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>

<ol>
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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.

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			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8154</td>
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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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Trasplante ortotópico aórtica: un modelo de rata para estudiar el desarrollo de la enfermedad vascular crónica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Investigación

Medicina

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.

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 ...

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 ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

El trasplante de la válvula pulmonar Utilizando un modelo de ratón de heterotópico trasplante cardiaco

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cateterismo cardíaco en ratones para medir la relación de volumen Presión: Investigar el efecto Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

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 ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Desarrollo de un Modelo Experimental No Invasivo, Asistido por Láser de Pérdida Celular Endotelial Corneal

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

Reducción de las complicaciones después de la reconexión arterial en un modelo de rata de trasplante hepático ortotópico

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

Autoaplano de riñón ortotópico en un modelo porcino utilizando 24 horas de preservación de órganos y telemetría continua

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Trasplante intramiocardio de hidrogeles inyectables msc-loading después de infarto de miocardio en un modelo murino

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...