Name: murine corneal transplantation: a model to study the most common form of solid organ transplantation
Uploaded: 2014-11-17T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

HINWEIS: Alle Tiere in diesem Verfahren verwendet werden, in Übereinstimmung mit der Association for Research in Vision and Ophthalmology Rechnung für den Einsatz von Tieren in Ophthalmic und Vision Research sowie den von der Tieraufsichtsgremium in Saint Louis University Leitlinien behandelt. HINWEIS: Alle chirurgischen Instrumenten und Lösungen werden vor der Operation sterilisiert, um mikrobielle Infektionen des Auges zu begrenzen. Es sei darauf hingewiesen, dass, während die Tiere tun erleben einige Schmerzen von diesem Verfahren, wissen wir nicht Analgetika beschäftigen werden. Der Grund dafür ist, dass alle Analgetika sind entzündungshemmende und da die Hornhauttransplantation Reaktionen beinhalten Entzündung, wäre die Verwendung von entzündungshemmenden Medikamenten unsere Fähigkeit zu bestimmen, welche Faktoren in Hornhauttransplantatversagen beteiligt beeinträchtigen. 1. Anästhesie <ol style="margin-left: 40px;"><li> Zeigen Spender und Empfänger-Mäuse unter Vollnarkose durch IP-Injektionen von Ketamin (86.98 mg / kg) und Xylazin (13,04 mg / kg). </li><li> Halten Sie die Empfängermaus unter Narkose während des gesamten Verfahrens die in der Regel dauert 30 min zu einer Stunde. Folglich ständig die Maus auf Anzeichen von Bewusstsein wiedererlangt zu überwachen. </li><li> Bewerben Puralube Salbe für die Augen, die nicht operiert werden, nachdem das Tier narkotisiert, um Trockenheit zu verhindern. </li></ol> 2. Hornhauttransplantation <ol style="margin-left: 40px;"><li> Erhalt der Spenderhornhaut-Taste. <ol><li> Sobald das Tier vollständig betäubt wird, erreichen eine ausreichende Mydriasis durch Verabreichung von einem Paar von Augentropfen, von 1% und 2,5% Tropicamid Phenylephrin-Hydrochlorid. </li><li> Positionieren Sie den Kopf des Spendertieres horizontal auf einem Brett auf einem stabilen beweglichen Gestell montiert. Fixieren den Kopf mit einem Streifen Klebeband über den Hals, um sicherzustellen, dass das Auge in einer horizontalen Position während des gesamten Betriebs. </li><li> Mit einem Durchmesser von 2 mm Trepan, dessen Spitze wurde mit Methylenblau gefärbt, aussäumen den zentralen Hornhaut Transplantatstelle. </li><li> Mit einer scharfen Klinge, die Hornhaut zu durchdringen und zu injizieren Healon in die vordere Kammer zu vertiefen, um die Wahrscheinlichkeit einer Beschädigung des Spenders Endothels und des darunterliegenden Linsen reduzieren. </li><li> Verbrauchsteuern Das Spendertransplantat mit Vannas Schere aufnehmen und in eine Schale mit Hanks &#39;ausgewogener Salzlösung bis zur Verwendung. </li><li> Nachdem das Spendertransplantat hat die entfernt worden, einschläfern die Spendermaus über CO 2 Einatmen. </li></ol></li><li> Vorbereiten des Transplantatbett. <ol><li> Wiederholen Sie die gleichen Schritte wie in 2.1.1 bis 2.1.2 für den Empfänger beschrieben. </li><li> Mit einem Durchmesser von 1,5 mm Trepan, skizzieren den Empfänger Transplantatstelle. </li><li> Mit einer scharfen Klinge, die Hornhaut zu durchdringen und zu injizieren Healon in die vordere Kammer zu vertiefen, um die Möglichkeit einer Beschädigung der darunter liegenden Linse zu verringern. </li><li> Entfernen Sie die skizzierten zentralen Hornhaut Taste vom Empfänger mit Vannas Schere und entsorgen. </li></ol></li><li> Naht derTransplantat <ol><li> Legen Sie die Spenderhornhaut über das Transplantat in der Hornhaut des Empfängers Bett. Seien Sie sicher, dass eine angemessene Healon ist unter der Spenderhornhaut, um die Spender endothelialen Zellen vor Schäden durch direkten Kontakt mit Objektiv zu schützen. </li><li> Bei Verwendung von feiner Spitze microforceps, platzieren Sie den ersten Bissen von 11-0 Nylonnaht in die Geberseite, durch den Spender mit 90% Tiefe der gesamten Dicke der Seiten Empfängers, dann binden Sie. </li><li> Sobald die Hornhaut an Stelle verankert, führen midcardinal Nähten, so dass die Hornhaut hat 8 bis 10 insgesamt Nähte und die Spenderhornhaut fest mit gefüttert und an den Empfänger Hornhauttransplantatbett befestigt. </li></ol></li><li> Vertiefung der Vorderkammer <ol><li> Vertiefung der Vorderkammer durch Einspritzen von HBSS oder Luftblase in die Vorderkammer und sanft überprüfen die Integrität der Hornhaut-Transplantat zur Leckage mit einem Celluloseschwamm. HINWEIS: Wenn die Vorderkammer nicht reformiert werden kann, dann gibt es eine hohe Wahrscheinlichkeit von cataract die machen künftige Bewertungen der transplantierten Hornhaut sehr schwierig und wird möglicherweise auch auf Spender Hornhautendothels Dysfunktion und damit Transplantatversagen führen. </li></ol></li><li> Abschließende Bewertung <ol><li> Beobachten des Auges zu bestimmen, dass die Pupille ist rund und die Tiefe der vorderen Augenkammer ist normal. HINWEIS: Wenn die Schüler nicht rund bedeutet dies, dass während der Naht die Iris beschädigt wurde und damit das Transplantat wird als technisches Versagen. </li><li> Gelten antibiotische Salbe ins Auge. Optional: Schließen Sie das Augenlid mit einem 7-0 Seidennaht. </li><li> Mäusen zu beobachten, bis sie vollständig wach und dann einzeln beherbergen sie für mindestens zwei Tage nach der Operation. </li></ol></li></ol> 3. Nahtentfernung <ol style="margin-left: 40px;"><li> In jenen Fällen, in denen Deckel Naht verwendet wird, betäuben Mäusen, wie oben beschrieben, und entfernen Sie den Deckel Naht bei 48 h. </li><li> Anesthetize Mäusen an Tag 7 nach der Operation. Entfernen Sie die SuturES Sicherung der Hornhauttransplantat. Sobald die Nähte entfernt und das Tier voll erwacht ist, bringen Sie es in seinen Käfig. </li></ol> 4. Klinische Bewertung <ol style="margin-left: 40px;"><li> Untersuchen Sie das Auge für Hinweise auf Verfahrens Komplikationen, die zählen, Katarakt (Trübung der Linse), hyphema (Blut in der vorderen Augenkammer), eine vordere Kammer, die nicht der richtige Tiefe, oder erhebliche Trübung der Hornhaut. Betrachten Sie jene, die diese Komplikationen als &quot;gefährdet&quot; zu demonstrieren und einschläfern ihnen von CO 2 Einatmen. <ol><li> Führen Sie alle Untersuchungen an narkotisierten Mäusen. Halten Sie die Maus mit einer Hand so Zurückhalten der Maus, so dass die andere Hand das Auge proptose um eine bessere Sicht des Auges zu ermöglichen. Sobald Beobachtungen abgeschlossen sind, senden Sie das Tier in seinen Käfig. </li></ol></li><li> Haben ein Beobachter nicht mit den Behandlungsgruppen bewerten 2 bis 3 mal pro Woche transplantierten Hornhäute auf Anzeichen von HornhauttransplantatAbstoßungsreaktionen oder Hornhauttransplantatversagen. Verwenden Sie entweder das Operationsmikroskop oder eine horizontale Spaltlampen Biomikroskop für diese Beobachtungen. <ol><li> Evaluierung jedes Horn Opazität unter Verwendung einer Skala von 0 bis 5. Die Skala ist wie folgt definiert: <ol><li> Weisen Sie eine Punktzahl von 0 bis diesen Hornhäuten, die keine Anzeichen von Trübung haben. </li><li> Weisen Sie eine Punktzahl von 1 bis diesen Hornhäuten, die minimale oberflächliche Trübung zeigen. </li><li> Vergeben Sie einen Score von 2 bis Hornhäute, die mild und tiefer Opazität anzeigen, sondern die zugrunde liegende Pupille und Iris sind noch erkennbar. </li><li> Vergeben Sie einen Score von 3 bis Hornhaut, die Stromazellen Opazität anzuzeigen, wobei die Iris kann nicht im Detail mit Ausnahme der Pupillenränder zu sehen. </li><li> Weisen Sie eine Punktzahl von 4 bis Hornhaut, die dichten Stroma Opazität anzuzeigen und wenn keine zugrunde liegenden Strukturen können eingesehen werden. </li><li> Weisen Sie eine Punktzahl von 5 bis Hornhaut, die vollständige Opazität und intensive Stromaödems mit Pupille und Iris anzuzeigen völlig verdunkelt. </li></ol></li><li><html> Auch bewerten jede Hornhaut für den Grad der Blutgefäßinfiltration (Neovaskularisation) unter Verwendung einer Bewertungsskala von 1 bis 8. Um dies zu erreichen, sehen die Hornhaut als bestehend aus 4 gleiche Quadranten und legt die Höhe der Blutgefäße in jedem dieser Quadranten mit einem Partitur, die die Reichweite von 0 (keine Schiffe) bis 2 für umfangreiche Vaskularisierung diesem Quadranten. Fügen Sie die einzelnen Punkte jedem Quadranten, um die endgültige Neovaskularisation Score berechnen. </li><li> Bewerte Hornhäute als akut abgelehnt, wenn sie einen Wert von 3 für zwei aufeinander folgende Beobachtungen für Zeitpunkten bis zu 5 Wochen. </li><li> Bewerte Mäusen, deren Hornhäute waren klar in 5 Wochen, aber entwickeln Trübung zu Zeiten&gt; 45 Tage nach der Transplantation, mit einem Score von 3 für zwei aufeinander folgende Zeitpunkte, als unterzogen wurden spät-Begriff Hornhauttransplantatabstoßung. Verwenden Kaplan-Meier-Überlebenskurven zu Transplantatüberleben zu analysieren. </li></ol></li></ol> 5. Manipulation des Modells in-left: 40px; &quot;&gt; <li> Herstellung einzelner Zellen aus der Milz. <ol><li> Um einzelne Zellen herzustellen, zunächst einschläfern die Spendermaus. Dann entfernen Sie die Milz. </li><li> Legen Sie die Milz in einer Zelle Sieb und stören sie mit einem Spritzenkolben aus einem 3-ml-Spritze. </li><li> Waschen Zellen und Resuspendieren in 10 ml Hanks Balanced Salt Solution. </li><li> Entfernen Sie 10 ul Zellsuspension und fügen 10 ul 0,4% Trypanblau und mischen. Hinzufügen, dass Hämozytometers und zählen Sie die Zellen in das zentrale Netz. Die Anzahl der Zellen in Rohr ist die Zellzahl x 10 4 x 2 (Verdünnungsfaktor in Trypanblau) x 10 (Volumen im Röhrchen). </li></ol></li><li> Injektion in die vordere Kammer. <ol><li> Betäuben Mäusen, wie zuvor beschrieben. </li><li> Führen Injektionen mit einem Binokular. Für jede intrakamerale Injektion, verwenden 10 6 Milzzellen in einem 0.005 ml Volumen und eine 0,25 ml Mikroliterspritze mit einer 33 G-Nadel ausgestattet. HINWEIS: Andere Manipulationendes Modells kann durch die Behandlung von Tieren mit Reagenzien, die entweder als Antagonisten oder Agonisten, die Rolle, die einen bestimmten Faktor kann folgende orthotope Hornhauttransplantatchirurgie spielen Bestimmung handeln geführt werden. </li></ol></li></ol>

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	<li>Farooq, A. V., Shukla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+epithelial+and+stromal+keratitis:+an+epidemiologic+update.">Herpes simplex epithelial and stromal keratitis: an epidemiologic update.</a> Surv. Ophthalmol. , 448-462 (2012).</li><li>Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+and+diseases+of+the+ocular+surface+and.">Age-related changes and diseases of the ocular surface and.</a> Invest. Opthlamol. Vis. Sci. 54, 48-53 (2013).</li><li>Edwards, 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=Indications+for+corneal+transplantation+in+New+Zealand:+1991-1999.">Indications for corneal transplantation in New Zealand: 1991-1999.</a> Cornea. 21, 152-155 (2002).</li><li>Thompson, R. W., Price, M. O., Bowers, P. J., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+survival+after+penetrating+keratoplasty.">Long-term survival after penetrating keratoplasty.</a> Ophthalmol. 110, 1396-1402 (2003).</li><li>Williams, K. A., Roder, D., Esterman, A., Muehlberg, S. M., Coster, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+predictive+of+corneal+graft+survival.+Report+form+the+Australian+Corneal+Graft+Registry.">Factors predictive of corneal graft survival. Report form the Australian Corneal Graft Registry.</a> Ophthalmology. 99, 403-414 (1992).</li><li>Larkin, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+allograft+rejection.">Corneal allograft rejection.</a> Br. J. Ophthalmol. 78, 649-652 (1994).</li><li>Boisjoly, H. 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=Risk+factors+of+corneal+graft+failure.">Risk factors of corneal graft failure.</a> Ophthalmol. 100, 1728-1735 (1993).</li><li>Sugar, A., 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=Recipient+Risk+Factors+for+Graft+Failure+in+the+Cornea+Donor+Study.">Recipient Risk Factors for Graft Failure in the Cornea Donor Study.</a> Ophthalmol. 116, 1023-1028 (2009).</li><li>Namba, K., Kitaichi, N., Nishida, T., Taylor, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+regulatory+T+cells+by+the+immunomodulating+cytokines+alpha-melanocyte-stimulating+hormone+and+transforming+growth+factor-beta2.">Induction of regulatory T cells by the immunomodulating cytokines alpha-melanocyte-stimulating hormone and transforming growth factor-beta2.</a> J. Leukoc. Biol. 72, 946-952 (2002).</li><li>Taylor, A. W., Yee, D. G., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+nitric+oxide+generated+by+inflammatory+macrophages+by+calcitonin+gene-related+peptide+in+aqueous+humor.">Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor.</a> Invest. Ophthalmol. Vis. Sci. 39, 1372-1378 (1998).</li><li>Wilbanks, G. A., Mammolenti, M., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+induction+of+anterior+chamber-associated+immune+deviation+(ACAID).+III.+Induction+of+ACAID+depends+upon+intraocular+transforming+growth+factor-beta.">Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-beta.</a> Eur. J. Immunol. 22, 165-173 (1992).</li><li>Volpert, O. 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=Inducer-stimulated+Fas+targets+activated+endothelium+for+destruction+by+anti-angiogenic+thrombospondin-1+and+pigment+epithelium-derived+factor.">Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor.</a> Nat. Med. , 8-349 (2002).</li><li>Apte, R. S., Sinha, D., Mayhew, E., Wistow, G. J., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+role+of+macrophage+migration+inhibitory+factor+in+inhibiting+NK+cell+activity+and+preserving+immune+privilege.">Cutting edge: role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege.</a> J. Immunol. 160, 5693-5696 (1998).</li><li>Kennedy, M. 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=Novel+production+of+interleukin-1+receptor+antagonist+peptides+in+normal+human+cornea.">Novel production of interleukin-1 receptor antagonist peptides in normal human cornea.</a> J. Clin. Invest. 95, 82-88 (1995).</li><li>Shimmura-Tomita, M., Wang, M., Taniguchi, H., Akiba, H., Yagita, H., Hori, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-9-mediated+protection+from+allo-specific+T+cells+as+a+mechanism+of+immune+privilege+of+corneal+allografts.">Galectin-9-mediated protection from allo-specific T cells as a mechanism of immune privilege of corneal allografts.</a> PLoS One. 8, (2013).</li><li>Goldberg, M. F., Ferguson, T. A., Pepose, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cellular+adhesion+molecules+in+inflamed+human+corneas.">Detection of cellular adhesion molecules in inflamed human corneas.</a> Ophthalmol. 101, 161-168 (1994).</li><li>Stuart, P. M., Griffith, T. S., Usui, N., Pepose, J. S., Yu, X., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD95+ligand+(FasL)-induced+apoptosis+is+necessary+for+corneal+allograft+survival.">CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival.</a> J Clin Invest. 99, 396-402 (1997).</li><li>Yamagami, 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=Role+of+Fas-Fas+ligand+interactions+in+the+immunorejection+of+allogeneic+mouse+corneal+transplants.">Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplants.</a> Transplantation. 64, 1107-1111 (1997).</li><li>Stuart, P. M., Pan, F., Plambeck, S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fas/Fas+ligand+interactions+regulate+neovascularization+in+the+cornea.">Fas/Fas ligand interactions regulate neovascularization in the cornea.</a> Invest. Ophthalmmol. Vis. Sci. 44, 93-98 (2003).</li><li>Stuart, P. M., Yin, X. T., Pan, F., Haskova, Z., Plambeck, S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+matrix+metalloproteinases+activity+prolong+corneal+allograft+acceptance+by+increasing+FasL+expression.">Inhibitors of matrix metalloproteinases activity prolong corneal allograft acceptance by increasing FasL expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1169-1173 (2004).</li><li>Joo, C. -. K., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+mediated+responses+in+a+murine+model+of+orthotopic+corneal+transplantation.">T-cell mediated responses in a murine model of orthotopic corneal transplantation.</a> Invest.Ophthalmol. Vis. Sci. 36, 1530-1540 (1995).</li><li>Sonoda, Y., Sano, Y., Ksander, B., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cell-mediated+immune+responses+elicited+by+orthotopic+corneal+allografts+in+mice.">Characterization of cell-mediated immune responses elicited by orthotopic corneal allografts in mice.</a> Invest. Ophthalmol. Vis. Sci. 36, 427-434 (1995).</li><li>Sano, Y., Osawa, H., Sotozono, C., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+expression+during+orthotopiccorneal+allograft+rejection+in+mice.">Cytokine expression during orthotopiccorneal allograft rejection in mice.</a> Invest. Ophthalmol. Vis. Sci. 39, 1953-1957 (1998).</li><li>Haskova, Z., Usui, N., Ferguson, T. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+cells+are+critical+in+corneal+but+not+skin+allograft+rejection.">CD4+ T cells are critical in corneal but not skin allograft rejection.</a> Transplantation. 69, 483-488 (2000).</li><li>Tan, Y., 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=Immunological+disruption+of+antiangiogenic+signals+by+recruited+allospecific+T+cells+leads+to+corneal+allograft+rejection.">Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.</a> J. Immunol. 188, 5962-5969 (2012).</li><li>Dana, M. R., Yamada, J., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+interleukin-1+receptor+antagonist+promotes+corneal+transplant+survival.">Topical interleukin-1 receptor antagonist promotes corneal transplant survival.</a> Transplantation. 63, 1501-1507 (1997).</li><li>Cunnusamy, K., Chen, P. W., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17A-dependent+CD4+CD25++regulatory+T+cells+promote+immune+privilege+of+corneal+allografts.">IL-17A-dependent CD4+CD25+ regulatory T cells promote immune privilege of corneal allografts.</a> J. Immunol. 186, 6737-6745 (2011).</li><li>Fu, H., 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=Arginine+depletion+as+a+mechanism+for+the+immune+privilege+of+corneal+allografts.">Arginine depletion as a mechanism for the immune privilege of corneal allografts.</a> Eur. J. Immunol. 41, 2997-3005 (2011).</li><li>Medina, C. A., Rowe, A. M., Yun, H., Knickelbein, J. E., Lathrop, K. L., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azithromycin+treatment+increases+survival+of+high-risk+corneal+allotransplants.Cornea.">Azithromycin treatment increases survival of high-risk corneal allotransplants.Cornea.</a> , 32-658 (2013).</li><li>Cho, Y. K., Zhang, X., Uehara, H., Young, J. R., Archer, B., Ambati, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Endothelial+Growth+Factor+Receptor+1+morpholino+increases+graft+survival+in+a+murine+penetrating+keratoplasty.">Vascular Endothelial Growth Factor Receptor 1 morpholino increases graft survival in a murine penetrating keratoplasty.</a> Invest. Ophthalmol. Vis. Sci. 53, 8458-8471 (2012).</li><li>Kim, H. K., Choi, J. A., Uehara, H., Zhang, X., Ambati, B. K., Cho, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presurgical+corticosteroid+treatment+improves+corneal+transplant+survival+in+mice.">Presurgical corticosteroid treatment improves corneal transplant survival in mice.</a> Cornea. 32, 1591-1598 (2013).</li><li>Yamazoe, K., Yamazoe, K., Shimazaki-Den, S., Shimazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+for+corneal+graft+recovery+after+severe+corneal+graft+rejection+following+penetrating+keratoplasty.">Prognostic factors for corneal graft recovery after severe corneal graft rejection following penetrating keratoplasty.</a> BMC Ophthalmol. 13, 5 (2013).</li><li>Panda, A., Vanathi, M., Kumar, A., Dash, Y., Priya, 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+graft+rejection.">Corneal graft rejection.</a> Surv. Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+keratoplasty:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

The authors have no competing financial interests.

Das Mausmodell der hier beschriebenen Hornhauttransplantation ermöglicht die Ermittler für die menschliche Hornhauttransplantatabstoßung in einem Modell, das Vorhersage, welche Faktoren am besten mit beiden Ablehnung 15,17,18,20, 26-30 und Akzeptanz 21-25 von Hornhaut assoziiert ist studieren Allografts. Im Gegensatz zu menschlichen Hornhauttransplantation, in denen die Patienten entweder topische oder systemische Behandlung mit Steroiden, entweder zu behandeln oder zu verhindern Ablehnung ...

Hornhauttransplantation ist eine der erfolgreichsten und häufigsten Arten der Transplantation in Menschen durchgeführt. Die Gründe, warum diese Operation wird durchgeführt, sind das Ergebnis einer Verletzung, Infektionskrankheit, 1, oder andere Formen von nicht-infektiösen Erkrankung der Hornhaut 2. Zahlen aus dem Eye Bank Association of America zeigen, dass mehr als 46.000 wurden im Jahr 2011 durchgeführt (siehe Website unter: restoresight.org/eye_banks/eye_banks.html). Ein Hinweis auf seinen Erfolg ist, dass ein Jahr Ausfallraten für allogene Hornhauttransplantate reichen von 10 bis 15% und nach 5 Jahren ist der Erfolg von über 70% 3-8. Wie viele Studien gezeigt haben, wird der Erfolg der Hornhauttransplantaten direkt auf der Tatsache, daß das Auge eine immunologisch privilegierte Stelle stehen. Faktoren für die Hornhäute Status als Immunprivileg Website verantwortlich sind das Fehlen beider Blut- und Lymphgefäße in der Hornhaut, eine relative Abwesenheit von Antigen-präsentierenden Zellen, Faktoren, die von der Hornhaut, die Entstörung produzierts Immuneffektorzelle funtions 9-15, niedrige Expression von MHC-Antigenen 16, und die Expression von FasL 17-20. Trotz dieser Faktoren prädisponierenden diese Transplantate für den Erfolg, sie durchlaufen die Ablehnung 3-7. Folglich Verständnis dieser Mechanismen, die diese Ablehnung zu vermitteln sowie das Testen verschiedener Therapien, um eine Abstoßung zu verhindern, ist von entscheidender Bedeutung. Zu diesem Zweck beschreiben wir hier ein murines Modell der Hornhauttransplantation, die seit mehr als 20 Jahren wurde die Hornhauttransplantation, in einer kontrollierten experimentellen Umgebung zu studieren. Da Transplantationsreaktionen beinhalten viele verschiedene Faktoren die im Konzert, die ultimative bestimmen, ob das transplantierte Gewebe ausfällt oder erfolgreich ist, ist es nicht möglich, die Wichtigkeit dieser Faktoren in jedem in vitro Modell zu verstehen. Infolgedessen werden Studien mit intakten Tieren erforderlich, um festzustellen, welche Faktoren wichtig, entweder Erfolg oder failu sindre des transplantierten Gewebes. Während andere Tierarten wurden verwendet, um die Hornhauttransplantation zu untersuchen, wurde die Maus-Modell im Vergleich zu der Verwendung anderer Arten mehrere Vorteile. Die erste ist die Existenz vieler Stämme von Mäusen, die bestimmte Transgene exprimieren oder wurden Gen-Targeting, um die Expression von spezifischen immunologischen Faktoren, deren Funktion in der Transplantations besser untersucht werden fehlt. Darüber hinaus gibt es viele Reagenzien (sowohl rekombinante Faktoren und Antikörper, die Faktoren zu neutralisieren), die spezifisch für Mäuse sind, und die nicht für viele andere Arten von Tieren vorhanden sind. Aufgrund der Existenz dieser Faktoren ist dieses Modell wurde ausgiebig genutzt, um relevante Faktoren bei akuten Hornhauttransplantatreaktionen 15, 17,18,20 -29 beteiligt sind. Außerdem sind viele der Faktoren in die Hornhauttransplantation beteiligt sind auch bekannt Funktions in der Transplantation von anderen Geweben zu sein.

<table><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr></tbody></table>

murine corneal transplantation: a model to study the most common form of solid organ transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Das Mausmodell der Hornhauttransplantation ist seit über 20 Jahren verwendet worden, um erfolgreich zu charakterisieren Mechanismen sowohl Hornhauttransplantatabstoßung, 19-23 und Hornhauttransplantatannahme 13, 15,16,18, 24-27. Dieses Modell wurde verwendet, um die Bedeutung des FasL-Expression in Hornhauttransplantatakzeptanz zu schaffen, dass Tiere, die FasL fehlen nicht in der Lage, Hornhauttransplantate 15 zu akzeptieren. Es wurde auch verwendet, um zu zeigen, dass vaskuläre endo...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Murine Hornhauttransplantation: Ein Modell, das die häufigste Form der Organtransplantation Studieren

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

murine-corneal-transplantation-model-to-study-most-common-form-solid

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.6K Aufrufe.  Saint Louis University. Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Serie: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Maus Nierentransplantation: Modelle der Transplantatabstoßung

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Forschung

Medizin

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Murine Nierentransplantations Technik

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine Vollhauttransplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Immunologie und Infektion

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

A Novel Mikromodell für Heterotope, En Bloc Brustwand, Thymus und Herz-Transplantation bei Mäusen

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Orthotopische Hind Gliedmaßen Transplantation in der Maus

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Heterotope Renal Autotransplantation in einem Schweine-Modell: Eine Schritt-für-Schritt-Protokoll

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

Ein modifiziertes heterotopes Herztransplantationsprotokoll der Maus, das den heutigen Standards der aseptischen Technik, Anästhesie und Analgesie entspricht

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Ein Porcine Corneal Endothelial Organ Culture Modell mit Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

Orthotopische Nieren-Autotransplantation in einem Schweinemodell mit 24 Stunden Organkonservierung und kontinuierlicher Telemetrie

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.