Ce travail est soutenu par 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>

Dans les sections ci-dessus, nous avons décrit les étapes détaillées pour l&#39;isolement et la transplantation des îlots pancréatiques de souris. Ici, nous brièvement les étapes qui sont critiques pour le succès de la procédure. 1. L&#39;isolement Islet Les principales étapes sont l&#39;identification d&#39;un temps de digestion enzymatique optimale (1,4), le positionnement de la pince hémostatique sur l&#39;ouverture du duodénum du canal cholédoque (2.2.3), cannulating le cholédoque (2,3), une agitation vigoureuse du pancréas après le 37 ° C Digest (3,2), et la suppression de la contamination de cellules exocrines (3.3.7 et 3.3.16). Ces mesures ont une influence considérable sur la qualité, le rendement et la pureté des îlots. Peut-être l&#39;étape la plus difficile techniquement est la canulation du canal cholédoque. Dans beaucoup de souris, le canal cholédoque peut être difficile à visualiser en raison de fascia environnant. Par conséquent, il est fréquent que les tentatives de cathétériser le résultat du cholédoque dans la pénétration du tissu du fascia seulement. Dans ces cas, le Libérase commence à remplir le tissu conjonctif environnant et ne perfuser le pancréas. Si cela est observé, arrêter l&#39;écoulement de Libérase et repositionner l&#39;aiguille de sorte qu&#39;elle pénètre dans la lumière de la voie biliaire. Avec la pratique, il est possible d&#39;identifier rapidement la position optimale pour le placement d&#39;aiguilles et de compléter la canulation sans endommager le conduit friable. 2. La transplantation d&#39;îlots Les étapes essentielles sont l&#39;induction du diabète par STZ (5,1) et la prestation efficace des îlots à la poche capsulaire (5.3.14-5.3.16). STZ est un analogue du glucose d&#39;origine naturelle qui est sélectivement toxique pour les cellules sécrétant de l&#39;insuline des cellules β des îlots-9. Cependant, cette toxicité sélective se produit dans une gamme de concentration relativement étroite. Cela est attesté par le fait que des doses élevées de STZ peut entraîner rapidement (2-3 jours) de létalité en l&#39;absence d&#39;hyperglycémie. Par conséquent, des précautions doivent être prises pour identifier la dose optimale de STZ pour la souche et l&#39;âge des souris étant utilisés avant tout des expériences à grande échelle sont lancées. La livraison physique des îlots à la poche capsulaire est également une étape clé. Pour obtenir des résultats cohérents, il est impératif de minimiser la perte d&#39;îlots lors de leur livraison. Cela peut se produire si il ya des défauts ou des larmes dans la capsule du rein couvrant la poche ou si le volume injecté est trop grand. Dommages à la capsule peut être évité par une manipulation douce et attentive, et en utilisant la sonde en verre lisse pour préparer la poche sous-capsulaire. De plus, le volume d&#39;injection peuvent être minimisés par l&#39;élimination soigneuse du surnageant à l&#39;étape 5.3.6.3. Si le surnageant est éliminé, jusqu&#39;au niveau des îlots sédimenté, il est généralement plus assez de place dans la poche capsulaire au lieu de 400 à 500 îlots. Toutefois, si le volume d&#39;injection est trop importante, les îlots sont plus susceptibles de s&#39;échapper de la poche durant le processus de transplantation, compromettre la reproductibilité de l&#39;expérience. Dans ce protocole, il est possible de modifier certaines des étapes et toujours obtenir des résultats satisfaisants. Ces sont les suivants: (a) l&#39;utilisation d&#39;une enzyme différente pour digérer le pancréas, (b) une méthode différente pour la prestation de l&#39;enzyme du pancréas, (c) l&#39;utilisation d&#39;un processus continu de Ficoll-sodium-diatrizoate (DSE) gradient au lieu de la densité Histopaque1077 seule étape, et (d) la transplantation d&#39;îlots à des endroits autres que le subcapsule reins. <ol class="lalpha"><li> Dans ce protocole, Libérase est utilisée pour affaiblir les contacts cellule-cellule au sein du pancréas. Libérase est essentiellement un mélange de très purifiée collagénases qui se sont avérés utiles dans la libération des îlots du pancréas. Toutefois, moins purifié collagénases sont capables d&#39;accomplir un résultat similaire. Par conséquent, il est possible d&#39;utiliser différentes préparations enzymatiques disponibles commercialement pour digérer le pancréas, tant que la condition de digestion est optimisé pour obtenir une qualité élevée des îlots, le rendement et la pureté. </li><li> Il est également possible de digérer le pancréas sans perfusion d&#39;enzyme canalaire. La livraison des Libérase travers le canal cholédoque permet une exposition maximale de la surface du pancréas à l&#39;enzyme. Il en résulte une digestion plus du pancréas et une plus grande libération de îlots intacts. Toutefois, d&#39;autres mesures pour augmenter la surface du pancréas à l&#39;enzyme peut être utilisée. Par exemple, hacher le pancréas avant incubation dans Libérase ou directement injecter Libérase à travers la capsule du pancréas peut être utilisé pour isoler les îlots. Cependant, aucun autre mode de livraison Libérase est aussi efficace que la perfusion à travers le canal cholédoque. Par conséquent, l&#39;injection directe hachage ou devrait être réservée en dernier recours, si le cholédoque est endommagé et ne pas perfusable. </li><li> Une méthode alternative pour séparer les îlots à partir des cellules exocrines implique lal&#39;utilisation d&#39;un gradient de DSE au lieu d&#39;Histopaque1077 10. La séparation efficace des îlots de cellules exocrines dans ce protocole est en partie due à un différentiel de densité entre les deux types cellulaires. Histopaque1077 a une densité de 1,0771 g / ml à 25 ° C. A cette température, Histopaque est plus dense que les îlots, mais moins dense que les cellules exocrines. Par conséquent, sous la force de centrifugation, Histopaque1077 granules des cellules exocrines tout en soulevant les îlots à la surface. En principe, toute autre substance avec une densité qui peut séparer les îlots de cellules exocrines peuvent être utilisés dans cette étape et le gradient de DSE est l&#39;une de ces substances. </li><li> En ce qui concerne l&#39;emplacement des îlots de transplantation, la capsule du rein utilisé dans ce protocole est l&#39;un des rares sites possibles. Alors que le subcapsule rein est le site le plus fréquemment rapportés de la transplantation chez les souris, les sites de la transplantation d&#39;autres ont été trouvés pour être efficaces et qu&#39;ils comprennent la veine porte, l&#39;espace sous-rétinien, les testicules, l&#39;épididyme coussinet adipeux, la rate, du pancréas et même 11-14. Chacun de ces lieux a ses propres avantages et les défis. Par conséquent, la sélection d&#39;un site pour la transplantation d&#39;îlots doit être déterminée par les questions abordées et des circonstances particulières de l&#39;expérimentation. </li></ol> On croit que les stratégies qui peuvent dépasser les limites sur la transplantation d&#39;îlots va considérablement accroître son potentiel thérapeutique. Par conséquent, l&#39;utilisation d&#39;un modèle murin tel que détaillé par ce protocole offre une approche intéressante pour identifier de telles stratégies.

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<td>Gibco</td>
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<td>Liberase TL</td>
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<td>10771</td>
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<td>Penicillin Streptomycin </td>
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<td>Gibco</td>
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<td>S4641</td>
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<td>ISOSOL</td>
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<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22362574</td>
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<td>Insulin Syringe</td>
<td>Equipment</td>
<td>BD Falcon</td>
<td>329461</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>Hamilton Syringe #1702</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
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<td>NC9083963</td>
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<td>309603</td>
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<td>Equipment</td>
<td>BD Falcon</td>
<td>352098</td>
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<td>15ml Conical Tube</td>
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<td>BD Falcon</td>
<td>352097</td>
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<td>Fisher Scientific</td>
<td>22363549</td>
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<td>Newark Wire Cloth Company</td>
<td>0300241</td>
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<td>15-462-5</td>
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<td>TMS</td>
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<td>Napco</td>
<td>6300</td>
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<td>Kent Scientific</td>
<td>INS14393</td>
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<td>McPherson-Vannas Scissors</td>
<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>Dupont &#x2013; Sorvall</td>
<td>RT6000D</td>
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<td>Dupont &#x2013; Sorvall</td>
<td>H1000B</td>
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<td>Equipment</td>
<td>Mettler Toledo/ Fisher Scientific</td>
<td>09313509</td>
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<td>Equipment</td>
<td>Fisher Scientific</td>
<td>05408151</td>
<td>&nbsp;</td>
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<td>p200 Tips</td>
<td>Equipment</td>
<td>USA Scientific</td>
<td>111-0730</td>
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a method for murine islet isolation and subcapsular kidney transplantation

Depuis les premiers travaux de pionnier de Ballinger et Reckard démontrant que la transplantation d&#39;îlots de Langerhans dans les rongeurs diabétiques pourraient normaliser leurs taux de glycémie, la transplantation d&#39;îlots a été proposé pour être un traitement potentiel pour 1,2 diabète de type 1. Plus récemment, les progrès de la transplantation d&#39;îlots humains ont encore renforcé ce point de vue 1,3. Cependant, deux limitations majeures empêchent la transplantation d&#39;îlots d&#39;être une réalité largement répandue cliniques: (a) l&#39;obligation pour un grand nombre d&#39;îlots par patient, ce qui réduit considérablement le nombre de bénéficiaires potentiels, et (b) la nécessité d&#39;une immunosuppression lourde, qui affecte de manière significative la population pédiatrique de patients en raison de leur vulnérabilité à long terme immunosuppression. Les stratégies qui peuvent contourner ces limitations ont le potentiel d&#39;améliorer l&#39;utilité thérapeutique de la transplantation d&#39;îlots. La transplantation d&#39;îlots sous la capsule rénale de souris est un modèle largement accepté pour étudier les différentes stratégies pour améliorer la transplantation d&#39;îlots. Cette expérience nécessite l&#39;isolement des îlots de haute qualité et l&#39;implantation d&#39;îlots pour les bénéficiaires diabétiques. Ces deux procédures nécessitent des étapes chirurgicales qui peuvent être mieux démontré par la vidéo que par texte. Ici, nous documentons les étapes détaillées de ces procédures à la fois par la vidéo et de protocole écrit. Nous discuterons aussi brièvement les modèles de transplantation différents: syngéniques, allogéniques, syngéniques auto-immunes, et allogéniques auto-immunes.

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

La transplantation d&#39;îlots isolés a été proposé pour être un traitement potentiel pour les diabétiques de type 1. Nous décrivons ici une méthode pour isoler les îlots de souris pancréas et de les transplanter à l&#39;espace sous-capsulaire du rein.

Une méthode pour isoler l&#39;îlot murin et transplantation rénale sous-capsulaires

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

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Nanyang Technological University

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63.3K vues.  The Ohio State University. La transplantation d'îlots isolés a été proposé pour être un traitement potentiel pour les diabétiques de type 1. Nous décrivons ici une méthode pour isoler les îlots de souris pancréas et de les transplanter à l'espace sous-capsulaire du rein.

Vidéo: 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.

Développement de la bronchiolite oblitérante dans un modèle murin de transplantation pulmonaire orthotopique

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

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Médecine

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.

Murin col de l&#39;utérus Cœur, la transplantation en utilisant une technique de modification de manchette

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.

Murin de transplantation cardiaque hétérotopique 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 ...

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.

Murin de transplantation de la cornée: Un modèle pour étudier la forme la plus courante de la transplantation d&#39;organes solides

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.

Souris Transplantation rénale: Les modèles de rejet des allogreffes

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.

Rein murin 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 ...

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.

Normothermique<em&gt; Ex Vivo</em&gt; Kidney Perfusion pour la préservation du rein Greffes avant la transplantation

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.

Une méthode d’isolement roman de Grade clinique pour les cellules stromales de rein humain périvasculaires

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.

Greffe orthotopique de rein de rat: une approche chirurgicale nouvelle et simplifiée

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.

Effacement optique et imagerie du tissu rénal immunolabeled

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