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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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

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

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 ...

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 ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...