Name: rat heterotopic abdominal heart/single-lung transplantation in a volume-loaded configuration
Uploaded: 2015-05-29T13:00:00.000+00:00
Duration: 6 min 16 s
Description: Watch this Scientific Journal Video about Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration at JoVE.com

Mark J. Kearns riceve sostegno da parte del personale medico del programma UBC Investigator (aggiudicati attraverso il Royal College dei Medici Chirurghi del Canada), e la UBC 4YF Dottorato Fellowship. John H. Boyd è un Sanitorium associazione nazionale e Michael Smith Fondazione per la ricerca sulla salute Scholar. Finanziamenti attraverso CIHR. Gli autori desiderano ringraziare il Dr. M. Allard e Richard Wamboldt per la loro assistenza con l&#39;installazione e manutenzione di apparecchiature di perfusione.

Tutti gli animali sono stati alloggiati e curati in conformità con le linee guida istituzionali per la cura e l&#39;uso di animali da laboratorio nazionale e. Approvazione etica per questo protocollo è stato concesso dalla University of Comitato Animal Care della Columbia Britannica. Maschio, ratti Sprague-Dawley di peso compreso tra 300-450 g sono stati utilizzati per questo protocollo. 1. donatori Operazione <ol><li> Hanno circa 100 ml di cardioplegia (RT) in vaso cilindrico collegato alla (IV) tubi catetere endovenoso lungo da un rubinetto a 3 vie. Utilizzare un supporto per elevare il matraccio a circa 80 centimetri al di sopra della superficie operativa, consente la distribuzione cardioplegia per gravità. </li><li> Per visualizzare in modo adeguato le strutture, utilizzare un paio di occhialini per la chirurgia o un microscopio da dissezione. NOTA: Al momento stiamo utilizzando un microscopio operatorio binocolo con 3.4 - 21.3X ingrandimento. </li><li> Posizionare il donatore in una camera anestetico e indurre l&#39;anestesia con 4-5% isoflurano. </li&gt; <li> Trasferire il topo di una piattaforma operativa e mantenere l&#39;anestesia da naso-cono con 1-2% isoflurano. Applicare veterinario unguento per gli occhi degli animali per prevenire la secchezza. Somministrare midazolam (2 mg / kg) intra-peritoneally con un ago da 25 G. </li><li> Utilizzando tagliatori chirurgici, radere il donatore da xiphisternum a mandibola. Radere una piccola regione inguinale sinistra (per il successivo accesso IV). Applicare un prodotto depilazione per le superfici operative, attendere circa 5 minuti, e rimuovere i capelli con un pezzo di garza. </li><li> Prep i siti chirurgici con una soluzione basata su clorexidina povidone-iodio o (usiamo solo clorexidina). Infiltrati i siti incisionali con 0,1-0,5% lidocaina per via sottocutanea. </li><li> Fissare gli arti anteriori e degli arti posteriori a sinistra per la piattaforma operativa con del nastro adesivo, lasciando il arti posteriori destro libero per il monitoraggio della profondità dell&#39;anestesia e dei segni vitali. </li><li> Dopo aver verificato appropriato profondità dell&#39;anestesia dal pedale pizzico, fare un&#39;incisione nell&#39;inguine piega a sinistra conun bisturi 22-blade. Per via smussa, esporre la vena femorale comune sinistra e ottenere l&#39;accesso IV come segue: <ol><li> Ritrarre delicatamente il tessuto sovrastante la vena femorale, e cannulate la vena con un 24 G IV. Collegare il IV ad un breve tratto di tubo soluzione salina IV, e fissare il tubo in posizione con nastro adesivo. NOTA: La procedura per l&#39;incannulamento venoso femorale nei ratti è descritto altrove da Jespersen e colleghi 14. </li><li> Collegare una siringa da 10 ml di soluzione salina-riempita al tubo IV, e delicatamente aspirare sangue per assicurare il corretto posizionamento del IV. </li><li> Iniettare 300-500 UI di eparina non frazionata attraverso il tubo IV, e successivamente lavare il tubo con 3 - 5 ml di soluzione fisiologica. </li></ol></li><li> Successivamente, tracheotomize il donatore come segue: <ol><li> Eseguire un&#39;incisione mediana nei tessuti molli tra la tacca giugulare e della mandibola utilizzando un bisturi 22 pale. Penetrare la capsula della ghiandola tiroidea sulla linea mediana con Metzenforbici Baum, e separati i suoi lobi utilizzando scollamento. </li><li> Utilizzando smussa, separare i muscoli cinghia del collo sulla linea mediana per esporre la superficie anteriore della trachea. </li><li> Utilizzare una pinza curve per sezionare senza mezzi termini di un piano circonferenziale attorno alla trachea. Circondare la trachea con una cravatta di seta 4-0. </li><li> Usando forbici iris, fare una incisione trasversale nella trachea anteriore, circa 5 millimetri inferiore alla cartilagine tiroidea. Introdurre delicatamente la cannula tracheale (a 14 G IV) e fissarlo con la cravatta di seta 4-0. </li><li> Collegare la cannula tracheale di un ventilatore meccanico. Reindirizzare il flusso di ossigeno e isoflurano attraverso il circuito del ventilatore, e ventilare il donatore ad una velocità e il volume corrente previsto dal suo peso 13. </li></ol></li><li> Fare un&#39;incisione sulla linea mediana del torace (con un bisturi 22-lama), estendendo l&#39;incisione alla giugulare al di sotto del xipisternum. </li><li> Rimanendo sulla linea mediana, Eseguire una sternotomia mediana con un cutter osso. Ritrarre i bordi dello sterno con un divaricatore autoritenuta. Inserisci il pericardio e cavità pleuriche. </li><li> Eseguire un timectomia. E &#39;più facile per dividere prima senza mezzi termini il timo sulla linea mediana, e poi separarla dalle strutture circostanti utilizzando una combinazione di scollamento e tagliente. NOTA: L&#39;origine delle arterie toraciche interne può essere ferito quando sezionare il timo dai bordi sternali superiori. Per prevenire il sanguinamento, clip emostatici possono essere applicate prima di rimuovere il timo in questi punti. </li><li> Utilizzando forbici Metzenbaum e / o di un forte Lauer, sezionare peri-cavale grasso dalla vena cava inferiore (IVC). Assicurarsi che la IVC è relativamente priva di grassi e tessuto connettivo dalla giunzione cavo-atriale superiormente, inferiormente al diaframma. </li><li> Utilizzando un forte Lauer, circonferenzialmente libera vena cava superiore (SVC) e circondare con una cravatta 4-0 seta. </li><li> Avanti, sezionare ilsinistra vena cava libera da strutture circostanti, e legare esso prossimale e distale con 4-0 cravatte di seta. Resecare la parte intervenendo di cava per esporre l&#39;arteria succlavia sinistra. </li><li> Circonferenzialmente libera i vasi dell&#39;arco aortico con un forte Lauer. Applicare clips chirurgiche prossimale e distale della arteria anonima, e dividerlo tra i clip. Lasciare l&#39;arteria carotide comune sinistra e succlavia sinistra arteria un-ritagliato. </li><li> Successivamente, cannulate la SVC con un catetere 24 G IV. Fissare il catetere con la precedentemente posizionato 4-0 cravatta di seta. </li><li> Preparati per un raccolto trapianto ripiego, ma gentile. </li><li> Utilizzando forbici affilate, dividere la IVC appena superiore alla superficie diaframmatica. Divide la SVC superiore al suo sito incannulazione, assicurando che le strutture delle vie aeree nei dintorni non sono feriti. </li><li> Spegnere il ventilatore e staccare la cannula tracheale. Transetto la trachea prossimale. </li><li> Afferrare la trachea sezionato con una pinza, e togliere il cuoree polmoni en-bloc. Ciò richiederà una leggera trazione con dissezione tagliente come il trapianto cuore-polmone è stato rimosso. Separare la trachea dall&#39;esofago sottostante. Evitare lesioni dell&#39;aorta discendente, in modo che una porzione lunga di esso rimane intatto dopo la raccolta. </li></ol> 2. Preparazione del cuore-polmoni Graft NOTA: mentre il completamento di questa parte della procedura, un assistente dovrebbe essere anestetizzare il roditore destinatario e la preparazione per trapianto impianto. <ol><li> Posizionare il trapianto cuore-polmoni su garza chirurgica con la superficie anteriore del cuore rivolto verso il basso. Utilizzando dissezione tagliente, rimuovere eventuali residui di esofago, evitando lesioni alle vie respiratorie posteriori. </li><li> Individuare l&#39;aorta toracica discendente e inserire una punta smussata cannula 16 G. Fissare la cannula in posizione con un pareggio 4-0 in seta. </li><li> Individuare i vasi dell&#39;arco aortico, e applicare una clip chirurgico al succlavia sinistra. Lasciare la carotide sinistra un-tagliati per subsequent de-aerazione. </li><li> Somministrare la prima dose di cardioplegia collegando il tubo cardioplegia alla cannula aortica 16 G. L&#39;arteria carotide deve essere occluso temporaneamente con una pinza per garantire un&#39;adeguata fornitura di cardioplegia. NOTA: Un vantaggio di avere la cannula aortica allegato è che cardioplegia può essere somministrato in modo continuo e / o periodicamente, se lo desideri. Per il dosaggio intermittente, di solito consegniamo cardioplegia ogni 10 - 15 minuti in 5 ml boli over 30 - 45 sec. </li><li> Usando forbici Metzenbaum e / o un forte Lauer, sezionare grasso peri-aortico lontano dall&#39;aorta. Assicurarsi che l&#39;aorta è relativamente priva di grassi e tessuto connettivo della succlavia sinistra (arco distale) al suo sito cannulazione. </li><li> Avanti, esporre il bronco principale di sinistra con un forte Lauer, e legare è prossimale con un pareggio 4-0 in seta. Transetto il bronco distale sinistra per la legatura con forbici iris. Eseguire un pneumonectomy lasciato legando polmonare sinistraarteria e vena con 4-0 cravatte di seta. Transect distale alla legatura e rimuovere il polmone sinistro. </li><li> Come sopra, rimuovere tutte le altre lobi polmonari con l&#39;eccezione del diritto lobi superiori e medie. Evitare ferendo la IVC nel processo di esecuzione lobectomie. </li><li> Collegare il tubo cardioplegia alla cannula aortica e amministrare cardioplegia continuo mentre il destinatario è in preparazione. Posizionare il trapianto cuore-polmone in un contenitore sterile (ad esempio, un becher). </li></ol> 3. Destinatario Operazione <ol><li> Anestetizzare il destinatario secondo l&#39;operazione donatore, sopra. Utilizzare veterinario pomata per proteggere gli occhi dell&#39;animale da secchezza. Confermare anestesia spesso a pedale pizzico. </li><li> Posizionare l&#39;animale come per l&#39;operazione del donatore, questa volta lasciando la zampa anteriore destra libera di monitorare i segni vitali e profondità dell&#39;anestesia. </li><li> Radere l&#39;addome da xiphisternum a pene. Radere una piccola regione inguinale sinistra (per il successivo accesso IV). Applicare unaagente depilating le superfici operative, attendere circa 5 minuti, e rimuovere i capelli con un pezzo di garza. </li><li> Prep i siti chirurgici con povidone-iodio o soluzione basata clorexidina-. Infiltrati i siti incisionali con 0,1-0,5% lidocaina per via sottocutanea. </li><li> Tracheotomize e ventilare il destinatario come indicato sopra. In alternativa, mantenere il destinatario in anestesia naso-cono. </li><li> Introdurre un catetere nella vena femorale come descritto sopra. Iniettare 300-500 UI di eparina non frazionata attraverso il tubo IV, e successivamente lavare il tubo con 3 - 5 ml di soluzione fisiologica. </li><li> Eseguire una laparotomia facendo una incisione addominale mediana con un bisturi a lama da 22 xiphisternum a pene. Ritrarre la parete addominale con un divaricatore di auto-mantenimento. Avanti, ritrarre le viscere superiormente e per il lato sinistro del topo. Avvolgere in caldo, garza imbevuta di soluzione salina. </li><li> Esporre l&#39;aorta addominale IVC e da forte dissezione attraverso la sovrastante regrasso tro-peritoneale. </li><li> Avere un morsetto nave curvo disponibili e pronti. </li><li> Circonferenzialmente liberare l&#39;IVC e dell&#39;aorta prossimale e distale e circondare con 4-0 cravatte di seta. Ci dovrebbe essere di circa 2-3 cm di spazio tra questi siti. </li><li> Applicare con cura il morsetto nave curvo, in modo che una porzione sufficiente sia IVC e dell&#39;aorta sono esposti sopra le ganasce del morsetto. </li><li> Fare un&#39;incisione nella parete anteriore della IVC con un ago 25 G collegato ad una siringa di soluzione salina 1 ml. Estendere l&#39;incisione con le forbici Potts in base alla lunghezza del donatore IVC orifizio. </li><li> Rimuovere il trapianto cuore-polmone dal suo contenitore e scollegarlo dalla cardioplegia. Posizionare l&#39;innesto nell&#39;addome del destinatario in una posizione ottimale per eseguire l&#39;anastomosi venosa. NOTA: L&#39;innesto cuore-polmone alla fine sarà orientata leggermente obliquo, con la punta apice verso l&#39;addome di sinistra quadrante inferiore. </li><li> Fissare il taccoe punta estremità della anastomosi con 9-0 sutura in nylon. Fare un nodo sicuro ad ogni estremità, lasciando l&#39;ago di un lungo braccio di sutura, e una breve fine di sutura per essere legato a un secondo momento. Somministrare una dose di cardioplegia. </li><li> In modo esecuzione, completa la metà della linea di sutura e cravatta al braccio di sutura breve opposte. Somministrare una dose di cardioplegia. </li><li> Completa l&#39;altra metà della linea di sutura e legarlo giù. L&#39;anastomosi venosa è completo. Somministrare una dose di cardioplegia </li><li> Orientare il trapianto cuore-polmone con puntamento apice del cuore verso l&#39;addome di sinistra quadrante inferiore. Assicurarsi che l&#39;anastomosi venosa non sia piegato o attorcigliato. </li><li> Valutare la lunghezza dell&#39;aorta donatore che sarà richiesto per raggiungere l&#39;aorta destinatario, mantenendo sia IVC e anastomosi aortica in configurazioni tension-free e non-piegati. </li><li> Dividete l&#39;aorta discendente (distale succlavia sinistra) con una forbici iris. </li><li> Fai incision nella parete anteriore dell&#39;aorta destinatario con un ago da 25 G collegato ad una siringa di soluzione salina 1 ml. Estendere l&#39;incisione con le forbici Potts in base alla lunghezza dell&#39;orifizio aortico donatore. </li><li> Ancorare la punta (aspetto superiore) dell&#39;aorta donatore al ricevente aorta usando una sutura 9-0 nylon. Quindi, passare l&#39;ago al mediale dell&#39;aorta e completare la metà delle anastomosi in esecuzione moda. </li><li> Al tallone del anastomosi aortica, invertire l&#39;orientamento della linea di sutura (una transizione nella direzione di cucitura), e completare la metà laterale della anastomosi in esecuzione moda. </li><li> Rimuovere lentamente il catetere IV nel SVC, e legare l&#39;SVC con una clip chirurgica. </li><li> De-air l&#39;aorta individuando il non-ritagliato sinistra arteria carotide comune. Tenere aperta l&#39;arteria, in posizione verticale che permetterà all&#39;aria di essere liberamente evacuato. </li><li> Brevemente aprire le ganasce della pinza vaso curvo e consentire l&#39;arteria carotide sanguinare (de-air) per 2-3 sec. Riapplicare il morsetto nave. </li><li> Applicare una clip chirurgica alla carotide. Rimuovere la fascetta vaso curvo. </li><li> Verificare la presenza di emorragie alle linee di sutura. Se presente, applicare lieve compressione con garza o riparazione con una lunghezza di 9-0 sutura di nylon (a seconda della gravità del sanguinamento). </li><li> Il cuore del donatore dovrebbe riprendere battendo in pochi minuti. </li><li> Prestare particolare attenzione ai segni vitali del destinatario, profondità dell&#39;anestesia, e amministrare il volume rianimazione, se necessario. </li><li> A seconda dei punti finali sperimentali, o chiudere l&#39;addome dell&#39;animale e farlo recuperare da anestesia, o prepara l&#39;animale per la valutazione cardiovascolare. NOTA: Gli esempi di valutazione del trapianto sono in misure in vivo di emodinamica in funzione del carico e indipendente dal carico, misure in vitro della funzione in Langendorff e modalità di lavoro, e il cuore (in chirurgia sopravvivenza) ecocardiografici o indagini di risonanza magnetica. </li><li> Alfine degli esperimenti terminali acuti, gli animali sono eutanasia per dissanguamento. </li></ol>

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Il successo con la tecnica descritta qui basarsi su diversi fattori. Key tra i quali sarà garantire la stabilità di entrambi i donatori e riceventi, adottando meticolosa tecnica operatoria che è sicuro e associata con una minima perdita di sangue, garantendo completo arresto cardioplegico con raffreddamento trapianto uniforme, riducendo al minimo il tempo ischemico totale, e adeguatamente de-onda l&#39;innesto. Come riconosciuto in precedenza, la complessità tecnica della tecnica è il suo limite principale. Abbiamo avanzato le tecniche HAHLT precedenti sotto diversi aspetti. Le modifiche descritte nelle operazioni di donatore e ricevente sono un mezzo di esecuzione necessarie fasi operative in modo controllato ed efficiente. Il protocollo di protezione miocardica descritto è un mezzo ideale di ridurre al minimo pregiudizio durante il periodo ischemico. Il risultato finale è un innesto HAHLT in cui geometria è stata ottimizzata, il pregiudizio è stato ridotto al minimo, e la funzione del trapianto intrinseca è stato preservito nella maggior misura possibile. La tecnica sopra descritta produce un innesto HAHLT con condizioni ventricolare a caricamento dinamico e potenzialmente quasi fisiologici. Una volta che la tecnica è stato masterizzato, i permessi di configurazione trapianto completo di caratterizzazione vivo di funzione del trapianto. Come notato, le condizioni di precarico e postcarico possono essere modificati acuta o cronica, e del polmone del donatore possono essere acutamente nuovamente ventilato. Gli investigatori possono facilmente e in generale applicare questo modello allo studio di molte condizioni mediche, pur mantenendo la capacità di studiare la struttura e la funzione del trapianto.

The first rodent model of heterotopic abdominal heart transplantation (HAHT) was described by Abbott and colleagues in 19641. This technique, and subsequent modifications have been widely applied to characterize transplant graft function and immunologic status. The majority of HAHT techniques described involve a non-volume loaded heart2,3. Models of HAHT involving volume-loaded ventricles have been described, but they are frequently limited in one or more respects.
Heterotopic abdominal heart-lung transplantation (HAHLT) with a volume-loaded left ventricle (LV) has been described previously. Chen and colleagues4, and subsequently Ibrahim and colleagues5 described HAHLT with a single aorto-aortic (donor ascending to recipient abdominal aorta) anastomosis. The only volume load presented to the ventricle in this circulation is the coronary venous return. Asfour and colleagues described a HAHT technique in which the lung circuit was eliminated by anastomosing donor pulmonary artery (PA) to donor left atrium (LA)6. In this circulation, venous inflow to right ventricle (RV) occurs via a donor SVC to recipient IVC anastomosis, and the subsequent LV load is ejected into the aorto-aortic anastomosis. Cardiac function was partially assessed in vivo, and also in vitro using a Langendorff rig. Figueiredo and colleagues described a HAHLT model similar to our own7, but in mice. Venous inflow to the RV occurs via donor SVC to recipient IVC anastomosis. Blood subsequently passes through the single lung circulation and LV load is ejected into the aorto-aortic anastomosis. Cardiac function in their study was assessed by magnetic resonance imaging (MRI). Wen and colleagues described a unique HAHT technique in which the LV is loaded by means of a recipient aorta to donor LA anastomosis8. The LV, therefore, fills at systemic pressures. Cardiac function, and whether LV stroke volume is ejected antegradely in their model was not assessed.
Many of the techniques referenced above involve non-physiologic LV loading conditions, including the techniques whose partial LV load is represented only by coronary venous return. On the other hand, many techniques do approach physiologic LV loading. The majority of these techniques, as with the technique of Asfour and colleagues, omit the pulmonary circulation and utilize a donor PA to donor LA anastomosis6,9. The circulation described by Galinanes and colleagues10 employs a direct recipient cava to donor LA anastomosis, omitting the pulmonary circulation and the right heart. Yokoyama and colleagues achieve the same effect by ligating the donor PA and creating an interatrial communication in the donor heart (omitting donor lung and right heart circulations)11. The circulation of Maruyama and colleagues12 involves an anastomosis between donor left PA and recipient Ao, which permits LV filling via the pulmonary circulation as a conduit, but effectively excludes the right heart.
In cases where near physiologic loading conditions were met, we advance the technique of HAHLT in 2 major respects. First, to our knowledge, the exact configuration we report has not been described in rats. It is possibly the most versatile circulation for investigators wishing to study the physiology, structure, and immunology of the transplanted heart-lung graft. Second, we describe how the function of the transplant graft can be directly characterized in vivo. For this application, pressure-volume conductance catheters can be introduced directly into the LV apex of the transplant graft, which allows for complete cardiac functional characterization. 
The technique described here can be applied to both acute and chronic studies of transplant graft function, while the functional assessment may be performed either in vivo or in vitro. We present a model in which the loading conditions can be near physiologic, however the degree of ventricular loading may be manipulated both acutely and chronically by diverting venous return towards or away from the graft. Afterload conditions can also be manipulated. Because the lung and its airway are retained in this transplant configuration, investigators can re-ventilate the donor lung acutely. Uniquely, lung re-ventilation changes the composition of blood perfusing the transplant coronary arteries. Under non-ventilated conditions, blood ejected from the donor aorta is deoxygenated, and mixes with oxygenated blood in the recipient aorta. Under acutely ventilated conditions, ejected blood becomes oxygenated. Thus, transplant graft function can be compared under ventilated and non-ventilated conditions, and also under variably loaded conditions.
The protocol below describes important modifications to previously described HAHLT donor and recipient operations. It also describes an optimal technique for protecting the transplant graft throughout the period of ischemia (time between donor explant and recipient implant). Advantages of this technique include physiologic conditions potentially approaching that of an orthotopically transplanted graft, and a wide range of investigative applications. An important limitation is its technical complexity. With adequate mentoring and practice, the advantages of this technique will likely outweigh the challenges in adopting it.

<table><tbody><tr><td>Celsior Cardioplegic Soution</td><td>Genzyme</td><td></td><td>The solution is kept on ice throughout the procedure. We prepare our own solution, with slight modifications.</td></tr><tr><td>Rodent Ventilator</td><td>Harvard Apparatus</td><td>Model 683</td><td></td></tr><tr><td>Vital Sign Monitor</td><td>Nonin</td><td>Model 9847V</td><td>Displays SpO&lt;sub&gt;2&lt;/sub&gt; and heart rate.</td></tr><tr><td>IV Cannulae</td><td>Jelco</td><td>3063</td><td>24 - 26 G x 3/4&quot; cannulae.</td></tr><tr><td>IV Tubing</td><td>CareFusion</td><td>MP9259-C</td><td>Short-length connector tubing (18 cm).</td></tr><tr><td>Surgical Clips</td><td>Teleflex Medical</td><td>001204</td><td>Horizon titanium ligating clips.</td></tr><tr><td>Sutures</td><td>Ethicon, Sharpoint</td><td>LA54G, AK-0107</td><td>3-0 silk reel, and 9-0 prolene suture (single-armed, DR5 needle).</td></tr><tr><td>Surgical Instruments</td><td>Not Applicable</td><td>Not Applicable</td><td>The instruments used are generic, and can be purchased from any surgical supply company.</td></tr></tbody></table>

rat heterotopic abdominal heart/single-lung transplantation in a volume-loaded configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

La tecnica HAHLT sopra descritta è molto tecnico e richiede grande attenzione ai dettagli. Tabella 1 evidenzia alcuni dei fattori chiave associati con successo contro le procedure fallimentari, e può essere utilizzato come una guida per la risoluzione di problemi tecnici. Dopo l&#39;aorta destinatario è sbloccata, l&#39;innesto arterie coronarie dovrebbero essere considerati a riempirsi di sangue ossigenato. Di conseguenza, il miocardio dovrebbe diventare rosa e ben irrorato. Negli esperimenti tecnicamente successo, il cuore inizia a battere poco dopo trapianto riperfusione. L&#39;innesto deve essere lasciato in uno stato scaricato (con ricevente IVC bloccato) per un periodo di almeno 20 - 30 minuti per consentire il recupero funzionale. In seguito a questo, le condizioni di carico del innesto può essere modificato per soddisfare scopi sperimentali. Ulteriori misure quantitative della funzione del trapianto (e un esito positivo) possono essere impiegate, se lo desideri. Come indicato nel protocollo, in vivo </em&gt; e in vitro funzionali, nonché ecocardiografica e ricerche MRI può fornire tali informazioni. figure 1 e 2 sono esempi di base in vitro e dati pressione-volume che possono essere derivate acutamente con questa metodologia-occlusione precaricare. Dati emodinamici di questi studi in grado di fornire agli investigatori la gittata cardiaca, stroke volume, volume della camera, la frequenza cardiaca, frazione di eiezione, elastanza telesistolico, e precarico lavoro ictus recruitable. Diversi altri parametri possono essere quantificati, come desiderato. <img alt="Figura 1" src="/files/ftp_upload/52418/52418fig1.jpg" /> Figura 1. basali di pressione-volume di dati. <a href="https://www.jove.com/files/ftp_upload/52418/52418fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> La figura 1 mostra stampa di basedati ure volumi da una valutazione cuore in vitro di lavoro della funzione cardiaca. EDV, volume telediastolico; EDP, pressione telediastolica; ESV, volume telesistolico; ESP, pressione di fine sistolica. <img alt="Figura 2" src="/files/ftp_upload/52418/52418fig2.jpg" /> Figura 2. precarico-occlusione pressione-volume di dati. <a href="https://www.jove.com/files/ftp_upload/52418/52418fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> La figura 2 mostra precaricare-occlusione dati pressione-volume da una valutazione del cuore in vitro di lavoro della funzione cardiaca. EDPVR, relazione pressione-volume telediastolico; ESPVR, relazione pressione-volume telesistolico. <table border="0" cellpadding="0" cellspacing="0"><colgroup><col span="2" /></colgroup><tbody><tr><td></td><td> Procedura di successo</td><td> Procedura non riuscita </td></tr><tr><td> Donatore </td><td></td><td></td></tr><tr><td> Donatori di stabilità </td><td> Stabile </td><td> Instabile </td></tr><tr><td> Preparatorio Dissection </td><td> Efficiente, Limited </td><td> Inefficiente, eccessiva </td></tr><tr><td> Perdita di sangue </td><td> Minimo </td><td> Eccessivo </td></tr><tr><td> Tempo di espianto </td><td> Ridotti al minimo </td><td> Prolungata </td></tr><tr><td> Arresto cardioplegica </td><td> Veloce </td><td> Ritardato </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Trapianto </td><td></td><td></td></tr><tr><td> Totale ischemica tempo </td><td> &lt;90 min </td><td> &gt; 90 min </td></tr><tr><td> Arresto cardioplegica </td><td> Mantenuto Periodicamente </td><td> Incompleto </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Destinatario </td><td></td&gt; <td></td></tr><tr><td> Stabilità del destinatario </td><td> Stabile </td><td> Instabile </td></tr><tr><td> Preparatorio Dissection </td><td> Efficiente </td><td> Inefficiente </td></tr><tr><td> Pinza Volte </td><td> &lt;45 min </td><td> &gt; 45 min </td></tr><tr><td> Anastomosi Tempo </td><td> &lt;30 min </td><td> &gt; 30 min </td></tr><tr><td> Perdita di sangue </td><td> Minimo </td><td> Eccessivo </td></tr><tr><td> Graft De-aerazione </td><td> Adeguato </td><td> Insufficiente </td></tr><tr><td> Volume Rianimazione </td><td> Adeguato </td><td> Insufficiente </td></tr><tr><td> Riperfusione successo </td><td> Sì </td><td> No </td></tr><tr><td> Ritorno di Stabile funzione cardiaca </td><td> Sì </td><td> No </td></tr></tbody></table> Tabella 1. Caratteristiche del successo Versus procedure HAHLT senza successo. Tabella 1 &lt;/ Strong&gt; fornisce esempi di fattori associati con successo e insuccesso donatore, trapianto, e le operazioni di destinatari.

Watch this Scientific Journal Video about Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration at JoVE.com

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Rat eterotopico addominale Cuore / Trapianti Single-polmone in una configurazione del volume-caricato

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

rat-heterotopic-abdominal-heartsingle-lung-transplantation-volume

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K Views.  UBC James Hogg Research Center, St. Paul's Hospital. We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Video: Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Il trapianto cardiaco eterotopico nel Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Ricerca

Biologia

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Eterotopica ausiliario Ratto trapianto di fegato con flusso regolato Portal Vein arterialization in acuta insufficienza epatica

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Medicina

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. 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 was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Murini eterotopica cuore Tecnica Trapianto

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Un modello immunologico per il trapianto di cellule cardiache e cardiache cardiache cardiache cardiache cardiache cardiache

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Immunologia e infezioni

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Ricostruzione del circuito sanguigno in un modello di trapianto di cuore di topo addominale

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

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.

Un modello semplificato per il trapianto di valvole cardiache eterotopiche nei roditori

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 ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Una tecnica di bracciale modificata per il modello di trapianto di cuore eterotopico cervicale del topo

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Modello di ratto del trapianto di cuore eterotopico perfuso ex-situ normothermic

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Trapianto di cuore addominale eterotopico modificato e un nuovo modello di rigurgito aortico nei ratti

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...