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.

NOTA: Tutti gli animali utilizzati in questa procedura sono trattati in conformità con l&#39;Associazione per la Ricerca e la Visione e la dichiarazione di Oftalmologia per l&#39;uso di animali in Oftalmica e Vision Research, nonché le linee guida stabilite dalla commissione di sorveglianza degli animali a Saint Louis University. NOTA: Tutti gli strumenti chirurgici e le soluzioni sono sterilizzate prima di un intervento chirurgico per limitare l&#39;infezione microbica degli occhi. Va notato che, mentre gli animali fanno vivere il dolore da questa procedura, non impieghiamo analgesici. La ragione di questo è perché tutti gli analgesici sono anti-infiammatori e da risposte trapianto di cornea coinvolgono l&#39;infiammazione, l&#39;uso di farmaci anti-infiammatori potrebbero compromettere la nostra capacità di determinare quali fattori sono coinvolti nel fallimento del trapianto di cornea. 1. Anestesia <ol style="margin-left: 40px;"><li> Mettere i topi donatori e beneficiari in anestesia generale con iniezioni IP di ketamina (86.98 mg / kg) e xilazina (13,04 mg / kg). </li><li> Tenere il mouse recipiente sotto anestesia durante tutta la procedura che richiede in genere 30 minuti a un ora. Di conseguenza, monitorare costantemente il mouse per eventuali segni di riprendere conoscenza. </li><li> Applicare puralube pomata per l&#39;occhio che non sottoporsi ad intervento chirurgico dopo che l&#39;animale è anestetizzato per prevenire la secchezza. </li></ol> 2. innesto corneale <ol style="margin-left: 40px;"><li> Ottenere il bottone corneale del donatore. <ol><li> Una volta che l&#39;animale è completamente anestetizzato, conseguire un&#39;adeguata midriasi mediante somministrazione di un paio di gocce oculari di 1% e 2,5% tropicamide fenilefrina cloridrato. </li><li> Posizionare la testa dell&#39;animale donatore orizzontalmente su una tavola posta su un robusto supporto mobile. Fissare la testa con una striscia di nastro sul collo per assicurare che l&#39;occhio è in posizione orizzontale durante l&#39;intera operazione. </li><li> Utilizzando un trapano 2 mm di diametro, la cui punta è stato tinto con blu metile, fuorilinea il sito del trapianto della cornea centrale. </li><li> Con una lama affilata, penetrare nella cornea e Healon iniettare nella camera anteriore di approfondire per ridurre la possibilità di danni all&#39;endotelio donatore e lente sottostante. </li><li> Accise l&#39;innesto del donatore con Vännäs forbici e riporre in un piatto contenente soluzione salina bilanciata Hanks &#39;fino al momento dell&#39;uso. </li><li> Dopo l&#39;innesto del donatore è stato il rimosso, eutanasia il mouse donatore tramite CO 2 inalazione. </li></ol></li><li> Preparare il letto trapianto. <ol><li> Ripetere la stessa procedura descritta al punto 2.1.1 tramite 2.1.2 per il destinatario. </li><li> L&#39;utilizzo di un trapano di diametro 1,5 mm delineare il sito ricevente del trapianto. </li><li> Con una lama affilata, penetrare nella cornea e Healon iniettare nella camera anteriore di approfondire per ridurre la possibilità di danni alla lente sottostante. </li><li> Rimuovere il pulsante corneale centrale delineato dal destinatario utilizzando Vännäs forbici e scartare. </li></ol></li><li> Sutura deltrapianto <ol><li> Posizionare la cornea del donatore nel trapianto letto nella cornea del ricevente. Assicurarsi che un&#39;adeguata Healon è sotto la cornea del donatore per proteggere le cellule endoteliali del donatore venga danneggiato da contatto diretto con lente. </li><li> Utilizzando Super Fine microforceps a punta, posizionare il primo morso di 11-0 in nylon sutura nel lato dei donatori, attraverso il donatore con il 90% della profondità del tutto spessore a lato del destinatario, quindi legare. </li><li> Una volta che la cornea è ancorato al suo posto, eseguire punti staccati midcardinal tale che la cornea ha da 8 a 10 punti di sutura totale e la cornea del donatore è rivestito in modo sicuro con e allegato al destinatario letto trapianto di cornea. </li></ol></li><li> Approfondire la camera anteriore <ol><li> Approfondire la camera anteriore iniettando HBSS o bolla d&#39;aria nella camera anteriore e controllare delicatamente l&#39;integrità del trapianto di cornea per perdite con una spugna di cellulosa. NOTA: Se la camera anteriore non può essere riformato allora c&#39;è un&#39;alta probabilità di cataract che renderà le future valutazioni della cornea trapiantata molto difficile e anche potenzialmente portare ad una disfunzione dell&#39;endotelio corneale del donatore e quindi il fallimento del trapianto. </li></ol></li><li> Valutazione finale <ol><li> Osservare l&#39;occhio per determinare che la pupilla è rotonda e la profondità della camera anteriore è normale. NOTA: Se l&#39;alunno non è tutto ciò indica che durante la sutura l&#39;iride è stato danneggiato e, quindi, l&#39;innesto è considerato un guasto tecnico. </li><li> Applicare una pomata antibiotica per gli occhi. Opzionale: Chiudere il coperchio occhio con una sutura di seta 7-0. </li><li> Osservare i topi fino a quando sono completamente sveglio e poi individualmente li ospitano per un minimo di due giorni post-operatorie. </li></ol></li></ol> 3. Sutura di rimozione <ol style="margin-left: 40px;"><li> Nei casi in cui viene utilizzato coperchio sutura, anestetizzare topi come descritto sopra e rimuovere la sutura coperchio a 48 ore. </li><li> Anestetizzare topi il giorno 7 dopo l&#39;intervento. Rimuovere il sutures garantire il trapianto di cornea. Una volta che i punti di sutura vengono rimossi e l&#39;animale si è completamente risvegliato, restituirlo alla sua gabbia. </li></ol> 4. Valutazione clinica <ol style="margin-left: 40px;"><li> Controllare l&#39;occhio per indicazioni di complicazioni procedurali che comprendono, cataratta (opacità del cristallino), ifema (sangue nella camera anteriore), una camera anteriore che non è di profondità adeguata, o significativo l&#39;opacità della cornea. Prendere in considerazione quelli che dimostrano queste complicazioni come &quot;compromesso&quot; e l&#39;eutanasia da CO 2 inalazione. <ol><li> Eseguire tutti gli esami sui topi non anestetizzati. Tenere il mouse con una mano trattenendo così il mouse in modo che l&#39;altra parte può proptose l&#39;occhio per consentire una migliore vista dell&#39;occhio. Una volta che le osservazioni sono stati completati, rispedire l&#39;animale verso la sua gabbia. </li></ol></li><li> Avere un osservatore non hanno familiarità con i gruppi di trattamento valutare cornee trapiantate 2 o 3 volte a settimana per i segni di trapianto di corneaepisodi di rigetto o di fallimento del trapianto di cornea. Utilizzare il microscopio operatorio o un biomicroscopio con lampada a fessura orizzontale per queste osservazioni. <ol><li> Valutare ogni cornea opacità usando una scala da 0 a 5. La scala è definito come segue: <ol><li> Assegnare un punteggio da 0 a quelle cornee che non hanno segni di opacità. </li><li> Assegnare un punteggio di 1 a quelle cornee che mostrano il minimo l&#39;opacità superficiale. </li><li> Assegnare un punteggio di 2 a cornee che visualizzano l&#39;opacità mite e più in profondità, ma la pupilla e l&#39;iride sottostante sono ancora distinguibile. </li><li> Assegnare un punteggio di 3 cornea da visualizzare stromale opacità in cui il diaframma non possono essere viste in dettaglio con l&#39;eccezione dei margini pupilla. </li><li> Assegnare un punteggio di 4 a cornea da visualizzare densa opacità stromale e se non strutture sottostanti possono essere visualizzati. </li><li> Assegnare un punteggio di 5 a cornea da visualizzare per l&#39;opacità completa ed intensa edema stromale, con la pupilla e l&#39;iride completamente oscurata. </li></ol></li><li><html> Inoltre valuta ogni cornea per il grado di infiltrazione dei vasi sanguigni (neovascolarizzazione) utilizzando una scala di valutazione da 1 a 8. Per eseguire ciò, visualizzare la cornea come costituito da 4 quadranti uguali e determinare la quantità di vasi sanguigni in ciascuno di questi quadranti con punteggio che spaziano volontà da 0 (nessun vasi) a 2 per esteso vascolarizzazione di quel quadrante. Aggiungere i singoli punteggi di ogni quadrante per calcolare il punteggio finale neovascolarizzazione. </li><li> Classificare cornee come acutamente rifiutati se hanno un punteggio di 3 per due osservazioni consecutive per tempo i punti fino a 5 settimane. </li><li> Classificare i topi le cui cornee erano chiari a 5 settimane, ma sviluppare opacizzazione a volte&gt; 45 giorni dopo l&#39;attecchimento, con un punteggio di 3 per due momenti consecutivi, ad avere subito in ritardo termine rigetto corneale. Utilizzare le curve di Kaplan-Meier di sopravvivenza per analizzare la sopravvivenza del trapianto. </li></ol></li></ol> 5. Manipolazione del Modello in-left: 40px; &quot;&gt; <li> Preparazione delle singole cellule dalla milza. <ol><li> Per preparare singole cellule, prima eutanasia il mouse donatore. Quindi, rimuovere la milza. </li><li> Posizionare la milza in un colino cellulare e interrompere con un pistone della siringa da una siringa da 3 ml. </li><li> Lavare le cellule e risospendere in 10 ml di soluzione salina bilanciata Hanks. </li><li> Rimuovere 10 ml di sospensione cellulare e aggiungere 10 ml di 0,4% trypan blu e mescolare. Aggiungere che emocitometro e contare le cellule nella griglia centrale. Il numero di cellule nel tubo è il numero di celle 10 x 4 x 2 (fattore di diluizione in blu tripan) x 10 (volume nel tubo). </li></ol></li><li> Iniezione nella camera anteriore. <ol><li> Anestetizzare topi come precedentemente descritto. </li><li> Eseguire iniezioni usando un microscopio da dissezione. Per ogni iniezione intracameral, utilizzare 10 6 cellule della milza di un volume di 0,005 ml e una siringa microlitro 0,25 ml con ago 33 G. NOTA: altre manipolazionidel modello può essere effettuata trattando gli animali con reagenti che agiscono sia come antagonisti o agonisti per determinare il ruolo che un determinato fattore può giocare dopo l&#39;intervento chirurgico ortotopico allogenico corneale. </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.

Il modello murino di trapianto di cornea qui descritta consente al ricercatore di studiare rigetto corneale umano in un modello che è predittivo di quali fattori sono più associati sia con il rifiuto 15,17,18,20, 26-30 e 21-25 accettazione di cornea trapianti. A differenza di trapianto di cornea umana, in cui i pazienti sono indicati sia per il trattamento steroideo topico o sistemico sia per trattare o prevenire il rigetto 31, questo modello è in genere utilizzato per determinare que...

Il trapianto corneale è uno dei tipi più comuni di successo e trapianti eseguiti negli esseri umani. Le ragioni per cui si esegue questa chirurgia sono il risultato di lesioni, malattie infettive 1, o di altre forme di malattie della cornea non infettive 2. I dati della Eye Bank Association of America indicano che più di 46.000 sono stati eseguiti nel 2011 (si veda il sito Web all&#39;indirizzo: restoresight.org/eye_banks/eye_banks.html). L&#39;indicazione del suo successo è che i tassi di fallimento di un anno per gli innesti corneali allogenici vanno dal 10 al 15% a 5 anni e il successo è superiore al 70% 3-8. Come molti studi hanno dimostrato, il successo di alloinnesti corneali è direttamente legato al fatto che l&#39;occhio è un sito immunologicamente privilegiato. I fattori responsabili dello stato cornee come sito privilegio immunitario comprendono la mancanza di entrambi i vasi sanguigni e linfatici nella cornea, una relativa assenza di cellule presentanti l&#39;antigene, fattori prodotta dalla cornea che Abbateres funtions effettrici del sistema immunitario 9-15, bassa espressione di MHC antigeni 16, e l&#39;espressione di FasL 17-20. Tuttavia, nonostante questi fattori predisponenti questi innesti di successo, essi vanno incontro a rigetto 3-7. Di conseguenza, la comprensione quei meccanismi che mediano questo rifiuto e testare varie terapie per prevenire il rigetto è di importanza critica. A tal fine, si descrive qui un modello murino di trapianto di cornea, che è stato in uso per oltre 20 anni per studiare il trapianto di cornea in un ambiente controllato sperimentale. Poiché risposte trapianto coinvolgono diversi fattori che lavorano in concerto che sarà definitiva determinare se il tessuto trapiantato riesce o riesce, non è possibile comprendere l&#39;importanza di tali elementi in qualsiasi modello in vitro. Di conseguenza, gli studi su animali intatti sono necessari per determinare quali sono i fattori importanti per il successo o il failure del tessuto trapiantato. Mentre altre specie di animali sono stati utilizzati per studiare trapianto di cornea, il modello murino ha diversi vantaggi rispetto all&#39;utilizzo di altre specie. Il primo è l&#39;esistenza di molti ceppi di topi che esprimono alcuni transgeni o sono stati dei geni-bersaglio a non avere l&#39;espressione di specifici fattori immunologici la cui funzione nel trapianto può essere meglio studiata. In aggiunta, ci sono molti reagenti (entrambi i fattori ricombinanti e anticorpi che neutralizzano i fattori) che sono specifici per i topi e che non esistono per molte altre specie di animali. A causa dell&#39;esistenza di questi fattori, questo modello è stato ampiamente utilizzato per identificare i fattori rilevanti che partecipano acute risposte trapianto allogenico corneali 15, 17,18,20 -29. Inoltre, molti dei fattori coinvolti nel trapianto di cornea sono anche noti per essere funzionale nel trapianto di altri tessuti.

<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.

Il modello murino di trapianto di cornea è stato utilizzato da oltre 20 anni con successo per caratterizzare i meccanismi di entrambi corneale rigetto del trapianto corneale 19-23 e accettazione 13, 15,16,18, 24-27. Questo modello è stato utilizzato per stabilire l&#39;importanza di FasL nella cornea del trapianto di accettazione, in quanto gli animali che non hanno FasL non erano in grado di accettare trapianti corneali 15. E &#39;stato anche utilizzato per dimostrare che la crescita ...

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.

Murini trapianto di cornea: Un modello per studiare la forma più comune di trapianto di organi solidi

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 Views.  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.

Video: 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.

Mouse trapianto renale: Modelli di Allograft Rifiuto

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

Ricerca

Medicina

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.

Rene Murine Tecnica trapianto

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 tutto spessore trapianto della pelle

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

Immunologia e infezioni

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 microchirurgica modello per eterotopica, in blocco parete toracica, Timo, e trapianto cardiaco nei topi

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).

Ortotopico degli arti posteriori del trapianto di mouse

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.

Eterotopico renale Autotrapianto in un modello di suino: un protocollo Step-by-Step

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.

Un protocollo di trapianto di cuore eterotopico murino modificato che corrisponde agli standard contemporanei di tecnica asettica, anestesia e analgesia

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.

Un modello di cultura dell'organo endoteliale porcina utilizzando i pulsanti corneali divisi

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.

Auto-trapianto di rene ortotopico in un modello suino utilizzando la conservazione degli organi 24 ore su 24 e la telemetria continua

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.