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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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.

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			<td height="21" style="height:21px;width:216px;">Zeiss Surgical Microscope</td>
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			<td style="width:119px;">Rebuilt</td>
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			<td height="21" style="height:21px;">1 mL Syringe</td>
			<td>BD</td>
			<td>305122</td>
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			<td height="21" style="height:21px;">3 mL Syringe</td>
			<td>BD</td>
			<td>309657</td>
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			<td height="21" style="height:21px;">10 mL Syringe</td>
			<td>BD</td>
			<td>309602</td>
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			<td height="21" style="height:21px;width:216px;">Vannus Scissors</td>
			<td style="width:71px;">Stortz</td>
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			<td height="21" style="height:21px;width:216px;">11-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">717939M</td>
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			<td height="21" style="height:21px;width:216px;">Trephine 2.0mm</td>
			<td style="width:71px;">Katena</td>
			<td style="width:119px;">K 2-7520</td>
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			<td height="21" style="height:21px;width:216px;">Trephine 1.5 mm</td>
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			<td height="41" style="height:41px;width:216px;">Tricaine Hydrochloride 0.5%</td>
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			<td style="width:71px;">FST</td>
			<td style="width:119px;">11251-20</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
			<td align="right" style="width:119px;">8065</td>
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			<td height="41" style="height:41px;width:216px;">2.5% Phenylephrine HCl</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">NDC 61314-342-02</td>
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			<td height="41" style="height:41px;width:216px;">1% Tropicamide</td>
			<td style="width:71px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">NDC-24208-585-59</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Hamilton Syringe</td>
			<td style="width:71px;">Hamilton</td>
			<td style="width:119px;">7654-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">33 gauge needle</td>
			<td style="width:71px;">Hamilton</td>
			<td align="right" style="width:119px;">90033</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cell Strainer (100 &#956;m nylon)</td>
			<td style="width:71px;">BD Falcon</td>
			<td align="right" style="width:119px;">352360</td>
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			<td height="41" style="height:41px;width:216px;">Hemocytometer</td>
			<td style="width:71px;">Cardinal Health</td>
			<td style="width:119px;">B3175</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8154</td>
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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

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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Orthotopen Aorten-Transplantation: Ein Rattenmodell die Entwicklung von chronischen Vaskulopathie Study

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Forschung

Medizin

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Eine Methode zur Murine Islet Isolation und subkapsulären Nierentransplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murine Cervical Herztransplantation Modell mithilfe eines modifizierten Cuff-Technik

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Transplantation von Pulmonalklappe Mit einem Maus-Modell der heterotopen Herztransplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Herzkatheter in Mäuse, um den Druck Volumen Relationship Maßnahme: Untersuchung der Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

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 ...

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

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

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

Entwicklung eines nichtinvasiven, laserunterstützten Versuchsmodells des cornealen Endotheliaalzellverlustes

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

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

Reduzierte Komplikationen nach arterieller Wiederverbindung in einem Rattenmodell der orthotopischen Lebertransplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

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 ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Intramyokardiale Transplantation von MSC-Loading injizierbaren Hydrogelen nach Myokardinfarkt in einem Murine-Modell

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...