Los autores desean agradecer FORTE proyecto, financiado por la Región Toscana (POR CREO FESR 2007-2013, Bando Unico R &amp; S 2012), los de la UE 7PM ECHORD ++ Experimento LA-ROSAS que parcialmente apoyaron las actividades de investigación y el 7PM BiophotonicPlus Proyecto &quot;LITE&quot; otorgado por la Región Toscana.

El estudio se realizó con la aprobación del Comité de Ética prospectivamente del hospital; Se obtuvo el consentimiento informado. El estudio estaba en la adhesión a los principios de la Declaración de Helsinki. 1. Preparación de Donantes endotelio <ol><li> Utilice una córnea donante preparado por el banco de ojos local en la temperatura ambiente. </li><li> En la sala de cirugía, tire de la córnea del donante de su contenedor de entrega y apartado de la solución para la conservación de tejidos y la nutrición que se utiliza para el transporte de la córnea. </li><li> Coloque el borde corneo-escleral donante a través de una cámara anterior artificial, en el que un líquido puede fluir y crear una presión variable; cubrir la córnea con la cabeza de retención del tejido. </li><li> Conectar la cámara anterior artificial (AAC) a través de un conector de 3 vías a una jeringa llena con la preservación córnea y líquido nutrición. </li><li> Utilice el anillo de compresión para sostener el retén en su lugar. </li><li> Utilice la syrInge conectado a la AAC para mantener la presión intracameral: llenar la cámara anterior con la solución de preservación del tejido hasta alcanzar la presión óptima (ver 1.7). Cierre el conector. </li><li> Prueba de la presión dentro de la cámara anterior con un dedo. Cambiar la presión en el interior hasta alcanzar la presión interna derecha (en el rango de 60-70 mmHg). Cierre el conector de 3 vías. </li><li> Mida el espesor de la córnea donante con coherencia óptica Thomography (OCT). Mantenga la cámara anterior con esa córnea en una posición fija en el frente de la óptica del instrumento y luego tomar una adquisición de espesor completo-octubre. </li></ol> 2. Preparación láser femtosegundo del endotelio de los donantes <ol><li> Realizar aplanación de la córnea del donante bajo el dispositivo de láser de femtosegundo. Utilice el láser femtosec para cortar el tejido del donante con tres cortes posteriores: corte lateral posterior, llena laminar y cortar lado anterior. Ajuste los siguientes parámetros para los tres cortes.<ol><li> Para el corte completo lamelar (patrón de trama, empezar), ajustar la profundidad de corte correspondiente al punto de la córnea donante (medido con la OCT como se describe en 1.8) más delgado, siempre restando 95 micras. Establecer la energía de pulso en el rango de 0,8-0,9 mu J (dependiendo de la profundidad de trabajo). Ajuste el diámetro de 8,7 mm. Ajuste la separación punto tangencial a 2 micras. Ajuste la separación radial al punto 2 micras. </li><li> Para el corte de cara anterior, establezca la profundidad posterior 30 m más profundo que el corte lamelar completa anterior. Establezca la energía de impulso a 2,10 mu J; el diámetro a 8,6 mm; la separación lugar a 3 m, mientras que la separación de la capa a 3 micras. </li><li> Para el corte lateral posterior, localice la profundidad anterior de la cara posterior cortó 30 micras en sentido anterior de corte lamelar completa. Ajuste la profundidad posterior a 900 micras. Establezca la energía de impulso a 2,10 mu J; el diámetro a 8,3 mm; la separación lugar a 2 micras; la separación de la capa a 2 micras. </li></ol></li&gt; </ol> 3. Destinatario Preparación de ojos <ol><li> Preparar al paciente para la cirugía. Hacer una incisión corneal 4.00 mm a las 12 horas con 4,0 mm hoja precalibrados. Hacer una paracentesis limbal con una cuchilla precalibrado 1,2 mm, colocado en dos. Haz otra paracentesis limbal a las 6 horas con un cuchillo puñalada 30 °. </li><li> Insertar un mantenedor de cámara anterior en la cámara anterior del paciente, a través de las 2 en punto paracentesis. </li><li> Lleve a cabo una de diámetro 8,2 mm descemetorexis circular con el gancho dedicado. </li><li> Pele la membrana de Descemet y el endotelio de la estroma posterior y quitar los tejidos con un gancho de Descemet. </li></ol> 4. Preparación cromóforo <ol><li> Ponga 1 mg de polvo verde de indocianina en un tubo de microcentrífuga de 1,5 ml. </li><li> Añadir 9 mg de agua estéril (la solución de agua ICG es 10% w / w). </li><li> Mezclar manualmente el polvo de ICG y el agua con un agitador de metal. </li&gt; </ol> 5. La tinción de endotelio del donante <ol><li> Ponga el endotelio del donante sobre la parte más amplia de una Busin-inyector, con el lado interior en contacto con la superficie del inyector. </li><li> Teñir el lado interior del endotelio del donante con la solución de cromóforo, en su parte periférica, usando una espátula. El tejido manchado correctamente tiene un color verdoso homogénea. Espere 3 minutos antes de iniciar el paso 5.3. </li><li> Tire del endotelio de los donantes en la parte anterior de la Busin-inyector, laminado. </li></ol> 6. Insertar el endotelio de los donantes <ol><li> Sujete e inserte el endotelio donante plegado usando un fórceps atraumáticos coaxiales para lentícula endotelial. </li><li> Quite el mantenedor de cámara anterior. </li><li> Suturar la incisión corneal y la paracentesis (realizada en el paso 3.1) con un solo Stich Nylon 10.0. </li><li> Inyectar una burbuja de aire para desplegar el tejido del donante y presionar contra la córnea receptora. La burbuja de aire debe compensarmevisi- bles a llenar el espacio de la cámara anterior. </li></ol> 7. Soldadura Láser <ol><li> Coloque la lentícula donante en el centro de la córnea del receptor, desde el interior con un gancho o mover la burbuja de aire desde el exterior de la córnea con una espátula. </li><li> Usa un láser de diodo que emite a 810 nm, equipado con un diámetro de 300 micras núcleo de la fibra óptica estéril, con 0,22 como una apertura numérica (NA). </li><li> Mantenga la punta de la fibra fuera del globo ocular, y entregar la luz láser hacia el endotelio manchado, a través del tejido de la córnea transparente. La punta de la fibra está en la configuración sin contacto. </li><li> Utilice los siguientes valores para el láser: modo de emisión solo punto, 70-80 duración del impulso ms, 35-40 mJ por impulso. He-Ne con el objetivo de haz en. </li><li> Entregar puntos de láser individuales en la periferia manchado del endotelio donante. Entregar los puntos secuencialmente: el aspecto final es un anillo de puntos en la periferia de la lentícula donante. La distancia entre dos adjacentro manchas ciento es el doble de un diámetro de punto. </li><li> Aplicar una lente de contacto en el ojo del paciente, junto con 0,3% de tobramicina y dexametasona 0,1% suspensión oftálmica. </li></ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, 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+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

El &quot;todo láser&quot; trasplante endotelial es un enfoque original de invasión mínima trasplante de córnea. Todos los procedimientos descritos en el protocolo se realizaron en la sala de cirugía, observando el procedimiento de higiene y esterilización que son prácticas comunes durante las cirugías, tales como el uso de esterilizar los guantes, bata, mascarilla y gorro. La solución de ICG se preparó de la sala de cirugía, poco antes de su aplicación en la tinción del endoteli...

En este trabajo se presenta un enfoque original de la queratoplastia endotelial, basado en el uso de un láser de femtosegundo para preparar tejido del donante y un láser de diodo de infrarrojo cercano para soldar sobre la cama destinatario. Medición intraoperatoria de la córnea donante es necesario diseñar correctamente las dimensiones del tejido donante. Queratoplastia endotelial se ha propuesto en los últimos años para sustituir a la queratoplastia penetrante en el tratamiento de la enfermedad de 1,2 endotelial. La principal ventaja de esta técnica es una recuperación más rápida visual, con respecto a la queratoplastia penetrante, la anestesia durante la cirugía reducido, una disminución del riesgo de rechazo del injerto y la preservación de la integridad de los ojos. El principal factor de riesgo es la luxación postoperatoria lentícula donante. La técnica estándar se realiza mediante la inserción del endotelio del donante en su posición final donde se mantiene mediante la inyección de una burbuja de aire: no hay suturas se utilizan debido a la chara mecánica, biofísica y dimensionalcteristics del endotelio. Por otra parte, la recuperación de la agudeza visual puede estar limitada principalmente a causa de una falta de coincidencia entre los tejidos del donante y del receptor debido a un tejido grueso trasplantado. Aquí presentamos un procedimiento en la realización de la queratoplastia endotelial que puede superar los principales problemas. El endotelio del donante se puede asegurar en su posición final mediante el uso de la técnica de soldadura láser. Este es un proceso fototérmico controlada y localizada: puede ser inducida en la interfase con el donante / receptor. Se ha estudiado en los últimos diez años y se propone en la queratoplastia penetrante y en el trasplante de endotelio 3-5. La luz casi infrarroja (longitud de onda: 810 nm) emitida por un láser de baja diodo de potencia se entrega hacia el tejido biológico en el sitio de la herida. La córnea es naturalmente transparente a esta longitud de onda: con el fin de hacer de este tejido para absorber la luz del láser, es necesario para teñir con un cromóforo. El tinte propuesto es una saturar estérild solución acuosa de indocianina verde (ICG). Hemos demostrado que cuando el tejido de la córnea se tiñe correctamente con esta preparación ICG, que muestra un pico de absorción a 810 nm 6. Además, el ICG se utiliza ampliamente en el diagnóstico clínico y su seguridad ha sido ya demostrado en sujetos humanos. La córnea manchado absorbe la energía de la luz láser de diodo y el efecto resultante principal es un aumento de la temperatura controlada en el lugar de soldadura. No hay efectos térmicos son inducidas en los tejidos sin teñir. La mejora de la temperatura induce la desnaturalización térmica reversible en el colágeno del estroma, con un closuring inmediata de las paredes de la herida tras el enfriamiento. Este efecto de la soldadura láser se demostró en primer lugar en la cirugía de cataratas 7,8 y queratoplastia penetrante 9,10. Un enfoque optimizado que presentamos en este trabajo se ha estudiado para su aplicación en la queratoplastia endotelial. En la cirugía propuesta, puntos de láser individuales (decenas duraderos de mseg) are entregado al tejido, resultando en un efecto fototérmica localizada dentro de la dimensión punto (unos pocos cientos de micras de diámetro): el efecto inducido es una soldadura láser duro, que consiste en una fotocoagulación del colágeno confinado en la interfase con el donante / host. El resultado de la desnaturalización del colágeno en el sitio soldada es una fuerte adhesión entre los tejidos del donante y del huésped, proporcionando así un efecto de sutura que es imposible de obtener con la técnica estándar (puntos). El tejido recupera su aspecto natural en un seguimiento corto (1 mes) y la adhesión entre tejidos de donantes / receptores se mejora la soldadura proporcionado en la etapa muy temprana de la fase de curación. Para evitar el otro riesgo principal de la queratoplastia endotelial, es decir, el trasplante de un tejido de donante de espesor, intraoperatorio tomografía de coherencia óptica (OCT) se utiliza: un dispositivo comercial mide el grosor de la córnea del donante, de modo que un perfil de corte correcta puede ser diseñado con laláser femtosec. Así pues, la propuesta de &quot;todo láser&quot; trasplante endotelial parece mejorar los resultados clínicos de este tipo de cirugía mínimamente invasiva.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pulsion Medical Systems, Germany</td>
			<td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td>
			<td>Alternative product: IC-GREEN, Akorn&#160; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td>
		</tr>
		<tr>
			<td>Femtosecond Laser&#160;</td>
			<td>Abbott Medical Optics,&#160; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td>
			<td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Coherence Tomography (OCT)</td>
			<td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td>
			<td>Visante</td>
			<td></td>
		</tr>
		<tr>
			<td>Diode Laser</td>
			<td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td>
			<td>Mod. WELD 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Anterior Chamber</td>
			<td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td>
			<td>&#160;Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_anterior_chamber.html)</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution for tissue preservation and nutrition&#160;&#160;</td>
			<td>&#160;AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&#242; - PD 
			&#160;ITALY</td>
			<td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&#176;-c-eb/carry-c-eb)</td>
			<td></td>
		</tr>
	</tbody>
</table>

an &#8220;all-laser&#8221; endothelial transplant

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

Se propone el procedimiento quirúrgico &quot;todo laser&quot; para realizar mínimamente invasiva trasplante de córnea. El procedimiento es fácil de realizar (véase la Figura 1): con respecto a un trasplante endotelial estándar sólo las etapas de medir el grosor corneal, manchando el tejido de donante y la entrega de la luz láser se añaden. Las ventajas conseguidas compensan en gran medida un mayor tiempo quirúrgico de unos pocos min. El uso de intraoperatoria octubre para medir el espesor de l...

Watch this Scientific Journal Video about An â€œAll-laserâ€ Endothelial Transplant at JoVE.com

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

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

Un &quot;All-láser&quot; Trasplante endotelial

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

Research

JoVE Journal

Medicine

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

9.3K vistas.  Italian National Research Council. An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

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

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando cuantitativa en tiempo real PCR para determinar quimerismo de células donantes en un modelo competitivo Trasplante de médula ósea murina

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Investigación

Medicina

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 ...

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

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

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

Técnica de Adquisiciones de hígado porcino y trasplante ortotópico usando un Active Porto-Caval derivación

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

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

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

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

Modelo de ratón de rechazo vascular inducida por aloinmune y Arteriosclerosis Trasplante

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

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 ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

Reacción de griess optimizada para UV-Vis y determinación a vista desnuda de primaquina antipalúdica

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lesión de isquemia-reperfusión de órganos mediante la simulación de cambios hemodinámicos en el modelo de trasplante de hígado de rata

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

TBase - an Integrated Electronic Health Record and Research Database for Kidney Transplant Recipients

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory values, medical reports, radiology and pathology data) via standardized interfaces with manual data entry during routine treatment (e.g., clinical notes, medication list, and transplantation data). By this means, a comprehensive database for KTR is created with benefits for routine clinical care and research. It enables both easy everyday clinical use and quick access for research questions with highest data quality. This is achieved by the concept of data validation in clinical routine in which clinical users and patients have to rely on correct data for treatment and medication plans and thereby validate and correct the clinical data in their daily practice. This EHR is tailored for the needs of transplant outpatient care and proved its clinical utility for more than 20 years at Charit&#233; - Universit&#228;tsmedizin Berlin. It facilitates efficient routine work with well-structured, comprehensive long-term data and allows their easy use for clinical research. To this point, its functionality covers automated transmission of routine data via standardized interfaces from different hospital information systems, availability of transplant-specific data, a medication list with an integrated check for drug-drug interactions, and semi-automated generation of medical reports among others. Key elements of the latest reengineering are a robust privacy-by-design concept, modularity, and hence portability into other clinical contexts as well as usability and platform independence enabled by HTML5 (Hypertext Markup Language) based responsive web design. This allows fast and easy scalability into other disease areas and other university hospitals. The comprehensive long-term datasets are the basis for the investigation of Machine Learning algorithms, and the modular structure allows to rapidly implement these into clinical care. Patient reported data and telemedicine services are integrated into TBase in order to meet future needs of the patients. These novel features aim to improve clinical care as well as to create new research options and therapeutic interventions.

TBase combines an electronic health record with an innovative research database for kidney transplant recipients. TBase is built upon an in-memory database platform, connected to different hospital systems, and used for regular outpatient care. It automatically integrates all relevant clinical data including transplantation-specific data creating a unique research database.

TBase - una base de datos integrada de investigación y registro electrónico de salud para receptores de trasplante de riñón

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Ensayo de transcitosis de células endoteliales como modelo in vitro para evaluar la permeabilidad de la barrera interna sangre-retina

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...