Vorremmo riconoscere Gundula Hertl per il suo sostegno.

Tutte le procedure in questo protocollo sono state approvate da linee guida e normativa sul benessere degli animali di tedesco. Considerare l'utilizzo di topi nudi di 10 a 12-week-old NMRI e topi C57Bl/6 (donatore: vecchia femmina; destinatario: maschio) durante gli esperimenti. Utilizzare topi nudi NMRI per isolamento dell'isolotto. Tenere i topi in condizioni definite secondo le normative di struttura animale locale.
1. induzione di diabete da Streptozotocin
<ol><li>Il giorno -4, misurare il peso corporeo di 10 a 12 settimana-vecchio NMRI nudi topi e topi C57Bl/6 utilizzando una macchina di pesatura.</li>
	<li>Secondo il peso corporeo misurato, preparare una dose di 180 mg/kg dello streptozotocin in 0,1 mL di soluzione fisiologica per topo. Nota: Streptozotocin dovrebbe essere appena preparata prima dell'uso e coperto con carta stagnola, in quanto è sensibile alla luce.</li>
	<li>Iniettare i topi syngeneic e allogenici ciascuno con una singola dose di 0,1 mL dello streptozotocin (180 mg/kg) per via intraperitoneale utilizzando un ago 26-G.</li>
	<li>Il giorno -3, -2 e -1 raccogliere 2-4 µ l di sangue da puntura le vene di coda dei topi non a digiuno.</li>
	<li>Utilizzare 2-4 µ l di campione di sangue a striscia reattiva per misurare il glucosio nel sangue in topi nudi NMRI e topi C57Bl/6 Nota: A questo punto, l'80% dei topi dovrebbe diventare diabetico (20 mmol/L).<ol>
<li>Utilizzare topi per trapianto dell'isolotto quando loro livelli di glucosio nel sangue raggiungono 20 mmol/L.</li>
	</ol>
</li>
</ol>2. isolamento degli isolotti pancreatici dai topi
<ol><li>Anestetizzare i topi con ketamina (100 mg/kg di peso corporeo) e xilazina (20 mg/kg di peso corporeo) tramite l'iniezione intraperitoneale con un ago 26-G. Nota: I volumi di ketamina e xilazina sono stati determinati secondo il peso corporeo degli animali (0,10 mL/10 g).</li>
	<li>Tenere i topi in posizione supina e disinfettare con soluzione povidone-iodio. Confermare l'eutanasia corretto controllando il riflesso di pedale. Nota: In caso di C57Bl/6 radere la regione addominale.</li>
	<li>Tenete la pelle con una pinzetta e fare un'incisione del midline con le forbici per esporre la cavità addominale. Posto il situs dell'intestino a sinistra e di fuori della cavità addominale su un impacco di garza sterile.</li>
	<li>Regolare lo stomaco in un'altra direzione per individuare il pancreas e gli altri associati tessuti.</li>
	<li>Raggiungere l'aorta e fare un piccolo taglio per drenare il sangue. Utilizzare garza sterile per assorbimento di sangue.</li>
	<li>Rimuovere con cura l'intestino. Separare il pancreas dallo stomaco e milza.</li>
	<li>Asportare il pancreas e metterlo in una capsula Petri fresco. Rimuovere tutti i materiali indesiderati, quali grassi, usando il forcipe.</li>
	<li>Riempire una siringa da 5 mL con 4 mL di collagenasi e iniettare nel pancreas con un ago da 26 G (soluzione: 30 mg di collagenasi in 12 mL di soluzione di Hank).</li>
	<li>Tritare il pancreas con le forbici per aumentare la superficie per una migliore digestione e trasferirlo in una provetta tappo rosso 12 mL. Nota: Soluzione salina equilibrata di Hank: HBSS 10 x (100 mL), penicillina-streptomicina (10ml), gentamicina (1 mL), ciprofloxacina (10ml), HEPES (35 mL), distillato di H2O (900 mL)</li>
	<li>Eseguire una digestione per 10 min a 37 ° C in un bagnomaria con agitazione. Vortexare il campione tre volte a intervalli regolari per 15 s.</li>
	<li>Mescolare energicamente il pancreas digerito a mano per 3 min e posizionarlo sul ghiaccio per 10 s.</li>
	<li>Aggiungere 6 mL di soluzione di Hank fredda per fermare la reazione di digestione e centrifugare a 560 x g per 3 min a temperatura ambiente.</li>
	<li>Rimuovere il supernatante e sciogliere la pastiglia in 10 mL di terreno di siero (P/FCS) vitello Parker/fetale a temperatura ambiente. Nota: Medio P/FCS: TCM-199 10 x (100 mL), FCS (50 mL), penicillina-streptomicina (10ml), HEPES (20 mL), distillato di H2O (900 mL)</li>
	<li>Identificare gli isolotti sulla base della loro morfologia (sferoide, colore bruno-dorato) sotto un microscopio chiaro. Evitare di isolotti con centri di scuri. Verifica per purezza e vitalità eseguendo Ditizone macchiatura sotto un microscopio chiaro. Nota: Ditizone si lega allo ione zinco e fornisce la macchia di colore rosso agli isolotti.</li>
	<li>Utilizzare una pipetta 1 mL per contare e accuratamente isolotti sotto un microscopio chiaro e trasferirli su una piastra Petri fresca (60 x 15 mm) contenente 4 mL di terreno di P/FCS. Nota: Il volume di P /medie FCS di Petri non devono superare i 4 mL al fine di garantire la disponibilità di ossigeno.</li>
	<li>Mantenere gli isolotti isolati in 4 mL di terreno di P/FCS durante la notte a 37 ° C in un incubatore.</li>
</ol>3. preparazione degli animali per trapianto di insule pancreatiche
<ol><li>Rimuovere gli isolotti isolati da incubatore 37 ° C e metterli sotto una panchina di flusso. Centro gli isolotti con un movimento oscillante circolare del piatto Petri.</li>
	<li>Riempire una siringa 1 mL (ago 23-G blu) con 0,1 mL di terreno di P/FCS. Quindi, aggiungere 350 isolotti in 0,3 mL di soluzione di Hank. Tenere la siringa in posizione verticale per consentire gli isolotti di stabilirsi.</li>
	<li>Diminuire il volume a 0,05 mL e sostituire l'ago 23-G blu con un ago 26G marrone. La stessa siringa, aggiungere 0,15 mL di Ficoll (medio gradiente di densità) verso l'alto per raggiungere un volume finale di 0,2 mL in siringa. Evitare le bolle d'aria.</li>
	<li>Tenere la siringa in posizione verticale e ruotare leggermente per evitare l'agglutinamento di isolotti. Le isolette si depositerà presso il cono della siringa entro 2-3 min.</li>
	<li>Misurare i livelli di glucosio nel corpo peso e sangue dei topi destinatari; i livelli di glucosio nel sangue dovrebbero essere circa 20 mmol/L.</li>
	<li>Anestetizzare i topi con ketamina (100 mg/kg di peso corporeo) e xilazina (20 mg/kg di peso corporeo) tramite l'iniezione intraperitoneale con ago 26-G.</li>
	<li>Posizionare i mouse in posizione supina su una piastra termica calda (37 ° C). Controllare la profondità dell'anestesia pizzicando la punta.</li>
	<li>Pulire la regione addominale con soluzione povidone-iodio (disinfettante della pelle). Per evitare che gli occhi secchi, tenerli umidi con unguento oculare. Nota: In caso di C57Bl/6 radere la regione addominale.</li>
</ol>4. islet trapianto Intraportal per via
<ol><li>Il giorno 0, iniziare con una laparotomia tenendo la pelle con il forcipe e fare un 2-3 cm-incisione del midline lungo con una forbice. Posto una garza sterile bagnata comprimere intorno ai bordi della ferita per mantenerla umida.</li>
	<li>Posto il situs dell'intestino a sinistra e di fuori della cavità addominale sull'impacco di garza sterile. Nota: Mantenere umido durante tutta la procedura con soluzione di NaCl 0,9% pre-riscaldata o garza sterile imbevuto in una soluzione salina normale dell'intestino.</li>
	<li>Spostare il duodeno e individuare il pancreas. Individuare la vena portale situata presso il sito ventrale spingendo verso l'alto il pancreas. Utilizzare un dito e il pollice per esporre la vena portale e, una volta esposto, perforarla vicino al fegato.</li>
	<li>Entro 1 min, lentamente e costantemente iniettare gli isolotti (0,2 mL dal punto 3.4) nella vena portale sotto l'ispezione di lente di ingrandimento per evitare eventuali perdite di isolotti. Nota: Dopo l'iniezione, controllare sempre per isolotti rimanenti nella siringa. Prendere 1 mL di media P/FCS in una siringa, a filo in un fresco di Petri e conteggio al microscopio.</li>
	<li>Posizionare un misuratore sterile sul sito di puntura e applicare una pressione con il dito indice (profondità di penetrazione: 0,5-1 cm) per 6 min fermare l'emorragia. Nota: Il volume iniettato non deve superare 0,2 mL. Pressione e volume eccessivo può danneggiare le isolette a causa della sollecitazione di taglio.</li>
	<li>Posizionare con attenzione il situs dell'intestino, peritoneo, strato del muscolo addominale e fascia posteriore in posizione.</li>
	<li>Usando il forcipe, sollevare la pelle, chiudere l'incisione del muscolo con il suturare assorbibile di seta e strato superiore della pelle con 2-3 clip.</li>
	<li>Pulire la superficie con un disinfettante della pelle, applicare un guaritore ferita e posizionare i topi in gabbie individuali. Guaritore ferita fornisce un strato di acrilico resistente all'acqua e proteggere da macerazione e infezione secondaria. Nota: Osservare i topi fino a quando essi hanno ripreso conoscenza sufficiente, fornire tramadol (0,2 mg per topo) insieme a fluidi. In questo protocollo, topi recupero dall'anestesia rapidamente e tramadol amministrazione ha ridotto il dolore. Dopo 7 giorni, dovrebbe guarire la ferita.</li>
	<li>Nutrire i topi con acqua e cibo normale. L'escrezione dell'urina e del consumo di cibo e acqua aumenterà dopo l'iniezione di streptozotocin.</li>
	<li>Misurare i livelli di glucosio nel sangue ogni giorno per la prima settimana e poi una volta a settimana in topi syngeneic. In topi allogenici, misurare i livelli di glucosio nel sangue tre volte a settimana per fino a 15 giorni.</li>
</ol>

<ol>
	<li>McCall, M., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), a007823 (2012).</li><li>Shapiro, A. 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=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>Cantarelli, E., 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=Bone+marrow+as+an+alternative+site+for+islet+transplantation.">Bone marrow as an alternative site for islet transplantation.</a> Blood. 114 (20), 4566-4574 (2009).</li><li>Echeverri, G. 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=Endoscopic+gastric+submucosal+transplantation+of+islets+(ENDO-STI):+technique+and+initial+results+in+diabetic+pigs.">Endoscopic gastric submucosal transplantation of islets (ENDO-STI): technique and initial results in diabetic pigs.</a> American Journal of Transplantation. 9 (11), 2485-2496 (2009).</li><li>Korsgren, 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=Optimising+islet+engraftment+is+critical+for+successful+clinical+islet+transplantation.">Optimising islet engraftment is critical for successful clinical islet transplantation.</a> Diabetologia. 51 (2), 227-232 (2008).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95 (12), 1449-1461 (2008).</li><li>Lingwal, 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=Inhibition+of+gelatinase+B+(matrix+metalloprotease-9)+activity+reduces+cellular+inflammation+and+restores+function+of+transplanted+pancreatic+islets.">Inhibition of gelatinase B (matrix metalloprotease-9) activity reduces cellular inflammation and restores function of transplanted pancreatic islets.</a> Diabetes. 61 (8), 2045-2053 (2012).</li><li>Chen, 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=Improved+intraportal+islet+transplantation+outcome+by+systemic+IKK-beta+inhibition:+NF-kappaB+activity+in+pancreatic+islets+depends+on+oxygen+availability.">Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-kappaB activity in pancreatic islets depends on oxygen availability.</a> American Journal of Transplantation. 11 (2), 215-224 (2011).</li><li>Wang, Y., Wang, T., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+aspects+are+critical+for+carrying+out+intraportal+islet+transplants+successfully+in+a+diabetes+mouse+model.">Three aspects are critical for carrying out intraportal islet transplants successfully in a diabetes mouse model.</a> Experimental and Clinical Transplantation. 10 (3), 302-304 (2012).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments. (50), (2011).</li><li>Rother, K. I., Harlan, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+facing+islet+transplantation+for+the+treatment+of+type+1+diabetes+mellitus.">Challenges facing islet transplantation for the treatment of type 1 diabetes mellitus.</a> Journal of Clinical Investigation. 114 (7), 877-883 (2004).</li><li>Su, 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=Small+islets+are+essential+for+successful+intraportal+transplantation+in+a+diabetes+mouse+model.">Small islets are essential for successful intraportal transplantation in a diabetes mouse model.</a> Scandinavian Journal of Immunology. 72 (6), 504-510 (2010).</li><li>Rekittke, N. E., Ang, M., Rawat, D., Khatri, R., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Therapy+of+Type+1+Diabetes+Mellitus:+From+Pancreatic+Islet+Transplantation+to+Mesenchymal+Stem+Cells.">Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells.</a> Stem Cells International. 2016, 22 (2016).</li></ol>

Tutti gli autori dichiarano di non avere alcun conflitto di interessi.

In questo studio, il percorso intraportal di trapianto dell'isolotto è esplorato. Questo protocollo è altamente efficace nell'alleviamento dello stress diabetico e fornisce un'opportunità di approfondimento per esplorare studi di trapianto dell'isolotto. Questo protocollo conferma che circa 350 isolotti murini sono in grado di invertire lo stato iperglicemico. Inoltre, nessuno dei topi morti durante la procedura e controllo glicemico è stato realizzato in tutti i destinatari. Livelli di glucosio nel sangue e peso del...

Trapianto dell'isolotto è un approccio promettente per il trattamento di tipo 1 diabete mellito1. Il primo tentativo di trasferire un frammento del pancreas di pecore in un paziente diabetico è stato effettuato da Watson Williams nel 1893. Tuttavia, un importante passo avanti nel trapianto di insule pancreatiche clinica è stato realizzato con il protocollo di Edmonton, e da allora in poi, una serie di programmi nazionali sono stati sviluppati2. In passato, diversi siti, quali il midollo osseo, borsa omentale, regione intramuscolare, superficie mucosa gastrica, milza e capsula del rene, sono stati esplorati in modelli preclinici, ma il sistema venoso portale è considerato come uno del sito adatto ed efficace per programmi clinici3,4,5,6.
Somministrazione esogena di insulina può essere sostituito dall'infusione dell'isolotto nella vena portale. Questo è considerato un sito preferito, perché l'apporto di ossigeno è paragonabile a quella del pancreas nativo a causa della posizione a valle della confluenza del portale dell'arteria e della vena. Inoltre, c'è una grande superficie, così che la struttura tridimensionale degli isolotti può essere conservata e vascolarizzazione potrebbe essere agevolato5. In un destinatario topo diabetico, 2.000 equivalenti di isolotto umana o suina o mouse 350 isolotti sono adeguati per invertire il stato iperglicemico7,8. Livelli di euglycemic sono stati segnalati per 15 giorni nel modello murino destinatario degli isolotti xenogeneic e nel modello di trapianto allogeneic del mouse-a-mouse e per più di 120 giorni nel trapianto syngeneic del mouse-a-mouse.
Fattori pertinenti per l'efficacia di trapianto dell'isolotto via vena portale sono appropriata anestesia, metodo di puntura e di emostasi9. L'anestesia può essere indotta da inalazione (5% isoflurano) o iniezione (ketamina, xilazina o pentobarbital), e le sostanze sono spesso combinate. Per il controllo della profondità e tempo di anestesia, si dovrebbe prestare attenzione allo stato del mouse, ad esempio il colore della membrana mucosa, palpebra, riflesso corneale, frequenza respiratoria e temperatura corporea. È importante garantire che l'animale non è in difficoltà e che sopravvive l'operazione. La temperatura può essere mantenuta dai dispositivi di riscaldamento differenti, quali termofori, rosso lampadine, ecc A caldo piastra termica è stato utilizzato per mantenere una temperatura di 25-30 ° C tra il mouse e la tabella di funzionamento, che impedisce il verificarsi di ipotermia. I dosaggi degli anestetici sono importanti in quanto tutti loro sono metabolizzati dal fegato, e funzione epatica può essere transitoriamente disordinati dall'infusione della sospensione dell'isolotto. Il punto di puntura ideale per vena portale è la posizione tra la prima e la seconda vena tributaria.
Il numero di isolotti e il volume di iniezione prevista anche influenza l'esito del trapianto, come un volume eccessivo può aumentare la sollecitazione di taglio. Un volume di 0,2 mL è considerato appropriato per trapianto dell'isolotto nella vena portale. La ferita di perforatura (ago 26-gauge) induce emorragia dalla vena portale, che deve essere fermato in modo tempestivo ed efficace. Garza sterile o un dito può essere applicato con una pressione minima presso il sito della puntura per circa 6 minuti per fermare l'emorragia. Presi insieme, portale isolotto iniezione è efficace e fornisce il regolamento sopra i livelli della glicemia in modelli di topo diabetico chimicamente indotta.

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="20">
			<td>C57Bl/6 mice&#160;</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>BALB/c mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td>NMRI nude mice</td>
			<td>Charles River (Sulzfeld, Germany)</td>
			<td></td>
			<td>(10-12 weeks)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine</td>
			<td>Bela-pharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine</td>
			<td>CEVA TIERGESUNDHEIT GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe, 23G and 26G needle</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Falcon</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Carl Roth</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Gauze</td>
			<td>Fuhrmann</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shaking Water bath</td>
			<td>Kotterman</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Braun</td>
			<td>1501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose Meter&#160;</td>
			<td>One Touch</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm Thermal Plate</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Braunol (povidone-iodide solution)</td>
			<td>B.Braun Melsungen AG</td>
			<td>3864154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liposic Edo - Sprinkled Ointment (eye ointment)</td>
			<td colspan="2">Dr. G Mann Chem.-Pharm.&#160; GmbH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Fa. Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Flow Bench</td>
			<td>Herdus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ficoll</td>
			<td>Sigma-Aldrich</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-0 Silk Suture Vicryl</td>
			<td>Braun</td>
			<td></td>
			<td>(7.5 *1.75mm)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Michel Clamps (clips)</td>
			<td>Aesculap&#160;</td>
			<td>BN507R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P/FCS Solution</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCM-199 (10x)</td>
			<td>Gibco</td>
			<td>21180-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1 M)</td>
			<td>Biochrom</td>
			<td>L 1613</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamycin</td>
			<td>Ratiopharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ciprofloxacin</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#39;s Solution</td>
			<td>Gibco</td>
			<td>14060-040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Roche</td>
			<td>11213865001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptozotocin</td>
			<td>Calbiochem</td>
			<td>572204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPSITE-spray (wound healer)</td>
			<td>Smith and nephew</td>
			<td>66004978</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Reugular food</td>
			<td>Altromin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Head light LED 16042 (magnifying glass)</td>
			<td>Eschenbach</td>
			<td>16042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM-1(CD31) monoclonal primary antibody</td>
			<td>Millipore, Chemicon</td>
			<td>CBL1337</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-rat secondary antibody</td>
			<td>Dianova</td>
			<td>712095153</td>
			<td>Dilution (1:400)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyclonal guinea pig anti-insulin primay antibody</td>
			<td>Dako</td>
			<td>A0564</td>
			<td>Dilution (1:500)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-guinea pig secondary antibody</td>
			<td>Dianova</td>
			<td>706-259-148</td>
			<td>Dilution (1:400)</td>
		</tr>
	</tbody>
</table>

intraportal transplantation of pancreatic islets in mouse model

Trapianto di isole pancreatiche per ridurre l'iperglicemia è altamente-riuscito nei roditori con diabete indotto chimicamente. Il sito più comune di trapianto trapianto di insule sperimentale è la capsula del rene. Tuttavia, come di meno è conosciuto circa l'interazione degli isolotti pancreatici con costituenti del sangue, ha anche senso di utilizzare l'approccio della vena portale nel trapianto di insule pancreatiche sperimentale.
Questo protocollo dimostra una tecnica di trapianto dell'isolotto intraportal in topi nudi NMRI. Streptozotocin (180 mg/kg) è iniettato intraperitonealmente per indurre l'iperglicemia nei topi di destinatario. Sono considerati come diabetico a un livello di glucosio nel sangue non a digiuno superiore a 20 mmol/L. Un giorno prima del trapianto, isolotti pancreatici del topo sono isolati dal pancreas donatore di digestione della collagenosi; un minimo di 350 isolotti sono utilizzata per ogni destinatario diabetica. A seconda del rendimento di isolamento dell'isolotto, due o più topi donatore sono utilizzati per ogni destinatario. Dopo coltura durante la notte a 37 ° C, isolotti sono amministrati nel destinatario fegato tramite la vena portale. Dopo la chirurgia, i topi sono protetti in case rosse Makrolon e osservati fino a sono svegli. Questo protocollo gestisce controllo glycemic per 120 giorni in topi syngeneic e 15 giorni in topi allogenici.

La competenza delle insule trapiantate è stata studiata in topi diabetici chimicamente indotti. Gli isolotti sono stati trapiantati tramite il sistema venoso portale nel modello di topi diabetici. Il rapporto donatore-destinatario era 2:1. Nel modello syngeneic del topo, due topi di donatore sono stati utilizzati per ottenere 350 isolotti. Gli isolotti sono stati poi trapiantati in topi diabetici. Stata osservata una riduzione nel livello di glucosio nel sangue il giorno 1 dopo trapianto...

Watch this Scientific Journal Video about Intraportal Transplantation of Pancreatic Islets in Mouse Model at JoVE.com

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Trapianto di isole pancreatiche è un modo per raggiungere normoglycemia in diabete di tipo 1. Questo articolo si concentra sulla tecnica di trapianto attraverso il percorso intraportal per mantenere i livelli normali del glucosio nei topi diabetici.

Intraportal trapianto di isole pancreatiche nel modello del topo

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

intraportal-transplantation-of-pancreatic-islets-in-mouse-model

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JoVE Journal

Medicine

9.4K Views.  Clinical Research Lab. Trapianto di isole pancreatiche è un modo per raggiungere normoglycemia in diabete di tipo 1. Questo articolo si concentra sulla tecnica di trapianto attraverso il percorso intraportal per mantenere i livelli normali del glucosio nei topi diabetici.

Video: Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Mouse Model of in Utero Transplantation

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

A Mouse Model of in Utero Transplantation

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

Un modello murino di Nell&#39;utero Trapianto

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

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Medicina

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Modello murino di endometriosi chirurgicamente indotto da Auto-trapianto di tessuto uterino

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolli di colorazione di isole pancreatiche umane

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Procedure chirurgiche per un modello di Rat parziale trapianto di fegato con la ricostruzione arteriosa epatica

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Protocollo migliorato per microdissezione laser di isole pancreatiche umane da campioni chirurgici

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

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

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

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

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

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

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

LEPR<sup&gt; Db</sup&gt; Mouse Modello di diabete di tipo 2: pancreas isolamento Islet e live-cella 2-Photon Imaging di isolotti Intact

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

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.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (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 ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Utilizzando la tecnica di manica in un modello murino di trapianto aortico - un Video didattico

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Intraportal trapianto di isole pancreatiche nel modello del topo

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Un modello murino di Nell'utero Trapianto