Ce travail a été financé en partie par 1R03DK096151.

Toutes les souris ont été achetés auprès de The Jackson Laboratory (Bar Harbor, ME) et ont été logés dans des conditions exemptes d&#39;organismes pathogènes à l&#39;Université du Colorado à Denver, Facilité Barbara Davis Animal Center conformément aux directives du NIH et avec l&#39;approbation de l&#39;Université du Colorado à Denver IACUC. 1. donneur de rein récolte <ol><li> Stériliser tous les instruments, porter des gants stériles tout au long de la procédure et de maintenir un champ stérile. Effectuer toutes les interventions chirurgicales à l&#39;aide d&#39;un microscope opératoire. </li><li> Enlever chirurgicalement reins de donneurs de souris à partir de pentobarbital anesthésiés (60mg / kg ip) les bailleurs de fonds. Déterminer profondeur de l&#39;anesthésie par pincement pincée, et observer la fréquence respiratoire. </li><li> Clip de la fourrure puis immobiliser la souris en 4 voies restrictions. Préparer la peau avec de la povidone-iode et le drapé de la souris de façon stérile. </li><li> Faire une ligne médiane verticale 2 cm incision abdominale et entrer dans la cavité abdominale. Rentrer l&#39;intestin supérieurement et extériorisersur la poitrine. Gardez l&#39;intestin enveloppé dans de la gaze stérile humide tout au long de la procédure. </li><li> Identifier les grands vaisseaux de l&#39;abdomen et de les mobiliser, d&#39;identifier les branches lombaires et soit cautériser ou de les ligaturer avec 10/0 suture en nylon. </li><li> Immédiatement en aval des vaisseaux rénaux gauche taquiner le veine cave inférieure (VCI) et l&#39;aorte abdominale (AA) en dehors par dissection sur environ 2mm. Ligaturer avec 10/0 nylon et diviser petite artériel et veineux branches des vaisseaux rénaux. </li><li> Maintenant séparer la veine rénale de l&#39;artère rénale attentivement par dissection de ces structures. Cela permet la création d&#39;un timbre précis Carrel à l&#39;extrémité proximale de la veine rénale, qui sera utilisé pour former une anastomose termino-latérale aux destinataires IVC. </li><li> Identifier, ligaturer et de diviser les vaisseaux surrénales gauche. Ceci permet l&#39;accès à l&#39;aorte sus-rénale et une suture en soie 6-0 de est placé autour de l&#39;aorte en vue de la ligature, mais pas lié à ce moment. </li&gt; <li> Mobiliser les reins, les vaisseaux et de l&#39;uretère de la planche de bord environnante. Tourner le rein à la droite et ligaturer / diviser des branches postérieures. Puis retour du rein vers la gauche. </li><li> Maintenant diriger l&#39;attention vers l&#39;uretère. Sans perturber le hile rénal libérer l&#39;uretère de la planche de bord environnante en prenant soin de préserver les vaisseaux de l&#39;uretère. Diviser l&#39;uretère au niveau du canal déférent. Le rein est maintenant prêt pour la récupération. </li><li> Injecter lentement 300 unités d&#39;héparine dans le IVC distale héparinisation ainsi le donateur. Attacher le fil de suture en soie 6-0 de placée autour de la surrénale AA et perfuser le rein gauche via l&#39;aorte abdominale distale avec 0,8 ml de solution saline d&#39;héparine (100 U / ml). </li><li> Une fois la perfusion a cessé de créer un patch Carrel veineuse immédiatement pour éliminer refoulement dans le rein. Rétracter la veine rénale vers le rein révélant l&#39;AA et l&#39;artère rénale dessous. </li><li> Diviser l&#39;aorte adjacente à l&#39;artère rénale la création d&#39;un talon et àe brassard, comme montré dans la figure 1. Retirez les reins, les vaisseaux et de l&#39;uretère du donneur. </li><li> Retirer le rein directement à un destinataire pré-établi (0 min temps froid ischémie) ou stockées à 4 ° C dans une solution de choix pour un temps prédéterminé jusqu&#39;au moment de l&#39;implantation. Euthanasiés par les bailleurs de fonds et exsanguanation dislocation cervicale. Temps total de récupérer donneur de rein est d&#39;env. 15-20 min. </li></ol> 2. Rein Implant Technique <ol><li> Anesthésier la souris receveuse avec du pentobarbital (60 mg / kg de la dose initiale IP, dose supplémentaire / kg IP de 25mg si nécessaire). Déterminer profondeur de l&#39;anesthésie par pincement pincée, et observer la fréquence respiratoire. Coupé la fourrure puis immobiliser la souris en 4 voies contraintes et appliquer pommade ophtalmique pour les yeux. Préparer la peau avec de la povidone-iode et le drapé de la souris de façon stérile. </li><li> Faire une ligne médiane verticale 2 cm incision abdominale et entrer dans la cavité abdominale. Retrait de l&#39;intestinsupérieurement et extérioriser sur la poitrine. Gardez l&#39;intestin enveloppé dans de la gaze stérile humide tout au long de la procédure. </li><li> A ce moment effectuer une néphrectomie droite. Ligaturer l&#39;artère et la veine rénales avec 6-0 sutures de soie. Exciser le rein distale par rapport à la suture. Ligaturer l&#39;uretère avec soie 6-0 et ensuite diviser proximale du rein. </li><li> Isoler l&#39;aorte abdominale et veine cave inférieure (VCI) au-dessous des artères rénales. Placez 4-0 liens de coton autour de l&#39;aorte et IVC supérieure puis inférieure vers le site d&#39;anastomose. Identifier et ligaturer des vaisseaux lombaires dans le domaine de nylon 10-0 suture. </li><li> Nouer les liens de coton, d&#39;abord l&#39;inférieur, suivie par le supérieur. De cette manière, un peu de sang est retenu dans la prise de l&#39;aorte aortotomie plus facile. </li><li> Former le aortotomie avec une aiguille de 30G pour entrer dans la lumière de l&#39;aorte. Elargir l&#39;incision avec de fines micro ciseaux pour une longueur d&#39;environ 2mm. </li><li> Faire une fin anastomose de la coiffe aortique donateurs talon-orteill&#39;aorte bénéficiaire de la façon suivante. Placez un nylon 10-0 séjour de suture point dans l&#39;aorte des bailleurs de fonds et de l&#39;angle inférieur de l&#39;incision dans l&#39;aorte bénéficiaire et une cravate. </li><li> Placez un second nylon 10-0 opposée à la première dans l&#39;aorte des bailleurs de fonds et le coin supérieur de l&#39;incision dans l&#39;aorte abdominale et une cravate. Faire une ligne de suture allant de supérieure à inférieure dans la paroi latérale de l&#39;aorte et une cravate contre le séjour point précédemment placé. Puis suturer la paroi interne d&#39;une manière courante et une cravate. </li><li> Faire une fin anastomose de la veine rénale de donneur au receveur IVC de la façon suivante. Piquer la IVC avec une aiguille de 30G et d&#39;étendre l&#39;incision avec de beaux ciseaux micro. Attachez la veine rénale des bailleurs de fonds dans le coin inférieur de l&#39;incision dans l&#39;IVC, avec nylon 10-0. Faire une ligne de suture courant entre la veine rénale et la veine cave inférieure et une cravate. NOTE: Il est également extrêmement important que toute l&#39;épaisseur passe de l&#39;aiguille de suture compris le adventit vasculaireIA et l&#39;intima sont atteints. Éversion des bords garantit également qu&#39;il n&#39;y a intima à intima contact, ce qui contribue à l&#39;étanchéité et à la guérison des anastomoses. Alors un agent de coagulation hémostatique peut être utile pour réduire les fuites, nous recommandons que le chirurgien place reposent sur une bonne technique. </li><li> Veiller à ce que les anastomoses sont «propres». Autrement dit, que les parois opposées sont pas pris en plaçant des points de suture. Cela entraînera une constriction importante à l&#39;écoulement qui se traduira par un greffage en panne et dans des cas extrêmes, à la paralysie des membres postérieurs. NOTE: Un autre facteur extrêmement important est de veiller à ce que la tension des lignes de suture anastomotiques est également optimale. Trop lâche et il y aura une fuite irréversible, trop serré et sténose de circuler en résultera. Si artérielle sur le côté cela se traduira par une mauvaise perfusion de la greffe, si sur le côté veineux d&#39;un rein congestionné en résultera. </li><li> Relâchez le flux veineux distale 4-0 coton cravate rétablissement. Une fois l&#39;hémostase du veinonous anastomose a été observé progressivement desserrer la cravate 4-0 de coton proximale et observer l&#39;anastomose artérielle pour l&#39;hémostase. </li><li> Retirez les liens de coton de la souris une fois les deux anastomoses sont considérés comme sûrs. Pierce la vessie avec une aiguille 20 G création de deux trous. </li><li> Passer les conseils de pince courbe à travers les trous et tirez l&#39;uretère des bailleurs de fonds à travers la vessie avec la pince avec l&#39;extrémité proximale de l&#39;uretère étant ancrée à la paroi de la vessie avec deux fils de nylon 10-0. Coupez l&#39;excès de longueur de l&#39;uretère saillie de la deuxième trou permettant l&#39;uretère se rétracter dans la vessie et fermer le trou avec deux fils de nylon 10-0. </li><li> Retour de l&#39;intestin à l&#39;abdomen. Fermez la paroi abdominale en deux couches en utilisant 5-0 suture soie dans un mode en cours d&#39;exécution. </li><li> Administrer un bolus de 1,0 ml solution saline stérile normale chaud dans l&#39;abdomen comme la réanimation liquidienne à la clôture, et injecter 0,8 ml de solution saline sous-cutanée post-opératoire normal. Aucune autre supportive mesures sont nécessaires pendant la chirurgie. </li><li> Récupérer l&#39;animal sur une couverture chauffante. Temps implant totale est d&#39;env. 35 à 45 min. Administrer des analgésiques tels que la buprénorphine, 0,05 mg / kg, SC, 0,1-0,2 ml au début de la procédure et tous les 6-12 h pour 72 h post-op. </li></ol> 3. néphrectomie controlatérale <ol><li> Selon les exigences du protocole, plusieurs jours après la procédure d&#39;implantation, effectuent une néphrectomie controlatérale l&#39;isoflurane (5% de l&#39;isoflurane inhalation pour l&#39;induction, 1,5-2,0% pour la maintenance). </li><li> Entrez la cavité abdominale et se rétracter doucement l&#39;intestin à la droite de l&#39;animal. Exposer le rein gauche et de disséquer émoussée du fascia environnantes. </li><li> Ligaturer l&#39;artère rénale, la veine et de l&#39;uretère avec 6-0 sutures de soie puis exciser le rein au-dessus des points de suture et enlever le rein. Le temps opératoire total est d&#39;env. 10 min. </li></ol> 4. Évaluation Graft <ol><li> Mesure sérum creatineuf en utilisant le procédé au picrate alcalin (réaction de Jaffe). </li></ol>

<ol>
	<li>Skoskiewicz, M., Chase, C., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+transplants+between+mice+of+graded+immunogenetic+diversity.">Kidney transplants between mice of graded immunogenetic diversity.</a> Transplant Proc. 5 (1), 721-725 (1973).</li><li>Kalina, S. L., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microsurgical+technique+for+renal+transplantation+in+mice.">A microsurgical technique for renal transplantation in mice.</a> Aust N Z J Surg. 63 (3), 213-216 (1993).</li><li>Han, W. R., Murray-Segal, L. J., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+for+kidney+transplantation+in+mice.">Modified technique for kidney transplantation in mice.</a> Microsurgery. 19 (6), 272-274 (1999).</li><li>Ge, F., Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+successfully+establishing+a+kidney+transplant+in+a+mouse+model.">Strategies for successfully establishing a kidney transplant in a mouse model.</a> Exp Clin Transplant. 9 (5), 287-294 (2011).</li><li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+curve,+surgical+results+and+operative+complications+for+kidney+transplantation+in+mice.">Learning curve, surgical results and operative complications for kidney transplantation in mice.</a> Microsurgery. 26 (8), 590-593 (2006).</li><li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Zhang, Z., Chen, H., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+Transplantation+in+Mice.">Kidney Transplantation in Mice.</a> Experimental Organ Transplantation. , 45-64 (2013).</li><li>Zhang, L., Moskovitz, M., Piscatelli, S., Longaker, M. T., Siebert, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+study+of+different+angled+end-to-side+anastomoses.">Hemodynamic study of different angled end-to-side anastomoses.</a> Microsurgery. 16 (2), 114-117 (1995).</li><li>Liu, Q., Mirc, D., Fu, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+mechanisms+of+thrombosis+in+intact+bent+microvessels+of+rat+mesentery.">Mechanical mechanisms of thrombosis in intact bent microvessels of rat mesentery.</a> J Biomech. 41 (12), 2726-2734 (2008).</li><li>Chesnutt, J. K., Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tortuosity+triggers+platelet+activation+and+thrombus+formation+in+microvessels.">Tortuosity triggers platelet activation and thrombus formation in microvessels.</a> J Biomech Eng. 133 (12), 121004 (2011).</li><li>Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+buckling+within+soft+surrounding+tissue+generates+tortuosity.">Blood vessel buckling within soft surrounding tissue generates tortuosity.</a> J Biomech. 42 (16), 2797-2801 (2009).</li><li>Gutierrez-Diaz, J. 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=Intraluminal+thrombus+and+neointimal+hyperplasia+after+microvascular+surgery.">Intraluminal thrombus and neointimal hyperplasia after microvascular surgery.</a> Surg Neurol. 24 (2), 153-159 (1985).</li><li>Khouri, R. K., Cooley, B. C., Kenna, D. M., Edstrom, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis+of+microvascular+anastomoses+in+traumatized+vessels:+fibrin+versus+platelets.">Thrombosis of microvascular anastomoses in traumatized vessels: fibrin versus platelets.</a> Plast Reconstr Surg. 86 (1), 110-117 (1990).</li><li>Johnson, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+platelet+deposition+at+the+human+microvascular+anastomosis:+effect+on+downstream+platelet+deposition+to+intact+and+injured+vessels.">Initial platelet deposition at the human microvascular anastomosis: effect on downstream platelet deposition to intact and injured vessels.</a> Plast Reconstr Surg. 90 (4), 650-658 (1992).</li></ol>

The authors have nothing to disclose.

La maîtrise de cette technique de greffe est difficile, mais une fois accomplie, il est un outil de recherche très puissant. Le patient chirurgien / chercheur sera récompensé par une attention aux détails et la cohérence de la technique, qui est la clé de la maîtrise de toute intervention chirurgicale, plus encore dans de petits modèles animaux. Les difficultés techniques de maîtrise de la greffe de rein de souris sont nombreux plis, et il est hautement probable que l&#39;expérience dans d&#39;autres petits ...

Depuis 1973, le modèle de greffe de rein chez la souris a été un précieux outil de recherche, mais des problèmes techniques ont entravé son utilisation généralisée. Au cours des années, plusieurs articles ont été publiés en détail les améliorations / améliorations apportées à cette procédure. Comme un modèle de transplantation d&#39;organes solides principalement vascularisé cette procédure est probablement le deuxième seulement pour le modèle de transplantation cardiaque hétérotopique qui a également été conçu par le laboratoire Russell en 1973 6. Les deux modèles se prêtent à la recherche sur les réponses de rejet allogénique, le développement de la fonction du greffon retardée et les lésions d&#39;ischémie reperfusion. Une des questions les plus courantes à être rapportés avec la transplantation rénale est l&#39;incidence relativement élevée de thrombose artérielle 4,5,7 que nous avons également vécu dans notre laboratoire. Nous mettons donc à effectuer une revue de la littérature de la formation de thrombus et peut-être trouver la cause de ce problème technique et d&#39;élaborer aussi unsolution possible. La cause la plus probable de la thrombose est le chemin tortueux peu le sang prend de l&#39;aorte bénéficiaire, dans l&#39;aorte rénale des bailleurs de fonds lors de l&#39;artère rénale des bailleurs de fonds. Cette voie provoque une turbulence dans l&#39;artère rénale qui peut conduire à l&#39;activation des plaquettes et la formation de thrombus. Basé sur les observations récentes et une recherche de la littérature pertinente 8-14 nous sommes arrivés avec une nouvelle technique qui a réduit la thrombose de 0%. La technique décrite ici varie de techniques précédemment rapportées dans la formation d&#39;une artère talon-orteil qui brassard installations améliorées circulation sanguine et réduit considérablement la formation de thrombus. La manchette est formée en divisant l&#39;aorte sous-rénale sur la face de l&#39;ostium des artères rénales à un angle inférieur à 45 ° à l&#39;axe longitudinal de l&#39;aorte (figure 1A et 1B). Cela se traduit par une manchette d&#39;environ 2 mm de longueur. Un patch Carrel veineuse est formé par sectionnant la rénal la veine IVC en augmentant ainsi le diamètre du brassard. Le donneur aorte abdominale talon-orteil brassard sous-rénale est de bout en côté anastomosée au destinataire aorte abdominale et la veine rénale des bailleurs de fonds / patch IVC est de bout en côté anastomosée au destinataire abdominale veine cave inférieure (VCI) . L&#39;uretère est ensuite introduit dans et ancrée à la vessie telle que décrite par Han et al trois. Pour cette étude des greffes non traités avec des temps d&#39;ischémie chaude seulement (ie., Aucune ischémie froide) sont comparées. Dans ce cas ischémie chaude se réfère au moment de la cessation du flux sanguin dans le rein d&#39;un donneur (étape 1.11 ci-dessous) et la reperfusion du greffon chez le receveur (étape 2.11 ci-dessous). Ischémie froide se réfère au temps que le rein perfusé est pas et est conservé en chambre froide jusqu&#39;à ce que le début de la procédure d&#39;implantation.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Instrument</td>
			<td>Roboz #</td>
			<td>Fine Science Tools #</td>
			<td>Arosurgical #</td>
		</tr>
		<tr>
			<td>Straight micro-dissecting forcep #5</td>
			<td>RS-5015</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved micro-dissecting forcep #7</td>
			<td>RS-5047</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved serrated forcep</td>
			<td>RS-5137</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas micro-dissecting scissors, short</td>
			<td>RS-5610</td>
			<td></td>
			<td>09.140.08</td>
		</tr>
		<tr>
			<td colspan="2">Micro-dissecting scissors, straight, sharp, long</td>
			<td></td>
			<td>11.602.11</td>
		</tr>
		<tr>
			<td>Micro spring handle needle holder</td>
			<td></td>
			<td></td>
			<td>11.549.15</td>
		</tr>
		<tr>
			<td>Straight mosquito forcep</td>
			<td></td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors, straight, blunt</td>
			<td>RS-5962</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors, curved, blunt</td>
			<td>RS-5981</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro retractor</td>
			<td>RS-6540</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument tray, 10&#8221; x 6 &#189;&#8221; x &#190;&#8221;</td>
			<td>RT-1350S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silk suture, 5/0, 22.5m spool</td>
			<td></td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10/0 nylon</td>
			<td></td>
			<td></td>
			<td>T4A10Q07</td>
		</tr>
		<tr>
			<td>5/0 silk</td>
			<td></td>
			<td></td>
			<td>E19A05N</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td>Drapes</td>
			<td></td>
		</tr>
		<tr>
			<td>Biogel from Medex Supply</td>
			<td></td>
			<td>Precept, #64-9012-9</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td>Cotton applicators</td>
			<td></td>
		</tr>
		<tr>
			<td>B-D 1cc insulin, #329424</td>
			<td></td>
			<td colspan="2">Fisher-brand, #23-400-100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDI, #B40600</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4/0 Cotton ties</td>
			<td></td>
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			<td colspan="2">Domestic cotton autoclaved with instruments</td>
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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.

Cette technique chirurgicale permet pour des études de survie / de rejet de greffe, simples ou protocoles expérimentaux assez complexes. Dans les figures ci-dessous, nous démontrons les avantages de l&#39;utilisation de cette technique améliorée d&#39;anastomose artérielle. En utilisant cette technique, nous avons considérablement réduit l&#39;incidence de la thrombose artérielle de 35% à 0% augmentant ainsi la productivité. Nous avons utilisé cette technique pour plus d&#39;un an avec le même résultat de ...

Watch this Scientific Journal Video about Murine Kidney Transplant Technique at JoVE.com

Murine Kidney Transplant Technique

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 ...

Research

JoVE Journal

Medicine

15.8K vues.  University of Colorado, Denver. 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. 

Vidéo: Murine Kidney Transplant Technique

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

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 ...

Recherche

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 ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Technique de Subnormothermic<em&gt; Ex Vivo</em&gt; Foie de perfusion pour l&#39;entreposage, de l&#39;évaluation et de réparation des marginaux greffes du foie

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

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 ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

Technique de porc approvisionnement du foie et la transplantation orthotopique utilisant un Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

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

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

Modèle murin de rejet vasculaire allo-induite et Transplant artériosclérose

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

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 ...

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

Un «All-laser&quot; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

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

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

Modèle de souris pour une Transplantation du pancréas en utilisant une Technique mis à jour le brassard

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Blessure axée sur le ballon pour provoquer l’hyperplasie Myointimal dans l’aorte abdominale de souris

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...