Questo lavoro è supportato da RO1 DK064938 (TH).

<ol>
	<li>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=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> N Engl J Med. 343, 230-238 (2000).</li><li>Weir, G. C., Bonner-Weir, S. <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+as+a+treatment+for+diabetes.">Islet transplantation as a treatment for diabetes.</a> J Am Optom Assoc. 69, 727-732 (1998).</li><li>Gangemi, A. <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+for+brittle+type+1+diabetes:+the+UIC+protocol.">Islet transplantation for brittle type 1 diabetes: the UIC protocol.</a> Am J Transplant. 8, 10-1111 (2008).</li><li>Zmuda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+ATF3,+an+adaptive-response+gene,+protects+islets+and+ameliorates+inflammation+in+a+syngenic+transplantation+model.">Deficiency of ATF3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngenic transplantation model.</a> Diabetologia. , (2010).</li><li>Bottino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Human+Islets+to+Isolation+Stress+and+the+Effect+of+Antioxidant+Treatment.">Response of Human Islets to Isolation Stress and the Effect of Antioxidant Treatment.</a> Diabetes. 53, 2559-2568 (2004).</li><li>Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+islet+cell+culture+in+vitro+at+24+degrees+C+on+graft+survival+and+MHC+antigen+expression.">The effect of islet cell culture in vitro at 24 degrees C on graft survival and MHC antigen expression.</a> Transplantation. 49, 272-277 (1990).</li><li>Terasaka, R., Lacy, P. E., Hauptfeld, V., Bucy, R. P., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cyclosporin-A,+low-temperature+culture,+and+anti-Ia+antibodies+on+prevention+of+rejection+of+rat+islet+allografts.">The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts.</a> Diabetes. 35, 83-88 (1986).</li><li>Ricordi, C., Lacy, P. E., Sterbenz, K., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+culture+of+human+islets+or+in+vivo+treatment+with+L3T4+antibody+produces+a+marked+prolongation+of+islet+human-to-mouse+xenograft+survival.">Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.</a> Proc Natl Acad Sci U S A. 84, 8080-8084 (1987).</li><li>Schnedl, W. J., Ferber, S., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STZ+transport+and+cytotoxicity.+Specific+enhancement+in+GLUT2-expressing+cells.">STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells.</a> Diabetes. 43, 1326-1333 (1994).</li><li>Eckhard, M., Brandhorst, D., Brandhorst, H., Brendel, M. D., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+in+osmolality+and+range+of+density+of+a+continuous+ficoll-sodium-diatrizoate+gradient+for+isopycnic+purification+of+isolated+human+islets.">Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.</a> Transplantation Proceedings. 36, 2849-2854 (2004).</li><li>Gores, P. F., Rabe, F., Sutherland, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+survival+of+intraportal+versus+subrenal+capsular+transplanted+islet+allografts.">Prolonged survival of intraportal versus subrenal capsular transplanted islet allografts.</a> Transplantation. 43, 747-749 (1987).</li><li>Nasr, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+Immune+Privilege+Promotes+Transplantation+Tolerance+by+Altering+the+Balance+between+Memory+and+Regulatory+T+Cells.">Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells.</a> J Immunol. 174, 6161-6168 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. J. <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, 1449-1461 (2008).</li><li>Inoue, M., Maeno, T., Hatchell, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+allografted+pancreatic+islets+in+the+subretinal+space+in+rats.">Survival of allografted pancreatic islets in the subretinal space in rats.</a> Ophthalmic Res. 35, 48-53 (2003).</li></ol>

Nessun conflitto di interessi dichiarati.

Nelle sezioni di cui sopra, abbiamo descritto la procedura dettagliata per l&#39;isolamento e trapianto delle isole pancreatiche del mouse. Qui, brevemente evidenziare i passaggi che sono critico per il successo delle procedure. 1. Isolotto isolamento I passi principali sono identificare un momento ottimale digestione enzimatica (1,4), il posizionamento della pinza emostatica sopra l&#39;apertura duodenale del dotto biliare comune (2.2.3), cannulating il dotto biliare comune (2,3), agitazione vigorosa del pancreas dopo i 37 ° C digerire (3,2), e la rimozione di contaminazione delle cellule esocrine (3.3.7 e 3.3.16). Questi passaggi hanno un influsso drammatico sulla qualità della resa, e la purezza di isolotti. Forse il punto tecnicamente più impegnativo è l&#39;incannulazione del dotto biliare comune. In molti topi, il dotto biliare comune possono essere difficili da visualizzare a causa della fascia circostante. Pertanto, è comune che i tentativi di cannulate il dotto biliare comune risultato in termini di penetrazione del tessuto fascia unica. In questi casi, il Liberase comincia a riempire il tessuto connettivo circostante e non profumato al pancreas. Se questa è osservato, fermare il flusso di Liberase e riposizionare l&#39;ago in modo che penetri nel lume del dotto biliare. Con la pratica, è possibile identificare velocemente la posizione ottimale per il posizionamento dell&#39;ago e per completare la cannulazione senza danneggiare il condotto friabile. 2. Trapianto di isole I passi fondamentali sono l&#39;induzione STZ di diabete (5,1) e la consegna efficiente delle isole nella busta subcapsulare (5.3.14-5.3.16). STZ è un analogo del glucosio naturale che è selettivamente tossica per le cellule che secernono l&#39;insulina β-cellule delle isole 9. Tuttavia, questa tossicità selettiva avviene in un intervallo di concentrazione relativamente ristretti. Ciò è dimostrato dal fatto che alte dosi di STZ può provocare un rapido (2-3 giorni) letalità in assenza di iperglicemia. Pertanto, occorre prestare attenzione per identificare la dose ottimale di STZ per il ceppo di topi e l&#39;età in uso prima di ogni esperimento su larga scala sono iniziati. La consegna fisica delle isole nella busta sottocapsulare è anche un passo fondamentale. Per ottenere risultati costanti, è indispensabile ridurre al minimo la perdita di isolotti nel corso della loro consegna. Ciò può verificarsi se ci sono difetti o rotture della capsula del rene che copre il sacchetto o se il volume iniettato è troppo grande. Danni alla capsula può essere evitato con la manipolazione dolce e attenta, e utilizzando la sonda vetro liscio per preparare il sacchetto sottocapsulare. Inoltre, il volume di iniezione può essere minimizzato con rimozione accurata del surnatante al punto 5.3.6.3. Se il surnatante viene rimosso fino al livello degli isolotti sedimentate, non vi è in genere più di spazio sufficiente nel sacchetto subcapsulari al posto 400-500 isolotti. Tuttavia, se il volume di iniezione è troppo grande, gli isolotti hanno maggiori probabilità di fuoriuscire il sacchetto durante il processo di trapianto, compromettendo la riproducibilità dell&#39;esperimento. All&#39;interno di questo protocollo, è possibile modificare alcuni dei passaggi e ottenere comunque risultati soddisfacenti. Questi includono: (a) l&#39;utilizzo di un enzima diverso per digerire il pancreas, (b) un metodo diverso per la consegna l&#39;enzima al pancreas, (c) l&#39;uso di un continuo Ficoll-sodio-diatrizoato (FSD) gradiente al posto del singolo densità Histopaque1077 passo, e (d) trapianto di isole pancreatiche in luoghi diversi dal subcapsule rene. <ol class="lalpha"><li> In questo protocollo, Liberase è usato per indebolire la cellula-cellula contatti all&#39;interno del pancreas. Liberase è essenzialmente una miscela altamente purificato di collagenasi che si sono dimostrati utili nel liberare isole dal pancreas. Tuttavia, collagenasi meno purificati sono in grado di realizzare un risultato simile. Pertanto, è possibile utilizzare diversi preparati enzimatici disponibili in commercio per digerire il pancreas, fino a quando la condizione di digestione è ottimizzato per ottenere una qualità elevata delle isole, la resa e la purezza. </li><li> E &#39;anche possibile per digerire il pancreas senza perfusione duttale di enzima. Consegna del Liberase attraverso il dotto biliare comune permette di esposizione massima di zona del pancreas superficie per l&#39;enzima. Ciò si traduce in più anche la digestione del pancreas e una maggiore rilascio di isolette intatte. Tuttavia, altre misure per aumentare la superficie del pancreas per l&#39;enzima può essere utilizzato. Per esempio, macinazione del pancreas prima incubazione in Liberase o direttamente iniettando Liberase attraverso la capsula pancreatica può essere utilizzato per isolare isolotti. Tuttavia, nessun altro metodo di consegna Liberase è efficace come perfusione attraverso il dotto biliare comune. Pertanto, macinazione o iniezione diretta dovrebbe essere riservato come ultima risorsa, se il dotto biliare comune è danneggiato e non perfusable. </li><li> Un metodo alternativo per separare gli isolotti dalle cellule esocrine comporta lal&#39;uso di un gradiente FSD Histopaque1077 invece di 10. La separazione efficiente di isolotti dalle cellule esocrine in questo protocollo è in parte dovuto ad una differenza di densità tra i due tipi di cellule. Histopaque1077 ha una densità di 1,0771 g / ml a 25 ° C. A questa temperatura, HISTOPAQUE è più densa gli isolotti, ma meno densa rispetto alle cellule esocrine. Pertanto, sotto la forza di centrifugazione, Histopaque1077 pellet le cellule esocrine mentre si solleva il isolotti in superficie. In linea di principio, qualsiasi altra sostanza con una densità che che può separare isolotti dalle cellule esocrine possono essere utilizzati in questo passaggio e il gradiente FSD è una sostanza del genere. </li><li> Per quanto riguarda la posizione per il trapianto di isole, la capsula del rene usati in questo protocollo è uno dei pochi siti possibili. Mentre il subcapsule rene è il luogo più comunemente riportati di trapianto nei topi, il trapianto di altri siti sono stati trovati per essere efficace e comprendono la vena porta epatica, spazio sottoretinico, del testicolo, dell&#39;epididimo cuscinetto adiposo, milza e pancreas anche 11-14. Ognuno di questi luoghi, ha i propri vantaggi e le sfide. Pertanto, la scelta di un sito per il trapianto di isole deve essere determinato dal questioni affrontate e le circostanze specifiche degli esperimenti. </li></ol> Si ritiene che le strategie che possono superare i limiti sul trapianto di isole drasticamente aumentare il suo potenziale terapeutico. Pertanto, l&#39;uso di un modello murino come descritto da questo protocollo offre un approccio interessante per identificare tali strategie.

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<td>RPMI 1640</td>
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<td>Gibco</td>
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<td>Liberase TL</td>
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<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22362574</td>
<td>&nbsp;</td>
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<td>Insulin Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>329461</td>
<td>&nbsp;</td>
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<td>27 Gauge Syringe Needle</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>305109</td>
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<td>Equipment</td>
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<td>NC9083963</td>
<td>&nbsp;</td>
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<td>309603</td>
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<td>Equipment</td>
<td>BD Falcon</td>
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<td>22363549</td>
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<td>0300241</td>
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<td>6300</td>
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<td>INS14393</td>
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<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS500260</td>
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<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS15915</td>
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<td>Equipment</td>
<td>Kent Scientific</td>
<td>INS 500451</td>
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<td>USA Scientific</td>
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a method for murine islet isolation and subcapsular kidney transplantation

Dal momento che i primi lavori pionieristici di Ballinger e Reckard dimostrando che il trapianto di isole di Langerhans in roditori diabetici potrebbe normalizzare i loro livelli di glucosio nel sangue, il trapianto di isole è stato proposto di essere un potenziale trattamento per il diabete di tipo 1 1,2. Più recentemente, i progressi nella trapianto di isole umane hanno ulteriormente rafforzato questa visione 1,3. Tuttavia, due limitazioni principali impediscono il trapianto di isole ad essere una realtà diffusa clinico: (a) la necessità di un gran numero di isolotti per paziente, che riduce fortemente il numero dei potenziali destinatari, e (b) la necessità di immunosoppressione pesante, che colpisce in modo significativo la popolazione pediatrica di pazienti a causa della loro vulnerabilità a lungo termine immunosoppressione. Strategie che possono superare questi limiti hanno la potenzialità di migliorare l&#39;utilità terapeutica del trapianto di isole. Trapianto di isole sotto la capsula renale mouse è un modello ampiamente accettato di studiare strategie diverse per migliorare il trapianto di isole. Questo esperimento richiede l&#39;isolamento delle isole di alta qualità e l&#39;impianto di isolotti ai destinatari diabetica. Entrambe le procedure richiedono operazioni chirurgiche che possono essere meglio dimostrato dal video che per il testo. Qui, documentano la procedura dettagliata per queste procedure sia da video e protocollo scritto. Abbiamo anche brevemente il trapianto di modelli diversi: singenico, allogenico, autoimmuni singenici, e allogenico autoimmuni.

Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Trapianto di isole isolate è stato proposto di essere un potenziale trattamento per il diabete di tipo 1. Qui si descrive un metodo per isolare le isole dal pancreas del mouse e trapiantarle allo spazio sottocapsulare del rene.

Un metodo per isolamento Islet murini e trapianto renale subcapsulare

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

a-method-for-murine-islet-isolation-subcapsular-kidney

Nanyang Technological University

Research

JoVE Journal

Medicine

63.3K Views.  The Ohio State University. Trapianto di isole isolate è stato proposto di essere un potenziale trattamento per il diabete di tipo 1. Qui si descrive un metodo per isolare le isole dal pancreas del mouse e trapiantarle allo spazio sottocapsulare del rene.

Video: A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Sviluppo di bronchiolite obliterante in un modello murino di trapianto polmonare ortotopico

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Ricerca

Medicina

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murino cervicale Cuore trapianto Modello utilizzando una tecnica Cuff Modificato

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Murini eterotopica cuore Tecnica Trapianto

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Murini trapianto di cornea: Un modello per studiare la forma più comune di trapianto di organi solidi

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Mouse Kidney Transplantation: Models of Allograft Rejection

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

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

Mouse trapianto renale: Modelli di Allograft Rifiuto

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

Murine Kidney Transplant Technique

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

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

Rene Murine Tecnica trapianto

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

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

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

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

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

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

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

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Un metodo di isolamento di romanzo Clinical Grade per rene umano perivascolari cellule stromali

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

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

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

Trapianto di rene ortotopico ratto: un nuovo approccio chirurgico semplificato

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

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

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

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

Cancellazione ottica e imaging del tessuto renale immunoetichettato

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