Name: a novel microsurgical model for heterotopic, en bloc chest wall, thymus, and heart transplantation in mice
Uploaded: 2016-01-23T14:00:00.000+00:00
Duration: 12 min 24 s
Description: Watch this Scientific Journal Video about A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice at JoVE.com

This work was funded by the American Association of Plastic Surgeons 2014 Academic Scholar Award.

Tutte le procedure operative sono state completate in conformità con la Johns Hopkins University e il Dipartimento dell&#39;Agricoltura degli Stati Uniti e le esigenze di servizio di sanità pubblica. Questo protocollo segue il Hopkins University cura e l&#39;uso degli animali Comitato Johns, comitato istituzionale di revisione approvato le linee guida (numero di protocollo M013M490). Dati di sopravvivenza finale è stato registrato per le procedure chirurgiche descritte di seguito. Entrambi gli animali donatori e beneficiari ricevono l&#39;anestesia di prelazione con buprenorfina a 0.1 mg / kg sc un&#39;ora prima di un intervento chirurgico e la buprenorfina destinatario animale è ri-somministrato alla stessa dose dopo il trapianto e ri-dosato come necessario nelle prime 48 ore dopo l&#39;intervento chirurgico. 1. donatori Allograft recupero Nota: Inizia la parte del donatore del trapianto 40 minuti prima del trapianto destinatario di minimizzare il tempo di anestesia destinatario e per facilitare un tempo di fine simultaneo o leggermente orecchioora di fine lier contro la preparazione destinatario. <ol><li> Utilizzare strumenti di microchirurgia sterili standard e guanti sterili per la procedura. Il nostro laboratorio utilizza la sterilizzazione in autoclave di strumenti di microchirurgia. </li><li> Anestetizzare il mouse donatore (maschio) con isoflurano vaporizzatore induzione al 4%. Uso Clippers meccanici atraumatica rimuovere i capelli dalla cervicale, toracica, addominale e della regione. Posto l&#39;animale in posizione supina e mantenere isoflurane sul 1-2% attraverso un cono naso. Garantire un&#39;adeguata anestesia durante tutta la procedura, valutando periodicamente il riflesso di punta di ritiro pizzico. </li><li> Prima l&#39;incisione cutanea, ampiamente preparare il dispositivo applicando iodopovidone antisettico seguito da alcol isopropilico utilizzando un tampone di cotone sterile. </li><li> Inizia con una incisione cutanea superficiale trasversale con le forbici in tutta la pelle cervicale e addominale. Collegare i due incisioni bilateralmente lungo le linee ascellare media. </li><li> Utilizzando pinze microchirurgichesezionare la regione cervicale bilaterale per identificare, legare e dividere le vene giugulari esterne con 6-0 sutura di seta e forbici. Quindi, utilizzando elettrocauterizzazione dividere i muscoli sternocleidomastoideo per esporre le vene giugulari interne e le arterie carotidi comuni, a livello bilaterale. Passare una sutura 6-0 di seta sotto il lato sinistro e il lato destro carotide comune e le vene giugulari interne in modo sfuso. NOTA: Saranno legate e divise in seguito al passaggio 1.9. </li><li> Acutamente dividere i muscoli cinghia e associati tessuto areolare sciolto, che si trova anteriori alla trachea, con le forbici per liberare i restanti allegati della regione cervicale. </li><li> Utilizzando elettrocauterizzazione bipolare e la dissezione tagliente, dividere i pettorale muscoli principali e le clavicole per esporre i vasi succlavi e legare (6-0 sutura di seta) e dividere prossimale. </li><li> Poi, delicatamente, afferrare ed estrarre il pene dell&#39;animale. Lungo il dorso del pene visualizzare la vena dorsale del pene, e disinfettare laregione con alcol isopropilico. Utilizzando un ago 30 G, iniettare 30.000 unità di eparina per via endovenosa attraverso la vena dorsale e permettono al pene di rinculo indietro alla sua posizione originale. Può verificarsi perdite parziale della soluzione di eparina nel tessuto circostante. </li><li> Utilizzando i legami di massa precedentemente posizionati intorno carotide comune e la vena giugulare interna, legare e dividere le strutture, bilateralmente. </li><li> Successivo utilizzo forbici per rendere una incisione intraddominale trasversale. Sviscerare l&#39;intestino per esporre la infraepatica vena cava inferiore e iniettare 2 ml di soluzione di cardioplegia fredda Euro-Collins nel infraepatica vena cava inferiore. Garantire la corretta iniezione visualizzando scolorimento del fegato e la cessazione del battito cardiaco prima di passare al passo successivo. NOTA: la soluzione Euro-Collins è preparato nel nostro laboratorio, vedi tabella di reagenti e strumenti specifici. </li><li> Utilizzando le forbici accedono cavità intratoracica tramite un DIAFRAMMA bilateraleincisione ragmatic dall&#39;addome esposta. Estendere l&#39;incisione cefalica attraverso i muscoli intercostali e le costole. Riflettere parete toracica esponendo il cuore, il timo, e grandi vasi assicurando allo stesso tempo la conservazione dei vasi toracici interni lungo la parete toracica. </li><li> Iniettare la sovraepatica vena cava inferiore con 4 ml di soluzione di cardioplegia fredda Euro-Collins. </li><li> Identificare la radice dell&#39;aorta e tracciare distalmente alla aorta discendente. Acutamente tagliare l&#39;aorta discendente (conservando lunghezza massima). </li><li> Identificare il tronco polmonare e dividere appena prossimale al suo punto di filiale (conservando lunghezza massima). Quindi, utilizzando 2 ml di soluzione di cardioplegia fredda Euro-Collins, lavare il tronco polmonare e il cuore mettendo un morbido punta del catetere di plastica nel lume del tronco polmonare. </li><li> Utilizzando una sutura di seta 6-0, legare e sezionare la vena cava inferiore, confluenza delle vene polmonari, e rami accessorie del cava bilaterale cava superiore. Poi elevaree sezionare la cefalica cuore dagli attaccamenti lungo bronchi fusto principale e la trachea con attenzione a non inserire le vie respiratorie. Utilizzando elettrocauterio bipolare acuto e sezionare la parete toracica, il timo, e il cuore completamente liberandolo dal mouse donatore. </li><li> Infine, tagliare la parete toracica allogenico ex vivo a una dimensione inferiore, con le forbici, che attraversa lo sterno e costae laterali, con attenzione a non interrompere i vasi toracici interni (Figura 1A). Per ridurre al minimo emorragia dopo rivascolarizzazione, utilizzare electrocauterization bipolare lungo i confini dello sterno osteomusculocutaneous. </li><li> Posizionare il allogenico in 10 ml di freddo (4 o Celsius) soluzione Euro-Collins se il destinatario non è preparato per incasso. Tuttavia, se il destinatario è pronto per inserto, trasferire l&#39;alloinnesto direttamente al campo operatorio destinatario. </li></ol> 2. Destinatario Preparazione Nota: Per ridurre al minimo il tempo di anestesia destinatario,iniziare la preparazione destinatario in una stazione operativa separata circa 40 minuti prima del completamento del raccolto allograft donatore. <ol><li> Utilizzare un insieme separato di strumenti di microchirurgia sterili standard e guanti sterili per la procedura. </li><li> Anestetizzare il mouse destinatario (maschio o femmina) con isoflurano vaporizzatore induzione al 4%. Utilizzando Clippers meccanici atraumatica rimuovere i capelli dalla regione cervicale e toracica destra. </li><li> Posizionare il mouse in posizione supina e l&#39;angolo l&#39;arto superiore destro leggermente inferiormente formando un angolo di 110 gradi tra la testa e arto superiore destro. Mantenere l&#39;anestesia sul 1-2% isoflurano attraverso un cono naso. </li><li> Posizionare petrolio pomata oftalmica sugli occhi del mouse utilizzando un cotton-fioc. Prima l&#39;incisione cutanea, ampiamente preparare il sito operatorio utilizzando iodopovidone antisettico seguito da alcol isopropilico. </li><li> Utilizzando le forbici, fare una incisione cutanea dalla linea mediana lungo la destra bor inferioreder della mandibola e prolungare l&#39;incisione infero lateralmente alla regione toracica destra. Utilizzando smussa con pinze microvascolari, mobilitare la vena giugulare esterna circonferenziale libero nave da tessuti molli e avventizia. Dividere tutti i rami con elettrocauterizzazione, e rimuovere il lobo destro della ghiandola sottomandibolare utilizzando dissezione tagliente e elettrocauterizzazione di spazio libero per il trapianto allogenico. </li><li> Assicurare una lunghezza sufficiente della vena giugulare esterna per rovesciare su un bracciale, e legare la vena giugulare esterna usando una sutura 6-0 seta. Inserire la vena attraverso il lume di un bracciale pretagliati poliimmide e utilizzare un morsetto microvascolare bulldog per risolvere il complesso nave-polsino in posto. Poi con le forbici, prossimale dividere la vena giugulare esterna, Evert sopra il polsino, e fissare con una sutura in nylon 10-0. (Figura 1B) </li><li> Dividere il muscolo sternocleidomastoideo destro con elettrocauterizzazione bipolare per esporre l&#39;arteria carotide comune. Mob circonferenzialeilize l&#39;arteria cefalica al distale punto più all&#39;interno della regione cervicale. Questa operazione viene eseguita utilizzando scollamento della nave con una pinza per rimuovere tessuti molli e avventizia circostante. </li><li> Utilizzando sutura di seta 6-0, legare e sezionare l&#39;arteria carotide comune. Passare l&#39;arteria attraverso il lume di un bracciale pretagliati poliimmide e fissarlo con un bulldog microvascolare morsetto più vicino possibile al toracico possibile. Dividete il vaso distale, dilatare delicatamente il recipiente con un dilatatore microchirurgico, Evert sopra il polsino, e fissare con una sutura in nylon 10-0. (Figura 1B) NOTA: Il dilatatore microchirurgica specifico è descritto nella tabella di reagenti e strumenti specifici. </li></ol> 3. Allograft dell&#39;inserzione <ol><li> Mantenere la strumentazione sterili standard guanti sterili per posizionare il allotrapianto all&#39;interno della regione cervicale destinatario in un testa in giù e posizione obliqua. </li><li> Quindi, posizionare il aor donatore discendentelumen tic sul costrutto bracciale arteriosa del destinatario e fissarlo con una sutura di nylon 10-0 (Figura 1C e 1D). </li><li> Moda la stessa anastomosi come al punto 3.2 tra l&#39;arteria polmonare donatore e il estroflesso esterna giugulare vena-polsino costrutto del mouse destinatario (Figura 1C e 1D). </li><li> Innanzitutto rimuovere morsetto microvascolare venoso (morsetto vena giugulare esterna) e poi allentare il blocco arterioso (arteria carotide comune morsetto). Durante la riperfusione arteriosa, controllare la totalità del allogenico di affrontare qualsiasi emorragia. Se emorragia viene visualizzato, riapplicare la pinza arteriosa per ridurre al minimo la perdita di sangue e ridurre la fonte di sanguinamento con elettrocauterizzazione bipolare. </li><li> Controllare l&#39;innesto e di garantire l&#39;emostasi. Rilasciare e rimuovere completamente il morsetto microvascolare arteriosa. Osservare il cuore a mostrare segni di riperfusione, che saranno istantaneamente apparente con l&#39;espansione del volume rapida delle camere cardiache, e attendere bmangiare per iniziare entro 0,5-1 min. Utilizzare salina calda (35 ° C) per inumidire il cuore. </li><li> Telo parete toracica in posizione anatomica in modo da non indurre alcuna attorcigliamento o tensioni sui anastomosi. Chiudere la pelle della ferita chirurgica sta usando 6-0 punti di sutura in nylon continui (Figura 1E). </li></ol> 4. Cura postoperatoria <ol><li> Somministrare un 0,3 ml di soluzione salina normale bolo fluido intraperitoneale immediatamente dopo l&#39;intervento per la sostituzione del fluido. </li><li> Poi via sottocutanea iniettare buprenorfina (0,1 mg / kg) e enrofloxacina (5mg / kg) per la profilassi del dolore e infezione, rispettivamente. </li><li> Posto l&#39;animale sotto una lampada di calore fino risveglio dall&#39;anestesia e tornare al decubito sternale. Durante il recupero, ispezionare il collo per visualizzare il battito cardiaco fibrillazione del allotrapianto garantire un&#39;adeguata perfusione allogenico. </li><li> Una volta sveglio e in posizione supina, restituire il mouse in una gabbia separata (senza la compagnia di altri topi) Dove riceva cibo e acqua ad libitum. A causa di qualsiasi movimento restrittiva minore temporanea del arto superiore destro, lasciare una fonte di cibo gelatina sul pavimento della gabbia. </li><li> Osservare il mouse destinatario per 1 ora dopo l&#39;intervento e poi tornare alla struttura gabbia dove possa ricevere cibo e acqua ad libitum e viene ispezionato tre volte al giorno per le prime 24 ore di attività e di apporto nutrizionale. Monitorare i topi per i segni di dolore e di angoscia e ri-dosaggio con buprenorfina (0.1mg / kg) per via sottocutanea due volte al giorno come necessario per la prima 72 ore. Esaminare gli animali ogni giorno da allora in poi e pesare ogni settimana. </li><li> Consultare un membro del personale veterinario, se eventuali topi mostrano segni di dolore, angoscia, o diminuito l&#39;assunzione di cibo. Considerare presto l&#39;eutanasia (nel nostro protocollo la tecnica eutanasia impiega CO 2 overdose per 7 minuti, seguita da dislocazione cervicale). </li><li> Cessazione del battito cardiaco allogenico è definito come un endpoint specifico spingendo il mouse per essere sacrificed. </li></ol>

The authors do not have any conflicts of interest or financial disclosures to declare.

Ci sono una moltitudine di fenomeni che fattore nella ricerca immunologica di allotrapianto, che includono ma non sono limitati a meccanismi di rigetto acuto e cronico, la presentazione diretta e indiretta dell&#39;antigene, destinatario di sensibilizzazione o l&#39;induzione di chimerismo misto. 19 modelli animali sono diventati il gold standard per lo studio di Immunologia dei Trapianti, e modelli di topo sono comunemente implementate a causa del loro basso costo, la disponibilità di transgenici e gene top...

Cardiac transplantation is the treatment of choice for end-stage heart failure. Both technical advancements and pharmacological innovations have propelled the field to early graft acceptance rates above 90%.12,13 Despite this, 60-80% 5-year graft survival is at a standstill and chronic rejection, characterized by transplant vasculopathy, remains inevitable.14-16 Furthermore, patients are subjected to multiple surgical procedures and lifelong immunosuppression, which are associated with chest wall deformities and medical sequelae and toxicities, respectively. The need for innovative approaches to extend allograft survival, minimize the immunosuppressive requirements, and offer reconstructive options for anatomical deformities is pressing.
Vascularized composite allotransplantation offers a unique strategy for improving heart transplant outcomes both from an immunological aspect as well as a reconstructive perspective.17 Vascularized composite allografts are also unique in a way that they have an inherent source of donor-derived hematopoietic stem cells which has shown a favorable ability to reduce immunosuppression and induce and sustain mixed chimerism.1-8 Additionally, co-transplantation of the thymus has shown to prolong survival of both, solid organ transplants and vascularized composite allografts.2,9-11 Combining these strategies with heart transplantation offers a novel solution to the aforementioned challenges facing heart transplantation.18
Murine models serve as excellent platforms for mechanistic in vivo investigation because of the availability of antibodies and well-defined inbred and knockout strains.19-21 Although heart transplantation in mice is commonly studied using a heterotopic intraabdominal microsurgical suture transplant model22-25, a heterotopic, cervical, non-suture cuff technique model has shown to be extremely replicable, reliable, and carries fewer rates of thrombosis.19,26,27 The goal of this study is to develop a heterotopic en bloc osteomyocutaneous chest wall, thymus, and heart transplant technique in mice to study the immunological mechanisms of combined solid organ and vascularized composite allotransplantation using a cervical non-suture cuff technique. This cluster allograft is perfused through the anastomosis of the donor descending aorta to the right common carotid artery and the donor pulmonary artery to the right external jugular vein. Preservation of the internal thoracic vessels and associated thymus branches is paramount to perfusing the chest wall (sternum, ribs, muscles, and skin) and thymus.

<table><tbody><tr><td>Euro-Collins Solution</td><td></td><td></td><td>The solution is not commercially purchased but rather prepared in the laboratory. To make a 500 ml solution add the ingredient listed below to a 330 ml of double distilled water. Mix well, and then fill in the rest of the 170 ml of double distilled water into the solution to a final volume of 500 ml.&lt;br/&gt; Ingredients: 1.02 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 3.66 g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.56 g KCl, 0.42 g NaHCO&lt;sub&gt;3&lt;/sub&gt;, and 17.52 g of glucose.</td></tr><tr><td>Suture</td><td>Ethilon</td><td>MWI 72667</td><td>6-0 Ethilon


https://www.mwivet.com (MWI - Veterinary Supplies)</td></tr><tr><td>Polyimide Cuff Vein (21G)</td><td>Vention Medical</td><td>141-0043</td><td>http://www.ventionmedical.com/products-and-services/polyimide-tubing/</td></tr><tr><td>Polyimide Cuff Artery (24G)</td><td>Vention Medical</td><td>141-0027</td><td>http://www.ventionmedical.com/products-and-services/polyimide-tubing/</td></tr><tr><td>Soft plastic tip catheter</td><td>Terumo</td><td>SR*OX2419CA</td><td>24G x 3/4&quot;&amp;nbsp;</td></tr><tr><td>Microsurgical dilator</td><td>S&amp;amp;T</td><td>D-5a.1</td><td>Dilator, 11 cm, FH, 0.1 mm AT10d</td></tr></tbody></table>

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.

Singenico C57BL / 6 trapianti raggiunto sopravvivenza a lungo termine. Il disegno del eterologo (Figura 1) si sono dimostrati efficaci da una prospettiva di sopravvivenza degli animali e la capacità di valutare la sopravvivenza allograft corso. Questo è stato dimostrato attraverso la pelle sovrastante rimanente praticabile, crescita attiva dei capelli allogenico in corso, e battiti cardiaci sono stati in grado di valutare con la visualizzazione e il dito palpazione. Dati di sopravvivenza è rappresent...

Watch this Scientific Journal Video about A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice at JoVE.com

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

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 ...

a-novel-microsurgical-model-for-heterotopic-en-bloc-chest-wall-thymus

Research

JoVE Journal

Medicine

9.2K Views.  Johns Hopkins University School of Medicine. 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.

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

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

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

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

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

Murino cervicale Cuore trapianto Modello utilizzando una tecnica Cuff Modificato

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

Medicina

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Un metodo modificato per eterotopica mouse Cuore Trapianto

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

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 ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Eterotopico cervicale trapianto cardiaco nei topi

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Ottimizzazione della tecnica del bracciale per il trapianto di cuore di Murine

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Immunologia e infezioni

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Un modello pre-clinico di trapianto di cuore ortotopico

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Trapianto cardiaco cervicale eterotopico di topo utilizzando bracciali vascolari

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

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 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 ...