Questo lavoro è stato sostenuto, in parte, da una sovvenzione del NIH (RO1 HL098228) per CKB.

Nota: Tutte le procedure sugli animali sono state approvate dalla Hospital Institutional Animal Care ed uso Comitato dei Bambini di Nationwide. 1. Polmonare valvola cardiaca raccolta da un donatore Valvola cardiaca mouse <ol><li> Sterilizzare in autoclave tutti gli strumenti chirurgici prima della chirurgia: 1x forbici sottili, 3x micro pinze, pinze vascolari micro 2x, 1x morsetto pinze applicano, porta-aghi micro 1x, forbici primavera 1x, 1x divaricatore. </li><li> A 6-8 settimane di età femmina C57BL / 6 mouse viene utilizzato come donatore valvola cardiaca polmonare. Rimuovere il mouse dalla gabbia e si pesa, poi eutanasia con un cocktail di ketamina / xylazina (ketamina, 200 mg / kg e xylazina, 20 mg / kg, IP) overdose. </li><li> Agganciare la zona del torace e posizionare il mouse in una posizione prona dorsale su un taccuino. Poi fare la toracotomia. Esporre il cuore, fare un piccolo taglio sul atrio destro e profumato il ventricolo sinistro con ghiaccio soluzione fisiologica fredda. </li><li> Senza mezzi termini sezionare l&#39;arteria polmonare (PA) dal crescente unorta. Tagliare la valvola polmonare (PV) insieme a due millimetri bracciale dell&#39;arteria polmonare. Smaltire il resto del cuore. </li><li> Conservare il fotovoltaico in soluzione salina (100 unità / ml) di eparina freddo e. Nota: Il PV può essere mantenuto in soluzione per due ore prima del trapianto verso il cuore del donatore. </li></ol> 2. Preparazione donatore di cuore <ol><li> A 6-8 settimane di età femmina C57BL / 6 mouse viene utilizzato come un donatore di cuore. Rimuovere il mouse dalla gabbia e si pesa, poi eutanasia con un cocktail di ketamina / xylazina (ketamina, 200 mg / kg e xylazina, 20 mg / kg, IP) overdose. Questa è una procedura terminale. </li><li> Agganciare la zona del torace e posizionare il mouse in una posizione prona dorsale su un taccuino. Poi fare la toracotomia. Senza mezzi termini separare il cuore, vena cava inferiore (IVC), vena cava superiore (SVC), dell&#39;aorta ascendente, PA, e la vena polmonare. Profumato l&#39;IVC con il freddo ghiaccio soluzione salina sterile. </li><li> Legare l&#39;IVC, SVC, e vena polmonare con 6-0 sutura di seta poi tagliare superiore allegature. </li><li> Tagliare l&#39;aorta e PA 2 mm bracciale. </li><li> Tagliare il PV e smaltirlo. </li></ol> 3. Valvola cardiaca trapianto su un donatore di cuore <ol><li> Subito dopo la fase 2.5, posizionare la valvola cardiaca dal punto 1.5 nel cuore del donatore e orientare la valvola cardiaca. </li><li> Fissare la PV con un punto sul lato destro della valvola attraverso 10-0 monofilamento sugli aghi conici e inizia a suturare continuamente con 5-6 stiches dall&#39;altro lato del PV. </li><li> Dopo aver terminato il lato anteriore, ruotare cuore orizzontalmente e iniziare a suturare il lato posteriore del PV sul cuore del donatore. </li><li> Conservare il cuore in una soluzione fredda di eparina / soluzione salina sterile. Nota: Il cuore del donatore può essere mantenuto in soluzione per due ore prima dell&#39;impianto per il mouse destinatario. </li></ol> 4. Eterotopico trapianto cardiaco a un destinatario mouse <ol><li> A 6-8 settimane di età femmina C57BL / 6 topo è stato usato come un recipient. Rimuovere il mouse dalla gabbia e si pesa, poi anestetizzati con cocktail di ketamina / xilazina (ketamina, 100 mg / kg e xilazina 10 mg / kg). Ketoprofene (5 mg / kg) è usato come preanesthesia analgesico. </li><li> Dopo aver verificato il livello di sedazione per coda pizzicare, tagliare il addominale e peli del torace. Lubrificare gli occhi con pomata oftalmica sterile, e posizionare il mouse in una posizione prona dorsale su un taccuino. Disinfettare l&#39;addome con Betadine e alcool pastiglie. Poi coprire il mouse con un telino sterile ed esporre solo la zona di incisione. </li><li> Effettuare una laparotomia mediana incisione da sotto la xifoidea alla regione sovrapubica, e inserire un divaricatore auto ritegno. Avvolgere l&#39;intestino in soluzione salina inumidito garza. Senza mezzi termini definire l&#39;aorta sottorenale e la vena cava. </li><li> Inserire due 6-0 punti di sutura in seta prossimale e distale attorno alla aorta e IVC per limitare la circolazione del sangue. </li><li> Posizionare il cuore del donatore sul lato destro dell&#39;aorta addominale e coprire con gau sterileze. Idratare con soluzione fisiologica. </li><li> Effettuare una aortotomy nell&#39;aorta addominale utilizzando un ago G 30 ed estendere l&#39;apertura con le forbici alle dimensioni dell&#39;aorta donatore. </li><li> Eseguire un end-to side anastomosi con sterili suture monofilamento 10-0 sugli aghi conici. Fissare l&#39;aorta donatore con un punto sull&#39;estremità prossimale dell&#39;apertura nell&#39;aorta addominale e inizia a suturare continuamente con 4-5 stiches dall&#39;estremità distale dell&#39;aorta addominale. </li><li> Capovolgere il cuore a sinistra, coprire con garza salina infusa e iniziare a suturare continuamente con 4-5 stiches dall&#39;estremità distale dell&#39;aorta addominale. </li><li> Effettuare una venotomia nella IVC usando un ago G 30 ed estendere l&#39;apertura alla dimensione dell&#39;arteria polmonare donatore. </li><li> Eseguire la fine anastomosi lato utilizzando sterili suture monofilamento 10-0 sugli aghi conici. Fissare il donatore PA con un punto sull&#39;estremità prossimale dell&#39;apertura nella IVC e inizia a suturare continuamente con 4-5 stiches dal&#39;estremità distale della vena cava inferiore. Questa volta, perché l&#39;aorta è nel senso, si assicura la sutura della parete sinistra della PA del donatore è all&#39;interno della IVC. </li><li> Lavare lume IVC con eparina e soluzione salina (100 unità / ml). Chiudere la parete destra del donatore e del ricevente PA IVC suturando loro continuamente all&#39;estremità distale. </li><li> Togliere la legatura distale e controllare l&#39;emorragia applicando una topica agente emostatico sterile riassorbibile. Quando l&#39;emorragia si arresta completamente, rimuovere la sutura prossimale e controllare l&#39;emorragia allo stesso modo. </li><li> Ritorno intestini e chiudere la muscolatura addominale e la pelle in due strati con un 6-0 nero poliammide monofilamento di sutura. </li><li> Iniettare 0,5 ml di soluzione fisiologica per via sottocutanea e posizionare il mouse in una gabbia di recupero su un rilievo di riscaldamento fino a quando il mouse è completamente mobile. Al momento del recupero, ritorno il mouse in una nuova gabbia con biancheria da letto di carta. Dare farmaci antidolorifici (ibuprofene, 30 mg / kg, acqua potabile) per 48 ore. FareNon restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. </li></ol>

<ol>
	<li>Polito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+morbidity+and+mortality+in+very+preterm+VLBW+infants+with+congenital+heart+disease.">Increased morbidity and mortality in very preterm VLBW infants with congenital heart disease.</a> Intens Care Med. 39, 1104-1112 (2013).</li><li>Wren, C., Reinhardt, Z., Khawaja, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty+year+trends+in+diagnosis+of+life+threatening+neonatal+cardiovascular+malformations.">Twenty year trends in diagnosis of life threatening neonatal cardiovascular malformations.</a> Arch Dis Child Fetal. 93, F33-F35 (2008).</li><li>Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+transplantation+Third+annual+Samuel+Jason+Mixter+lecture.">Beyond transplantation Third annual Samuel Jason Mixter lecture.</a> Archives of surgery. 123, 545-549 (1960).</li><li>Tudorache, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+replacement+of+aortic+heart+valves+with+tissue-engineered+grafts.">Orthotopic replacement of aortic heart valves with tissue-engineered grafts.</a> Tissue engineering Part A. 19, 1686-1694 (2013).</li><li>van Geldorp, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+outcome+after+aortic+valve+replacement+with+a+mechanical+or+biological+prosthesis+weighing+lifetime+anticoagulant+related+event+risk+against+reoperation+risk.">Patient outcome after aortic valve replacement with a mechanical or biological prosthesis weighing lifetime anticoagulant related event risk against reoperation risk.</a> The Journal of thoracic and cardiovascular surgery. 137, 881-886 (2009).</li><li>El Oakley, R., Kleine, P., Bach, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choice+of+prosthetic+heart+valve+in+today's+practice.">Choice of prosthetic heart valve in today's practice.</a> Circulation. 117, 253-256 (2008).</li><li>Quinn, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Bench+to+Bedside+Translation+of+Regenerative+Cardiac+Constructs.">Animal Models for Bench to Bedside Translation of Regenerative Cardiac Constructs.</a> Progress in Pediatric cardiology. 35, (2013).</li><li>Shinoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+heart+valves+valve+leaflet+replacement+study+in+a+lamb+model.">Tissue engineering heart valves valve leaflet replacement study in a lamb model.</a> The Annals of thoracic surgery. 60, S513-S516 (1995).</li><li>Sodian, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+heart+valves+in+vitro+experiences.">Tissue engineering of heart valves in vitro experiences.</a> The Annals of thoracic surgery. 70, 140-144 (2000).</li><li>Shinoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineered+heart+valves.+Autologous+valve+leaflet+replacement+study+in+a+lamb+model.">Tissue engineered heart valves. Autologous valve leaflet replacement study in a lamb model.</a> Circulation. 94, II164-II168 (1996).</li><li>Sodian, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biodegradable+three+dimensional+matrices+for+tissue+engineering+of+heart+valves.">Evaluation of biodegradable three dimensional matrices for tissue engineering of heart valves.</a> ASAIO journal (American Society for Artificial Internal Organs. 46, 107-110 (1992).</li><li>Sodian, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+in+vivo+experience+with+tissue-engineered+trileaflet+heart+valves).">Early in vivo experience with tissue-engineered trileaflet heart valves).</a> Circulation. 102, III22-III29 (2000).</li></ol>

Non abbiamo nulla da rivelare.

Il tasso di mortalità di questa procedura è vicino al 20%, che è stato causato principalmente da emorragia nel sito di trapianto PV e anastomosi sull&#39;aorta donatore all&#39;aorta addominale destinatario. Nella maggior parte dei casi, il tasso di mortalità diminuisce significativamente chirurgia 48 hr postale. I topi hanno mostrato di sopravvivenza cuore batte forte e il flusso di sangue attraverso il fotovoltaico impiantato. L&#39;intero processo dura quattro ore per una micro chirurgo esperto. Ci vorranno circa...

Difetti cardiovascolari congenite sono una delle principali cause di mortalità infantile del 1,2 mondo occidentale. Tra questi, la stenosi valvolare polmonare e premolari difetti valvolari aortici sono una forma che si verificano di frequente 3. Intervento di sostituzione della valvola cardiaca è una scelta di routine di chirurgia ricostruttiva; Tuttavia, le complicanze, tra cui la stenosi e calcificazione della valvola cardiaca, e la dipendenza per tutta la vita su anticoagulanti sono una fonte significativa di malattie croniche e morte 4-7. Inoltre, la mancanza di potenziale di crescita richiede interventi di revisione, che aumenta ulteriormente la mortalità di questi pazienti giovani 4,8,9. Nel tentativo di sviluppare una valvola cardiaca sostituzione funzionale con potenziale di crescita, le cellule autologhe seminati Shinoka et al. Su una valvola cardiaca sintetico biodegradabile 8. La valvola sintetico trasformato in una valvola cardiaca nativa come la struttura con la crescita potenziometroal. Preliminari studi su animali di grandi dimensioni hanno dimostrato la possibilità di utilizzare questa metodologia per creare una valvola cardiaca funzionale 10. Tuttavia, gli studi di impianto lungo termine hanno dimostrato scarsa durata a causa del progressivo ispessimento della neotissue valvola con conseguente restringimento della valvola cardiaca. Lavorare da Sodian et al. Utilizzata la metodologia Shinoka, ma alla fine sostituito matrice PGA con un elastomero biodegradabile, che ha le proprietà biomeccaniche della valvola di ingegneria tessutale costruire un profilo più fisiologico 9,11,12. Nello studio in vivo, nonostante il successo del l&#39;impianto, un rivestimento delle cellule endoteliali confluenti non era formata che potrebbe limitare il successo a lungo termine di questo ponteggio 12. Al fine di progettare razionalmente una migliore valvola cardiaca sintetico di seconda generazione, un modello murino di trapianto di valvole cardiache è stato creato per studiare i meccanismi cellulari e molecolari underlyinformazione g neotissue, ispessimento della valvola, e lo sviluppo di stenosi. Modelli murini offrono una vasta gamma di reagenti molecolari, tra cui transgenici, che non sono facilmente disponibili in altre specie 7. In questo cuore modello di trapianto di valvole, un ex vivo singenico sostituzione della valvola cardiaca polmonare è stata eseguita prima; e poi il cuore con la valvola cardiaca impiantato è stato impiantato heterotopically in un ospite singenici utilizzando una tecnica microchirurgica. Questo modello permette la sostituzione della valvola cardiaca senza la necessità di bypass cardiopolmonare. In questo lavoro, una spiegazione dettagliata di un raccolto valvola cardiaca, preparazioni cuore del donatore, il trapianto di valvole cardiache, e il trapianto di cuore eterotopico è descritto. I risultati hanno mostrato un battito cardiaco continuo dal cuore del donatore, che era indipendente dal battito cardiaco destinatario. Il flusso sanguigno attraverso la valvola polmonare impiantato è stata misurata usando un sistema a ultrasuoni ad alta frequenza con onda pulsata Fareppler.

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		<tr height="20">
			<td height="20" style="height:20px;width:176px;">DPBS</td>
			<td style="width:167px;">gibco</td>
			<td style="width:133px;">14190-144</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:176px;">Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:133px;">M80</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:176px;">C57BL/6J (H-2b), Female</td>
			<td style="width:167px;">Jackson Laboratories&#160;</td>
			<td style="width:133px;">664</td>
			<td style="width:168px;">8-12 weeks</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Ketamine Hydrochloride Injection</td>
			<td style="width:167px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-2053</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Xylazine Sterile Solution</td>
			<td style="width:167px;">Akorn Inc.</td>
			<td style="width:133px;">NADA# 139-236</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">ketoprofen</td>
			<td style="width:167px;">Fort Dodge Animal Health</td>
			<td style="width:133px;">NDC 0856-4396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">Ibuprofen</td>
			<td style="width:167px;">PrecisionDose</td>
			<td style="width:133px;">NDC 68094-494-59</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Heparin Sodium</td>
			<td style="width:167px;">Sagent Pharmaceticals</td>
			<td style="width:133px;">NDC 25021-400</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Saline solution (Sterile 0.9% Sodium Chloride)</td>
			<td style="width:167px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-0138-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">0.9% Sodium Chloride Injection</td>
			<td style="width:167px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-4888-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:176px;">Petrolatum Ophthalmic Ointment</td>
			<td style="width:167px;">Dechra Veterinary Products</td>
			<td style="width:133px;">NDC 17033-211-38</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Iodine Prep Pads</td>
			<td style="width:167px;">Triad Disposables, Inc.</td>
			<td style="width:133px;">NDC 50730-3201-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Alcohol Prep Pads</td>
			<td style="width:167px;">McKesson Corp.</td>
			<td style="width:133px;">NDC 68599-5805-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Cotton tipped applicators</td>
			<td style="width:167px;">Fisher Sientific</td>
			<td style="width:133px;">23-400-118</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:176px;">Fine Scissor</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">14028-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Micro-Adson Forcep</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11018-12</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:176px;">Clamp Applying Forcep</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">00072-14</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">S&#38;T Vascular Clamp</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">00396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Spring Scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">15008-08</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Colibri Retractors</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">17000-04</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #5 Forcep</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11251-20&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #7 - Fine Forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11274-20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #5/45 Forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11251-35</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Tish Needle Holder/Forceps</td>
			<td style="width:167px;">Micrins</td>
			<td style="width:133px;">MI1540</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:176px;">Black Polyamide Monofilament Suture, 10-0</td>
			<td style="width:167px;">AROSurgical Instruments Corporation</td>
			<td style="width:133px;">TI638402</td>
			<td style="width:168px;">For sutureing the graft</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Black Polyamide Monofilament Suture, 6-0</td>
			<td style="width:167px;">AROSurgical Instruments&#160;</td>
			<td style="width:133px;">SN-1956</td>
			<td style="width:168px;">For musculature and skin closure</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Non-Woven Songes</td>
			<td style="width:167px;">McKesson Corp.</td>
			<td style="width:133px;">94442000</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Absorbable hemostat</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:133px;">1961</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:176px;">1 ml Syringe</td>
			<td style="width:167px;">BD</td>
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transplantation of pulmonary valve using a mouse model of heterotopic heart transplantation

Valvole cardiache di ingegneria tessutale, valvole soprattutto decellularized, stanno iniziando a guadagnare slancio in uso clinico di chirurgia ricostruttiva con risultati alterni. Tuttavia, i meccanismi cellulari e molecolari dello sviluppo neotissue, ispessimento della valvola, e lo sviluppo stenosi non sono studiati estesamente. Per rispondere a queste domande, abbiamo sviluppato una valvola cardiaca modello murino di trapianto eterotopico. Una valvola cardiaca è stato raccolto da un topo donatore valvola e trapiantato in un topo donatore di cuore. Il cuore con una nuova valvola è stato trapiantato heterotopically ad un mouse destinatario. Il cuore trapiantato ha mostrato il proprio battito cardiaco, indipendente battito cardiaco del destinatario. Il flusso di sangue è stato quantificato usando un sistema a ultrasuoni ad alta frequenza con un Doppler pulsato. Il flusso attraverso la valvola polmonare impiantata mostrato flusso in avanti con rigurgito minimo e il picco di flusso era vicino a 100 mm / sec. Questo modello murino di trapianto di valvola cardiaca è highly versatile, in modo che possa essere modificato e adattato per fornire diversi ambienti emodinamici e / o può essere usato con vari topi transgenici per studiare lo sviluppo neotissue in una valvola cardiaca ingegneria tessutale.

La Figura 1 illustra gli schemi della valvola modello di trapianto di cuore con trapianto di cuore eterotopico. La valvola cardiaca è stato raccolto da un donatore e impiantato su un cuore da un secondo mouse donatore. Poi il cuore con la nuova valvola cardiaca è stato impiantato all&#39;addome di un mouse destinatario. Figura 2 mostra una illustrazione del cuore impiantato nello spazio addominale (A), subito dopo trapianto cardiaco (B), e 5 minuti do...

Watch this Scientific Journal Video about Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation at JoVE.com

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

Al fine di comprendere i meccanismi cellulari e molecolari alla base neotissue formazione e lo sviluppo di stenosi in ingegneria tessutale valvole cardiache, è stato sviluppato un modello murino di trapianto di valvole cardiache eterotopico. Una valvola cardiaca polmonare è stato trapiantato al destinatario utilizzando la tecnica di trapianto di cuore eterotopico.

Il trapianto di valvola polmonare Utilizzando un modello murino di trapianto cardiaco eterotopico

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

transplantation-pulmonary-valve-using-mouse-model-heterotopic-heart

Research

JoVE Journal

Medicine

18.8K Views.  Nationwide Children's Hospital.  Al fine di comprendere i meccanismi cellulari e molecolari alla base neotissue formazione e lo sviluppo di stenosi in ingegneria tessutale valvole cardiache, è stato sviluppato un modello murino di trapianto di valvole cardiache eterotopico. Una valvola cardiaca polmonare è stato trapiantato al destinatario utilizzando la tecnica di trapianto di cuore eterotopico. 

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Un modello murino di Nell&#39;utero Trapianto

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Ricerca

Medicina

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 ...

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 ...

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.

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 ...

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 ...

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 ...

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 ...

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 Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Un protocollo di trapianto di cuore eterotopico suino per la somministrazione di terapie a un allotrapianto cardiaco

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

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 ...