Name: establishing a porcine ex vivo cornea model for studying drug treatments against bacterial keratitis
Uploaded: 2020-05-12T13:30:00
Description: Watch this Scientific Journal Video about Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis at JoVE.com

Forfatterne vil gerne takke Elliot Abattoir i Chesterfield for at give svin øjne. Glasringene er lavet på baggrund af vores design af glasblæseren Dan Jackson fra Institut for Kemi ved University of Sheffield. Forfatterne vil gerne takke Medical Research Council (MR/S004688/1) for finansieringen. Forfatterne vil også gerne takke fru Shanali Dikwella for teknisk hjælp med forberedelse af hornhinden. Forfatterne vil gerne takke Jonathan Emery for hjælp til formatering af billeder.

Albino laboratoriekaniner blev ofret i laboratoriet for andet planlagt eksperimentelt arbejde under hjemmekontor godkendte protokoller. Øjnene var ikke nødvendige for eksperimentel brug i disse undersøgelser, så de blev brugt til denne protokol.
1. Sterilisering
<ol><li>KRITISK TRIN: Desinficer alle pincet og saks ved iblødsætning i 1 time i 5% (v/v) opløsning af Distel i destilleret vand, rengør med en børste, skyl med vand fra hanen og steriliser i en ovn ved 185 °C i mindst 2 ti...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-Resistant+Pseudomonas+aeruginosa+Keratitis+Risk+Factors,+Clinical+Characteristics,+and+Outcomes.">Multidrug-Resistant Pseudomonas aeruginosa Keratitis Risk Factors, Clinical Characteristics, and Outcomes.</a> Ophthalmology. 122 (10), 2110-2114 (2015).</li><li>Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratitis.">Keratitis.</a> Bioscience Reports. 21 (4), 419-444 (2001).</li><li>Sharma, G., 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=Pseudomonas+aeruginosa+biofilm:+Potential+therapeutic+targets.">Pseudomonas aeruginosa biofilm: Potential therapeutic targets.</a> Biologicals. 42 (1), 1-7 (2014).</li><li>Ersoy, S. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+Characterization+of+an+Air-Liquid+Canine+Corneal+Organ+Culture+Model+To+Study+Acute+Herpes+Keratitis.">Establishment and Characterization of an Air-Liquid Canine Corneal Organ Culture Model To Study Acute Herpes Keratitis.</a> Journal of Virology. 88 (23), 13669-13677 (2014).</li><li>Madhu, S. N., Jha, K. K., Karthyayani, A. P., Gajjar, D. U. <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+Caprine+Model+to+Study+Virulence+Factors+in+Keratitis.">Ex vivo Caprine Model to Study Virulence Factors in Keratitis.</a> Journal of Ophthalmic &amp; Vision Research. 13 (4), 383-391 (2018).</li><li>Vermeltfoort, P. B. J., van Kooten, T. G., Bruinsma, G. M., Hooymans, A. M. M., vander Mei, H. C., Busscher, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+transmission+from+contact+lenses+to+porcine+corneas:+An+ex+vivo+study.">Bacterial transmission from contact lenses to porcine corneas: An ex vivo study.</a> Investigative Ophthalmology &amp; Visual Science. 46 (6), 2042-2046 (2005).</li><li>Duggal, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+oxide+tetrapods+inhibit+herpes+simplex+virus+infection+of+cultured+corneas.">Zinc oxide tetrapods inhibit herpes simplex virus infection of cultured corneas.</a> Molecular Vision. 23, 26-38 (2017).</li><li>Brothers, K., 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=Bacterial+Impediment+of+Corneal+Cell+Migration.">Bacterial Impediment of Corneal Cell Migration.</a> Investigative Ophthalmology &amp; Visual Science. 56 (7), (2015).</li><li>Alekseev, O., Tran, A. H., Azizkhan-Clifford, J. <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+Organotypic+Corneal+Model+of+Acute+Epithelial+Herpes+Simplex+Virus+Type+I+Infection.">Ex vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection.</a> Journal of Visualized Experiments. (69), (2012).</li><li>Sack, R. A., Nunes, I., Beaton, A., Morris, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-Defense+Mechanism+of+the+Ocular+Surfaces.">Host-Defense Mechanism of the Ocular Surfaces.</a> Bioscience Reports. 21 (4), 463-480 (2001).</li><li>Kunzmann, B. 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Den vigtigste drivkraft bag udviklingen af denne keratitis model ved hjælp af ex vivo svin hornhinden er at give forskere, der udvikler nye antimikrobielle stoffer med en repræsentativ in vitro model til mere præcist at bestemme antimikrobiel effekt på prækliniske stadier. Dette vil give forskere, der er involveret i udviklingen af nye antimikrobielle stoffer, større kontrol over lægemiddeldesign og -formulering på de prækliniske stadier, øge succesen ved kliniske forsøg, reducere brugen af dyr ved at muliggø...

Hornhindeinfektioner er vigtige årsager til blindhed og forekommer i epidemiske proportioner i lav- og mellemindkomstlande. Sygdommens ætiologi varierer fra region til region, men bakterier tegner sig for et stort flertal af disse tilfælde. Pseudomonas aeruginosa er et vigtigt patogen, der forårsager en hurtigt fremadskridende sygdom. I mange tilfælde er patienterne tilbage med stromal ardannelse, uregelmæssig anstiftelse, kræver transplantation eller i værste fald mister et øje1,2.
Bakteriel keratitis forårsaget af P. aeruginosa er en vanskelig øjeninfektion at behandle, især på grund af den stigende fremkomst af antimikrobielle resistente stammer af P. aeruginosa. Inden for det sidste årti er det blevet klart, at testning og udvikling af nye behandlinger for hornhindeinfektioner generelt og dem, der er forårsaget af Pseudomonas sp., i særdeleshed, er afgørende for at bekæmpe den nuværende tendens i antibiotikaresistens3.
Til test af effekten af nye behandlinger for hornhindeinfektioner er konventionelle in vitro mikrobiologiske metoder et dårligt surrogat på grund af forskellen i bakteriel fysiologi under laboratoriekultur og under infektioner in vivo samt på grund af manglen på værtsgrænsefladen4,5. In vivo-dyremodeller er imidlertid dyre, tidskrævende, kan kun levere et lille antal replikater og give anledning til bekymring for dyrevelfærden.
I denne artikel demonstrerer vi en enkel og reproducerbar organotypisk ex vivo porcine model af keratitis, der kan bruges til at teste forskellige behandlinger for akutte og kroniske infektioner. Vi har brugt P. aeruginosa til dette eksperiment, men modellen fungerer også godt sammen med andre bakterier og organismer som svampe og gær, der forårsager keratitis.

<table>
	<tbody>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>SLS</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellstar 12 well plate</td>
			<td>Greiner Bio-One</td>
			<td>665180</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma</td>
			<td>31425-100mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Distel</td>
			<td>Fisher Scientific</td>
			<td>12899357</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM + glutamax</td>
			<td>SLS</td>
			<td>D0819</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual Oven Incubator</td>
			<td>SLS</td>
			<td>OVe1020</td>
			<td>Sterilising oven</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>SLS</td>
			<td>E5036-200UG</td>
			<td></td>
		</tr>
		<tr>
			<td>F12 HAM</td>
			<td>Sigma</td>
			<td>N4888</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal calf serum</td>
			<td>Labtech International</td>
			<td>CA-115/500</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>15307805</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld homogeniser 220</td>
			<td>Fisher Scientific</td>
			<td>15575809</td>
			<td>Homogeniser</td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i</td>
			<td>Thermo Scientific</td>
			<td>15373212</td>
			<td>Tissue culture incubator</td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R</td>
			<td>VWR</td>
			<td>521-2242</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>Insulin, recombinant Human</td>
			<td>SLS</td>
			<td>91077C-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Sigma</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>Multitron</td>
			<td>Infors</td>
			<td>Not appplicable</td>
			<td>Bacterial incubator</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>SLS</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SLS</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>12664785</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 35x10mm CytoOne</td>
			<td>Starlab</td>
			<td>CC7672-3340</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td>Weldricks pharmacy</td>
			<td>2122828</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe 2020</td>
			<td>Fisher Scientific</td>
			<td>1284804</td>
			<td>Class II microbiology safety cabinet</td>
		</tr>
		<tr>
			<td>Scalpel blade number 15</td>
			<td>Fisher Scientific</td>
			<td>O305</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Swann Morton</td>
			<td>Fisher Scientific</td>
			<td>11849002</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing a porcine ex vivo cornea model for studying drug treatments against bacterial keratitis

Ved udvikling af nye antimikrobielle stoffer afhænger dyreforsøgenes succes af nøjagtig ekstrapolering af antimikrobiel effekt fra in vitro-test til dyreinfektioner in vivo. De eksisterende in vitro-test overvurderer typisk antimikrobiel effekt, da der ikke tages højde for tilstedeværelsen af værtsvæv som en diffusionsbarriere. For at overvinde denne flaskehals, har vi udviklet en ex vivo svin hornhinde model af bakteriel keratitis ved hjælp af Pseudomonas aeruginosa som en prototypisk organisme. Denne artikel beskriver forberedelsen af svin hornhinden og protokollen for etablering af infektionen. Skræddersyede glasforme muliggør ligetil opsætning af hornhinden til infektionsundersøgelser. Modellen efterligner in vivo infektion som bakteriel spredning er afhængig af bakteriens evne til at beskadige hornhindevæv. Infektionssted verificeres som en stigning i antallet af kolonidannende enheder, der vurderes ved hjælp af levedygtige pladetal. Resultaterne viser, at infektion kan fastslås på en meget reproducerbar måde i ex vivo corneas ved hjælp af den her beskrevne metode. Modellen kan udvides i fremtiden for at efterligne keratitis forårsaget af andre mikroorganismer end P. aeruginosa. Det endelige mål med modellen er at undersøge effekten af antimikrobiel kemoterapi på udviklingen af bakteriel infektion i et scenario, der er mere repræsentativt for in vivo-infektioner. Dermed vil den model, der er beskrevet her, reducere brugen af dyr til test, forbedre succesraten i kliniske forsøg og i sidste ende muliggøre hurtig oversættelse af nye antimikrobielle stoffer til klinikken.

Udformningen af glasforme er en innovativ og original idé, hvis brug gjorde det muligt for os at oprette modellen på en konsistent måde med minimal / ingen problemer med forurening. Formene blev udarbejdet af en glasblæser ved University of Sheffield baseret på et design (Figur 1A). Den eksperimentelle opsætning opretholder hornhindens konvekse form og holder bakterier på toppen af epitelet, hvor infektion finder sted (Figur 1B).
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Watch this Scientific Journal Video about Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis at JoVE.com

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

I denne artikel beskrives en trinvis protokol til opsætning af en ex vivo-svinemodel af bakteriel keratitis. Pseudomonas aeruginosa bruges som en prototypisk organisme. Denne innovative model efterligner in vivo infektion som bakteriel spredning er afhængig af bakteriens evne til at beskadige hornhindevæv.

Etablering af en Porcine Ex Vivo Hornhinde Model for at studere medicinske behandlinger mod bakteriel Keratitis

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

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