We thank Drs. Keping Xu (M.D. and O.D.) and Jia Yin (M.D. and Ph.D.) for their contributions to the development of bovine and porcine corneal organ culture and Ray Guo and Andy Wu of Troy High School for the artwork of Figure 1. Dr. Yu&#39;s lab research was funded by NIH grants (R01 EY010869, R01EY035785, P30 EY04068) and by Research to Prevent Blindness at Kresge Eye Institute.

Since fresh pig corneas are a byproduct of the food industry, the Institutional Animal Care and Use Committee did not need to approve their use for research. Unlike human corneas used in research, there are no biohazard concerns, and unused parts of pig eyes can be disposed of as regular garbage. The reagents and equipment used for this study are listed in the Table of Materials.
1. Preparation for organ culture
<ol>
	<li>Add penicillin-streptomycin to the Minimum essential medium (MEM) as supplements before use.</li>
	<li>Prepare high-glucose culture media by adding 3.6 g of D-glucose to 1 L of supplemented MEM, which contains 5 mM glucose (equal to 90 mg/dL), to reach a 25 mM (equal to 450 mg/dL) final glucose concentration, mimicking hyperglycemia in diabetic patients.</li>
	<li>Prepare 1% agarose in supplemented MEM with 5 mM or 25 mM glucose by adding 0.2 g agarose to 20 mL of MEM and heating it in a microwave until the agarose dissolves.</li>
	<li>Transfer the agarose-containing solution into a water bath maintained at 48 &#176;C.</li>
	<li>Before the experiment, autoclave all experimental reagents, such as distilled water, PBS, and surgical equipment: hemostat, forceps, scalpel handle, scissors, and trephine, as well as beakers, paper towels, lint-free wipes, gloves, and pipette tips. Soak the silicon mold, razor blade, and blade holder in 70% alcohol for 30 min and wash with sterile PBS three times.</li>
</ol>
2. Porcine eyeball preparation for corneal culture
<ol>
	<li>Obtain porcine eyeballs from a local abattoir and transport them to the laboratory on ice in a moist chamber. 
	NOTE: Bovine eyes can also be used in a similar fashion. However, bovine eyes and corneas differ from human eyes/corneas more remarkably. For example, human and porcine corneas have 5-7 layers of epithelial cells, while bovine corneas have about 20 layers13,14. Moreover, bovine eyes are more likely to be contaminated during corneal organ culture; hence, more bovine eyes are needed for statistical analysis.</li>
	<li>Place the porcine eyes in a 1 L beaker containing sterilized PBS. 
	NOTE: The porcine eyeballs are collected within 1 h after slaughter and transported to the lab in about 2 h. Overall, the eyes were used within 4 h (step 2.3).</li>
	<li>Hold an eyeball&#160;with tweezers and remove the extraocular tissues with a scissor in a sterile Petri plate.</li>
	<li>Rinse the eyeballs with distilled water once and PBS twice.</li>
	<li>Rinse the eye bulbs with a Povidone-Iodine antiseptic solution for 10 s, followed by sterilized PBS three times washing.</li>
	<li>Incubate the eyeballs in PBS containing 20 &#181;g/mL gentamicin for 30 min and rinse them twice with PBS.</li>
</ol>
3. Epithelium wounding
<ol>
	<li>Hold an eyeball with sterilized lint-free wipes and mark the center of the corneas with a 6 mm trephine.</li>
	<li>Gently scrape epithelial cells within the trephine-marked circle with a small scalpel or corner-blunted soft razor blade, remove all cell debris but leave the basement membrane intact, and clean the wound area with cotton swabs. 
	NOTE: The scalpel with collected epithelial cells&#160;can be transferred to a microcentrifuge tube placed on&#160;ice. The cells can be lysed immediately or stored in a -20 &#176;C freezer to serve as the controls..</li>
</ol>
4. Corneal organ culture and ex vivo hyperglycemia modeling
<ol>
	<li>Dissect the eyeball by cutting along corneal-scleral rims with a scalpel and scissors and rinse the corneas in a sterilized 1000 mL beaker containing PBS (pH 7.4).</li>
	<li>Place the excised corneas upside down into a sterile mold made from non-toxic liquid mold-making silicone. 
	NOTE: Pour the non-toxic silicone mold-making liquid into the holes of a 50 mL tissue culture/test tube rack, using the bottom of the 30 mL ultracentrifuge tube to make a half-round mold.</li>
	<li>Fill the endothelial corneal cavity with MEM containing 1% agarose maintained at 48 &#176;C and allow the mixture to gel at&#160;the room temperature..</li>
	<li>Invert and transfer the corneas to a 35 mm dish, and add 2 mL of MEM with or without testing agent(s) dropwise to the surface of the central cornea to cover the limbal conjunctiva region, leaving the epithelium exposed to the air (Figure 1).</li>
	<li>Place the culture dishes in a humidified 5% CO2 incubator at 37 &#176;C and replace the media with fresh MEM media with or without a testing agent(s) daily for 2 days.</li>
	<li>Establish an ex vivo hyperglycemia model by adding L-glucose to MEM to reach a total of 25 mM glucose, which is used for 1% agarose gel preparation and as culture media.</li>
</ol>
5. Corneal function assessment
<ol>
	<li>Determine the epithelial wound healing rate by staining the wounded corneas with Richardson&#39;s staining20 to mark the remaining wound area and photograph it (Figure 2 and Figure 3). Quantify the wound size with Image-J or Photoshop software (histogram).</li>
	<li>Process the corneas for histology, histochemistry, and immunohistochemistry analyses (Figure 2 and Figure 3) by embedding them in the OCT and cryostat sectioning. Fix the sections with ice-cold acetone, stain with H&#38;E staining, or block with 1% BSA for 1 h for TUNEL staining or immunohistochemistry to detect proteins and signaling molecules (such as phosphorylated Erk and ATK). 
	NOTE: Most commercially available antibodies were not tested for porcine antigens. However, if an antibody recognizes both human and mouse antigens, it can mostly be used to detect the corresponding porcine antigen Western blotting and immunohistochemistry.</li>
	<li>Wash the stained corneas with PBS and use the same-sized trephine to mark the original wound and scrap epithelial cells within the marker circles.
	<ol>
		<li>Collect epithelial cells with a small scalpel, immerse the scalpel with collected cells&#160;a centrifuge tube precooled on ice.</li>
		<li>Store the collected cells in -80&#160;&#176;C deep freezer or extract them immediately and perform lysis for Western blotting and/or ELISA or in RNA extraction buffer for PCR (if required)10,16,21.</li>
	</ol>
	</li>
</ol>

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

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V., 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=Human+corneal+epithelial+basement+membrane+and+integrin+alterations+in+diabetes+and+diabetic+retinopathy.">Human corneal epithelial basement membrane and integrin alterations in diabetes and diabetic retinopathy.</a> J Histochem Cytochem. 46 (9), 1033-1041 (1998).</li><li>Shah, R., 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=Reversal+of+dual+epigenetic+repression+of+non-canonical+Wnt-5a+normalizes+diabetic+corneal+epithelial+wound+healing+and+stem+cells.">Reversal of dual epigenetic repression of non-canonical Wnt-5a normalizes diabetic corneal epithelial wound healing and stem cells.</a> Diabetologia. 66 (10), 1943-1958 (2023).</li><li>Poe, A. J., 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=Regulatory+role+of+miR-146a+in+corneal+epithelial+wound+healing+via+its+inflammatory+targets+in+human+diabetic+cornea.">Regulatory role of miR-146a in corneal epithelial wound healing via its inflammatory targets in human diabetic cornea.</a> Ocul Surf. 25, 92-100 (2022).</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 (2), 306-314 (2000).</li><li>Wilson, S. L., Ahearne, M., Hopkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+current+techniques+for+ocular+toxicity+testing.">An overview of current techniques for ocular toxicity testing.</a> Toxicology. 327, 32-46 (2015).</li><li>Rose, J. 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=An+experimental+study+to+test+the+efficacy+of+mesenchymal+stem+cells+in+reducing+corneal+scarring+in+an+ex-vivo+organ+culture+model.">An experimental study to test the efficacy of mesenchymal stem cells in reducing corneal scarring in an ex-vivo organ culture model.</a> Exp Eye Res. 190, 107891 (2020).</li><li>Xu, K. P., Li, Y., Ljubimov, A. V., 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=High+glucose+suppresses+epidermal+growth+factor+receptor/phosphatidylinositol+3-kinase/Akt+signaling+pathway+and+attenuates+corneal+epithelial+wound+healing.">High glucose suppresses epidermal growth factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and attenuates corneal epithelial wound healing.</a> Diabetes. 58 (5), 1077-1085 (2009).</li><li>Seyed-Safi, A. G., Daniels, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+porcine+corneal+organ+culture+model+to+study+the+limbal+response+to+corneal+epithelial+injury.">A validated porcine corneal organ culture model to study the limbal response to corneal epithelial injury.</a> Exp Eye Res. 197, 108063 (2020).</li><li>Peng, M., 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+ex+vivo+model+of+human+corneal+rim+perfusion+organ+culture.">An ex vivo model of human corneal rim perfusion organ culture.</a> Exp Eye Res. 214, 108891 (2022).</li><li>Elsheikh, A., Alhasso, D., Rama, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+properties+of+human+and+porcine+corneas.">Biomechanical properties of human and porcine corneas.</a> Exp Eye Res. 86 (5), 783-790 (2008).</li><li>Zeng, Y., Yang, J., Huang, K., Lee, Z., Lee, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+biomechanical+properties+between+human+and+porcine+cornea.">A comparison of biomechanical properties between human and porcine cornea.</a> J Biomech. 34 (4), 533-537 (2001).</li><li>Yoon, C. H., Choi, H. J., Kim, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+xenotransplantation:+Where+are+we+standing.">Corneal xenotransplantation: Where are we standing.</a> Prog Retin Eye Res. 80, 100876 (2021).</li><li>Xu, K. P., Ding, Y., Ling, J., Dong, Z., 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=Wound-induced+HB-EGF+ectodomain+shedding+and+EGFR+activation+in+corneal+epithelial+cells.">Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells.</a> Invest Ophthalmol Vis Sci. 45 (3), 813-820 (2004).</li><li>Xu, K. P., Yin, J., Yu, F. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+epithelial+wound+healing+and+EGFR+signaling+pathways+in+the+corneas+of+diabetic+rats.">Impaired epithelial wound healing and EGFR signaling pathways in the corneas of diabetic rats.</a> Invest Ophthalmol Vis Sci. 52, 3301-3308 (2011).</li><li>Gao, N., Yin, J., Yoon, G. S., Mi, Q. S., 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=Dendritic+cell-epithelium+interplay+is+a+determinant+factor+for+corneal+epithelial+wound+repair.">Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair.</a> Am J Pathol. 179 (5), 2243-2253 (2011).</li><li>Yin, J., Yu, F. 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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 (2), 306-314 (2000).</li><li>Abhari, 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=Anatomic+studies+of+the+miniature+swine+cornea.">Anatomic studies of the miniature swine cornea.</a> Anat Rec (Hoboken). 301 (11), 1955-1967 (2018).</li><li>Araj, H., Tseng, H., Yeung, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+in+vivo+draize+rabbit+eye+and+skin+irritation+tests+with+a+focus+on+3D+reconstructed+human+cornea-like+epithelium+and+epidermis+models.">Alternatives to in vivo draize rabbit eye and skin irritation tests with a focus on 3D reconstructed human cornea-like epithelium and epidermis models.</a> Toxicol Res. 33 (3), 191-203 (2017).</li><li>Xu, K., McDermott, M., Villanueva, L., Schiffman, R. M., Hollander, D. 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Cultured bovine and mostly porcine corneas have been used to assess the toxicities of cosmetic chemicals, glaucoma medications, and nonsteroidal anti-inflammatory drugs21,26. Pig corneas have also been used as an ex vivo model of human diabetic keratopathy. Unlike rabbit eyes, the pig eye resembles the human eye anatomically, physiologically, and biomechanically27, hence, being used for xenotransplantation into h...

While cell models possess limited clonal populations and fail to reproduce an organism&#39;s in vivo architecture, organ culture or explant offers insights into organ function, development, disease mechanisms, and potential therapies while providing ethical and physiological advantages over other experimental models1,2. In addition to reducing the number of animals needed, culturing explants control and sensibly manipulate the surrounding conditions, which is ideal for a detailed exploration of factors controlling cell proliferation, migration, wound response, and cellular differentiation in an organ culture setting2,3. Among different tissues/organs, corneal explants, including that of humans4,5,6, have been used broadly for ocular toxicity, irritation assessments7,8, to study molecularly mechanisms underlying stem cell function9 and wound healing10,11, and to Primary open-angle glaucoma12.
Porcine corneas share several structural and physiological similarities with human corneas, making them an excellent model for studying human corneal biology and diseases. Structurally, both have Bowman&#39;s layer, 5-7 layers of epithelial cells, and similar curvature and diameter. Physiologically, they are highly transparent, have similar tear film composition and corneal hydration, exhibit comparable patterns and functions of corneal innervation, and follow similar wound healing processes, making them an excellent model for studying human corneal biology and diseases13,14. While human and porcine corneas have slight differences in collagen fibril arrangement and water content, their immune signaling and responses are not identical. These differences pose challenges for xenotransplantation15. Hence, species-specific differences must be considered while interpreting experimental data.
Compared to human corneas, porcine eyes are readily available as byproducts of the meat industry, making them cost-effective and easily accessible for research14. Using porcine corneas helps reduce the need for human donor corneas and minimizes the ethical concerns associated with animal testing. Moreover, the availability of many porcine corneas at one time allows for consistent and reproducible experiments, which is crucial for reliable research outcomes.
A porcine corneal organ culture system was initially used to replace animal tests of cosmetic chemicals and ocular drugs7. This system has been used to study corneal epithelial wound healing and to identify several important signal pathways such as HB-EGF ectodomain shedding,&#160;lipid mediator lysophosphatidic acid stimulation, and EGFR activation for corneal wound healing16,17. Using high glucose as a pathologic factor, an ex vivo model of hyperglycemia was established with delayed epithelial wound healing to mimic human diabetic keratopathy. Using this model, the balance expressions of IL-1&#946; versus IL-1Ra18 and TGF&#946;3 versus TGF&#946;119 were shown to be important factors for proper wound healing in the corneas, and manipulation of these balances may be used to treat diabetic keratopathy. Hence, porcine organ culture represents a relevant, economical, and manipulating experiment system with various applications in chemical toxicity tests, biomedical research, drug discovery, and assessing tissue damage and repair in response to ocular exposure to chemical weapons.
In this article, a detailed protocol of porcine corneal organ culture is described, and its applications for assessing the potential effects of ocular NSAID (NS) eye drops on corneal health and for determining signaling pathways and biological processes involved in the pathogenesis of diabetic keratopathy are illustrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL tubes</td>
			<td>Axygen</td>
			<td>AXYMCT175SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Thermo Scientific</td>
			<td>R0491</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromfenac (Prolensa) 0.09%&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Canon</td>
			<td>PowerShot A620</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose&#160;</td>
			<td>Sigma</td>
			<td>50-99-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Zeiss</td>
			<td>Stemi 2000c</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FisherScientific</td>
			<td>10-316A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>FisherScientific</td>
			<td>13-812-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketorolac (Acular) 0.45%</td>
			<td></td>
			<td>Kresge Clinic</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>LL-37</td>
			<td>Tocris</td>
			<td>5213/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)&#160;</td>
			<td>Gibco</td>
			<td>A1048901</td>
			<td></td>
		</tr>
		<tr>
			<td>Nepafenac (Ilevro) 0.1%&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin&#160;</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Pig eyes&#160;</td>
			<td>Bernthal Packing Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>VWR</td>
			<td>76322-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine corneas</td>
			<td>Bernthal Packing , Inc. Frankenmuth, MI&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine&#160;</td>
			<td>Betadine</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Q-Tips cotton swabs</td>
			<td>Q-Tips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72002-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade holder&#160;</td>
			<td>Stotz</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Bard-Parker</td>
			<td>377112</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>Bard-Parker</td>
			<td>#3</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FisherScientific</td>
			<td>08-951-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon mold</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culter enclosure</td>
			<td>Labconco</td>
			<td>5100000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trephine</td>
			<td>Acu.Punch</td>
			<td>3813775</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath&#160;</td>
			<td>VWR</td>
			<td>1235</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Cataract surgery is one of the most frequently performed procedures globally, and eye drops play a crucial role in post-surgery care. Applying eye drops after cataract surgery helps prevent complications such as eye infections, inflammation, and macular edema. NSAID (NS) eye drops, including ketorolac, bromfenac, and nepafenac, have commonly been used to treat pain and swelling of the eye before, during, and after cataract surgery. The long-term use of these eye drops that contain various amounts of preservatives, such a...

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.

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 ...

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Research

JoVE Journal

Medicine

6.1K Views.  Wayne State University School of Medicine. 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.

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

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 ...

Immunology and Infection

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

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.

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 ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

Biology

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

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/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.

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

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/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.

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