for up to 24 hours.
Discard the DMEM from the six-welled plate and add one milliliter of sterile PBS to each well to rinse it. Remove the PBS gently, without touching the central part of the corneoscleral button. Remove the glass mold using sterile forceps, and place it in 5% Distel. Gently rinse the top of the corneoscleral button twice with one milliliter of PBS.
Use fine-tip forceps to lift the edge of the corneoscleral button, detach it from the agar underneath, and transfer it to a 50-milliliter tube filled with one to two milliliters of ice-cold PBS. To help detach bacteria from the corneal epithelium and the cut area, add PBS to the tube and use a fine-tip homogenizer to shear the top of the infected cornea. The tissue does not have to be completely liquidized.
Vortex to resuspend settled bacteria, and add 20 microliters of the homogenized cornea to 180 microliters of PBS. Then, perform serial dilutions of the homogenate in a 96-well plate.
Pipette 10 microliters of the diluted homogenate onto a blood agar plate.
After incubating the plate for 16 hours, count the number of colony-forming units.

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

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

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

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

Plaquing of Herpes Simplex Viruses

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

Model rogówki ex vivo świńskiego bakteryjnego zapalenia rogówki: technika ustalania infekcji bakteryjnej w komórkach nabłonka rogówki

Selektywna ablacja wspomagana laserowo komórek śródbłonka rogówki świń: technika wywoływania skoncentrowanego uszkodzenia śródbłonka rogówki świni do badań ex vivo

Określanie skuteczności przeciwwirusowej leków eksperymentalnych za pomocą testu płytki nazębnej

Model rogówki ex vivo świńskiego zapalenia rogówki: technika ustalania infekcji bakteryjnej w komórkach nabłonka rogówki