Questo lavoro è stato supportato dal Esercito, Marina, NIH, Aeronautica, VA e Health Affairs per sostenere lo sforzo AFIRM II, sotto Premio n W81XWH-13-2-0053. L&#39;US Army Medical Research di acquisizione di attività, 820 Chandler Street, Fort Detrick MD 21.702-5.014 è l&#39;Ufficio acquisizione assegnazione e la gestione. Opinioni, interpretazioni, conclusioni e raccomandazioni sono quelle dell&#39;autore e non sono necessariamente condivise dal Dipartimento della Difesa. Gli autori desiderano ringraziare Jessica Izzi, DVM, Caroline Garrett, DVM e Julie Watson, DVM per il loro eccellente supporto veterinario nel corso di questo studio.

Tutti gli esperimenti sono stati condotti in conformità con la guida per la cura e l&#39;uso di animali da laboratorio del National Institute of Health (NIH) e sono stati approvati dal Comitato Johns Hopkins University Animal Care e Usa (JHUACUC). Le procedure specifiche sono state eseguite sotto la MO13M108 protocollo ACUC approvato. Operazione 1. donatore <ol><li> Somministrare l&#39;analgesia al punto momento opportuno per ogni formulazione farmacologica prima di un intervento chirurgico. Come per la cura degli animali approvato e l&#39;uso l&#39;uso del protocollo 0,1 mg / kg di peso corporeo di buprenorfina per via sottocutanea 1 ora prima della incisione cutanea. </li><li> Sedare il donatore con isoflurano applicato attraverso una camera collegata ad un vaporizzatore isoflurano al 4%; mantenere la sedazione e l&#39;anestesia al 2% attraverso un cono di naso. Eseguire punta pizzico ritiro di riflessione per monitorare la profondità dell&#39;anestesia prima dell&#39;inizio della procedura. </li><li> Indossare maschere, abiti di isolamento monouso e guanti. </li><li> Radere l&#39;ar chirurgicaEA, in particolare l&#39;arto posteriore e l&#39;inguine, e preparazione con il 10% Povidone - iodio. </li><li> Utilizzare un drappo campo sterile, strumenti in autoclave e un microscopio ad alto ingrandimento (40X). </li><li> Rendere all&#39;inguine incisione cutanea usando forbici prossimalmente alla metà coscia e circonferenzialmente collegare l&#39;incisione per delimitare l&#39;arto posteriore dal resto del corpo del mouse. </li><li> Identificare e sezionare l&#39;arteria femorale, vena e nervo. Separare tutte le tre strutture con pinze e micro-forbici. </li><li> Una volta che il peduncolo vascolare è sezionato dividere i vasi a livello del legamento inguinale con le forbici micro. </li><li> Successivamente, continuare a dividere i singoli ventrale (gracilis e muscoli della coscia mediale) e gruppi muscolari dorsali 20 prossimalmente a livello della metà coscia per separare l&#39;innesto dal donatore con le forbici. </li><li> Transect femore e tagliare a metà del femore con le forbici. </li><li> L&#39;eutanasia degli animali da isoflurano fol overdoselowed da dislocazione cervicale. Confermare la cessazione del battito cardiaco e la respirazione. </li><li> Lavare l&#39;arto con 2 ml eparinizzate (30 IE) fredda (4 ° C) saline utilizzando un 33 G lavaggio ago montato su una siringa (vedi Materiali Tabella). </li><li> Posizionare un bracciale in poliimmide la vena femorale e l&#39;arteria, rispettivamente. </li><li> Avvolgere trapianto in garza di cotone bagnato, posto nella capsula di Petri e conservare a 4 ° C fino a quando nel riquadro. </li></ol> 2. Destinatario Funzionamento <ol><li> La rimozione del dell&#39;arto posteriore <ol><li> Somministrare l&#39;analgesia al punto momento opportuno per ogni formulazione farmacologica prima di un intervento chirurgico. Come per la cura degli animali approvato e l&#39;uso l&#39;uso del protocollo 0,1 mg / kg di peso corporeo di Buprenorfina SC 1 ora prima della incisione cutanea. </li><li> Sedare il donatore con isoflurano applicato attraverso una camera collegata ad un vaporizzatore isoflurano al 4%; mantenere la sedazione e l&#39;anestesia al 2% attraverso un cono di naso. Eseguire punta pizzico ritiro di riflessione per monitorare il repartoh di anestesia prima dell&#39;inizio del procedimento. </li><li> Usare pomata veterinario gli occhi del mouse per prevenire la secchezza mentre sotto anestesia. </li><li> Radere l&#39;area chirurgica, in particolare l&#39;arto posteriore e l&#39;inguine e preparazione con il 10% Povidone - iodio. </li><li> Rendere all&#39;inguine incisione cutanea usando forbici prossimalmente alla metà coscia e circonferenzialmente collegare l&#39;incisione per delimitare l&#39;arto posteriore dal resto del corpo del mouse. </li><li> Identificare e sezionare l&#39;arteria femorale, vena e nervo e separare tutte le tre strutture con pinze e micro-forbici. </li><li> Una volta che il peduncolo vascolare è sezionato, bloccare i vasi femorali a livello del legamento inguinale. </li><li> Tagliare i vasi distale a livello dell&#39;arteria epigastrica superficiale. </li><li> Successivamente, continuare a dividere i singoli ventrale (gracilis e muscoli della coscia mediale) e dorsali gruppi muscolari 20 prossimalmente a livello della metà coscia per separare il hind li nativamb dagli animali destinatario utilizzando le forbici. </li><li> Transect il femore nel mezzo della diafisi femorale con le forbici. </li><li> Cauterize muscoli della coscia precedentemente sezionati per prevenire le emorragie del sito dissezione e, quindi, la perdita di sangue destinatario. </li></ol></li><li> Impianto <ol><li> Minimizzare la perdita di fluido irrigando il campo operatorio con soluzione fisiologica tiepida (37 ° C) e l&#39;iniezione di 0,3 ml di soluzione salina calda prima e dopo l&#39;operazione. </li><li> Inserire l&#39;innesto in modo da riflettere la posizione anatomica accurata dell&#39;arto posteriore nativa allineando dell&#39;osso femore del destinatario e l&#39;innesto e collegate con un ago spinale 20 G come un chiodo endomidollare. </li><li> Coapt i gruppi muscolari ventrale e dorsale con materiale di sutura riassorbibile (6-0 Polysorb). </li><li> Collegare vasi femorali con la tecnica bracciale non suture; in dettaglio, tirare il lato ricevente della nave sui polsini precedentemente montate sulla nave estremità del graft. Utilizzare una sutura in nylon 10-0 ed eseguire una cravatta circonferenziale per fissare la nave ricevente sul bracciale. </li><li> Successivo rilasciare le fascette. In questa fase verificare visivamente la rotazione bracciale e un posizionamento ottimale per evitare errori di rotazione e piegamento dei vasi. </li><li> Eseguire emostasi meticolosa con cauterizzazione electro con una particolare attenzione per l&#39;interfaccia del donatore ricevente muscolare e le estremità delle ossa. </li><li> Chiudere la pelle con punti di sutura in nylon non assorbibili (6-0 ETHILON). </li><li> Stabilire condizioni normotermici consentendo all&#39;animale di recuperare nella sua gabbia sotto una lampada riscaldante. Continuare monitoraggio regolare per almeno 4 ore prima di ritornare alla struttura alloggiativa. </li><li> Fornire analgesia post-operatoria con buprenorfina alla dose di 0,1 mg / kg SC ogni 6-8 ore per 3 giorni. </li></ol></li></ol>

<ol>
	<li>Khalifian, S., 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=Facial+transplantation:+the+first+9+years.">Facial transplantation: the first 9 years.</a> Lancet. , (2014).</li><li>Petruzzo, P., Dubernard, J. 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+International+Registry+on+Hand+and+Composite+Tissue+allotransplantation.">The International Registry on Hand and Composite Tissue allotransplantation.</a> Clin. Transpl. , 247-253 (2011).</li><li>Shores, J. T., Brandacher, G., Lee, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+and+Upper+Extremity+Transplantation:+An+Update+of+Outcomes+in+the+Worldwide+Experience.">Hand and Upper Extremity Transplantation: An Update of Outcomes in the Worldwide Experience.</a> Plast. Reconstr. Surg. , (2014).</li><li>Levi, D. 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=Transplantation+of+the+abdominal+wall.">Transplantation of the abdominal wall.</a> Lancet. 361, 2173-2176 (2003).</li><li>Strome, 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=Laryngeal+transplantation+and+40-month+follow-up.">Laryngeal transplantation and 40-month follow-up.</a> N.Engl.J. Med. 344, 1676-1679 (2001).</li><li>Rose, K. G., Sesterhenn, K., Wustrow, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+allotransplantation+in+man.">Tracheal allotransplantation in man.</a> Lancet. 1, 433 (1979).</li><li>Hofmann, G. O., 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=Allogeneic+vascularized+transplantation+of+human+femoral+diaphyses+and+total+knee+joints--first+clinical+experiences.">Allogeneic vascularized transplantation of human femoral diaphyses and total knee joints--first clinical experiences.</a> Transplant. Proc. 30, 2754-2761 (1998).</li><li>Hu, 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=A+preliminary+report+of+penile+transplantation.">A preliminary report of penile transplantation.</a> Eur. Urol. 50, 851-853 (2006).</li><li>Brannstrom, 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=Livebirth+after+uterus+transplantation.">Livebirth after uterus transplantation.</a> Lancet. , (2014).</li><li>Sarhane, K. 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=Diagnosing+skin+rejection+in+vascularized+composite+allotransplantation:+advances+and+challenges.">Diagnosing skin rejection in vascularized composite allotransplantation: advances and challenges.</a> Clin. Transplant. 28, 277-285 (2014).</li><li>Schneeberger, S., Khalifian, S., Brandacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+and+monitoring+of+rejection+in+hand+transplantation.">Immunosuppression and monitoring of rejection in hand transplantation.</a> Tech. Hand Up. Extrem. Surg. 17, 208-214 (2013).</li><li>Lee, W. P., 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=Relative+antigenicity+of+components+of+a+vascularized+limb+allograft.">Relative antigenicity of components of a vascularized limb allograft.</a> Plast. Reconstr. Surg. 87, 401-411 (1991).</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=Hemiface+allotransplantation+in+the+mouse.">Hemiface allotransplantation in the mouse.</a> Plast. Reconstr. Surg. 129, 867-870 (2012).</li><li>Ibrahim, Z., 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+modified+heterotopic+swine+hind+limb+transplant+model+for+translational+vascularized+composite+allotransplantation+(VCA)+research.">A modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (VCA) research.</a> J Vis Exp. , (2013).</li><li>Oberhuber, 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=Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique.">Murine cervical heart transplantation model using a modified cuff technique.</a> J Vis Exp. , (2014).</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=Mouse+hind+limb+transplantation:+a+new+composite+tissue+allotransplantation+model+using+nonsuture+supermicrosurgery.">Mouse hind limb transplantation: a new composite tissue allotransplantation model using nonsuture supermicrosurgery.</a> Transplantation. 90, 1374-1380 (2010).</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. , (2010).</li><li>Zou, Y., Brandacher, G., Margreiter, R., Steurer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cervical+heterotopic+arterialized+liver+transplantation+in+the+mouse.">Cervical heterotopic arterialized liver transplantation in the mouse.</a> J. Surg. Res. 93, 97-100 (2000).</li><li>Zhang, F., 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=Development+of+a+mouse+limb+transplantation+model.">Development of a mouse limb transplantation model.</a> Microsurgery. 19, 209-213 (1999).</li><li>Schneeberger, S., 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=Upper-extremity+transplantation+using+a+cell-based+protocol+to+minimize+immunosuppression.">Upper-extremity transplantation using a cell-based protocol to minimize immunosuppression.</a> Ann. Surg. 257, 345-351 (2013).</li><li>Azari, K., Brandacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+composite+allotransplantation.">Vascularized composite allotransplantation.</a> Curr Opin Organ Transplant. 18, 631-632 (2013).</li><li>Pomahac, B., Gobble, R. M., Schneeberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facial+and+hand+allotransplantation.">Facial and hand allotransplantation.</a> Cold Spring Harb. Perspect. Med. 4, (2014).</li><li>Chong, A. S., Alegre, M. L., Miller, M. L., Fairchild, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+and+limits+of+mouse+models.">Lessons and limits of mouse models.</a> Cold Spring Harb. Perspect. Med. 3, a015495 (2013).</li><li>Lin, C. H., 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+neck+as+a+preferred+recipient+site+for+vascularized+composite+allotransplantation+in+the+mouse.">The neck as a preferred recipient site for vascularized composite allotransplantation in the mouse.</a> Plast. Reconstr. Surg. 133, 133e-141e (2014).</li><li>Shapiro, R. I., Cerra, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+reimplantation+and+transplantation+of+a+complex+organ:+the+rat+hind+limb.">A model for reimplantation and transplantation of a complex organ: the rat hind limb.</a> J. Surg. Res. 24, 501-506 (1978).</li><li>Leto Barone, A. 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=The+gracilis+myocutaneous+free+flap+in+swine:+an+advantageous+preclinical+model+for+vascularized+composite+allograft+transplantation+research.">The gracilis myocutaneous free flap in swine: an advantageous preclinical model for vascularized composite allograft transplantation research.</a> Microsurgery. 33, 51-55 (2013).</li><li>Mathes, D. 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=A+preclinical+canine+model+for+composite+tissue+transplantation.">A preclinical canine model for composite tissue transplantation.</a> J. Reconstr. Microsurg. 26, 201-207 (2010).</li><li>Barth, R. N., 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=Prolonged+survival+of+composite+facial+allografts+in+non-human+primates+associated+with+posttransplant+lymphoproliferative+disorder.">Prolonged survival of composite facial allografts in non-human primates associated with posttransplant lymphoproliferative disorder.</a> Transplantation. 88, 1242-1250 (2009).</li><li>Brandacher, G., Grahammer, J., Sucher, R., Lee, W. P. <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+basic+and+translational+research+in+reconstructive+transplantation.">Animal models for basic and translational research in reconstructive transplantation.</a> Birth Defects Res C Embryo Today. 96, 39-50 (2012).</li><li>Foster, R. D., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+hindlimb+transplantation+in+the+mouse.">Orthotopic hindlimb transplantation in the mouse.</a> J. Reconstr. Microsurg. 19, 49 (2002).</li><li>Tung, T. H., Mohanakumar, T., Mackinnon, S. E. <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+a+Mouse+Model+for+Heterotopic+Limb+and+Composite-Tissue+Transplantation.">Development of a Mouse Model for Heterotopic Limb and Composite-Tissue Transplantation.</a> J. Reconstr. Microsurg. 17, 267-274 (2001).</li><li>Zhang, F., Shi, D. Y., Kryger, Z., Moon, W. <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+a+mouse+limb+transplantation+model.">Development of a mouse limb transplantation model.</a> Microsurgery. 19 (5), 209-213 (1999).</li><li>Schneeberger, S., 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=Atypical+acute+rejection+after+hand+transplantation.">Atypical acute rejection after hand transplantation.</a> Am. J. Transplant. 8, 688-696 (2008).</li><li>Mathes, D. 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=Stable+mixed+hematopoietic+chimerism+permits+tolerance+of+vascularized+composite+allografts+across+a+full+major+histocompatibility+mismatch+in+swine.">Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine.</a> Transpl. Int. 27, 1086-1096 (2014).</li><li>Yamada, 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=Use+of+CTLA4Ig+for+induction+of+mixed+chimerism+and+renal+allograft+tolerance+in+nonhuman+primates.">Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates.</a> Am. J. Transplant. 14, 2704-2712 (2014).</li><li>Sachs, D. H., Kawai, T., Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+tolerance+through+mixed+chimerism.">Induction of tolerance through mixed chimerism.</a> Cold Spring Harb. Perspect. Med. 4, a015529 (2014).</li><li>Kawai, 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=HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> N. Engl. J. Med. 358, 353-361 (2008).</li><li>Kawai, T., Sachs, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+induction:+hematopoietic+chimerism.">Tolerance induction: hematopoietic chimerism.</a> Curr Opin Organ Transplant. 18, 402-407 (2013).</li><li>Kawai, T., Sachs, D. H., Sykes, M., Cosimi, A. .. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-mismatched+renal+transplantation+without+maintenance+immunosuppression.">HLA-mismatched renal transplantation without maintenance immunosuppression.</a> N. Engl. J. Med. 368, 1850-1852 (2013).</li><li>Leventhal, J., 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=Chimerism+and+tolerance+without+GVHD+or+engraftment+syndrome+in+HLA-mismatched+combined+kidney+and+hematopoietic+stem+cell+transplantation.">Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation.</a> Sci. Transl. Med. 4, 124-128 (2012).</li><li>Cendales, L. C., 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+Banff+2007+working+classification+of+skin-containing+composite+tissue+allograft+pathology.">The Banff 2007 working classification of skin-containing composite tissue allograft pathology.</a> Am. J. Transplant. 8, 1396-1400 (2008).</li></ol>

The authors declare that they have no competing financial interest.

Vascolarizzato Composite allotrapianto, come ad esempio arti superiori e il trapianto di faccia per la ricostruzione di difetti del tessuto devastanti, si è evoluto come opzione di trattamento valido per i pazienti non modificabili a procedure ricostruttive tradizionali. I progressi tecnici nel campo della microchirurgia ricostruttiva, nonché una vasta esperienza con immunosoppressori potenti e le terapie modulatori del sistema immunitario a trapianto di organo solido, ora permette la sopravvivenza del trapianto a lungo termine in questa popolazione di pazienti unica 3,21. Tuttavia, significativi effetti collaterali di immunosoppressione a lungo termine necessari per la manutenzione e la sopravvivenza del trapianto limitano ancora la più ampia applicazione di questi migliorare la vita, ma non la vita di risparmio modalità ricostruttiva 3,22,23. Inoltre, il successo di VCA a differenza di trapianti di organi solidi dipende anche tempestiva rigenerazione dei nervi destinatari nella alloinnesto di ri-innervano entrambi i muscoli per la funzione motoria, nonchécomponenti sensoriali per il tatto e la sensazione della temperatura di recuperare la funzione. Dal punto di vista immunologico, come in tutti medicina dei trapianti, l&#39;obiettivo primario nel trapianto ricostruttiva è quello di raggiungere uno stato di tolleranza operativo che consente l&#39;accettazione di un allotrapianto senza la necessità di mantenimento a lungo termine immunosoppressione 23. A questo proposito, il topo è evoluta a rappresentare il principale sistema modello in vivo nella ricerca dei trapianti di studiare i mezzi per conseguire alloantigene tolleranza specifico in un sistema immunitario intatto. Inoltre, il complesso H2 del mouse assomiglia molto complesso MHC umana. Così, i ceppi inbred e di genotipi del mouse permettono di modellare scenari clinici di impostazioni di vita e di donatore cadavere correlate e non correlate impiegando vari gradi di alloantigene mismatching da un singenici a una combinazione ceppo completamente allogenico. La disponibilità di animali transgenici e di animali knockout specifici in aggiunta tuttiOWS indagini dei ruoli e degli effetti dei percorsi molecolari individuali e meccanismi di regolazione di accettazione immunitario e il rigetto, fornendo la possibilità di attivazione o l&#39;esaurimento delle componenti cellulari o proteine. Questo è accompagnato da un&#39;ampia disponibilità di agenti diagnostici e terapeutici (ad es., Anticorpi) sviluppati unicamente per il sistema di topo in vitro e in vivo studi 24. Nel complesso, questi aspetti rendono il sistema murino il &quot;gold standard&quot; per la ricerca di base trapianto di immunologia. Anche se ci sono stati diversi piccoli e grandi 13,16,18,20,25,26 14,27-29 modelli animali descritti per VCA come recentemente recensito da Brandacher et al. 30 solo pochissimi sono in realtà applicabile per la ricerca immunologica meccanicistica base 16 , 24,30. A causa del piccolo diametro del vaso del mouse arteria femorale e la vena, richiede advanced chirurgica e super-miformazione e le competenze per eseguire crosurgical anastomosi successo come il componente chiave del trapianto degli arti posteriori ortotopico. Mentre dissezione meticolosa e attenta delle strutture anatomiche è criticamente rilevante in questo modello come nei modelli precedentemente pubblicati 20,31,32, la tecnica bracciale ha mostrato una curva di apprendimento meno ripida rispetto alla sutura anastomosi di vasi sub-millimetriche 16,25 e può essere realizzato in un paio di mesi di formazione con rigorosa pratica quotidiana. Sulla base della nostra esperienza, un microsurgeon inesperto avrà bisogno di 30 -50 tentativi procedurali di acquisire competenze sufficienti e affidabili per eseguire questo modello con alte percentuali di successo. Per la microsurgeon esperto e altamente qualificato 15 - 30 tentativi dovrebbero essere sufficienti a padroneggiare questo mouse arto posteriore trapianto bracciale a base model.The ha ridotto la complessità della procedura qui presentata è ulteriormente testimoniata dal fatto che questo approccio richiede lunghezza dell&#39;imbarcazione limitata e quindi la dissezione. Abbiamo trovato prendendo tegli navi cedenti al legamento inguinale ei vasi riceventi a livello dei vasi epigastrici superficiali fornisce lunghezza sufficiente, nonostante la nozione comune che la tecnica bracciale richiede ampie lunghezza supplementare applicabile. Ad esempio, un approccio precedentemente pubblicato descrive la necessità di navi raccolto donatori a livello dei vasi iliaci e vasi riceventi esterni a livello dei vasi poplitei 32. Inoltre, con la tecnica bracciale, eseguire la anastomosi richiede meno tempo rispetto alla tecnica di sutura e porta ad un tempo di funzionamento ridotto complessiva. In mani esperte, entrambe le procedure donatore e ricevente possono essere completate in una media di 90 min. Questo è un miglioramento significativo rispetto ai metodi precedentemente riportati nel quale ha esteso la durata dell&#39;anestesia destinatario è stato un fattore determinante per il successo e la sopravvivenza perioperatoria; rendendo la necessità di un approccio a due chirurgo un prerequisito 31-33.Inoltre, minimo a nessun sanguinamento dal anastomosi contribuisce ulteriormente alla riduzione significativa perdita di sangue e, quindi, riduce la necessità di rianimazione fluida dei destinatari, come descritto in precedenza come un altro fattore determinante per il successo in questo modello 33. Infine, la tecnica bracciale rappresenta un metodo con un significativo vantaggio di costo rispetto agli elevati costi di 11-0 materiale micro-suture. Pertanto, la tecnica bracciale non sutura rappresenta la fase più critica del protocollo sottostante. In un elegante studio di Tung et al., Vari mio- diverso e lembi osteomyocutaneous tra cui un lembo cutaneo vascolarizzato inguine sono state descritte in base al largo del peduncolo del mouse nave femorale 32. Mentre il metodo presentato qui si concentra esclusivamente sul trapianto di tutto l&#39;arto del mouse posteriori, i principi presentati possono essere facilmente tradotti e impiegati in vari altri disegni anatomici lembo. Degno di nota è il fatto che, mentre gli autori precedenti riportano automutilazioneo autofagia dell&#39;innesto come complicazione significativa durante sopravvivenza post-operatorio 31,32 questo non è stato osservato nella nostra esperienza. Inoltre, il rapido recupero e la perdita di valore limitato di capacità dell&#39;animale di alimentazione e governare sottolinea che l&#39;inserto ortotopico della gamba trapiantato infligge solo minima morbilità destinatario. Il modello descritto in questo video pubblicazione presenta inoltre diversi vantaggi per l&#39;impostazione del mouse rispetto alle tecniche precedentemente pubblicati. Innanzitutto, come riportato in precedenza dal nostro gruppo 16, la tecnica bracciale può essere impiegato in serbatoi con un diametro del lume inferiore ad 1 millimetro, consentendo il trapianto ortotopico di un allotrapianto dell&#39;arto posteriore. Questo approccio fornisce la base per l&#39;impiego di questo modello combinato sia immunologico, nonché studi funzionali di rigenerazione nervosa relative al VCA 16,25. la rigenerazione del nervo è di importanza fondamentale per la Field del trapianto ricostruttiva, in quanto questi sono procedure di risparmio non vita il cui successo è determinato principalmente dal ripristino di difetti funzionali ed estetiche. L&#39;inserto eterotopico di un lembo osteomyocutaneous proveniente dal dell&#39;arto posteriore è un modello aggiuntivo a disposizione del chirurgo 15. In questo approccio tuttavia, l&#39;asportazione chirurgica di strutture di potenziale interesse, come le unghie e la pelle glabra della zampa, limita la versatilità di questo metodo rispetto al trapianto ortotopico di un anatomicamente inalterata allotrapianto arto posteriore. Questo è di particolare interesse nel trapianto ricostruttiva come queste strutture sono state descritte come destinazione di processi rigetto atipiche in pazienti infliggendo solleciti meccanicamente i allotrapianti mano 34. Intrinsecamente unica per un trapianto di tessuto composito vascolarizzato è la componente ossea, come un osso vascolarizzato contiene midollo osseo vitale ed è un constantly rinnovando fonte di componenti e precursori del sistema immunitario donatore. Mentre questo è stato cautamente considerato come una possibile fonte di graft-versus-host disease (GvHD), questo non è stato osservato sia in modelli animali 35,36 che nell&#39;uomo 37. Molto al contrario in realtà, come mostrato nella preclinici e clinici 36 studi 38-40, la combinazione di trapianto di organi con trapianto di midollo osseo del donatore o la trasfusione di osso selezionato derivate dal midollo prodotti cella mostra effettivamente effetti vantaggiosi per quanto riguarda la quantità di immunosoppressione necessaria 21 ; alcuni protocolli hanno addirittura dimostrato tolleranza operativa senza la necessità di immunosoppressione farmaci a base 41. Il modello e la metodologia mostrato da questa pubblicazione ha prodotto la sopravvivenza del 100% a lungo termine animale donatore singenici - combinazioni di destinatari (figura 1), così come ha dimostrato una ben caratterizzati modelli di trapianto acceptaSNO e rifiuto nelle combinazioni deformazione allogeniche come esposto nella figure 2 e 3. Inoltre, la pelle come obiettivo primario visibile del rifiuto in VCA, segue un modello riproducibile di 4 scenari clinici distinti di rifiuto correlazione con quelli VCA umana come indicato dal la classificazione di lavoro Banff 2007 della pelle contenenti VCA 42, rendendo questo modello particolarmente traducibile. Pertanto, questo modello di topo altamente affidabile per il trapianto ricostruttiva soprattutto, introduce la disponibilità di un versatile sistema modello immunologico dei ceppi di topi inbred e transgenici geneticamente definite che apre le possibilità di studi diagnostici e interventistiche con impatto traslazionale sulla VCA clinica. La necessità di competenze microchirurgia base prima di intraprendere controllare la tecnica descritta può essere considerato come una limitazione a questo modello, tuttavia, è la stessa principale ostacolo a qualsiasi modello di topo microchirurgico. gios, questo protocollo video è principalmente destinato a fornire al microsurgeon condito con un approccio alternativo al microvascolare anastomosi in un murino modello in vivo per vascolarizzato allotrapianto composito. Inoltre, il modello permette di trapianto di un componente di midollo osseo vascolarizzato intatto, così come l&#39;uso di un modello funzionale in ricerca rigenerazione nervosa. In conclusione, abbiamo stabilito un nuovo, versatile e modello affidabile del mouse per il trapianto degli arti posteriori ortotopico utilizzando una tecnica bracciale non sutura che apre la porta a meccanicistico di base, così come la ricerca traslazionale relative a qualsiasi aspetto della VCA.

The late nineties heralded the pioneering days of reconstructive transplantation with the first successful hand transplant performed in France in 1998. Since then, the use of VCAs for reconstruction of devastating tissue defects has been successfully employed in a wide spectrum of patients. To date, the world counts 76 recipients of 112 upper extremities as well as 31 faces 1-3. In addition, several other types of VCAs such as abdominal wall 4, larynx 5, trachea 6, vascularized joints 7, and even penis 8 have been performed. Furthermore, the live birth of a baby was recently reported after uterus transplantation 9. This growing world experience is indicative for how reconstructive transplantation has become a valid therapeutic option for patients suffering of significant functional tissue defects not amendable to conventional reconstructive and restorative surgery and treatment. 
While the idea of replacing "like with like" sparked clinical enthusiasm, initial skepticism still prevails with regards to side effects of conventional high-dose immunosuppression required to maintain allografts and their function 10,11. However, as shown by seminal work of Lee et al., these composite grafts are less likely to reject than its individual components, and furthermore, some of the tissue components such as the vascularized bone compartment have fueled optimism as they might exert unique immunological effects onto the balance of immune acceptance and rejection 12. 
Our group pioneered several microsurgical animal models for solid organ transplantation, as well as vascularized composite allotransplantation 13-19. Here we describe a novel surgical procedure using a non-suture cuff technique to perform super micro-vascular anastomosis in an orthotopic mouse hind limb transplantation model. This transplant model provides a useful tool for investigating immune acceptance and rejection mechanisms, as well as the role of individual tissue components, such as the vascularized bone marrow compartment, towards tolerance induction in the immunologically versatile setting of the mouse species. Additionally, the orthotopic placement of the limb opens the possibilities for nerve regeneration and functional outcome studies, which are critically important to the setting of VCA.

<table>
	<tbody>
		<tr>
			<td>Suture, 6-0 Nylon</td>
			<td>MWI</td>
			<td>31849</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 6-0 Polysorb</td>
			<td>MWI</td>
			<td>72667</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture, 10-0 Nylon</td>
			<td>Aero Surgical</td>
			<td>TK-107038</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Tubing, Size 25</td>
			<td>Vention Medical</td>
			<td>141-0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyimide Tubing, Size 27</td>
			<td>Vention Medical</td>
			<td>141-0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Clamps (Single)</td>
			<td>Synovis</td>
			<td>00396</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular Clamps (Double)</td>
			<td>Synovis</td>
			<td>00414</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Scissors</td>
			<td>Synovis</td>
			<td>SAS-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Forceps</td>
			<td>Synovis</td>
			<td>FRS-15 RM-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Dilators</td>
			<td>Synovis</td>
			<td>FRS-15 RM-8d.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Needledriver</td>
			<td>Synovis</td>
			<td>C-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Clamp Applicator</td>
			<td>Synovis</td>
			<td>CAF-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Flushing Needle</td>
			<td>Hamilton</td>
			<td>N/A</td>
			<td>10MM, 30&#176;, 33G</td>
		</tr>
		<tr>
			<td>Lactated Ringers Solution</td>
			<td>Fisher Scientific</td>
			<td>NC9968051</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>DEA Number required; Obtained from hosptial pharmacy.</td>
		</tr>
		<tr>
			<td>Enrofloxacin; Baytril</td>
			<td>Bayer Health Care</td>
			<td>186599</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Obtained from hosptial pharmacy</td>
		</tr>
	</tbody>
</table>

orthotopic hind limb transplantation in the mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

Esecuzione vascolarizzato allotrapianto composito in un modello di topo usando una tecnica bracciale non sutura consente di ottenere eccellenti e lungo graft termine e animali sopravvivenza come mostrato in Figura 1. Inoltre, rappresenta un metodo affidabile per ottenere risultati riproducibili di graduale rifiuto allograft in composito vascolarizzata allotrapianto come documentato dalle immagini mostrate in figura 2. H &amp; e istologico dei tessuti ottenuti da animali sottoposti a rifiuto sottolinea ulteriormente la dinamica riproducibili del rigetto dell&#39;organo trapiantato in questo modello murino (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/53483/53483fig1.jpg" /> Figura 1. Allograft sopravvivenza in un mouse Strain combinazione completamente H2-non corrispondenti [Balb / c (H2K d) in C57BL6 (H2K b)]. Mentre tutte Transpl singeniciformiche (n = 10) sono stati accettati a lungo termine, allotrapianti non trattati (n = 10) sono stati respinti acutamente dentro e media di 8 - 9 giorni. Pelle rifiuto di grado 3 secondo i criteri di Banff è stato considerato completo rifiuto in questo studio. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53483/53483fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/53483/53483fig2.jpg" /> Figura 2. Caratteristiche Rifiuto clinici e la dinamica di un Fully-H2 non corrispondenti [Balb / c (H2K d) in C57BL6 (H2K b)] murino ortotopico arto posteriore trapianto. (A) Grado Clinica 0, (B) grado clinico 1, ( C) di grado clinico 2, (D) di grado clinico 3, e (e) Clinical Grade 4 rifiuto, (F) termine sopravvivere trapianto lunga (POD 100) trattati con un costimulzione blocco (antiCD40 mAb + CTLA4Ig) regime di base. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53483/53483fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53483/53483fig3.jpg" /> Figura 3. H &amp; E colorazione di pelle e muscoli in un trapianto Singenico (A) e trapianto allogenico (B) su POD 8 così come H &amp; E colorazione di Footpad pelle e muscoli in un trapianto Singenico (C) e trapianto allogenico su POD 8 (D ) (bar Scala:. 0,1 micron) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53483/53483fig3large.jpg" target="_blank">clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Orthotopic Hind Limb Transplantation in the Mouse at JoVE.com

Orthotopic Hind Limb Transplantation in the Mouse

Questo nuovo modello per il trapianto degli arti posteriori ortotopico nel topo, applicando una tecnica bracciale non sutura per anastomosi super-microvascolare, fornisce un potente strumento per la ricerca in vivo meccanicistica immunologica relative al allotrapianto composito vascolarizzato (VCA).

Ortotopico degli arti posteriori del trapianto di mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

orthotopic-hind-limb-transplantation-in-the-mouse

Nanyang Technological University

Research

JoVE Journal

Medicine

11.6K Views.  Johns Hopkins University School of Medicine.  Questo nuovo modello per il trapianto degli arti posteriori ortotopico nel topo, applicando una tecnica bracciale non sutura per anastomosi super-microvascolare, fornisce un potente strumento per la ricerca in vivo meccanicistica immunologica relative al allotrapianto composito vascolarizzato (VCA). 

Video: Orthotopic Hind Limb Transplantation in the Mouse

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Trapianto ortotopico aortica: un modello di ratto per studiare lo sviluppo di vasculopatia cronica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Ricerca

Medicina

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

La valutazione longitudinale di perdita dell&#39;arto posteriore ossea del mouse dopo la lesione del midollo spinale tramite Novel, In vivo, Metodologia

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Ortotopico di intestino tenue di ratti

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Trapianto di fegato in ratti

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Aortica trapianto ortotopico in Mouse per lo studio della malattia vascolare

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Un modificata eterotopica suina Hind Arto Trapianto Modello traslazionale vascolarizzato composito allotrapianto (VCA) La ricerca

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Mouse trapianto renale: Modelli di Allograft Rifiuto

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

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;paziente-Like&quot; ortotopico Singenico modello murino della carcinoma epatocellulare Metastasi

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular disease is the development of ischemia. In severe cases, patients can develop critical limb ischemia in which they experience constant pain and an increased risk of limb amputation. Current therapies for peripheral ischemia include bypass surgery or percutaneous interventions such as angioplasty with stenting or atherectomy to restore blood flow. However, these treatments often fail to the continued progression of vascular disease or restenosis or are contraindicated due to the overall poor health of the patient. A promising potential approach to treat peripheral ischemia involves the induction of therapeutic neovascularization to allow the patient to develop collateral vasculature. This newly formed network alleviates peripheral ischemia by restoring perfusion to the affected area. The most frequently employed preclinical model for peripheral ischemia utilizes the creation of hind limb ischemia in healthy rabbits through femoral artery ligation. In the past, however, there has been a strong disconnect between the success of preclinical studies and the failure of clinical trials regarding treatments for peripheral ischemia. Healthy animals typically have robust vascular regeneration in response to surgically induced ischemia, in contrast to the reduced vascularity and regeneration in patients with chronic peripheral ischemia. Here, we describe an optimized animal model for peripheral ischemia in rabbits that includes hyperlipidemia and diabetes. This model has reduced collateral formation and blood pressure recovery in comparison to a model with a higher cholesterol diet. Thus, the model may provide better correlation with human patients with compromised angiogenesis from the common co-morbidities that accompany peripheral vascular disease.

We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion.

Modello preclinico di ischemia degli arti posteriori nei conigli diabetici

Peripheral vascular disease is a widespread clinical problem that affects millions of patients worldwide. A major consequence of peripheral vascular ...

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 ...

Ortotopico degli arti posteriori del trapianto di mouse

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