Este trabajo es apoyado por RO1 DK064938 (de 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>

En los apartados anteriores, se describen los pasos detallados para el aislamiento y el transplante de los islotes pancreáticos de ratón. En este sentido, destacar brevemente los pasos que son críticos para el éxito de los procedimientos. 1. Aislamiento de islotes Los pasos clave son la identificación de un momento óptimo digestión enzimática (1,4), el posicionamiento de la pinza hemostática sobre la abertura duodenal del conducto biliar común (2.2.3), canulación del conducto biliar común (2,3), una agitación vigorosa del páncreas después de los 37 ° C digerir (3,2), y la eliminación de la contaminación de células exocrinas (3.3.7 y 3.3.16). Estos pasos tienen una gran influencia en la calidad, el rendimiento y la pureza de los islotes. Quizás el paso más difícil técnicamente es la canalización del conducto biliar común. En muchos ratones, el conducto biliar común puede ser difícil de visualizar debido a la fascia que rodea. Por lo tanto, es común que los intentos para canalizar el resultado del conducto biliar común en la penetración del tejido facial solamente. En estos casos, la Liberase comienza a llenar el tejido conectivo circundante y no perfusión del páncreas. Si esto se detecta, detener el flujo de Liberase y la posición de la aguja para que penetre en el lumen de la vía biliar. Con la práctica, es posible identificar rápidamente la posición óptima para la colocación de la aguja y para completar la canalización sin dañar el conducto friable. 2. El trasplante de islotes Los pasos críticos son la inducción de la diabetes con STZ (5,1) y la prestación eficiente de los islotes de la bolsa subcapsular (5.3.14-5.3.16). STZ es un análogo de la glucosa natural que es selectivamente tóxica para las células de la secreción de insulina de las células β de los islotes 9. Sin embargo, esta toxicidad selectiva se produce en un rango de concentraciones relativamente estrecho. Prueba de ello es el hecho de que altas dosis de STZ puede resultar en un rápido (2-3 días) letalidad en ausencia de hiperglucemia. Por lo tanto, se debe tener cuidado para identificar la dosis óptima de STZ de la cepa y la edad de los ratones se utiliza antes de los experimentos a gran escala se inició. La entrega física de los islotes de la bolsa subcapsular es también un paso clave. Para lograr resultados consistentes, es imprescindible para reducir al mínimo la pérdida de islotes en su entrega. Esto puede ocurrir si hay defectos o roturas en la cápsula renal que cubre la bolsa o si el volumen inyectado es demasiado grande. El daño a la cápsula se puede evitar la manipulación suave y cuidadosa, y mediante el uso de la sonda de cristal liso para preparar la bolsa subcapsular. Además, el volumen de inyección puede ser minimizado con la extracción cuidadosa del sobrenadante en el paso 5.3.6.3. Si se retira el sobrenadante hasta el nivel de los islotes sedimentado, normalmente hay más que suficiente espacio en la bolsa subcapsular a cabo 400 a 500 islotes. Sin embargo, si el volumen de inyección es demasiado grande, los islotes son más propensos a fugas de la bolsa durante el proceso de trasplante, poniendo en peligro la reproducibilidad del experimento. Dentro de este protocolo, es posible modificar algunos de los pasos y aún así lograr resultados satisfactorios. Estos incluyen los siguientes: (a) el uso de una enzima diferente para digerir el páncreas, (b) un método diferente para la entrega de la enzima en el páncreas, (c) el uso de un continuo de Ficoll-diatrizoato de sodio (FSD) gradiente en lugar de la densidad de un solo paso Histopaque1077, y (d) trasplante de islotes a otros lugares de la subcapsule riñón. <ol class="lalpha"><li> En este protocolo, Liberase se utiliza para debilitar a los contactos célula-célula en el páncreas. Liberase es esencialmente una mezcla altamente purificada de colagenasas que han demostrado ser útiles en la liberación de los islotes del páncreas. No obstante, las colagenasas menos purificada son capaces de lograr un resultado similar. Por lo tanto, es posible utilizar diferentes preparados enzimáticos disponibles en el mercado para digerir el páncreas, siempre y cuando la condición de la digestión se ha optimizado para conseguir una calidad de islotes de alta, el rendimiento y la pureza. </li><li> También es posible para digerir el páncreas sin perfusión ductal de la enzima. La entrega de Liberase través del conducto biliar común permite una máxima exposición de la zona del páncreas superficie de la enzima. Esto da como resultado una digestión más aún del páncreas y una mayor liberación de los islotes intactos. Sin embargo, otras medidas para aumentar la superficie del páncreas para que la enzima se puede utilizar. Por ejemplo, picado el páncreas antes de incubar en Liberase o la inyección directa de Liberase través de la cápsula del páncreas puede ser usado para aislar los islotes. Sin embargo, ningún otro método de entrega Liberase es tan eficaz como la perfusión a través del conducto biliar común. Por lo tanto, la inyección de picado o directa debe reservarse como un último recurso, si el conducto biliar común está dañado y no perfusable. </li><li> Un método alternativo para la separación de los islotes de las células exocrinas implica lael uso de un gradiente de FSD en lugar de Histopaque1077 10. La separación eficiente de los islotes de células exocrinas en este protocolo se debe en parte a un diferencial de densidad entre los dos tipos de células. Histopaque1077 tiene una densidad de 1,0771 g / ml a 25 ° C. A esta temperatura, Histopaque es más denso que los islotes, pero menos densa que las células exocrinas. Por lo tanto, bajo la fuerza de centrifugación, Histopaque1077 gránulos de las células exocrinas, levantando los islotes a la superficie. En principio, cualquier otra sustancia con una densidad que puede separar los islotes de células exocrinas se pueden utilizar en este paso y el gradiente de FSD es una de esas sustancias. </li><li> Con respecto a la ubicación de los islotes del trasplante, la cápsula renal utilizados en este protocolo es uno de los pocos lugares posibles. Mientras que el subcapsule riñón es el sitio más frecuente de trasplante en ratones, el trasplante de otros sitios se han encontrado para ser eficaz y que incluyen la vena porta hepática, espacio subretiniano, testículos, almohadilla de grasa del epidídimo, el bazo, páncreas y hasta 11-14. Cada uno de estos lugares tiene sus propias ventajas y desafíos. Por lo tanto, la selección de un sitio para el trasplante de islotes debe ser determinado por las preguntas formuladas y las circunstancias específicas de los experimentos. </li></ol> Se cree que las estrategias que se pueden superar las limitaciones en el trasplante de islotes dramáticamente mejorar su potencial terapéutico. Por lo tanto, el uso de un modelo murino que se detallan en este protocolo ofrece un enfoque atractivo para la identificación de tales estrategias.

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<td>Equipment</td>
<td>Fisher Scientific</td>
<td>22362574</td>
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<td>BD Falcon</td>
<td>329461</td>
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<td>27 Gauge Syringe Needle</td>
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<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>Kent Scientific</td>
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<td>&nbsp;</td>
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<td>Equipment</td>
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<td>Equipment</td>
<td>Mettler Toledo/ Fisher Scientific</td>
<td>09313509</td>
<td>&nbsp;</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

Desde los primeros trabajos pioneros de Ballinger y Reckard lo que demuestra que el trasplante de islotes de Langerhans en roedores diabéticos podrían normalizar sus niveles de glucosa en la sangre, el trasplante de islotes se ha propuesto ser un tratamiento potencial para la diabetes tipo 1 1,2. Más recientemente, los avances en trasplante de islotes humanos han fortalecido aún más este punto de vista 1,3. Sin embargo, dos limitaciones importantes evitar el trasplante de islotes de ser una realidad clínica generalizada: (a) la necesidad de un gran número de islotes por paciente, lo cual reduce considerablemente el número de beneficiarios potenciales, y (b) la necesidad de inmunosupresión pesado, que afecta de manera significativa la población pediátrica de los pacientes debido a su vulnerabilidad a la inmunosupresión a largo plazo. Las estrategias que se pueden superar estas limitaciones tienen el potencial para mejorar la utilidad terapéutica del trasplante de islotes. El trasplante de islotes bajo la cápsula renal ratón es un modelo ampliamente aceptado para investigar varias estrategias para mejorar el trasplante de islotes. Este experimento requiere el aislamiento de islotes de alta calidad y la implantación de islotes a los destinatarios de la diabetes. Ambos procedimientos requieren pasos de la cirugía que puede ser mejor demostrado por el vídeo que por texto. En este sentido, el documento los pasos detallados para estos procedimientos por el vídeo y el protocolo escrito. También brevemente los diferentes modelos de trasplante: singénico, autoinmunes alogénico, singénico y alogénico autoinmunes.

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

El trasplante de islotes aislados se ha propuesto ser un tratamiento potencial para la diabetes tipo 1. Aquí se describe un método para aislar los islotes de páncreas de ratón y trasplantarlas en el espacio subcapsular del riñón.

Un método para el aislamiento de los islotes murinos y Trasplante Renal subcapsular

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

63.3K vistas.  The Ohio State University. El trasplante de islotes aislados se ha propuesto ser un tratamiento potencial para la diabetes tipo 1. Aquí se describe un método para aislar los islotes de páncreas de ratón y trasplantarlas en el espacio subcapsular del riñón.

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.

El desarrollo de bronquiolitis obliterante en un modelo murino de trasplante de pulmón ortotópico

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

Investigación

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 Cervical Trasplante Cardiaco Modelo Usando una técnica de Cuff Modificado

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.

Murino heterotópico Corazón Trasplante Técnica

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.

Murino Trasplante corneal: un modelo para estudiar la forma más común de trasplante de órganos sólidos

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.

Ratón Trasplante de Riñón: Modelos de rechazo del aloinjerto

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.

Riñón murino Trasplante Técnica

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.

Normotérmica<em&gt; Ex Vivo</em&gt; Perfusión renal para la Preservación de riñón Injertos antes del trasplante

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 método de aislamiento de grado clínico novedoso para Stromal células de riñón humano perivasculares

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.

Trasplante de riñón de rata ortotópica: un enfoque quirúrgico novedoso y simplificado

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.

Limpieza óptica e imágenes del tejido renal inmunoetiquetado

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