<meta charset="utf8"></head><body>Ex-vivo-Hornhautkultursystem für Schweine für Toxizitätstests und Wundheilung

<ol>
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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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid+promoting+corneal+epithelial+wound+healing+by+transactivation+of+epidermal+growth+factor+receptor.">Lysophosphatidic acid promoting corneal epithelial wound healing by transactivation of epidermal growth factor receptor.</a> Invest Ophthalmol Vis Sci. 48 (2), 636-643 (2007).</li><li>Yan, 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=Targeting+Imbalance+between+IL-1beta+and+IL-1+receptor+antagonist+ameliorates+delayed+epithelium+wound+healing+in+diabetic+mouse+corneas.">Targeting Imbalance between IL-1beta and IL-1 receptor antagonist ameliorates delayed epithelium wound healing in diabetic mouse corneas.</a> Am J Pathol. 186 (6), 1466-1480 (2016).</li><li>Gao, N., 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=Lack+of+elevated+expression+of+TGFbeta3+contributes+to+the+delay+of+epithelial+wound+healing+in+diabetic+corneas.">Lack of elevated expression of TGFbeta3 contributes to the delay of epithelial wound healing in diabetic corneas.</a> Invest Ophthalmol Vis Sci. 65 (3), 35 (2024).</li><li>Richardson, K. C., Jarett, L., Finke, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embedding+in+epoxy+resins+for+ultrathin+sectioning+in+electron+microscopy.">Embedding in epoxy resins for ultrathin sectioning in electron microscopy.</a> Stain Technol. 35, 313-323 (1960).</li><li>Xu, K., McDermott, M., Villanueva, L., Schiffman, R. M., Hollander, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+corneal+epithelial+wound+healing+following+exposure+to+ophthalmic+nonsteroidal+anti-inflammatory+drugs.">Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs.</a> Clinical Ophthalmol. 5, 269-274 (2011).</li><li>Goldstein, M. H., Silva, F. Q., Blender, N., Tran, T., Vantipalli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+benzalkonium+chloride+exposure:+problems+and+solutions.">Ocular benzalkonium chloride exposure: problems and solutions.</a> Eye (Lond). 36 (2), 361-368 (2022).</li><li>Xu, K., 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=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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LL-37+via+EGFR+transactivation+to+promote+high+glucose-attenuated+epithelial+wound+healing+in+organ-cultured+corneas.">LL-37 via EGFR transactivation to promote high glucose-attenuated epithelial wound healing in organ-cultured corneas.</a> Invest Ophthalmol Vis Sci. 51 (4), 1891-1897 (2010).</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>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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supporting+discovery+and+development+of+medical+countermeasures+for+chemical+injury+to+eye+and+skin.">Supporting discovery and development of medical countermeasures for chemical injury to eye and skin.</a> Exp Eye Res. 221, 109156 (2022).</li><li>Araj, H., Tumminia, S. J., Yeung, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+surface:+Merging+challenges+and+opportunities.">Ocular surface: Merging challenges and opportunities.</a> Transl Vis Sci Technol. 9 (12), 3 (2020).</li><li>Lee, M., Hwang, J. H., Lim, K. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+corneal+epithelial+wound+healing+following+exposure+to+ophthalmic+nonsteroidal+anti-inflammatory+drugs.">Ex vivo corneal epithelial wound healing following exposure to ophthalmic nonsteroidal anti-inflammatory drugs.</a> Clin Ophthalmol. 5, 269-274 (2011).</li><li>Bettahi, I., 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=Genome-wide+transcriptional+analysis+of+differentially+expressed+genes+in+diabetic,+healing+corneal+epithelial+cells:+Hyperglycemia-suppressed+TGFbeta3+expression+contributes+to+the+delay+of+epithelial+wound+healing+in+diabetic+corneas.">Genome-wide transcriptional analysis of differentially expressed genes in diabetic, healing corneal epithelial cells: Hyperglycemia-suppressed TGFbeta3 expression contributes to the delay of epithelial wound healing in diabetic corneas.</a> Diabetes. 63 (2), 715-727 (2014).</li><li>Yeung, D. T., Araj, H., Harper, J. R., Platoff, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+in+developing+medical+countermeasures+against+chemical+ocular+toxicity.">Considerations in developing medical countermeasures against chemical ocular toxicity.</a> Toxicol Lett. 334, 1-3 (2020).</li></ol>

<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

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.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Erforschung der angeborenen Immunität der Hornhaut und der verzögerten Wundheilung bei Diabetikern

Bewertung der therapeutischen und chemischen Toxizität anhand von organgezüchteten Schweinehornhäuten und der Heilung von Epithelwunden

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

evaluating-therapeutic-chemical-toxicity-using-organ-cultured-porcine

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Research

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

JoVE Journal

Medicine

4.4K Aufrufe.  Wayne State University School of Medicine. Wir untersuchen die angeborene Immunität der Hornhaut und die diabetische Wundheilung. Insbesondere wollen wir verstehen, warum Diabetiker anfälliger für bakterielle Infektionen sind und was eine verzögerte Wundheilung im Zusammenhang mit Hypoglykämie verursacht. Wir haben festgestellt, dass das Gleichgewicht zwischen IL-1 beta und IL-1-Rezeptorantagonisten sowie TGF Beta-1 und TGF Beta-3 für die Wundheilung der Hornhaut wichtig ist.Dies könnte ...

Serie: Autor im Rampenlicht: Erforschung der angeborenen Immunität der Hornhaut und der verzögerten Wundheilung bei Diabetikern

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.

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.