Name: murine kidney transplant technique
Uploaded: 2015-10-20T14:00:00.000+00:00
Duration: 8 min 58 s
Description: Watch this Scientific Journal Video about Murine Kidney Transplant Technique at JoVE.com

Questo lavoro è stato sostenuto in parte da 1R03DK096151.

Tutti i topi sono stati acquistati da The Jackson Laboratory (Bar Harbor, ME), e sono stati alloggiati in condizioni di libero patogeni presso l&#39;Università del Colorado Denver, Facility Barbara Davis Animal Center secondo Linee Guida NIH e con l&#39;approvazione della University of Colorado Denver IACUC. 1. Trapianto renale Harvest <ol><li> Sterilizzare tutti gli strumenti, indossare guanti sterili durante tutta la procedura e mantenere un campo sterile. Eseguire tutti gli interventi chirurgici con l&#39;uso di un microscopio operatorio. </li><li> Rimuovere chirurgicamente reni topo donatore da (kg IP 60mg /) donatori pentobarbital-anestetizzati. Accertare profondità dell&#39;anestesia da punta pizzico, e osservare la frequenza respiratoria. </li><li> Agganciare la pelliccia quindi immobilizzare il mouse da vincoli a 4 vie. Preparare la pelle con iodio-povidone e drappeggio il mouse in modo sterile. </li><li> Fare un 2 centimetri linea mediana verticale incisione addominale e entrare nella cavità addominale. Ritrarre l&#39;intestino superiormente ed esternare laal petto. Mantenere l&#39;intestino avvolto in una garza sterile umido durante tutta la procedura. </li><li> Identificare i grossi vasi addominali e mobilitare, identificare eventuali rami lombari e sia cauterizzare o legare con 10/0 sutura in nylon. </li><li> Immediatamente distale vasi renali sinistra stuzzicare la vena cava inferiore (IVC) e aorta addominale (AA) a parte per via smussa su circa 2mm. Legare con 10/0 nylon e dividere piccolo arterioso e rami venosi dai vasi renali. </li><li> Ora separare la vena renale dalla arteria renale attentamente per via smussa di queste strutture. Questo consente la creazione accurata di una patch Carrel all&#39;estremità prossimale della vena renale, che verrà utilizzata per formare una anastomosi end-to-side ai destinatari IVC. </li><li> Identificare, legare e sezionare i vasi surrenali sinistra. Questo consente l&#39;accesso all&#39;aorta surrenale e una sutura di seta 6-0 è collocato intorno l&#39;aorta in preparazione per legatura, ma non legato in questo momento. </li&gt; <li> Mobilitare il rene, i vasi e dell&#39;uretere dalla plancia circostante. Ruotare il rene a destra e legare / dividere i rami posteriori. Quindi tornare il rene di sinistra. </li><li> Ora dirigere l&#39;attenzione per l&#39;uretere. Senza scomodare l&#39;ilo renale liberare l&#39;uretere dal circostante plancia facendo attenzione a preservare i vasi ureterali. Dividere l&#39;uretere a livello del dotto deferente. Il rene è ora pronto per il recupero. </li><li> Iniettare lentamente 300 unità di eparina nel distale IVC heparinizing così il donatore. Fissare la sutura di seta 6-0 disposte intorno al AA surrenale e profumato rene sinistro tramite l&#39;aorta addominale distale con 0.8ml di soluzione di eparina salina (100 U / ml). </li><li> Una volta che la perfusione ha cessato creare una patch Carrel venoso immediatamente per eliminare il riflusso nel rene. Ritrarre la vena renale verso il rene rivelando l&#39;AA e dell&#39;arteria renale sotto. </li><li> Dividere l&#39;aorta adiacente dell&#39;arteria renale creando un tacco ee bracciale come mostrato in Figura 1. Rimuovere i reni, vasi e dell&#39;uretere dal donatore. </li><li> Rimuovere il rene direttamente a un destinatario pre-preparati (0 min tempo di ischemia fredda) o conservati a 4 ° C in una soluzione di scelta per un tempo predeterminato fino al momento dell&#39;impianto. Eutanasia donatori di exsanguanation e dislocazione cervicale. Il tempo totale per il recupero del rene del donatore è di circa. 15-20 min. </li></ol> 2. Rene tecnica di impianto <ol><li> Anestetizzare il mouse destinatario con pentobarbital (60 mg / kg IP dose iniziale, 25 mg dose supplementare / kg IP se richiesto). Accertare profondità dell&#39;anestesia da punta pizzico, e osservare la frequenza respiratoria. Clip della pelliccia quindi immobilizzare il mouse da vincoli a 4 vie e applicare pomata oftalmica per gli occhi. Preparare la pelle con iodio-povidone e drappeggio il mouse in modo sterile. </li><li> Fare un 2 centimetri linea mediana verticale incisione addominale e entrare nella cavità addominale. Ritrarre l&#39;intestinosuperiormente e externalize al petto. Mantenere l&#39;intestino avvolto in una garza sterile umido durante tutta la procedura. </li><li> A questo punto eseguire una nefrectomia destra. Legare l&#39;arteria e la vena renale con 6-0 punti di sutura in seta. Accise distale rene per le suture. Legare l&#39;uretere con 6-0 seta e poi dividere prossimale al rene. </li><li> Isolare l&#39;aorta addominale e vena cava inferiore (IVC) sotto i vasi renali. Mettere 4-0 legami di cotone attorno l&#39;aorta e vena cava inferiore superiore poi inferiore al sito anastomosi. Identificare e legare eventuali vasi lombari all&#39;interno del campo con 10-0 sutura in nylon. </li><li> Annodare i legami di cotone, prima l&#39;inferiore, seguito dal superiore. In questo modo viene mantenuta del sangue nell&#39;aorta rendendo il aortotomy facile. </li><li> Formare la aortotomy con un ago 30G per entrare nel lume dell&#39;aorta. Estendere l&#39;incisione con sottili forbici micro ad una lunghezza di circa 2 mm. </li><li> Effettuare la fine anastomosi lato della cuffia aortico donatore tacco e punta al&#39;aorta destinatario nel seguente modo. Inserire un 10-0 nylon soggiorno suture stitch nell&#39;aorta donatore e all&#39;angolo inferiore dell&#39;incisione nell&#39;aorta destinatario e cravatta. </li><li> Posizionare un secondo 10-0 nylon opposto al primo nell&#39;aorta donatore e l&#39;angolo superiore dell&#39;incisione in aorta addominale e cravatta. Fare una linea sutura continua da superiore a inferiore nella parete laterale dell&#39;aorta e legare contro la permanenza punto precedentemente posizionato. Poi suturare la parete mediale in modo esecuzione e cravatta. </li><li> Effettuare la fine anastomosi lato della vena renale donatore al ricevente IVC nel seguente modo. Forare il IVC con un ago 30G ed estendere l&#39;incisione con sottili forbici micro. Legare la vena renale donatore verso l&#39;angolo inferiore della incisione nel IVC con 10-0 di nylon. Fare una linea di sutura che corre tra la vena renale e l&#39;IVC e cravatta. NOTA: E &#39;di vitale importanza che tutto spessore passa l&#39;ago di sutura tra cui il adventit vascolareia e l&#39;intima sono raggiunti. Eversion dei bordi assicura anche che c&#39;è intima-to-intima contatto, che aiuta nella chiusura e guarigione delle anastomosi. Mentre un agente di coagulazione emostatico può essere utile per ridurre le perdite, si raccomanda che un chirurgo invece contare su una buona tecnica. </li><li> Assicurarsi che le anastomosi sono &quot;puliti&quot;. Vale a dire, che le pareti opposte, non sono catturati durante il posizionamento di punti. Questo provocherà un restringimento significativo di fluire che si tradurrà in un innesto fallito e, in casi estremi arto posteriore paralisi. NOTA: Un altro fattore estremamente importante è garantire che la tensione delle linee di sutura anastomotica anche ottimale. Troppo lenta e non ci saranno perdite irreversibili, troppo stretto e stenosi di fluire si tradurrà. Se sul lato arterioso questo si tradurrà in scarsa perfusione dell&#39;innesto, se sul lato venoso comporterà un rene congestionata. </li><li> Rilasciare il distale flusso venoso 4-0 cotone tie ristabilire. Una volta emostasi del venonoi anastomosi è stata osservata gradualmente allentare la prossimale pareggio 4-0 in cotone e osservare la anastomosi arteriosa per l&#39;emostasi. </li><li> Rimuovere le fascette di cotone dal mouse una volta entrambe le anastomosi sono considerati sicuri. Pierce vescica con un ago 20 G creando due fori. </li><li> Far passare la punta delle pinze curve attraverso i fori e tirare l&#39;uretere donatore attraverso la vescica con la pinza con l&#39;estremità prossimale dell&#39;uretere essere ancorato alla parete della vescica con due 10-0 suture in nylon. Tagliare la lunghezza in eccesso dell&#39;uretere sporge dal secondo foro permettendo l&#39;uretere ritragga all&#39;interno della vescica e chiudere il foro con due 10-0 suture in nylon. </li><li> Ritorna l&#39;intestino all&#39;addome. Chiudere la parete addominale in due strati con 5-0 sutura di seta in modo esecuzione. </li><li> Somministrare un bolo 1,0 ml di soluzione sterile, caldo normale soluzione fisiologica nell&#39;addome come rianimazione fluido alla chiusura, e iniettare 0,8 ml di soluzione fisiologica normale per via sottocutanea nel post-operatorio. Nessun altro Supportigiocare cinque misure durante l&#39;intervento sono obbligatori. </li><li> Recuperare l&#39;animale su una coperta di riscaldamento. Tempo totale dell&#39;impianto è di circa. 35-45 min. Somministrare analgesici come buprenorfina, 0,05 mg / kg, SC, 0,1-0,2 ml all&#39;inizio della procedura e ogni 6-12 ore per 72 ore post-op. </li></ol> 3. controlaterale Nefrectomia <ol><li> A seconda dei requisiti di protocollo, diversi giorni dopo la procedura di impianto, eseguire una nefrectomia controlaterale sotto anestesia isoflurano (5% isoflurano inalato per induzione, 1,5-2,0% per la manutenzione). </li><li> Inserire la cavità addominale e ritrarre delicatamente l&#39;intestino al diritto degli animali. Esporre il rene sinistro e sezionare smussato dalla fascia circostanti. </li><li> Legare l&#39;arteria renale, vena e dell&#39;uretere con 6-0 punti di sutura in seta e poi asportare il rene sopra le suture e rimuovere il rene. Tempo operatorio totale è di circa. 10 min. </li></ol> 4. Graft Assessment <ol><li> Misura siero Creatinove utilizzando il metodo del picrato alcalino (reazione Jaffe). </li></ol>

<ol>
	<li>Skoskiewicz, M., Chase, C., 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=Kidney+transplants+between+mice+of+graded+immunogenetic+diversity.">Kidney transplants between mice of graded immunogenetic diversity.</a> Transplant Proc. 5 (1), 721-725 (1973).</li><li>Kalina, S. L., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microsurgical+technique+for+renal+transplantation+in+mice.">A microsurgical technique for renal transplantation in mice.</a> Aust N Z J Surg. 63 (3), 213-216 (1993).</li><li>Han, W. R., Murray-Segal, L. J., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+for+kidney+transplantation+in+mice.">Modified technique for kidney transplantation in mice.</a> Microsurgery. 19 (6), 272-274 (1999).</li><li>Ge, F., Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+successfully+establishing+a+kidney+transplant+in+a+mouse+model.">Strategies for successfully establishing a kidney transplant in a mouse model.</a> Exp Clin Transplant. 9 (5), 287-294 (2011).</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=Learning+curve,+surgical+results+and+operative+complications+for+kidney+transplantation+in+mice.">Learning curve, surgical results and operative complications for kidney transplantation in mice.</a> Microsurgery. 26 (8), 590-593 (2006).</li><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=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Zhang, Z., Chen, H., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+Transplantation+in+Mice.">Kidney Transplantation in Mice.</a> Experimental Organ Transplantation. , 45-64 (2013).</li><li>Zhang, L., Moskovitz, M., Piscatelli, S., Longaker, M. T., Siebert, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+study+of+different+angled+end-to-side+anastomoses.">Hemodynamic study of different angled end-to-side anastomoses.</a> Microsurgery. 16 (2), 114-117 (1995).</li><li>Liu, Q., Mirc, D., Fu, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+mechanisms+of+thrombosis+in+intact+bent+microvessels+of+rat+mesentery.">Mechanical mechanisms of thrombosis in intact bent microvessels of rat mesentery.</a> J Biomech. 41 (12), 2726-2734 (2008).</li><li>Chesnutt, J. K., Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tortuosity+triggers+platelet+activation+and+thrombus+formation+in+microvessels.">Tortuosity triggers platelet activation and thrombus formation in microvessels.</a> J Biomech Eng. 133 (12), 121004 (2011).</li><li>Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+buckling+within+soft+surrounding+tissue+generates+tortuosity.">Blood vessel buckling within soft surrounding tissue generates tortuosity.</a> J Biomech. 42 (16), 2797-2801 (2009).</li><li>Gutierrez-Diaz, J. 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=Intraluminal+thrombus+and+neointimal+hyperplasia+after+microvascular+surgery.">Intraluminal thrombus and neointimal hyperplasia after microvascular surgery.</a> Surg Neurol. 24 (2), 153-159 (1985).</li><li>Khouri, R. K., Cooley, B. C., Kenna, D. M., Edstrom, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis+of+microvascular+anastomoses+in+traumatized+vessels:+fibrin+versus+platelets.">Thrombosis of microvascular anastomoses in traumatized vessels: fibrin versus platelets.</a> Plast Reconstr Surg. 86 (1), 110-117 (1990).</li><li>Johnson, P. 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=Initial+platelet+deposition+at+the+human+microvascular+anastomosis:+effect+on+downstream+platelet+deposition+to+intact+and+injured+vessels.">Initial platelet deposition at the human microvascular anastomosis: effect on downstream platelet deposition to intact and injured vessels.</a> Plast Reconstr Surg. 90 (4), 650-658 (1992).</li></ol>

The authors have nothing to disclose.

Padroneggiare questa tecnica di trapianto è difficile, ma una volta realizzato, è uno strumento di ricerca molto potente. Il paziente chirurgo / ricercatore sarà ricompensato dalla cura dei dettagli e la coerenza della tecnica, che è la chiave per padroneggiare qualsiasi procedura chirurgica, a maggior ragione nei piccoli modelli animali. Le difficoltà tecniche di padroneggiare il trapianto di rene di topo sono molte pieghe, ed è molto probabile che l&#39;esperienza in altri modelli di piccole dimensioni trapianto...

Dal 1973 il modello di trapianto di rene nei topi è stato un valido strumento di ricerca, ma problemi tecnici hanno ostacolato la sua diffusione. Nel corso degli anni numerosi lavori sono stati pubblicati in dettaglio i miglioramenti / perfezionamenti a questa procedura. Come modello di principalmente vascolarizzato trapianto di organo solido questa procedura è probabilmente secondo solo al modello di trapianto di cuore eterotopico che è stato anche ideato dal laboratorio di Russell nel 1973 6. Entrambi i modelli si prestano alla ricerca sulle risposte di rigetto allogenico, lo sviluppo di ritardata funzionalità del trapianto e danno da ischemia riperfusione. Uno dei problemi più comuni da segnalare con trapianto renale è relativamente alta incidenza di trombosi arteriosa 4,5,7 che abbiamo anche sperimentato nel nostro laboratorio. Pertanto abbiamo deciso di effettuare una revisione della letteratura di formazione di trombi e forse trovare la causa di questo problema tecnico e di elaborare anche unapossibile soluzione. La causa più probabile di trombosi è il percorso alquanto tortuosa sangue prende dall&#39;aorta destinatario, nell&#39;aorta renale donatore poi all&#39;arteria renale donatore. Questo percorso provoca turbolenza nella arteria renale che può portare all&#39;attivazione delle piastrine e formazione di trombi. Sulla base delle recenti osservazioni e una ricerca di letteratura 8-14 siamo arrivati ​​a una nuova tecnica che ha ridotto la trombosi a 0%. La tecnica qui descritta varia da tecniche precedentemente riportati nella formazione di un bracciale tallone e punta arteriosa che strutture migliorate flusso sanguigno e riduce notevolmente la formazione di trombi. Il polsino è formata dividendo dell&#39;aorta infra-renale attraverso la faccia della ostio arterioso renale con un angolo inferiore a 45 ° rispetto all&#39;asse longitudinale dell&#39;aorta (Figura 1A e 1B). Ciò si traduce in un bracciale circa 2mm di lunghezza. Una patch Carrel venoso è formata da sezionare la rvena Enal nella IVC aumentando il diametro del bracciale. Il donatore addominale dell&#39;aorta polsino tacco e punta infra-renale è end-to-side anastomizzata all&#39;aorta addominale destinatario e la vena renale donatore / patch IVC è end-to-side anastomizzata al destinatario addominale vena cava inferiore (IVC) . L&#39;uretere viene poi introdotto in ed ancorata alla vescica come descritto da Han et al 3. Per questo studio i trapianti non trattati con soli tempi di ischemia calda (es., Senza di ischemia fredda) si confrontano. In questo caso ischemia calda si riferisce al tempo dalla cessazione del flusso di sangue attraverso il rene donatore (passo 1.11) e riperfusione del graft nel ricevente (passo 2.11 sotto). Ischemia fredda si riferisce al tempo che il rene non è perfuso e si mantiene in celle frigorifere fino all&#39;inizio della procedura di impianto.

<table><tbody><tr><td>Instrument</td><td>Roboz #</td><td>Fine Science Tools #</td><td>Arosurgical #</td></tr><tr><td>Straight micro-dissecting forcep #5</td><td>RS-5015</td><td>11295-51</td><td></td></tr><tr><td>Curved micro-dissecting forcep #7</td><td>RS-5047</td><td>11297-00</td><td></td></tr><tr><td>Curved serrated forcep</td><td>RS-5137</td><td>11052-10</td><td></td></tr><tr><td>Vannas micro-dissecting scissors, short</td><td>RS-5610</td><td></td><td>09.140.08</td></tr><tr><td>Micro-dissecting scissors, straight, sharp, long</td><td></td><td>11.602.11</td><td></td></tr><tr><td>Micro spring handle needle holder</td><td></td><td></td><td>11.549.15</td></tr><tr><td>Straight mosquito forcep</td><td></td><td>91308-12</td><td></td></tr><tr><td>Micro-dissecting scissors, straight, blunt</td><td>RS-5962</td><td>14078-10</td><td></td></tr><tr><td>Micro-dissecting scissors, curved, blunt</td><td>RS-5981</td><td>14079-10</td><td></td></tr><tr><td>Micro retractor</td><td>RS-6540</td><td></td><td></td></tr><tr><td>Instrument tray, 10&amp;rdquo; x 6 &amp;frac12;&amp;rdquo; x &amp;frac34;&amp;rdquo;</td><td>RT-1350S</td><td></td><td></td></tr><tr><td>Silk suture, 5/0, 22.5m spool</td><td></td><td>18020-50</td><td></td></tr><tr><td>Suture</td><td></td><td></td><td></td></tr><tr><td>10/0 nylon</td><td></td><td></td><td>T4A10Q07</td></tr><tr><td>5/0 silk</td><td></td><td></td><td>E19A05N</td></tr><tr><td>Gloves</td><td></td><td>Drapes</td><td></td></tr><tr><td>Biogel from Medex Supply</td><td></td><td>Precept, #64-9012-9</td><td></td></tr><tr><td>Syringes</td><td></td><td>Cotton applicators</td><td></td></tr><tr><td>B-D 1cc insulin, #329424</td><td></td><td>Fisher-brand, #23-400-100</td><td></td></tr><tr><td>Povidone-Iodine swabs</td><td></td><td></td><td></td></tr><tr><td>PDI, #B40600</td><td></td><td></td><td></td></tr><tr><td>4/0 Cotton ties</td><td></td><td></td><td></td></tr><tr><td>Domestic cotton autoclaved with instruments</td><td></td><td></td><td></td></tr></tbody></table>

murine kidney transplant technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. 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 using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

Questa tecnica chirurgica consente sia per i semplici studi di sopravvivenza / rigetto dell&#39;organo o protocolli sperimentali piuttosto complessi. Nelle figure di seguito dimostriamo i vantaggi di utilizzare questo miglioramento tecnica anastomosi arteriosa. Utilizzando questa tecnica abbiamo ridotto significativamente l&#39;incidenza di trombosi arteriosa dal 35% allo 0%, aumentando così la produttività. Abbiamo usato questa tecnica per oltre un anno con lo stesso risultato trombosi 0% mantenuto. ...

Watch this Scientific Journal Video about Murine Kidney Transplant Technique at JoVE.com

Murine Kidney Transplant Technique

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Rene Murine Tecnica trapianto

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Research

JoVE Journal

Medicine

15.9K Views.  University of Colorado, Denver. The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis. 

Video: Murine Kidney Transplant Technique

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Ricerca

Medicina

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murino procedura trapianto renale

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Biologia

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Isolamento e Trapianto di diversi invecchiato murini del timo innesti.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Immunologia e infezioni

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Mappatura destino di cellule staminali embrionali umane per la formazione teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Eterotopico renale Autotrapianto in un modello di suino: un protocollo Step-by-Step

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

A Modified Surgical Technique for Kidney Transplantation in Mice

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure.

Una tecnica chirurgica modificata per il trapianto di rene nei topi

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Trapianto di rene ortotopico ratto: un nuovo approccio chirurgico semplificato

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Extracting Intact Kidney from Mouse: A Technique to Obtain Kidney Without Renal Capsule from Murine Models

Source: Grainger, N. et al. <a href="https://www.jove.com/video/62040" target="_blank">Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning.</a> J. Vis. Exp. (2021)
In this video, we describe the procedure for kidney extraction from the retroperitoneal space of the mouse. The extracted kidney can further be microdissected to obtain an intact kidney without a renal capsule and the surrounding fat layer.

Estrazione di rene intatto dal topo: una tecnica per ottenere rene senza capsula renale da modelli murini

Source: Grainger, N. et al. Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning. J. Vis. Exp. (2021)
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