Finanziamento da Rene Research UK, The Royal College of Surgeons di Edimburgo e la Società Europea di Trapianto sostenuto questo studio.

Adeguati etica istituzionali nazionali e locali dovrebbero essere a posto prima di eseguire esperimenti su animali. In particolare nel Regno Unito i seguenti esperimenti sono stati intrapresi sotto gli Animali (Scientific Procedures) Act 1986, in cui sono disponibili ad operare contemporaneamente due microsurgeons il chirurgo donatore deve eseguire i passaggi 1,1-1,16 poi da 3.1 a 3.5, mentre il chirurgo destinatario esegue 2.1 a 2.8 . Per un singolo operatore le fasi possono essere seguite in modo sequenziale. 1 donatore Preparazione Nota: La procedura qui presentata è un donatore C57BL / 6 BM12 e destinatario C57BL / 6 topi di sesso maschile di età compresa tra 8 a 16 settimane di vita con un peso corporeo superiore a 20 g. Tuttavia questa procedura può essere eseguita in modo riproducibile su una varietà di ceppi di topi. I dati presentati nella sezione risultati rappresentativi sono stati ottenuti da C57BL / 6, C57BL / 6 BM12 e topi BALB / c. <ol><li> Procedure di Condotta Using strumenti chirurgici sterili e di consumo (autoclave), con sforza di mantenere il campo operatorio sterile come possibile. Eseguire la preparazione del donatore in contemporanea con la preparazione destinatario, se due chirurghi sono disponibili. </li><li> Anestetizzare il mouse con un&#39;iniezione intraperitoneale (31G ago) di medetomidina (0,5 mg / kg) e ketamina cloridrato (200 mg / kg). Questo si traduce in un piano anestetico che viene mantenuta per 4 ore fornire tempo sufficiente per l&#39;intera procedura da eseguire. Anestesia supplementare non è necessario. </li><li> Verificare che il mouse è anestetizzato (nessuna risposta a pizzico tep). </li><li> Shave addome del mouse e rimuovere tutti i capelli sciolti con nastro adesivo. </li><li> Posizionare il mouse sulla sua schiena su una stuoia riscaldata sterilmente drappeggiato e vagamente immobilizzare gli arti con nastro adesivo sterile. </li><li> Monitorare il mouse durante tutta la procedura per le ustioni termiche. Se possibile, utilizzare una fonte di calore non elettriche. </li><li> Applicare un lubrificante occhio e sanitize la parete addominale con una soluzione diluita di iodio. </li><li> Eseguire un&#39;incisione mediana nell&#39;addome per entrare nella cavità peritoneale e inserire un tre centimetri Calibri divaricatore addominale. </li><li> Applicare salina riscaldata per mantenere l&#39;intestino e l&#39;area chirurgica umida ed evitare inutili essiccazione dei visceri. </li><li> Coprire il mouse con teli sterili e spostare l&#39;intestino alla sinistra dell&#39;operatore (a destra del mouse) per esporre l&#39;aorta, vena cava e il rene sinistro. </li><li> Applicare una pinza sfogo allo stomaco e tirare superiormente per esporre i principali vasi e rene sinistro completamente. Confezione umidi tamponi sterili (da 2 mm x 2 mm) nell&#39;addome di ritrattare tessuti lontano dalla zona chirurgica, come ad esempio i lobi del fegato, vescicole seminali, e intestino. </li><li> Isolare il rene sinistro da nell&#39;avventizia circostante, il grasso e la ghiandola surrenale sinistra nella cavità peritoneale senza mezzi termini dalla dissezione del tessuto connettivo con pinze punta fine. Posizionare le punte chiuse della pinza tra le aree che neEd da separare e lentamente lasciare le punte forcep per aprire a sezionare lo spazio. </li><li> Isolare la vena renale sinistra legando e dividendo la vena surrenalica e gonadica sinistra vena sinistra con 9 / O nylon. Posizionare la sutura vicino alla vena renale. </li><li> Legare e sezionare l&#39;uretere con 7 / O sutura di seta vicino alla vescica lasciando la sutura termina lungo. Queste estremità lunga di sutura verranno utilizzati quando il rene è raccolto e sono necessari per l&#39;anastomosi ureterale. </li><li> Per mobilitare e sezionare completamente l&#39;aorta e vena cava inferiore e superiormente l&#39;arteria renale e la vena utilizzare pinze punta fine di sezionare senza mezzi termini vasi linfatici e il grasso dalla parte anteriore e sui lati dei vasi. </li><li> Trovare il piano di tessuto tra l&#39;aorta e vena cava e diffondere lentamente il forcipe. Aprire lentamente le punte delle belle pinze a punta a diffondere il tessuto con il minimo trauma. </li><li> Legare un sciolto 7 / O seta intorno all&#39;aorta superiore ed inferiore per l&#39;arteria renale passando un bel angolatopinze punta intorno alla parte posteriore dei vasi e di disegno attraverso una sutura. Questi punti di sutura saranno serrati prima di recupero rene per consentire la perfusione retrograda della University of Wisconsin soluzione. </li></ol> 2 Destinatario Preparazione Come per la preparazione donatore follow passi da 1.2 a 1.8. <ol><li> Spostare l&#39;intestino alla destra dell&#39;operatore (a sinistra del mouse) per esporre l&#39;aorta, vena cava. Coprire l&#39;intestino con una soluzione salina sterile tampone imbevuto. </li><li> Eseguire una nefrectomia destra legando l&#39;arteria renale destra e la vena con 7/0 sutura di seta e poi dividere. </li><li> Legare l&#39;uretere destro con 7 / O sutura di seta e dividere. </li><li> Mobilitare e sezionare completamente l&#39;aorta e vena cava inferiore per l&#39;arteria renale e la vena come descritto al punto 1.14 e 1.15. Garantire la completa dissezione tra l&#39;aorta e vena cava. Fare attenzione a preservare l&#39;arteria spermatica interna che corre anteriormente alla vena cava e aortun insieme con il fascio linfatico. </li><li> Identificare i vasi lombari esecuzione in retroperitoneo dalla vena cava e vena alla colonna vertebrale, legare con 9 / O seta in continuità, non c&#39;è bisogno di dividere i vasi. </li><li> Identificare lo spazio sufficiente per posizionare i morsetti microvascolari con spazio tra di anastomosi vascolari. </li><li> Somministrare eparina per via endovenosa (5 unità) con pene dorsale vena. </li></ol> 3. donatori di rene Retrieval Serrare i 7 / O cravatte di seta che sono stati posti in giro per l&#39;aorta inferiore e superiore per isolare il rene dalla circolazione arteriosa. <ol><li> Infondere retrograde freddo 0,2-0,5 ml della University of Wisconsin soluzione con un ago (31G) nell&#39;aorta. </li><li> Dividere l&#39;aorta entro le suture e dividere la vena renale alla sua giunzione con la vena cava per rimuovere il rene e l&#39;arteria renale con una lunghezza dell&#39;aorta. Dividere vasi lombari derivanti dall&#39;aorta, se presenti, wenza legatura. </li><li> Posizionare il rene in soluzione salina fredda su un tampone sterile in un piatto di cultura. </li><li> Euthanize il topo donatore per dislocazione cervicale. </li></ol> 4. Trapianto di Rene - Rene Preparazione <ol><li> Creare una patch aortica dividendo la parete dell&#39;aorta longitudinalmente di fronte l&#39;arteria renale. Identificare i lumi dei vasi nella patch che devono essere ligato o evitati quando si esegue l&#39;anastomosi arteriosa. </li><li> Collocare una sutura 10 / O dall&#39;esterno all&#39;interno del lume della vena renale superiormente ed una seconda sutura separata inferiormente. Le suture utilizzati per dividere la vena surrenale e vena gonadica possono essere utilizzati per orientare la nave. </li><li> Posizionare il rene nel fianco destro del destinatario (Figura 1.1) e verificare che i punti di sutura sono in grado di garantire la anastomosi vascolari possono essere completate senza suture diventare intrecciati in tamponi o altri strumenti. </li></ol> 5. Rene Transplanzione - anastomosi vascolare Applicare fascette microvascolari prima inferiormente poi superiormente che comprende la vena cava e aorta. <ol><li> Fare un ventomy con un ago (31G) per pungere la parete anteriore della vena cava. Lavare il sangue dalla vena cava iniettando circa 50 microlitri di 0.9% NaCl. </li><li> Ampliare la venotomy utilizzando una pinza punta fine con la loro apertura nel venotomy modo che la lunghezza è equivalente al diametro della vena renale donatore. </li><li> Inserire la sutura superiore 10 / O (che è già nel lume della vena renale) prima all&#39;apice del venotomy (Figura 1.2) ed entra nella parete posteriore della anastomosi in una sutura continua fino a raggiungere l&#39;apice inferiore (Figura 1.3). </li><li> Posizionare il 10 / O sutura inferiore e legare prima con un singolo nodo (Figura 1.4), poi legare la sutura che va dalla parete di fondo. </li><li> Utilizzando la sutura inferiore creare il fronte wall della anastomosi (Figura 1.5) con una sutura continua e legare alla fine sutura superiore al vertice superiore (Figura 1.6). </li><li> Crea un aortotomy sollevando la parete dell&#39;aorta con una pinza e taglio di una patch ellittica con le forbici (circa il aortotomy dovrebbe essere un quinto della circonferenza dell&#39;aorta e tre volte il lume dell&#39;arteria renale di lunghezza). </li><li> Inserire un 10 / O sutura nel punto superiore della patch aortico dall&#39;esterno all&#39;interno (Figura 1.7) e passare attraverso l&#39;aorta per legare fuori dei vasi all&#39;apice superiore della aortotomy. </li><li> Creare l&#39;anastomosi arteriosa con una sutura continua a partire superiormente (Figura 1.8) anastomosing la patch donatore al destinatario aorta, fare attenzione a non costringere l&#39;anastomosi legando la sutura troppo stretto (Figura 1.9). </li><li> Rimuovere le pinze vascolari inferiori prima poi il morsetto superiore al reperfuse rene (Figura 1.10 e 1.11). Visibile peristalsi dell&#39;uretere forse vede se viene raggiunto un adeguato apporto di sangue al uretere. </li></ol> 6 Trapianto di Rene - Anastomosi Ureteric <ol><li> Dividere eventuali allegati della vescica dalla parete addominale. </li><li> Far passare un ago (21G) da sinistra a destra della vescica attraverso entrambe le pareti. </li><li> Inserire punte forcep diritte nel lume dell&#39;ago e passare sia indietro attraverso la vescica tale che le pinze sono ora passando da destra a sinistra dal lato sinistro della vescica. </li><li> Tracciare la sutura sul uretere donatore attraverso la vescica tale che l&#39;uretere passa nel difetto vescica sinistra poi dal lato destro. </li><li> Suturare la avventizia dell&#39;uretere alla avventizia della vescica con tre singoli punti staccati in giro per il punto di ingresso con 9 / O nylon sutura su un ago rotondo e corposo. </li><li> Tagliare il prossimale dell&#39;uretere alla legatura, aprendo così the uretere per consentire l&#39;urina di fluire e permettere al fine uretere per ritrarre nel corpo della vescica. Visibile produzione di urina può essere osservata con sanguinamento da peri-ureterali vasi. </li><li> Chiudere il diritto difetto vescicale laterale con un solo interrotto 9 / O sutura di seta. </li></ol> 7 Recupero e assistenza post-operatoria Sostituire i visceri gastro-intestinale in addome secondo l&#39;orientamento originale e chiudere la parete addominale mediante il ravvicinamento delle muscoli retti con 6 / O sutura assorbibile. <ol><li> Approssimare la pelle con clip in metallo pelle. </li><li> Parzialmente invertire l&#39;anestesia con una iniezione sottocutanea di cloridrato atipamazole (10 ml / g). </li><li> Somministrare analgesia sottocutanea buprenorfina cloridrato (0,05 mg / kg) e il supporto per l&#39;iniezione di fluido 1 ml di sottocutanea 0,9% NaCl. </li><li> Recuperare il mouse in un armadio riscaldamento a 28 ° C per 24-48 ore. Osservare il mouse per malattia fino allacompletamento dell&#39;esperimento. </li><li> Rimuovere le clip di pelle metallo 7 - 10 giorni dopo l&#39;intervento. </li><li> Una volta che l&#39;esperimento è completo eutanasia il mouse per dislocazione cervicale. </li><li> Togliere e raccogliere il rene controlaterale e allogenico per l&#39;analisi istologica. </li></ol>

<ol>
	<li>Guild, W. R., Harrison, J. H., Merrill, J. P., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+homotransplantation+of+the+kidney+in+an+identical+twin.">Successful homotransplantation of the kidney in an identical twin.</a> Trans. Am. Clin. Climatol Assoc. 67, 167-173 (1955).</li><li>Wolfe, R. 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=Comparison+of+mortality+in+all+patients+on+dialysis,+patients+on+dialysis+awaiting+transplantation,+and+recipients+of+a+first+cadaveric+transplant.">Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant.</a> N. Engl. J. Med. 341, 1725-1730 (1999).</li><li>Nankivell, B. J., Alexander, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rejection of the Kidney Allograft. N. Engl. J. Med. 363, 1451-1462 (2010).</li><li>Tse, G. H., Hughes, J., Marson, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+mouse+kidney+transplantation.">Systematic review of mouse kidney transplantation.</a> Transplant International. 26, 1149-1160 (2013).</li><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.+Transplant.">Kidney transplants between mice of graded immunogenetic diversity. Transplant.</a> Proc. 5, 721-725 (1973).</li><li>Zhang, 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=Pattern+of+liver,+kidney,+heart,+and+intestine+allograft+rejection+in+different+mouse+strain+combinations.">Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combinations.</a> Transplantation. 62, 1267-1272 (1996).</li><li>Qi, 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=Depletion+of+cells+of+monocyte+lineage+prevents+loss+of+renal+microvasculature+in+murine+kidney+transplantation.">Depletion of cells of monocyte lineage prevents loss of renal microvasculature in murine kidney transplantation.</a> Transplantation. 86, 1267-1274 (2008).</li><li>Dang, Z., Mackinnon, A., Marson, L. P., Sethi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+atrophy+and+interstitial+fibrosis+after+renal+transplantation+is+dependent+on+galectin-3.">Tubular atrophy and interstitial fibrosis after renal transplantation is dependent on galectin-3.</a> Transplantation. 93, 477-484 (2012).</li><li>Jabs, W. 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=Heterogeneity+in+the+Evolution+and+Mechanisms+of+the+Lesions+of+Kidney+Allograft+Rejection+in+Mice.">Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice.</a> Am. J. Transplant. 3, 1501-1509 (2003).</li><li>Lin, 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=Deficiency+of+C4+from+Donor+or+Recipient+Mouse+Fails+to+Prevent+Renal+Allograft+Rejection.">Deficiency of C4 from Donor or Recipient Mouse Fails to Prevent Renal Allograft Rejection.</a> Am. J. Pathol. 168, 1241-1248 (2006).</li></ol>

Gli autori non hanno nulla da rivelare.

Il modo più ben descritto effettuare l&#39;anastomosi arteriosa è utilizzare l&#39;aorta distale del donatore, con l&#39;arteria renale in continuazione, in modo end-to-side all&#39;aorta destinatario. Descriviamo l&#39;uso di una patch aortica, simile al mirroring &#39;Carrell cerotto&#39; che ha effettuato nel trapianto di rene umano che riteniamo essere più conveniente. Anche se segnalazioni in letteratura di tempo donatore e ricevente operativa sono scarsi crediamo che utilizzando una patch aortico al destinatari...

Trapianto renale con successo per il trattamento di insufficienza renale è stata descritta per la prima nel 1955 tra gemelli monozigoti 1, da allora è diventato un rivoluzionario trattamento per i pazienti con insufficienza renale allo stadio terminale in tutto il mondo, che offre sia un miglioramento nella durata e la qualità della vita 2. Tuttavia la sopravvivenza del trapianto a lungo termine è stata ostacolata da una moltitudine di processi patologici con conseguente danno cronica del trapianto 3. Rifiuto del rene trapiantato negli esseri umani rimane una delle principali cause di morbilità, nonostante i significativi miglioramenti nei regimi immunosupporessive. L&#39;obiettivo di sviluppare un modello murino di trapianto renale è replicare da vicino il processo e la patologia trovato nel trapianto renale umano 4. Skoskiewicz et al. Prima descritto il modello murino di trapianto di rene nel 1973 5. Anche se sono richieste competenze avanzate microchirurgiche, è una preziosa tool per diversi motivi: il genoma del topo è stato ben caratterizzato e c&#39;è una grande varietà di metodi e tecniche disponibili per studi sui topi sperimentali. Molti gruppi che utilizzano il modello murino di trapianto renale hanno usato il rene trapiantato come un organo di supporto vitale, ma in altri studi e nella nostra metodologia descritta uno dei reni nativi del topo destinatario viene lasciato in situ per tutta la durata dell&#39;esperimento 4. Il vantaggio è che il mouse subisce una sola anestesia e il funzionamento riducendo la morbilità per il mouse e il rischio di morte per una seconda procedura. Inoltre, il mouse non soffre gli effetti negativi di insufficienza renale progressiva. Sebbene esistano modelli di rigetto allogenico in altri organi come il cuore e la pelle, queste non sono sempre direttamente rilevanti per il trapianto renale. Ci sono prove che questi modelli suscitano diverse modalità e dynamics di rifiuto, per esempio, l&#39;andamento nel tempo del rigetto in allotrapianto cardiaco e trapianto renale differisce in modo significativo in determinate combinazioni di deformazione 6. Abbiamo descritto i modelli renale acuta rigetto allogenico in BALB donatori / c in non-transgenici topi FVB / NJ, questo modello ha mostrato lesioni mediato cellulare con accumulo di cellule T e macrofagi 7. In alternativa abbiamo anche descritto un modello di danno cronica del trapianto che presenta fibrosi interstiziale e atrofia tubulare, questo risulta dal trapianto di un rene da C57BL / 6 BM12 donatori in C57BL / 6 destinatari, in quanto questi mouse sono caratterizzati da un unico MHC di classe II mis loci -match 8. Molteplici aspetti del trapianto sono stati studiati utilizzando il modello murino di trapianto renale tra cui rigetto acuto, rigetto cellulare e umorale, ischemia riperfusione, e sperimentando nuovi agenti terapeutici. Abbiamo modificato la t chirurgicaecnica per ridurre il tempo di funzionamento e migliorare la facilità di intervento chirurgico. In particolare abbiamo descritto donatore simultanea e la preparazione destinatario e un semplificato tecnica di anastomosi vascolare utilizzando un continuo aortica patch di anastomosi. Questo video e manoscritto forniranno punti chiave per aiutare nella creazione di questa tecnica.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Blunt Dissecting Scissors</td>
			<td>Fine Science Tools</td>
			<td>&#160;14072-10</td>
			<td>For skin cutting</td>
		</tr>
		<tr>
			<td>Curved Castoviejo scissors</td>
			<td>Fine Science Tools</td>
			<td>15017-10</td>
			<td>For tissue cutting</td>
		</tr>
		<tr>
			<td>Spring Scissors &#8211; straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td>For suture cutting</td>
		</tr>
		<tr>
			<td>Toothed forceps 1x2 teeth</td>
			<td>Fine Science Tools</td>
			<td>11021-12</td>
			<td></td>
		</tr>
		<tr>
			<td>2 x Fine Tip forceps (Dumont No.5)</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled Fine Tip forceps (Dumont No. 5/45)</td>
			<td>Fine Science Tools</td>
			<td>11253-25</td>
			<td>For blunt dissecting</td>
		</tr>
		<tr>
			<td>Curved Fine Tip forcep (Dumont No.7)</td>
			<td>Fine Science Tools</td>
			<td>11273-22</td>
			<td>Useful to pass around vessels</td>
		</tr>
		<tr>
			<td>Curved Crile Haemostat</td>
			<td>Fine Science Tools</td>
			<td>1300-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip applicator with lock</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
		</tr>
		<tr>
			<td>2 x Micro serrefines spring width 2mm, jaw length 4mm</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Microvascular clamps</td>
		</tr>
		<tr>
			<td>2 x Colibri 3cm wire retractor</td>
			<td>Fine Science Tools</td>
			<td>17000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo needle holder with lock</td>
			<td>Fine Science Tools</td>
			<td>120660-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clip applicator</td>
			<td>Fine Science Tools</td>
			<td>12031-07</td>
			<td></td>
		</tr>
		<tr>
			<td>7mm wound clips</td>
			<td>Fine Science Tools</td>
			<td>12032-07</td>
			<td>Remove 7 to 10 days after surgery</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>OPMI pico microscope</td>
			<td>Carl Zeiss</td>
			<td>S100</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cautery unit with fine tip</td>
			<td>Geiger</td>
			<td>150A</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat electronic pad</td>
			<td>Cozee Cumfort</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Euroklav 23-S</td>
			<td>Melag</td>
			<td>n/a</td>
			<td>Autoclave</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Disposable equipment</td>
		</tr>
		<tr>
			<td>7/O Silk braided suture</td>
			<td>Pearsall</td>
			<td>30514</td>
			<td></td>
		</tr>
		<tr>
			<td>10/O Dafilon (polyamide) suture</td>
			<td>B-Braun</td>
			<td>&#160;G1118099</td>
			<td></td>
		</tr>
		<tr>
			<td>6/O Vicryl (plygalectin)</td>
			<td>Ethicon</td>
			<td>W9537</td>
			<td></td>
		</tr>
		<tr>
			<td>Regular bevel needle, 1 inch, 21G</td>
			<td>Bection, Dickinson and Company</td>
			<td>305175</td>
			<td>For ureteric anastamosis</td>
		</tr>
		<tr>
			<td>Regular bevel needle, 5/8 inch, 25G</td>
			<td>Bection, Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>Regular bevel needle, 1/2 inch, 30G</td>
			<td>Bection, Dickinson and Company</td>
			<td>304000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin needle 1ml, 29G</td>
			<td>Bection, Dickinson and Company</td>
			<td>324827</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin needle 0.3ml, 30G</td>
			<td>Bection, Dickinson and Company</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe slip tip</td>
			<td>Bection, Dickinson and Company</td>
			<td>300184</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe slip tip</td>
			<td>Bection, Dickinson and Company</td>
			<td>302187</td>
			<td></td>
		</tr>
		<tr>
			<td>Wypall paper swabs</td>
			<td>Kimberley-Clark</td>
			<td>L40</td>
			<td>sterilised by autoclave</td>
		</tr>
		<tr>
			<td>Cotton wool buds</td>
			<td>Johnson and Johnson</td>
			<td>n/a</td>
			<td>sterilised by autoclave</td>
		</tr>
		<tr>
			<td>Plain drapes</td>
			<td>Guardian</td>
			<td>CB03</td>
			<td>sterilised by autoclave</td>
		</tr>
		<tr>
			<td>Cell culture dish 60mm x 15mm</td>
			<td>Corning Incorporated</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensing Pin</td>
			<td>B-Braun</td>
			<td>DP3500L / 413501</td>
			<td>Used with NaCl 0.9%</td>
		</tr>
		<tr>
			<td>Re-agents and Drugs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2%</td>
			<td>Allergan Ltd</td>
			<td>21956GB10X</td>
			<td></td>
		</tr>
		<tr>
			<td>(Videne) Povidone-iodine 10%</td>
			<td>Ecolab Ltd</td>
			<td>PL 04509/0041</td>
			<td></td>
		</tr>
		<tr>
			<td>(Vetalar V) Ketamine hydrochloride</td>
			<td>Pfizer Animal Health</td>
			<td>Vm 42058/4165</td>
			<td>100mg/ml solution (dose 200mg/kg)</td>
		</tr>
		<tr>
			<td>(Domitor) Medetomidine hydrochloride&#160;</td>
			<td>Orion Pharma</td>
			<td>Vm 06043/4003</td>
			<td>1mg/ml (dose 0.5mg/kg)</td>
		</tr>
		<tr>
			<td>(Vetergesic) Bupernorphine hydrochloride&#160;</td>
			<td>Alsto Animal Health</td>
			<td>Vm 00063/4002</td>
			<td>0.3mg/ml (dose 0.05mg/kg)</td>
		</tr>
		<tr>
			<td>(Antisedan) Atipamezole hydrochoride</td>
			<td>Orion Pharma</td>
			<td>Vm 06043/4004</td>
			<td>5mg/ml (dose 2mg/kg)</td>
		</tr>
		<tr>
			<td>University of Wisconsin Solution</td>
			<td>Belzer Bridge to Life</td>
			<td>n/a</td>
			<td>dose approximately 500 microlitres/mouse</td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Baxter</td>
			<td>FKE1323</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sulphate</td>
			<td>non-proprietary</td>
			<td>n/a</td>
			<td>5000units/ml (dose 5units/mouse)</td>
		</tr>
	</tbody>
</table>

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.

Renale rigetto del trapianto allogenico può essere valutata mediante analisi istologica di sezioni di tessuto incluse in paraffina methacarn-fisso del rene trapiantato (Figura 2). Trapianto di reni tra Isograft risultati topi singenici in renale danno da riperfusione ischemica, comunque da 4 settimane di tubuli hanno recuperato e sono istologicamente paragonabili a reni nativi. Rigetto acuto può essere modellato da C57BL / 6 trapianto renale in riceventi BALB / c, entro 1 settimana non vi è diffusa i...

Watch this Scientific Journal Video about Mouse Kidney Transplantation: Models of Allograft Rejection at JoVE.com

Mouse Kidney Transplantation: Models of Allograft Rejection

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 ...

mouse-kidney-transplantation-models-of-allograft-rejection

Research

JoVE Journal

Medicine

20.4K Views.  The University of Edinburgh. 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.

Video: Mouse Kidney Transplantation: Models of Allograft Rejection

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

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

Imaging non invasivo di acuta Alloinnesto rigetto dopo trapianto renale Rat Utilizzo<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

Ad alta frequenza di Ecografia del mouse cervicali Linfonodi

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Modello murino della alloimmune indotta Rifiuto vascolare e trapianto Arteriosclerosi

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

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.

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 ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotermica<em&gt; Ex Vivo</em&gt; Rene perfusione per la Preservazione della Kidney Innesti prima del trapianto

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Valutazione della funzione del rene in modelli murini di malattia glomerulare

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

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 ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Cancellazione ottica e imaging del tessuto renale immunoetichettato

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Digital Home-Monitoring of Patients after Kidney Transplantation: The MACCS Platform

The MACCS (Medical Assistant for Chronic Care Service) platform enables secure sharing of key medical information between patients after kidney transplantation and physicians. Patients provide information such as vital signs, well-being, and medication intake via smartphone apps. The information is transferred directly into a database and electronic health record at the kidney transplant center, which is used for routine patient care and research. Physicians can send an updated medication plan and laboratory data directly to the patient app via this secure platform. Other features of the app are medical messages and video consultations. Consequently, the patient is better-informed, and self-management is facilitated. In addition, the transplant center and the patient's local nephrologist automatically exchange notes, medical reports, laboratory values, and medication data via the platform. A telemedicine team reviews all incoming data on a dashboard and takes action, if necessary. Tools to identify patients at risk for complications are under development. The platform exchanges data via a standardized secure interface (Health Level 7 (HL7), Fast Healthcare Interoperability Resources (FHIR)). The standardized data exchange based on HL7 FHIR guarantees interoperability with other eHealth solutions and allows rapid scalability to other chronic diseases. The underlying data protection concept is in concordance with the latest European General Data Protection Regulation. Enrollment started in February 2020, and 131 kidney transplant recipients are actively participating as of July 2020. Two large German health insurance companies are currently funding the telemedicine services of the project. The deployment for other chronic kidney diseases and solid organ transplant recipients is planned. In conclusion, the platform is designed to enable home monitoring and automatic data exchange, empower patients, reduce hospitalizations, and improve adherence, and outcomes after kidney transplantation.

The MACCS platform&#160;is a comprehensive telemedicine concept aiming at better outcomes after kidney transplantation by sharing key medical information between patients and physicians. A telemedicine team reviews incoming data to detect potential complications and to improve adherence in kidney transplant recipients to achieve better long-term outcomes.