Name: murine corneal transplantation: a model to study the most common form of solid organ transplantation
Uploaded: 2014-11-17T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

NOTA: Todos los animales utilizados en este procedimiento son tratados de acuerdo con la Asociación para la Investigación en Visión y Oftalmología declaración para el uso de animales en Oftálmica y Visión de Investigación, así como las directrices establecidas por el comité de supervisión de los animales en la Universidad de Saint Louis. NOTA: Todos los instrumentos y soluciones quirúrgicas se esterilizan antes de la cirugía para limitar la infección microbiana del ojo. Cabe señalar que, si bien los animales experimentan un poco de dolor de este procedimiento, no nos empleamos a los analgésicos. La razón de esto se debe a que todos los analgésicos son antiinflamatorios y desde las respuestas de trasplante de córnea involucran la inflamación, el uso de medicamentos anti-inflamatorios podrían comprometer nuestra capacidad para determinar qué factores están implicados en el fracaso del injerto corneal. 1. Anestesia <ol style="margin-left: 40px;"><li> Coloque donantes y receptores ratones bajo anestesia general por inyecciones IP de ketamina (86.98 mg / kg) y xilazina (13,04 mg / kg). </li><li> Mantenga el ratón receptor bajo anestesia durante todo el procedimiento que suele durar de 30 minutos a una hora. En consecuencia, monitorear constantemente el ratón para detectar cualquier signo de recuperar la conciencia. </li><li> Aplicar ungüento puralube al ojo que no se someterá a la cirugía después de que el animal está anestesiado para evitar la sequedad. </li></ol> 2. El injerto de córnea <ol style="margin-left: 40px;"><li> Obtención del botón corneal donante. <ol><li> Una vez que el animal esté totalmente anestesiado, midriasis adecuada lograr mediante la administración de un par de gotas oculares de tropicamida al 1% y 2,5% de hidrocloruro de fenilefrina. </li><li> Coloque la cabeza del animal donante en posición horizontal sobre un tablero colocado sobre un soporte móvil robusto. Fijar la cabeza con una tira de cinta a través del cuello para asegurar que el ojo está en una posición horizontal a lo largo de toda la operación. </li><li> El uso de un trépano 2 mm de diámetro, cuya punta estaba teñido con azul de metilo, fueraalinear el sitio central del injerto de córnea. </li><li> Con una cuchilla afilada, penetrar en la córnea y Healon inyectar en la cámara anterior para profundizar en ella para reducir la posibilidad de daños en el endotelio del donante y la lente subyacente. </li><li> Escindir el injerto de donante con Vannas tijeras y colocar en un plato que contiene solución salina equilibrada de Hanks hasta su uso. </li><li> Después de que el injerto de donantes ha sido la retirada, la eutanasia del ratón donante mediante inhalación de CO2. </li></ol></li><li> Preparación del lecho del injerto. <ol><li> Repita los mismos pasos como se describe en 2.1.1 al 2.1.2 para el destinatario. </li><li> El uso de un trépano de diámetro 1,5 mm, esbozar el sitio del injerto destinatario. </li><li> Con una cuchilla afilada, penetrar en la córnea y Healon inyectar en la cámara anterior para profundizar en ella para reducir la posibilidad de daño a la lente subyacente. </li><li> Quite el botón corneal central esbozado por el destinatario utilizando Vannas tijeras y deseche. </li></ol></li><li> La sutura de lainjerto <ol><li> Coloque la córnea del donante sobre el injerto cama en la córnea del receptor. Asegúrese de que Healon adecuada está bajo la córnea del donante para proteger las células endoteliales de los donantes de los daños por el contacto directo con la lente. </li><li> Usando microforceps reciben propinas súper finas, colocar el primer bocado de sutura de nylon 11-0 en el lado de los donantes, a través de la donante con el 90% de profundidad de todo el espesor de lado del destinatario, luego atar. </li><li> Una vez que la córnea está anclado en su lugar, realizar suturas interrumpidas midcardinal de tal manera que la córnea tiene de 8 a 10 el total de puntos de sutura y la córnea donante esté bien alineado con y unido al receptor cama injerto corneal. </li></ol></li><li> La profundización de la cámara anterior <ol><li> Profundizar en la cámara anterior mediante la inyección de HBSS o burbuja de aire en la cámara anterior y comprobar con cuidado la integridad del injerto corneal de fugas con una esponja de celulosa. NOTA: Si la cámara anterior no puede ser reformado, entonces hay una alta probabilidad de cataract lo que hará que las futuras evaluaciones de la córnea trasplantada muy difícil y será también potencialmente conducir a la disfunción del endotelio corneal donante y el fracaso de este modo de injerto. </li></ol></li><li> Evaluación final <ol><li> Observe el ojo para determinar si el alumno es redondo y la profundidad de la cámara anterior es normal. NOTA: Si el alumno no es redonda, esto indica que durante la sutura de iris fue dañado y por lo tanto el injerto se considera un fallo técnico. </li><li> Aplique ungüento antibiótico en el ojo. Opcional: Cierre la tapa del ojo con una sutura de seda 7-0. </li><li> Observar los ratones hasta que estén completamente despierto y luego individualmente alojarlos durante un mínimo de dos días después de la cirugía. </li></ol></li></ol> 3. La eliminación de la sutura <ol style="margin-left: 40px;"><li> En los casos en que se utiliza la sutura tapa, anestesiar a los ratones como se describió anteriormente y retire la tapa de sutura en 48 hr. </li><li> Anestesiar ratones en el día 7 después de la operación. Retire la Sutures asegurar el injerto corneal. Una vez que las suturas se retiran y el animal ha despertado completamente, devolverlo a su jaula. </li></ol> 4. Evaluación clínica <ol style="margin-left: 40px;"><li> Examine el ojo para las indicaciones de complicaciones de procedimientos que incluyen, cataratas (opacidad del cristalino), hifema (sangre en la cámara anterior), una cámara anterior que no es de la profundidad adecuada, o la opacidad significativa de la córnea. Considere los que demuestran estas complicaciones como &quot;en peligro&quot; y la eutanasia por inhalación de CO2. <ol><li> Lleve a cabo todos los exámenes en los ratones no anestesiados. Mantenga el ratón con una parte de restricción de este modo el ratón de manera que la otra parte puede proptose el ojo para permitir una mejor visión del ojo. Una vez que las observaciones se han completado, devolver el animal a su jaula. </li></ol></li><li> Haga que un observador familiarizado con los grupos de tratamiento valorar las córneas trasplantadas de 2 a 3 veces a la semana para detectar signos de injerto de córneaepisodios de rechazo o fracaso del injerto corneal. Use el microscopio quirúrgico o una horizontal lámpara de hendidura para estas observaciones. <ol><li> Evaluar cada córnea para la opacidad usando una escala de 0 a 5. La escala se define como sigue: <ol><li> Asigne una puntuación de 0 a esos córneas que no tienen signos de opacidad. </li><li> Asigne una puntuación de 1 a esas córneas que muestran opacidad superficial mínima. </li><li> Asigne una puntuación de 2 a córneas que muestran opacidad leve y profunda pero la pupila y el iris subyacentes son todavía discernible. </li><li> Asignar una puntuación de 3 a la córnea que deben mostrarse opacidad estromal en el que el iris no pueden verse en detalle con la excepción de los márgenes de la pupila. </li><li> Asigne una puntuación de 4 a córnea que deben mostrarse densa opacidad estromal y si no hay estructuras subyacentes se pueden ver. </li><li> Asigne una puntuación de 5 a córnea que deben mostrarse opacidad completa y edema estromal intensiva, con la pupila y el iris totalmente oscurecido. </li></ol></li><li><html> También evaluar cada córnea para el grado de infiltración de los vasos sanguíneos (neovascularización) utilizando una escala de calificación de 1 a 8. Para lograr esto, ver la córnea como un conjunto de 4 cuadrantes iguales y determinar la cantidad de vasos sanguíneos en cada uno de estos cuadrantes con una anotar ese rango voluntad de 0 (no hay vasos) a 2 para una amplia vascularización de ese cuadrante. Añadir las puntuaciones individuales de cada cuadrante para calcular la puntuación neovascularización final. </li><li> Clasifica las córneas como agudamente rechazados si tienen una puntuación de 3 para dos observaciones consecutivas por tiempo señala hasta 5 semanas. </li><li> Clasificar los ratones cuyas córneas eran claras a las 5 semanas, pero el desarrollo de la opacificación a veces&gt; 45 días después del injerto, con una puntuación de 3 para dos puntos de tiempo consecutivos, como haber sufrido tardío rechazo del aloinjerto corneal. Utilice las curvas de supervivencia de Kaplan-Meier para analizar la supervivencia del injerto. </li></ol></li></ol> 5. La manipulación de la Modelo en-left: 40px; &quot;&gt; <li> Preparación de células individuales a partir del bazo. <ol><li> Para preparar las células individuales, primero la eutanasia del ratón donante. Entonces, extirpar el bazo. </li><li> Coloque el bazo en un filtro de células y alterar con un émbolo de jeringa de una jeringa de 3 ml. </li><li> Lavar las células y resuspender en 10 ml de solución salina equilibrada de Hanks. </li><li> Retire 10 l de suspensión celular y añadir a 10 l de 0,4% azul de tripano y mezclar. Añadir que a hemocitómetro y contar las células en el Sistema Interconectado Central. El número de células en el tubo es el recuento de células x 10 4 x 2 (factor de dilución en azul de tripano) x 10 (volumen en el tubo). </li></ol></li><li> La inyección en la cámara anterior. <ol><li> Anestesiar a los ratones como se describió anteriormente. </li><li> Realizar inyecciones utilizando un microscopio de disección. Para cada inyección intracameral, utilizar 10 6 células de bazo en un volumen de 0.005 ml y una jeringa de microlitro 0,25 ml equipada con una aguja 33 G. NOTA: Otras manipulacionesdel modelo se puede realizar mediante el tratamiento de los animales con reactivos que actúan ya sea como antagonistas o agonistas para determinar el papel que un factor determinado puede desempeñar siguiente ortotópico cirugía aloinjerto corneal. </li></ol></li></ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presurgical+corticosteroid+treatment+improves+corneal+transplant+survival+in+mice.">Presurgical corticosteroid treatment improves corneal transplant survival in mice.</a> Cornea. 32, 1591-1598 (2013).</li><li>Yamazoe, K., Yamazoe, K., Shimazaki-Den, S., Shimazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+for+corneal+graft+recovery+after+severe+corneal+graft+rejection+following+penetrating+keratoplasty.">Prognostic factors for corneal graft recovery after severe corneal graft rejection following penetrating keratoplasty.</a> BMC Ophthalmol. 13, 5 (2013).</li><li>Panda, A., Vanathi, M., Kumar, A., Dash, Y., Priya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+graft+rejection.">Corneal graft rejection.</a> Surv. Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+keratoplasty:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

The authors have no competing financial interests.

El modelo murino de trasplante de córnea aquí descrito permite al investigador estudiar el rechazo del aloinjerto corneal humano en un modelo que es predictivo de los factores que se asocian tanto con mejor rechazo 15,17,18,20, 26-30 y 21-25 de la aceptación de la córnea aloinjertos. A diferencia de trasplante de córnea humana, en el que los pacientes reciben tratamiento con esteroides, ya sea tópica o sistémica para tratar o prevenir el rechazo 31, este modelo se utiliza típicam...

El trasplante de córnea es uno de los tipos más comunes de éxito y el trasplante realizados en seres humanos. Las razones por las que se realiza esta cirugía son resultado de una lesión, enfermedad infecciosa 1, u otras formas de enfermedad de la córnea no infecciosa 2. Las cifras de la Asociación de Bancos de Ojos de América indican que más de 46.000 se realizaron en 2011 (véase el sitio web en: restoresight.org/eye_banks/eye_banks.html). Una indicación de su éxito es que las tasas de fracaso durante un año por injertos corneales alogénicas van desde 10 a 15% a los 5 años y el éxito es superior al 70% 3-8. Como muchos estudios han demostrado, el éxito de los aloinjertos de córnea está directamente relacionado con el hecho de que el ojo es un sitio inmunológicamente privilegiado. Los factores responsables de la situación córneas como un sitio de privilegio inmune incluyen la falta de ambos vasos sanguíneos y linfáticos en la córnea, una relativa ausencia de las células presentadoras de antígenos, factores producidos por la córnea que una supresión ens funtions de efectoras inmunes 9-15, baja expresión de antígenos de MHC de 16, y la expresión de FasL 17-20. Sin embargo, a pesar de estos factores predisponentes estos injertos para el éxito, que no se someten a rechazo 3-7. En consecuencia, la comprensión de los mecanismos que median este rechazo, así como probar distintas terapias para prevenir el rechazo es de importancia crítica. Para ello, se describe aquí un modelo murino de trasplante de córnea, que ha estado en uso durante más de 20 años para estudiar el trasplante de córnea en un entorno experimental controlado. Desde respuestas trasplante implican muchos factores diferentes que trabajan en concierto que será definitiva determinar si el tejido trasplantado falla o tiene éxito, no es posible entender la importancia de estos factores en cualquier modelo in vitro. En consecuencia, se requieren estudios que utilizan animales intactos para determinar qué factores son importantes para el éxito o failure de tejido trasplantado. Mientras que otras especies de animales se han utilizado para estudiar el trasplante de córnea, el modelo murino tiene varias ventajas cuando se compara con el uso de otras especies. La primera es la existencia de muchas cepas de ratones que expresan ciertos transgenes o han sido dirigidos-gen a carecer de la expresión de factores inmunológicos específicos cuya función en el trasplante puede ser mejor estudiado. Además, hay muchos reactivos (ambos factores recombinantes y anticuerpos que neutralizan factores) que son específicos para los ratones y que no existen para muchas otras especies de animales. Debido a la existencia de estos factores, este modelo ha sido utilizado ampliamente para identificar los factores relevantes implicados en las respuestas de aloinjertos corneales agudas 15, 17,18,20 -29. Además, muchos de los factores que intervienen en el trasplante corneal también son conocidos por ser funcional en el trasplante de otros tejidos.

<table><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr></tbody></table>

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.

El modelo murino de trasplante de córnea ha sido utilizado durante más de 20 años con éxito para caracterizar los mecanismos tanto de rechazo del aloinjerto corneal 19-23 y la aceptación del aloinjerto de la córnea 13, 15,16,18, 24-27. Este modelo se utilizó para establecer la importancia de la expresión de FasL en la aceptación del aloinjerto corneal, en que los animales que carecen de FasL no fueron capaces de aceptar aloinjertos de córnea 15. También se ha utilizado para de...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

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

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 ...

murine-corneal-transplantation-model-to-study-most-common-form-solid

Research

JoVE Journal

Medicine

9.6K vistas.  Saint Louis University. 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.

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

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 ...

Investigación

Medicina

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 ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murino de grosor completo Trasplante de Piel

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Inmunología e Infección

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Un microquirúrgica Modelo Novela de heterotópico, en bloque, pared torácica, el timo y el trasplante cardiaco en ratones

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Ortotópico de la extremidad posterior de trasplante en el ratón

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex Vivo modelo de cultura órgano córneo para la curación de heridas estudios

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Modelo de trasplante de aórtica cervical murino utilizando una técnica de manguito no sutura modificada

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

Un protocolo modificado de trasplante de corazón heterotópico murino que coincide con los estándares contemporáneos de técnica aséptica, anestesia y analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Un modelo de cultivo de órgano endotelial Corneal porcino usando botones de córnea divididos

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...