Questo lavoro è stato supportato dalla concessione core ricevuta dal dipartimento di biotecnologia, governo dell'India per l'Istituto nazionale di immunologia, Nuova Delhi. Indirizzo attuale di Dr. Bhattacharjee è divisione di gastroenterologia, Epatologia e nutrizione, Ospedale Los Angeles dei bambini.

Le procedure presentate in questo protocollo sono state approvate dal comitato di etica animale istituzionale dell'Istituto nazionale di immunologia, Nuova Delhi. Il numero di riferimento seriale dell'approvazione è IAEC n. 319/13.
Nota: Ci sono ottime risorse su procedure di chirurgia generale20 e protocolli specifici per la chirurgia del roditore21. Per quelli che fanno chirurgia animale per la prima volta, è consigliabile per ampiamente pratica procedure chirurgiche su manichini prima di operare sugli animali.
1. preparazione
<ol><li>Prima dell'esperimento, mantenere la soluzione salina sterile tamponata al fosfato (PBS) o salino pronto.</li>
	<li>Assemblare un kit chirurgico contenente forbici, forcipe dentato, pinze, cotone, cotton fioc, fili di nylon e porta aghi micro differenti. Sterilizzare in autoclave il kit chirurgico. Dovrebbe prestare particolare attenzione se un cenno di immuno-compromessi. Mouse di SCID è incluso nel protocollo.</li>
	<li>Eseguire la procedura sperimentale completa, dalla preparazione alla conclusione della chirurgia, della bio-sicurezza classe gabinetto.</li>
	<li>Pesare un cenno del capo. Mouse di SCID di 6-8 settimane prima dell'intervento. In questo studio vengono utilizzati topi pesano fra 14-18 g.</li>
	<li>Radere la regione centrale e ipocondriaca addominale superiore del mouse con il tagliacapelli. Applicare la crema in modo uniforme in tutta la regione di epilazione con una spatola per rimuovere completamente i peli tagliati. Rimuovere i peli delicatamente con l'aiuto di un pezzo di cotone sterile bagnato dopo 2-5 min.</li>
	<li>Posizionare il mouse nella camera di isoflurano e sbloccare la valvola della bombola di ossigeno. Mantenere il flusso di ossigeno al ritmo di 4 L/min e la vaporizzazione di isoflurane al 4% per indurre l'anestesia.<ol>
<li>Assicurarsi che il mouse è stato anestetizzato correttamente pizzicando punta delicata.</li>
	</ol>
</li>
	<li>Collocare una tavola di chirurgia all'interno di una biosicurezza armadio. Metti l'animale sul Consiglio di chirurgia, tale che la porzione ventrale del mouse è rivolto verso l'alto e la parte anteriore del mouse è posizionata all'interno del cono di naso collegato all'alimentazione isoflurano e ossigeno.</li>
	<li>Ridurre la vaporizzazione di isoflurane al 2% e mantenerla durante tutta la procedura chirurgica.</li>
	<li>Disinfettare la pelle del mouse e sterilizzarlo strofinando con cotone sterile imbevuto di etanolo al 70%.</li>
</ol>2. intervento chirurgico
<ol><li>
Epatectomia parziale
	<ol>
<li>Fare un'incisione trasversale di circa 1 cm nella pelle appena sotto il sterno, perpendicolare al processo xifoideo e parallelamente la gabbia toracica, con l'aiuto di Forbici rette operative.</li>
		<li>Separare delicatamente la pelle attaccata con lo strato del muscolo addominale in prossimità della zona incisa con forcipe o punte sterili di cotone inumidito di distinguere tra pelle e strato del muscolo addominale. Godetevi la regione intradermica con PBS usando suggerimenti di cotone sterile per evitare il disseccamento.</li>
		<li>Esporre l'area del lobo sinistro senza problemi facendo un'incisione trasversale attraverso lo strato peritoneale appena sotto al xifoideo. Utilizza due punte di cotone inumidito per esporre e sollevare il lobo sinistro del fegato.</li>
		<li>Inserire una punta di cotone sul lato addominale del taglio e inserire un'altra punta di cotone sul lato del diaframma. Delicatamente premere la punta posizionata verso il diaframma e dare una Spinta scorrevole dall'altra punta per sollevare il lobo sinistro del fegato.</li>
		<li>Infilare un filo di nylon con un ciclo attraverso il lobo sinistro sollevato, quindi il ciclo verso la base del lobo di sinistra vicino al hilum con l'aiuto delle punte micro-forcipe o cotone. Spingere delicatamente verso il basso il loop di filo di nylon alla base del lobo di sinistra.</li>
		<li>Legare due estremità del filo di nylon nella parte superiore del lobo di sinistra usando il forcipe micro e un porta-aghi microchirurgia. Fare due nodi aggiuntivi sul lato opposto.</li>
		<li>Sezionare il lobo legato con l'aiuto di forbici. Non tentare di tagliare molto vicino il thread. Nel caso in cui la procedura dura più di 5 min, mantenere la cavità peritoneale e organi umido con PBS sterile per evitare disidratazione a causa della perdita di liquidi.</li>
		<li>Cucire il peritoneo di sutura continua utilizzando una sutura in Catgut 4-0. Successivamente, chiudere la pelle suturando discontinuo nel minor tempo possibile.</li>
	</ol>
</li>
	<li>
Trapianto di cellule Nota: Gli epatociti che esprimono GFP sono stati isolati da topi transgenici GFP (C57BL/6-Tg (UBC-GFP) 30 Scha/J), secondo la procedura descritta da Lee et al. 22 e Shen et al. 23 hepatocyte-come le cellule (NeoHep) di origine umana differenziati dai monociti24 sono state utilizzate anche per il trapianto. Tuttavia, le cellule derivate da altre fonti possono essere utilizzate anche nel protocollo.<ol>
<li>Sospendere 0,2 milioni cellule vitali in circa 50 µ l di per volta Dulbecco di Iscove Medium (IMDM) e aspirare in una siringa da insulina 1 mL con ago 30g. Tenere la siringa freddo mettendo sul ghiaccio.</li>
		<li>Posizionare il mouse in modo che la parte laterale sinistra rivolto verso l'alto verso la persona che effettua l'intervento chirurgico. Identificare l'area splenica e sezionare trasversalmente la pelle vicino alla regione ipocondriaca, seguita da una breve incisione attraverso lo strato peritoneale solo per esporre la milza.</li>
		<li>La milza di sollevare delicatamente e tenere di fuori della cavità con l'aiuto di due PBS inumidito punte in cotone.</li>
		<li>Tenere la milza attentamente con due punte in cotone in una mano e inserire l'ago della siringa esattamente verticale alla milza. Delicatamente bucare la milza e spingere l'ago molto lentamente all'interno; l'ago non dovrebbe ottenere più profondo di 2 mm.</li>
		<li>Abbassare lentamente il pistone della siringa per iniettare le cellule della milza. Dopo il trapianto, mantenere stabile l'ago della siringa e rimuoverlo lentamente dalla milza per evitare sanguinamenti o perdita di cellule.</li>
		<li>Dopo aver posizionato la milza indietro nella cavità peritoneale con le punte di cotone, chiudere il livello peritoneale suturando continuo con una sutura in Catgut 4-0. Cucire la pelle in maniera discontinua con la sutura stessa. Evitare l'uso di ferita clip per la chiusura della pelle; invece, chiuderlo facendo una sutura 4-0. Le clip di ferita limitano il naturale movimento del mouse, e spesso clip si allentano e uscire rapidamente.</li>
	</ol>
</li>
</ol>3. post-operatorio
<ol><li>Dopo aver chiuso la pelle, pulire i dintorni di entrambi i siti di sutura con soluzione di iodio (betadine) utilizzando una punta di cotone sterile.</li>
	<li>Iniettare una dose di antibiotica cefotaxime come 600 mg/kg di peso (in genere 12 mg cefotaxime in 100 µ l di soluzione salina/mouse) corporeo intraperitonealmente usando la siringa da 1 mL.</li>
	<li>Dare dosi giornaliere di Meloxicam analgesico come 1 mg/kg di peso corporeo (in genere 12 µ g Meloxicam in 100 µ l di soluzione salina/mouse) all'animale intraperitonealmente, fino a tre giorni dopo l'intervento chirurgico.</li>
	<li>Dopo il completamento della chirurgia, fermare il flusso di gas isoflurano e posizionare il mouse nella gabbia individualmente ventilata.</li>
</ol>4. euthanization e caratterizzazioni
<ol><li>Dopo i punti finali sperimentali (1 giorno e 10 giorni dopo l'intervento), eutanasia i topi secondo le linee guida istituzionali etica animale.</li>
	<li>Raccogliere il sangue mediante terminalmente sanguinamento gli animali, perforando il plesso terminale oculare.</li>
	<li>Isolare il siero dal sangue.</li>
</ol>

<ol>
	<li>Nussler, 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=Present+status+and+perspectives+of+cell-based+therapies+for+liver+diseases.">Present status and perspectives of cell-based therapies for liver diseases.</a> J. Hepatol. 45 (1), 144-159 (2006).</li><li>Ponder, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+hepatocytes+migrate+to+liver+parenchyma+and+function+indefinitely+after+intrasplenic+transplantation.">Mouse hepatocytes migrate to liver parenchyma and function indefinitely after intrasplenic transplantation.</a> Proc. Natl. Acad. Sci. U.S.A. 88 (4), 1217-1221 (1991).</li><li>Kokudo, N., Horimoto, H., Ishida, K., Takahashi, S., Nozawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+hepatocyte+and+fetal+liver+transplantation+and+xenogeneic+hepatocyte+transplantation+for+Nagase's+analbuminemic+rats.">Allogeneic hepatocyte and fetal liver transplantation and xenogeneic hepatocyte transplantation for Nagase's analbuminemic rats.</a> Cell Transplant. 5 (5 Suppl 1), S21-S22 (1996).</li><li>Christ, B., Bruckner, S., Stock, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+transplantation+of+mesenchymal+stem+cells+in+rodent+animal+models.">Hepatic transplantation of mesenchymal stem cells in rodent animal models.</a> Methods Mol. Biol. 698, 315-330 (2011).</li><li>Fox, I. J., Roy-Chowdhury, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+transplantation.">Hepatocyte transplantation.</a> J. Hepatol. 40 (6), 878-886 (2004).</li><li>Glanemann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+monocyte-derived+hepatocyte-like+cells+(NeoHeps)+improves+survival+in+a+model+of+acute+liver+failure.">Transplantation of monocyte-derived hepatocyte-like cells (NeoHeps) improves survival in a model of acute liver failure.</a> Ann. Surg. 249 (1), 149-154 (2009).</li><li>Rahman, T. M., Hodgson, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+hepatic+failure.">Animal models of acute hepatic failure.</a> Int. J. Exp. Pathol. 81 (2), 145-157 (2000).</li><li>Zhang, L., 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=Granulocyte+colony-stimulating+factor+treatment+ameliorates+liver+injury+and+improves+survival+in+rats+with+D-galactosamine-induced+acute+liver+failure.">Granulocyte colony-stimulating factor treatment ameliorates liver injury and improves survival in rats with D-galactosamine-induced acute liver failure.</a> Toxicol. Lett. 204 (1), 92-99 (2011).</li><li>Gardner, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+acetaminophen-induced+hepatotoxicity+in+the+rat.">Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat.</a> Hepatology. 27 (3), 748-754 (1998).</li><li>Nardo, B., 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=Successful+treatment+of+CCL4-induced+acute+liver+failure+with+portal+vein+arterialization+in+the+rat.">Successful treatment of CCL4-induced acute liver failure with portal vein arterialization in the rat.</a> Transplant Proc. 38 (4), 1187-1189 (2006).</li><li>Sathyasaikumar, K. V., 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=Fulminant+hepatic+failure+in+rats+induces+oxidative+stress+differentially+in+cerebral+cortex,+cerebellum+and+pons+medulla.">Fulminant hepatic failure in rats induces oxidative stress differentially in cerebral cortex, cerebellum and pons medulla.</a> Neurochem. Res. 32 (3), 517-524 (2007).</li><li>Wu, 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=Laennec+protects+murine+from+concanavalin+A-induced+liver+injury+through+inhibition+of+inflammatory+reactions+and+hepatocyte+apoptosis.">Laennec protects murine from concanavalin A-induced liver injury through inhibition of inflammatory reactions and hepatocyte apoptosis.</a> Biol. Pharm. Bull. 31 (11), 2040-2044 (2008).</li><li>Kaur, G., Tirkey, N., Chopra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effect+of+hesperidin+on+lipopolysaccharide-induced+hepatotoxicity.">Beneficial effect of hesperidin on lipopolysaccharide-induced hepatotoxicity.</a> Toxicology. 226 (2-3), 152-160 (2006).</li><li>Rupertus, K., 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=Major+but+not+minor+hepatectomy+accelerates+engraftment+of+extrahepatic+tumor+cells.">Major but not minor hepatectomy accelerates engraftment of extrahepatic tumor cells.</a> Clin. Exp. Metastasis. 24 (1), 39-48 (2007).</li><li>Selden, C., Casbard, A., Themis, M., Hodgson, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+long-term+survival+of+syngeneic+hepatocytes+in+rat+peritoneum.">Characterization of long-term survival of syngeneic hepatocytes in rat peritoneum.</a> Cell Transplant. 12 (6), 569-578 (2003).</li><li>Tang, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+donor+hepatocytes+and/or+splenocytes+pre-injection+in+reducing+islet+xenotransplantation+rejection.">The role of donor hepatocytes and/or splenocytes pre-injection in reducing islet xenotransplantation rejection.</a> Hepatobiliary. Pancreat. Dis. Int. 2 (3), 344-350 (2003).</li><li>Goto, Y., Ohashi, K., Utoh, R., Yamamoto, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+transplantation+through+the+hepatic+vein:+a+new+route+of+cell+transplantation+to+the+liver.">Hepatocyte transplantation through the hepatic vein: a new route of cell transplantation to the liver.</a> Cell Transplant. 20 (8), 1259-1270 (2011).</li><li>Gabelein, G., 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=Intrasplenic+or+subperitoneal+hepatocyte+transplantation+to+increase+survival+after+surgically+induced+hepatic+failure?.">Intrasplenic or subperitoneal hepatocyte transplantation to increase survival after surgically induced hepatic failure?.</a> Eur. Surg. Res. 41 (3), 253-259 (2008).</li><li>Bhattacharjee, 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=Autologous+NeoHep+Derived+From+Chronic+Hepatitis+B+Virus+Patients'+Blood+Monocytes+by+Upregulation+of+cMET+Signaling.">Autologous NeoHep Derived From Chronic Hepatitis B Virus Patients' Blood Monocytes by Upregulation of cMET Signaling.</a> Stem Cells Transl. Med. , (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic surgical skills. Emergency and Essential Surgical Care (EESC) programme. , (2017).</li><li>Pritchett-Corning, K. R., Luo, Y., Mulder, G. B., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+new+surgeon.">Principles of rodent surgery for the new surgeon.</a> J. Vis. Exp. (47), (2011).</li><li>Lee, S. M., Schelcher, C., Demmel, M., Hauner, M., Thasler, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+hepatocytes+by+a+two-step+collagenase+perfusion+procedure.">Isolation of human hepatocytes by a two-step collagenase perfusion procedure.</a> J. Vis. Exp. (79), (2013).</li><li>Shen, L., Hillebrand, A., Wang, D. Q., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+hepatic+cells.">Isolation and primary culture of rat hepatic cells.</a> J. Vis. Exp. (64), (2012).</li><li>Bhattacharjee, 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=Autologous+NeoHep+Derived+from+Chronic+Hepatitis+B+Virus+Patients'+Blood+Monocytes+by+Upregulation+of+c-MET+Signaling.">Autologous NeoHep Derived from Chronic Hepatitis B Virus Patients' Blood Monocytes by Upregulation of c-MET Signaling.</a> Stem Cells Transl. Med. 6 (1), 174-186 (2017).</li><li>Hori, 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=Simple+and+sure+methodology+for+massive+hepatectomy+in+the+mouse.">Simple and sure methodology for massive hepatectomy in the mouse.</a> Ann. Gastroenterol. 24 (4), 307-318 (2011).</li><li>Hori, 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=Simple+and+reproducible+hepatectomy+in+the+mouse+using+the+clip+technique.">Simple and reproducible hepatectomy in the mouse using the clip technique.</a> World J. Gastroenterol. 18 (22), 2767-2774 (2012).</li><li>Vidal, I., Richert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nude+mouse+as+model+for+liver+deficiency+study+and+treatment+xenotransplantation.">The nude mouse as model for liver deficiency study and treatment xenotransplantation.</a> Int. J. Hepatol. , 1400147 (2012).</li><li>Mitchell, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+and+well-tolerated+method+for+2/3+partial+hepatectomy+in+mice.">A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.</a> Nat. Protoc. 3 (7), 1167-1170 (2008).</li><li>Ahmed, S. U., 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=Generation+of+subcutaneous+and+intrahepatic+human+hepatocellular+carcinoma+xenografts+in+immunodeficient+mice.">Generation of subcutaneous and intrahepatic human hepatocellular carcinoma xenografts in immunodeficient mice.</a> J. Vis. Exp. (79), e50544 (2013).</li><li>Krikri, 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=Laparoscopic+vs.+open+abdominal+surgery+in+male+pigs:+marked+differences+in+cortisol+and+catecholamine+response+depending+on+the+size+of+surgical+incision.">Laparoscopic vs. open abdominal surgery in male pigs: marked differences in cortisol and catecholamine response depending on the size of surgical incision.</a> Hormones (Athens). 12 (2), 283-291 (2013).</li></ol>

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Hepatectomy parziale è una tecnica consolidata per inquirenti rigenerazione del fegato e hepatectomy eccessivo è segnalato per imitare il modello AHF. Tra modelli animali di AHF, roditori, soprattutto topi, sono il modello più ricercato. Per ottenere un modello di disturbo al fegato in topi, fino a un hepatectomy di 70% è stato segnalato con una buona sopravvivenza tasso25,26. Tuttavia in altri mouse immunodeficiente e nudo, un hepatectomy di 70% è stato seg...

Attualmente, il trapianto dell'epatocita è proposto come alternativa al trapianto d'organo intero per trattare i pazienti con gravi disturbi del fegato. Si ritiene che esso possa colmare pazienti per trapianto di organo intero1. Oltre il trapianto allogenico epatociti2, epatociti xenogenica3 ed epatociti derivati da cellule staminali4 sono anche indagati in modelli animali. In questo contesto, l'homing e attecchimento potenziale delle cellule trapiantate nel destinatario è un criterio importante per la terapia cellulare basata nell'insufficienza epatica acuta (AHF).
Per indagare il trapianto di epatociti o dell'epatocita-come le cellule5, AHF viene creato in un modello animale da chirurgica6 o farmacologici7 procedure, seguite da trapianto di cellule. Per rendere un modello animale di AHF di reagenti farmacologici, molti epatotossine come d-galattosamina8, acetaminofene9, tetracloruro di carbonio10, thioacetamide11, concanavalina A12, Lipopolisaccaride13 , ecc., sono stati utilizzati. Da questa lista, ogni reagente genera un insieme unico di funzionalità per AHF, ma purtroppo nessun singolo reagente imita gli AHF umano. Inoltre, gli AHF indotto da epatotossine richiede molto tempo, che mette gli animali in condizioni di stress cronico, e risultati riproducibili sono difficili da ottenere.
D'altra parte, l'intervento chirurgico di epatectomia parziale (PHx) è dipendente di abilità e risultati riproducibili sono facili da ottenere dopo lo sviluppo di competenze richieste. Per indurre AHF tramite intervento chirurgico da solo, la resezione di più del 70% del fegato è richiesta; Tuttavia, meno di un'epatectomia 70% ancora possa essere utilizzata per studiare l'attecchimento e la proliferazione di trapiantate cellule nel fegato per analizzare le loro capacità terapeutiche durante i danni al fegato14. Il trapianto di epatociti sono stati eseguiti post epatectomia attraverso il peritoneo15, coda vena16, vena epatica17o la milza18. Attualmente, infusione della vena epatica e intrasplenic trapianto di epatociti sono le procedure di comodo, come sono più facili da riprodurre.
In questo articolo, abbiamo descritto una procedura per una 30% di epatectomia parziale in NOD. SCID (CENNO DEL CAPO. CB17-Prkdcscid/J) topi in cui viene asportato il lobo sinistro del fegato. È seguito da trapianto di epatociti di mouse (C57BL/6-Tg (UBC-GFP) 30Scha/J) che esprimono GFP 0,2 milioni nonché di origine umana NeoHep19 nella milza. Questa procedura conduce all'attecchimento delle cellule trapiantate nel fegato. Questa procedura è meno invasiva e una tecnica minimamente dolorosa.

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			<td height="21" style="height:21px;width:411px;">Gas Anesthesia System</td>
			<td style="width:255px;">Ugo Basile; Italy</td>
			<td style="width:119px;">211000</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weighing machine</td>
			<td style="width:255px;">Goldtech ; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biological safety cabinet ( Class I)</td>
			<td style="width:255px;">Kartos international;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair Trimmer</td>
			<td>Panasonic ;&#160; Japan&#160;</td>
			<td style="width:119px;">ER-GY10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight operating scissor with sharp /sharp blades</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps with Serrations</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders&#160; straight &#38; curved&#160;</td>
			<td>Mercian ;&#160; England&#160;</td>
			<td style="width:119px;">BS-13-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml insulin syringe with 30G *5/16 needles&#160;</td>
			<td>Dispo Van; India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringe with 26 G * 1/2 needle</td>
			<td>BD ; US&#160;</td>
			<td>REF 303060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon Threads&#160;&#160;</td>
			<td style="width:255px;">Mighty ; India</td>
			<td style="width:119px;"></td>
			<td>(1-0) Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MERSUTURES 4-0 Sterilised Surgical Needled Suture</td>
			<td style="width:255px;">Ethicon, Johnson &#38; Johnson, India</td>
			<td style="width:119px;">NW 5047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRUGUT 76 cm 4-0 absorbable surgical suture</td>
			<td>Sutures India Pvt. Ltd; India</td>
			<td>SN 5048</td>
			<td>Sterilised Surgical Needled Suture Catgut Chromic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Buds</td>
			<td style="width:255px;">Pure Swabs Pvt Ltd ;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Tape</td>
			<td style="width:255px;">3M India ; India</td>
			<td style="width:119px;">1530-1</td>
			<td>Micropore Surgical Tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microtome</td>
			<td style="width:255px;">Histo-Line Laboratories, Italy</td>
			<td style="width:119px;">MRS3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shandon Cryotome E Cryostat</td>
			<td style="width:255px;">Thermo Electron Corporation ; US</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Confocal laser scanning microscope</td>
			<td style="width:255px;">Carl Zeiss ; Germany&#160;</td>
			<td style="width:119px;">LSM 510 META</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bright Field Microscope</td>
			<td style="width:255px;">Olympus, Japan</td>
			<td style="width:119px;">LX51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Automated analyser</td>
			<td>Tulip, Alto Santracruz, India</td>
			<td style="width:119px;">Screen Maaster 3000</td>
			<td>Biochemical analyser for liver functional test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Flow Cytometer</td>
			<td style="width:255px;">BD ; US&#160;</td>
			<td style="width:119px;">BD FACSverse</td>
			<td>Assesment of presence of cells post transplantation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veet hair removal cream&#160;</td>
			<td>Reckitt Benckiser , India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FORANE</td>
			<td style="width:255px;">Abbott ; US</td>
			<td style="width:119px;"></td>
			<td>isoflurane USP 99.9%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxim</td>
			<td style="width:255px;">AlKem ; India</td>
			<td style="width:119px;"></td>
			<td>cefotaxime sodium injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution&#160;</td>
			<td style="width:255px;">Win-Medicare;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Betadine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Paraformaldehyde</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">GRM 3660</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">12200-036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sucrose</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue-Tek</td>
			<td style="width:255px;">Sakura; US</td>
			<td>25608-930</td>
			<td>O.C.T compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAPI</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">MB 097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Albumin goat Polyclonal</td>
			<td style="width:255px;">Thermo Scientific,Pierce, US</td>
			<td style="width:119px;">PA126081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-connexin 32/GJB1 Polyclonal</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab64609-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">antiGFP rabbit polyclonal&#160;</td>
			<td style="width:255px;">Santa Cruz biotechnology; US</td>
			<td style="width:119px;">SC 8334</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 donkey anti-goat</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 488 donkey anti-sheep</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 chicken anti rabbit</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A21442</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Goat anti rabbit IgG HRP</td>
			<td style="width:255px;">Invitrogen, Thermo Fisher Scientific; US&#160;</td>
			<td>65-6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Ki67 antibody</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab15580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Antigen Unmasking Solution, Citric acid base</td>
			<td style="width:255px;">Vector laboratories, US</td>
			<td style="width:119px;">H-3300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ProLong Diamond antifade mountant</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">P36966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGOT (ASAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGPT (ALAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Alkaline Phosphatase Kit (DEA)</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hematoxylin Solution, Mayer&#39;s</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">MHS16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Eosin Y solution, alcoholic</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">HT110132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DPX Mountant&#160;</td>
			<td style="width:255px;">Sigma ; US</td>
			<td align="right" style="width:119px;">6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Melonex (Pain Killer)</td>
			<td style="width:255px;">Intas Pharmaceuticals Ltd; India</td>
			<td style="width:119px;"></td>
			<td>Meloxicam injection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAB enhanced liquid substrate system tetrahydrochloride</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">D3939</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice

Hepatectomy parziale è un metodo versatile e riproducibile per studiare la rigenerazione del fegato e l'effetto di terapeutica delle cellule basata in varie circostanze patologiche. Epatectomia parziale facilita anche la maggiore attecchimento e la proliferazione delle cellule trapiantate da neovascolarizzazione accelerante e migrazione delle cellule verso il fegato. Qui, descriviamo un semplice protocollo per eseguire 30% hepatectomy e trapianto di cellule nella milza di un diabetico non obeso/grave combinata NOD immunodeficienti. SCID (CENNO DEL CAPO. CB17-Prkdcscid/j) mouse.
In questa procedura, due piccole incisioni sono fatte. La prima incisione è quello di esporre e resecare il lobo sinistro del fegato, e un'altra piccola incisione è fatta per esporre la milza per il trapianto di cellule intrasplenic. Questa procedura non richiede alcuna abilità chirurgiche specializzate, e può essere completato in 5-7 minuti con meno stress e dolore, recupero più veloce e migliore sopravvivenza. Abbiamo dimostrato il trapianto di epatociti isolati da una proteina fluorescente verde (GFP) esprimendo mouse (transgenici C57BL/6-Tg (UBC-GFP) 30Scha/J), come pure dell'epatocita come cellule di origine umana (NeoHep) in parzialmente epatectomizzati NOD. Topi SCID.

Proliferazione degli epatociti dopo hepatectomy parziale del 30%: La proliferazione degli epatociti nei rimanenti del fegato dopo epatectomia 30% è stata esaminata secondo immunoistochimica (IHC) per un indicatore di proliferazione delle cellule, Ki-67. Un giorno post epatectomia, i topi sono stati sacrificati, i lobi del fegato restanti sono stati asportati e sezioni della paraffina sono state ottenute. Le sezioni sono state macchiate con anticorpo Ki-67, seguita da etichettatura con anticorpo secondar...

Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Abbiamo descritto un protocollo per l'esecuzione di epatectomia parziale (PHx) e trapianto di cellule via della milza nel cenno del capo. SCID (CENNO DEL CAPO. CB17-Prkdcscid/j) topi. In questo protocollo, un'incisione è fatta per esporre e resecare il lobo sinistro del fegato seguito da un'altra incisione per il trapianto di cellule intrasplenic.

Intrasplenic trapianto di epatociti dopo epatectomia parziale in NOD. Topi SCID

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

intrasplenic-transplantation-hepatocytes-after-partial-hepatectomy

Research

JoVE Journal

Medicine

13.9K Views.  National Institute of Immunology. Abbiamo descritto un protocollo per l'esecuzione di epatectomia parziale (PHx) e trapianto di cellule via della milza nel cenno del capo. SCID (CENNO DEL CAPO. CB17-Prkdcscid/j) topi. In questo protocollo, un'incisione è fatta per esporre e resecare il lobo sinistro del fegato seguito da un'altra incisione per il trapianto di cellule intrasplenic.

Video: Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Trapianto mammaria di cellule stromali e cellule di carcinoma in topi C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Ricerca

Medicina

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

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

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

Aortica trapianto ortotopico in Mouse per lo studio della malattia vascolare

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

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 ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Eterotopico cervicale trapianto cardiaco nei topi

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

Visualizzazione dei Vascolare e parenchimale rigenerazione dopo il 70% epatectomia parziale in topi normali

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Ri-Arterialized trapianto parziale di fegato del ratto con un in vivo vaso-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Trapianto di cellule staminali di tessuto adiposo-derivato foglio per ridurre le perdite dopo la colectomia parziale in un modello del ratto

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Lentivirali terapia genica mediata da vettore di epatociti Ex Vivo per trapianto autologo nei suini

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Generazione di un modello di ratto di insufficienza epatica acuta combinando il 70% di epatitectomia parziale e acetaminofene

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

Complicazioni ridotte dopo la riconnessione arteriosa in un modello di ratto di trapianto di fegato ortotopico

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...