M.-A. Deutsch is supported by Dr. Rusche Forschungsprojekt (2014) Deutsche Stiftung f&#252;r Herzforschung and Deutsche Gesellschaft f&#252;r Thorax- Herz- und Gef&#228;&#223;chirurgie. M. Krane is supported by Deutsche Stiftung f&#252;r Herzforschung (F/37/11), Deutsches Zentrum f&#252;r Herz Kreislauf Forschung (DZHK B 13-050A; DZHK B 14-013SE) and Deutsche Forschungsgemeinschaft (KR3770/7-1; KR3770/9-1)

Tutti gli esperimenti sono stati approvati dal tedesco Heart Center di Monaco di Baviera e il governo della Baviera, Germania (Gz. 55.2-1-54-2532.3-17-13). Gli animali sono stati alloggiati in una specifica struttura gratuito patogeno e hanno ricevuto assistenza umana in conformità ai &quot;Principi di laboratorio cura degli animali&quot; formulate dalla Società Nazionale per la ricerca medica e la &quot;Guida per la cura e l&#39;uso di animali da laboratorio&quot; fornita dalla National Academy di Scienze e pubblicato dal National Institutes of Health (NIH pubblicazione n. 86-23, rivisto 1985). NOTA: Un set completo di strumenti di microchirurgia sterili, tra cui due pinze curve e dilatatori nave, è necessario per la procedura (Figura 2a). La profilassi antibiotica perioperatoria non è di routine necessario ma può essere indicato in animali immunocompromessi. Gli animali sono stati sacrificati con il morsetto inalazione isoflurano. Donatori Preparazione e Graft Harvest &lt;ol&gt; <li> Anestetizzare l&#39;animale intraperitoneale (ip) iniezione di midazolam (5,0 mg / kg), medetomidina (0,5 mg / kg) e fentanyl (0,05 mg / kg di peso corporeo). Posizionare il mouse in posizione supina sul campo operatorio e confermare la tolleranza chirurgica dal riflesso recesso pedale. </li><li> Disinfettare la pelle tre volte con clorexidina e di eseguire una incisione addominale mediana. Ritrarre l&#39;intestino con garza umida verso sinistra per esporre la vena cava inferiore. </li><li> Iniettare 50 UI di eparina diluito in 0,4 ml di soluzione salina attraverso la vena cava inferiore per anticoagulante. Incidere la nave 1 min dopo eparinizzazione. </li><li> Aprire il diaframma ed eseguire una toracotomia bilaterale per esporre il cuore. La parete toracica anteriore si riflette superiormente e tenuta in posizione con una fascetta. </li><li> Rimuovere il timo e senza mezzi termini sezionare il tessuto connettivo tra aorta e tronco polmonare. Incidere la giusta vena cava superiore. </li><li> Forare il aorta ascendente mobilitato a livello dellatronco brachiocefalico con un ago da 30 G montato su una siringa da 5 ml e lentamente defluire in cuore retrogradually senza pressione eccessiva con 3 ml di 4 ° C la soluzione cardioplegica fredda. Evitare l&#39;ingresso di aria in circolo coronarico. Goccia soluzione salina ghiacciata sul trapianto per migliorare ulteriormente l&#39;arresto cardiaco. </li><li> Transect l&#39;aorta prossimalmente all&#39;origine del tronco brachiocefalico. </li><li> Legare il cavae inferiore ed entrambi Venae superiore vicino al cuore con 8-0 seta e transetto loro distalmente alle legature. </li><li> Sezionare gratis il tronco polmonare distale possibile. Legare le arterie polmonari vicino alla biforcazione con 8-0 seta e transetto li distale. </li><li> Legare le vene polmonari tutti insieme con un unico legatura 8-0 seta e transetto. </li><li> Rimuovere il cuore del donatore e conservarla in soluzione cardioplegica ghiacciata fino impianto. </li></ol> 2. Destinatario Preparazione <ol><li> Prepararepolsini per anastomosi tagliando il tubo poliimmide con un bisturi No. 11 o una forbice sotto il microscopio. Il bracciale è costituito da un corpo cilindrico ed una estensione (1/3 della circonferenza) per il posizionamento della pinza vascolare, ciascuna di una lunghezza di 1 mm (Figura 2b). </li><li> Anestetizzare l&#39;animale e confermare la tolleranza chirurgica come descritto sopra. Iniettare 0,25 ml di soluzione salina calda per via sottocutanea (sc) per la sostituzione del fluido. </li><li> Rimuovere i capelli della regione cervicale destra con un rasoio elettrico e posto l&#39;animale in posizione supina su un tappetino caldo. Fissare le gambe con fili di nastro adesivo, evitando pressioni eccessive gli arti anteriori come questo può compromettere la respirazione. Coprire gli occhi con pomata oftalmica per prevenire la secchezza. Disinfettare il sito chirurgico tre volte con clorexidina. </li><li> Fare un incisione cutanea trasversale dall&#39;angolo mandibolare diritto alla tacca giugulare. </li><li> Senza mezzi termini mobilitare il external vena giugulare. Cauterize rami laterali con una pinza bipolare e transetto. </li><li> Dividere la vena giugulare esterna tra legature a livello della confluenza con la vena dalla ghiandola sottomandibolare. </li><li> Estrarre la vena giugulare esterna attraverso il bracciale e occludere il vaso prossimalmente posizionando un morsetto vascolare sull&#39;estensione del bracciale. Togliere la legatura ed irrigare il lume del vaso con soluzione salina eparinizzata 01:10. </li><li> Evert la parete del vaso sopra il bracciale e fissarlo con un preset circonferenziale 8-0 legatura di seta. </li><li> Togliere la parte superficiale del muscolo sternocleidomastoideo. </li><li> Mobilitare il diritto carotide comune e transetto della nave tra le legature di sotto della biforcazione carotidea. </li><li> Estrarre carotide attraverso il bracciale e occludere il vaso prossimalmente posizionando un morsetto Yasargil sull&#39;estensione del bracciale. Togliere la legatura ed irrigare il lume del vaso con soluzione salina eparinizzata 01:10. </li><li> Delicatamente Dilate il lume arterioso con un dilatatore vaso, Evert la parete del vaso sopra il bracciale e risolvere il problema con un preset circonferenziale 8-0 legatura di seta. </li><li> Per guadagnare spazio sufficiente per innesto impianto, rimuovere il lobo destro della ghiandola sottomandibolare. </li><li> Mantenere il campo operatorio umido finché l&#39;impianto. </li></ol> 3. Graft impianto <ol><li> Posizionare l&#39;innesto nella regione cervicale destinatario testa in giù con l&#39;aorta orientato medialmente e l&#39;arteria polmonare lateralmente. </li><li> Estrarre l&#39;aorta dell&#39;innesto sul bracciale con la carotide e fissare la anastomosi con una legatura circonferenziale 8-0 seta. </li><li> Incidere il tronco polmonare sul suo aspetto anteriore e tirarlo sopra il bracciale con la vena giugulare esterna. Anche in questo caso garantire l&#39;anastomosi con una legatura circonferenziale 8-0 seta. </li><li> Rimuovere la fascetta sulla vena giugulare esterna, seguendo dal sbloccaggio della carotide. I cuori riempie immediatamente con il sangue. Per migliorare reperfusion goccia 37 ° C soluzione salina calda sul trapianto. Normalmente il cuore si sviluppa ritmo sinusale entro un minuto. Rimuovere l&#39;estensione bracciale al sito venoso. </li><li> Garantire la posizione dell&#39;innesto corretta, anastomosi devono essere privi di tensione e di navi senza torsione. Non vasi cauterizzata della pelle in quanto ciò può compromettere la guarigione della ferita. Il sanguinamento di solito si arresta spontaneamente. Chiudere la pelle con un solo 8-0 continuo di sutura. </li></ol> 4. Cura postoperatoria <ol><li> Iniettare 0,25 ml soluzione salina calda sc per la sostituzione del fluido. </li><li> Inimicarsi narcotici iniettare naloxone (1,2 mg / kg), flumazenil (0,5 mg / kg) e atipamezol (2,5 mg / kg di peso corporeo) sc sinistra l&#39;animale sul pad caldo fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Osservare l&#39;animale per almeno un&#39;ora aggiuntiva di registrare un comportamento anomalo. Non restituire un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino al completo Recoverosso. </li><li> Fornire preemptive analgesia con buprenorfina (0,05 mg / kg sc tre volte al giorno) o un&#39;analgesia alternativa secondo un protocollo istituzionale locale per una durata minima di 72 ore dopo la procedura. </li><li> Il chirurgo e un veterinario dovrebbe vedere regolarmente animali. In caso di perdita di peso&gt; 15%, apatia, gonfiore o chirurgiche animali di infezione lato sono eutanasia con il morsetto inalazione isoflurano. </li></ol>

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	<li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+transplantation+in+congenic+strains+of+mice.">Heart transplantation in congenic strains of mice.</a> Transplant Proc. 5, 733-735 (1973).</li><li>Chen, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+of+cervical+heterotopic+heart+transplantation+in+mice.">A technique of cervical heterotopic heart transplantation in mice.</a> Transplantation. 52, 1099-1101 (1991).</li><li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+graft+function+in+rodent+models+of+heart+transplantation.">Assessment of graft function in rodent models of heart transplantation.</a> Microsurgery. 28, 565-570 (2008).</li><li>Schramm, 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=The+subepicardial+microcirculation+in+heterotopically+transplanted+mouse+hearts:+an+intravital+multifluorescence+microscopy+study.">The subepicardial microcirculation in heterotopically transplanted mouse hearts: an intravital multifluorescence microscopy study.</a> J Thorac Cardiovasc Surg. 134, 210-217 (2007).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> J Vis Exp : JoVE. 238, (2007).</li><li>Gong, 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=Introduction+of+modified+cervical+cardiac+transplant+model+in+mice.">Introduction of modified cervical cardiac transplant model in mice.</a> Exp Clin Transplant. 10, 158-162 (2012).</li><li>Matsuura, A., Abe, T., Yasuura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+mouse+cervical+heart+transplantation+using+a+cuff+technique.">Simplified mouse cervical heart transplantation using a cuff technique.</a> Transplantation. 51, 896-898 (1991).</li><li>Chen, H., Zhang, Y., Zheng, D., Praseedom, R. K., Dong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+kidney+transplantation+in+mice:+technique+using+cuff+for+renal+vein+anastomosis.">Orthotopic kidney transplantation in mice: technique using cuff for renal vein anastomosis.</a> PloS one. 8, e77278 (2013).</li><li>Maglione, M., 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=A+novel+technique+for+heterotopic+vascularized+pancreas+transplantation+in+mice+to+assess+ischemia+reperfusion+injury+and+graft+pancreatitis.">A novel technique for heterotopic vascularized pancreas transplantation in mice to assess ischemia reperfusion injury and graft pancreatitis.</a> Surgery. 141, 682-689 (2007).</li><li>Sucher, 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> J Vis Exp : JoVE. , (2010).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47-50 (1979).</li><li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+rats.">Orthotopic liver transplantation in rats.</a> J Vis Exp : JoVE. , (2012).</li><li>Okazaki, M., 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=A+mouse+model+of+orthotopic+vascularized+aerated+lung+transplantation.">A mouse model of orthotopic vascularized aerated lung transplantation.</a> Am J Transplant. 7, 1672-1679 (2007).</li><li>Suzuki, H., Fan, L., Wilkes, 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=Development+of+obliterative+bronchiolitis+in+a+murine+model+of+orthotopic+lung+transplantation.">Development of obliterative bronchiolitis in a murine model of orthotopic lung transplantation.</a> J Vis Exp : JoVE. , (2012).</li><li>Zou, Y., 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=Mouse+model+of+venous+bypass+graft+arteriosclerosis.">Mouse model of venous bypass graft arteriosclerosis.</a> Am J Pathol. 153, 1301-1310 (1998).</li><li>Zhou, Y., Gu, X., Xiang, J., Qian, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+suture+versus+cuff+anastomosis+in+mouse+cervical+cardiac+transplant.">A comparative study on suture versus cuff anastomosis in mouse cervical cardiac transplant.</a> Exp Clin Transplant. 8, 245-249 (2010).</li><li>Niimi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+for+heterotopic+cardiac+transplantation+in+mice:+experience+of+3000+operations+by+one+surgeon.">The technique for heterotopic cardiac transplantation in mice: experience of 3000 operations by one surgeon.</a> J Heart Lung Transplant. 20, 1123-1128 (2001).</li><li>Arras, M., Autenried, P., Rettich, A., Spaeni, D., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intraperitoneal+injection+anesthesia+in+mice:+drugs,+dosages,+adverse+effects,+and+anesthesia+depth.">Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.</a> Comp Med. 51, 443-456 (2001).</li></ol>

The authors have nothing to disclose.

Abbiamo completato oltre 200 trapianti cardiaci con una percentuale di successo superiore al 90% in diversi contesti sperimentali. Con la tecnica qui descritta, la procedura completa può essere eseguita entro 60 minuti da un chirurgo esperto. Preparando i vasi cervicali prima reperimento di organi, il tempo totale ischemia innesto può essere limitata a 20 min. Tempo per l&#39;impianto (tempo di ischemia calda) è molto più breve con la tecnica gambetto rispetto alla tecnica di sutura 16. <p class="jove...

Il modello di trapianto di cuore è spesso utilizzato per studiare / R-infortunio io, rigetto post-trapianto vasculopatia e l&#39;efficienza degli agenti immunomodulatori. Modelli murini uniscono numerosi vantaggi come la immunologico noto e background genetico, la disponibilità di molti ceppi inbred e transgenici e relativamente bassi costi sperimentali. Nel 1973, una tecnica di trapianto di cuore addominale nei topi è stato descritto da Corry et al 1. In questo modello innesti vengono rivascolarizzati entro end-to-side anastomosi dell&#39;aorta ascendente alla aorta addominale del destinatario e della principale arteria polmonare per la vena cava inferiore. Nel 1991, una tecnica di sutura per il trapianto di cuore cervicale nei topi è stato descritto da Chen ed altri 2. In questo modello un retrogrado perfusione coronarica è stabilito da anastomosi della carotide del destinatario per l&#39;aorta ascendente del grpoppa. Gli scarichi sangue venoso attraverso il seno coronarico nell&#39;atrio destro, il ventricolo destro e viene espulso attraverso l&#39;arteria polmonare nel ricevente vena giugulare esterna (Figura 1). In confronto al trapianto di impianto addominale il modello cervicale è meno invasiva e la posizione superficiale del trapianto permette un&#39;accessibilità molto facile per gli esami di follow-up aggiuntivi (ad esempio, la palpazione, l&#39;ecocardiografia, la microscopia a fluorescenza intravitale) 3,4. Nonostante i miglioramenti tecnici 5,6, un uso più diffuso di questo modello è stato limitato a causa di difficoltà tecniche per eseguire anastomosi suturati dei piccoli vasi con un elevata incidenza delle perdite anastomosi e trombosi. Nel 1991, Matsuura et al. introdotto la tecnica bracciale in cui la parete del vaso è estroflesso su un cilindro sintetico, che facilita notevolmente il modello di trapianto di cuore cervicale 7. La tecnica era Further adottato per vari modelli sperimentali di trapianto renale, tra cui 8, 9 del pancreas, degli arti 10, epatica 11,12, 13,14 e trapianto polmonare vascolare 15. Rispetto alla tecnica di sutura il tempo richiesto per anastomosi e, quindi, il tempo di ischemia calda è più breve e ci sono complicazioni vascolari significativamente meno con la tecnica bracciale 16. Inoltre, esso consente al chirurgo con formazione minima in microchirurgia per eseguire l&#39;operazione con un alto tasso di successo. Uno svantaggio di questa tecnica è il bracciale legatura prerequisito della carotide comune, che possono alterare la perfusione cerebrale. In questo articolo il video vi presentiamo una tecnica bracciale migliorato e semplificato per il trapianto di cuore cervicale nei topi.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps JFC-7.2</td>
			<td>S&#38;T</td>
			<td>P-00036</td>
			<td>Curved tip 0.20 mm</td>
		</tr>
		<tr>
			<td>Vessel dilatator D-5a</td>
			<td>S&#38;T</td>
			<td>S-00124</td>
			<td></td>
		</tr>
		<tr>
			<td>Adventita scissor</td>
			<td>S&#38;T</td>
			<td>S-00102</td>
			<td></td>
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		<tr>
			<td>Vascular clamp B-1V</td>
			<td>S&#38;T</td>
			<td>S-00396</td>
			<td></td>
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		<tr>
			<td>Yasargil Clip</td>
			<td>Peter Lazic</td>
			<td>65,097</td>
			<td>3.5 mm lenght</td>
		</tr>
		<tr>
			<td>Bipolar forceps</td>
			<td>Micromed</td>
			<td>148-100-011</td>
			<td>tip 0.25 mm</td>
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		<tr>
			<td>Polyimide tubes</td>
			<td>River Tech Medical</td>
			<td></td>
			<td>19-24 gauge</td>
		</tr>
		<tr>
			<td>8.0 silk ligatures</td>
			<td>Catgut</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Custodiol HTK solution</td>
			<td>Essential Pharmaceuticals</td>
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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.

La nostra squadra ha potuto svolgere con successo la tecnica descritta dopo circa 20 interventi chirurgici di formazione. La parte più difficile è per rovesciare la parete del vaso della carotide sul bracciale, perché gira facilmente avanti e tentativi multipli può portare alla lacerazione della parete. Dopo il periodo di formazione, il tempo operatorio totale media era di 60 ± 8 min. Circa 20 minuti sono necessari per la raccolta dei donatori, 25 minuti per la preparazione dei vasi cervicali e 15 minuti per anasto...

Watch this Scientific Journal Video about Heterotopic Cervical Heart Transplantation in Mice at JoVE.com

Heterotopic Cervical Heart Transplantation in Mice

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 ...

heterotopic-cervical-heart-transplantation-in-mice

Research

JoVE Journal

Medicine

11.1K Views.  German Heart Center Munich, Technische Universität München. 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.

Video: Heterotopic Cervical Heart Transplantation in Mice

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controllata cervicale lesioni lacerazione nei topi

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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 ...

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 ...

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 ...

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

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

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

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 ...

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 ...

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

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

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

Cateterizzazione cardiaca nei topi per misurare il rapporto pressione del volume: Indagare l&#39;effetto Bowditch

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

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 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.

Eterotopico cervicale trapianto cardiaco nei topi

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