Este trabajo fue apoyado por la concesión de la base recibida del Departamento de biotecnología, gobierno de la India en el Instituto Nacional de Inmunología, en Nueva Delhi. Dirección actual del Dr. Bhattacharjee es la división de Gastroenterología, Hepatología y nutrición, Hospital Los Ángeles de los niños.

En este protocolo los procedimientos han sido aprobados por el Comité institucional de ética Animal del Instituto Nacional de Inmunología, en Nueva Delhi. El número de serie de referencia de la aprobación es AICE #319/13.
Nota: Hay excelentes recursos en procedimientos de cirugía general20 y protocolos específicos para roedores cirugía21. Para aquellos haciendo cirugía animal por primera vez, se recomienda ampliamente practicar procedimientos quirúrgicos en maniquíes antes de operar a los animales.
1. preparación
<ol><li>Antes del experimento, se mantenga estéril tamponada de fosfato salino (PBS) o solución salina preparada.</li>
	<li>Montar un kit de cirugía contiene tijeras, pinzas dentadas, pinzas de tejido, algodón, bastoncillos de algodón, hilos de nylon y portaagujas diferentes micro. Autoclave el kit de cirugía. Debe tener especial cuidado si un GUIÑO inmuno-comprometidos. Ratón SCID está incluido en el protocolo.</li>
	<li>Realizar el procedimiento experimental completo, desde la preparación hasta el final de la cirugía, en la bio-seguridad clase I gabinete.</li>
	<li>Pesar un GUIÑO. Ratón SCID de 6-8 semanas antes de la cirugía. Ratones que pesen entre 14-18 g son utilizados en este estudio.</li>
	<li>Afeitado de la región abdominal central e hipocondríaco superior del ratón con el recortador de vellos. Aplicar el cabello eliminación crema uniformemente en toda la región con una espátula para eliminar completamente el vello recortado. Quite los pelos con la ayuda de un trozo de algodón estéril húmedo después de 2-5 minutos.</li>
	<li>Colocar el ratón en la cámara de isoflurano y abrir la válvula de la botella de oxígeno. Mantener el flujo de oxígeno a razón de 4 L/min y la vaporización de isoflurano en el 4% para inducir anestesia.<ol>
<li>Asegúrese de que el ratón ha sido anestesiado correctamente pellizcando suave del dedo del pie.</li>
	</ol>
</li>
	<li>Coloque un tablero de cirugía dentro de una bioseguridad gabinete. Coloque el animal en el tablero de la cirugía, tal que la porción ventral del ratón es hacia arriba y la porción anterior del ratón se coloca dentro del cono de nariz conectado a la fuente de isoflurano y el oxígeno.</li>
	<li>Reducir la vaporización de isoflurano al 2% y mantener durante todo el procedimiento quirúrgico.</li>
	<li>Desinfectar la piel del ratón y esterilizar limpiándolo con algodón estéril empapado etanol al 70%.</li>
</ol>2. quirúrgico
<ol><li>
Hepatectomía parcial
	<ol>
<li>Realizar una incisión transversal de aproximadamente 1 cm en la piel justo debajo del esternón, perpendicular en el proceso de xiphoid y paralelo a la caja torácica, con la ayuda de tijeras de operación recta.</li>
		<li>Separe suavemente la piel atada con la capa del músculo abdominal en las cercanías de la zona Incisa con puntas de algodón estéril humedecido para distinguir entre la piel y la capa de músculo abdominal o fórceps. Empape la región intradérmica con PBS utilizando puntas de algodón estéril para evitar la desecación.</li>
		<li>Exponga el área del lóbulo izquierdo suavemente haciendo una incisión transversal a través de la capa peritoneal justo por debajo del xifoides. Utilizar dos puntas de algodón humedecido para exponer y levantar el lóbulo izquierdo del hígado.</li>
		<li>Una de las puntas de algodón en el lado abdominal del corte y otra punta de algodón en el lado del diafragma. Suavemente presione la punta a hacia el diafragma y dar un impulso corredero por el otro extremo para levantar el lóbulo izquierdo del hígado.</li>
		<li>Deslice un hilo de nylon con un bucle el lóbulo izquierdo levantado y deslice el lazo hacia la base del lóbulo izquierdo cerca del hilio con la ayuda de los consejos micro pinzas o algodón. Presione suavemente el bucle de hilo de nylon a la base del lóbulo izquierdo.</li>
		<li>Atar dos extremos del hilo de nylon en la parte superior del lóbulo izquierdo con un Portaagujas de microcirugía y las micro pinzas. Hacer dos nudos adicionales en el otro lado.</li>
		<li>Disecan hacia fuera del lóbulo atado con la ayuda de tijeras. No intente cortar muy cerca del hilo. En caso de que el procedimiento dura más de 5 min, mantener la cavidad peritoneal y órganos húmedos con PBS estéril para evitar la desecación debido a la pérdida de líquidos.</li>
		<li>Coser el peritoneo utilizando sutura continua sutura Catgut 4-0. Posteriormente, cerca de la piel por la sutura discontinua lo antes posible.</li>
	</ol>
</li>
	<li>
Trasplante de la célula Nota: Los hepatocitos expresan GFP fueron aislados de ratones transgénicos GFP (C57BL/6-Tg (UBC-GFP) 30 Scha/J), según el procedimiento descrito por Lee et al. 22 y Shen et al. 23 hepatocito-como las células (NeoHep) de origen humano diferenciado de monocitos24 también fueron utilizadas para el trasplante. Sin embargo, las células derivadas de otras fuentes pueden utilizarse también en el protocolo.<ol>
<li>Suspender las células viables 0,2 millones en alrededor de 50 μl de medio de modificado Dulbecco de Iscove (IMDM) y aspirar en una jeringa de insulina 1 mL con aguja de 30 G. Mantenga la jeringa fría puesta en hielo.</li>
		<li>Colocar el ratón de manera que la parte lateral izquierda se enfrenta hacia la persona que realiza la cirugía. Identificar el área esplénico y transversalmente disecar la piel cerca de la región hipocondríaco, seguida de una corta incisión a través de la capa peritoneal para exponer el bazo.</li>
		<li>Levante el bazo suavemente y mantener que fuera de la cavidad con la ayuda de dos PBS humedecido puntas de algodón.</li>
		<li>Sostenga el bazo cuidadosamente con las dos puntas de algodón en una mano y coloque la aguja de la jeringa vertical exactamente en el bazo. Perforar el bazo y empuje la aguja muy lentamente en el interior; la aguja no debe recibir más de 2 mm.</li>
		<li>Empuje el émbolo de la jeringa lentamente para inyectar las células en el bazo. Después del trasplante, mantener estable la aguja de la jeringa y retire lentamente desde el bazo para evitar sangrado o pérdida de las células.</li>
		<li>Después de colocar el bazo hacia la cavidad peritoneal con las puntas de algodón, cerca de la capa peritoneal continua de sutura con una sutura de Catgut 4-0. Coser la piel discontinuo con la misma sutura. Evitar el uso de clips de herida para el cierre de la piel; en cambio, cerrarla por una sutura de 4-0. Los clips de herida restringen el movimiento natural del ratón, y a menudo clips soltarse y salen rápidamente.</li>
	</ol>
</li>
</ol>3. postoperatorio cuidado
<ol><li>Tras el cierre de la piel, limpie los alrededores de los sitios de sutura con solución de yodo (betadine) usando una punta de algodón estéril.</li>
	<li>Inyectar una dosis de antibiótico cefotaxima como 600 mg/kg de peso (típicamente 12 mg cefotaxima en 100 μl de solución salina y el ratón) corporal por vía intraperitoneal utilizando jeringa de 1 mL.</li>
	<li>Dar dosis diarias de analgésico Meloxicam 1 mg/kg peso corporal (típicamente 12 μg Meloxicam en 100 μl de solución salina y el ratón) a los animales por vía intraperitoneal, hasta tres días después de la cirugía.</li>
	<li>Después de la terminación de la cirugía, detener el flujo de gas isoflurano y volver a colocar el ratón en la jaula individualmente ventilada.</li>
</ol>4. euthanization y caracterizaciones
<ol><li>Después de las variables experimentales (1 día y 10 días post cirugía), eutanasia a los ratones según las pautas del ética animal institucional.</li>
	<li>Recoger la sangre por hemorragia terminal los animales, pinchar el plexo terminal ocular.</li>
	<li>Aislar el suero de la sangre.</li>
</ol>

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Los autores no tienen nada que revelar.

Hepatectomía parcial es una técnica establecida para investigar la regeneración hepática y hepatectomía excesiva se divulga para imitar el modelo de AHF. Entre los modelos animales de AHF, roedores, especialmente ratones, son el modelo más investigado. Para obtener un modelo de daño hepático en ratones, hasta una hepatectomía del 70% se ha divulgado con una buena supervivencia tasa25,26. Sin embargo en nude y otro ratón inmunodeficiente, una hepatectom?...

Actualmente, trasplante de hepatocitos es propuesto como una alternativa al trasplante de órgano entero para tratar a los pacientes tener severos trastornos hepáticos. Se cree que puede superar a los pacientes de trasplante de órgano entero1. Además de los hepatocitos alogénicos2, hepatocitos xenogénica3 y hepatocitos derivados de las células4 también están siendo investigados en modelos animales. En este contexto, el engraftment y autoguiado hacia el blanco potencial de las células trasplantadas en el receptor es un criterio importante para la terapia celular basada en la insuficiencia hepática aguda (AHF).
Para investigando el trasplante de hepatocitos o de hepatocitos como las células5, AHF se crea en un modelo animal por quirúrgico6 o procedimientos farmacológicos7 , seguidos de transplante de células. Para hacer un modelo animal de la AHF por reactivos farmacológicos, muchas hepatotoxins como la d-galactosamina8, acetaminofén9, tetracloruro de carbono10, tioacetamida11, concanavalina A12, lipopolysaccharide13 , etcetera., se han utilizado. De esta lista, cada reactivo genera un conjunto de características únicas para AHF, pero lamentablemente no solo reactivo imita el humano AHF. Por otra parte, el AHF inducido por hepatotoxins lleva mucho tiempo, que pone a los animales bajo estrés crónico, y son difíciles de obtener resultados reproducibles.
Por otra parte, la intervención quirúrgica de hepatectomía parcial (PHx) es dependiente de la habilidad y la reproducibilidad es fácil de obtener después de desarrollar las habilidades necesarias. Para inducir AHF por solo la intervención quirúrgica, la resección de más del 70% del hígado se requiere; sin embargo, menos de una hepatectomía del 70% todavía puede ser utilizada para estudiar el engraftment y proliferación de trasplantar las células en el hígado para analizar su capacidad terapéutica en daño hepático14. El trasplante de hepatocitos han sido realizadas post hepatectomía por el peritoneo15, cola vena16, vena hepática17o el18del bazo. Actualmente, la infusión de la vena hepática y intrasplenic trasplante de hepatocitos son los procedimientos preferidos, ya que son fáciles de reproducir.
En este trabajo hemos descrito un procedimiento de una hepatectomía parcial del 30% en cabeceo. SCID (CABECEO. CB17-Prkdcscid/J) ratones en el que el lóbulo izquierdo del hígado es suprimido. Es seguida por el trasplante de hepatocitos de ratón (C57BL/6-Tg (UBC-GFP) 30Scha/J) expresando GFP 0,2 millones así como origen humano NeoHep19 en el bazo. Este procedimiento conduce al engraftment de células trasplantadas en el hígado. Este procedimiento es menos invasivo y una técnica mínimamente dolorosa.

<table border="0" cellpadding="0" cellspacing="0" width="1002">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Gas Anesthesia System</td>
			<td style="width:255px;">Ugo Basile; Italy</td>
			<td style="width:119px;">211000</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weighing machine</td>
			<td style="width:255px;">Goldtech ; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biological safety cabinet ( Class I)</td>
			<td style="width:255px;">Kartos international;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair Trimmer</td>
			<td>Panasonic ;&#160; Japan&#160;</td>
			<td style="width:119px;">ER-GY10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight operating scissor with sharp /sharp blades</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps with Serrations</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders&#160; straight &#38; curved&#160;</td>
			<td>Mercian ;&#160; England&#160;</td>
			<td style="width:119px;">BS-13-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml insulin syringe with 30G *5/16 needles&#160;</td>
			<td>Dispo Van; India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringe with 26 G * 1/2 needle</td>
			<td>BD ; US&#160;</td>
			<td>REF 303060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon Threads&#160;&#160;</td>
			<td style="width:255px;">Mighty ; India</td>
			<td style="width:119px;"></td>
			<td>(1-0) Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MERSUTURES 4-0 Sterilised Surgical Needled Suture</td>
			<td style="width:255px;">Ethicon, Johnson &#38; Johnson, India</td>
			<td style="width:119px;">NW 5047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRUGUT 76 cm 4-0 absorbable surgical suture</td>
			<td>Sutures India Pvt. Ltd; India</td>
			<td>SN 5048</td>
			<td>Sterilised Surgical Needled Suture Catgut Chromic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Buds</td>
			<td style="width:255px;">Pure Swabs Pvt Ltd ;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Tape</td>
			<td style="width:255px;">3M India ; India</td>
			<td style="width:119px;">1530-1</td>
			<td>Micropore Surgical Tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microtome</td>
			<td style="width:255px;">Histo-Line Laboratories, Italy</td>
			<td style="width:119px;">MRS3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shandon Cryotome E Cryostat</td>
			<td style="width:255px;">Thermo Electron Corporation ; US</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Confocal laser scanning microscope</td>
			<td style="width:255px;">Carl Zeiss ; Germany&#160;</td>
			<td style="width:119px;">LSM 510 META</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bright Field Microscope</td>
			<td style="width:255px;">Olympus, Japan</td>
			<td style="width:119px;">LX51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Automated analyser</td>
			<td>Tulip, Alto Santracruz, India</td>
			<td style="width:119px;">Screen Maaster 3000</td>
			<td>Biochemical analyser for liver functional test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Flow Cytometer</td>
			<td style="width:255px;">BD ; US&#160;</td>
			<td style="width:119px;">BD FACSverse</td>
			<td>Assesment of presence of cells post transplantation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veet hair removal cream&#160;</td>
			<td>Reckitt Benckiser , India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FORANE</td>
			<td style="width:255px;">Abbott ; US</td>
			<td style="width:119px;"></td>
			<td>isoflurane USP 99.9%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxim</td>
			<td style="width:255px;">AlKem ; India</td>
			<td style="width:119px;"></td>
			<td>cefotaxime sodium injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution&#160;</td>
			<td style="width:255px;">Win-Medicare;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Betadine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Paraformaldehyde</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">GRM 3660</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">12200-036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sucrose</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue-Tek</td>
			<td style="width:255px;">Sakura; US</td>
			<td>25608-930</td>
			<td>O.C.T compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAPI</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">MB 097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Albumin goat Polyclonal</td>
			<td style="width:255px;">Thermo Scientific,Pierce, US</td>
			<td style="width:119px;">PA126081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-connexin 32/GJB1 Polyclonal</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab64609-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">antiGFP rabbit polyclonal&#160;</td>
			<td style="width:255px;">Santa Cruz biotechnology; US</td>
			<td style="width:119px;">SC 8334</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 donkey anti-goat</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 488 donkey anti-sheep</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 chicken anti rabbit</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A21442</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Goat anti rabbit IgG HRP</td>
			<td style="width:255px;">Invitrogen, Thermo Fisher Scientific; US&#160;</td>
			<td>65-6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Ki67 antibody</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab15580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Antigen Unmasking Solution, Citric acid base</td>
			<td style="width:255px;">Vector laboratories, US</td>
			<td style="width:119px;">H-3300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ProLong Diamond antifade mountant</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">P36966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGOT (ASAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGPT (ALAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Alkaline Phosphatase Kit (DEA)</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hematoxylin Solution, Mayer&#39;s</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">MHS16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Eosin Y solution, alcoholic</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">HT110132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DPX Mountant&#160;</td>
			<td style="width:255px;">Sigma ; US</td>
			<td align="right" style="width:119px;">6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Melonex (Pain Killer)</td>
			<td style="width:255px;">Intas Pharmaceuticals Ltd; India</td>
			<td style="width:119px;"></td>
			<td>Meloxicam injection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAB enhanced liquid substrate system tetrahydrochloride</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">D3939</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice

Hepatectomía parcial es un método versátil y reproducible para el estudio de la regeneración hepática y el efecto de la terapéutica celular basado en diversas condiciones patológicas. Hepatectomía parcial también facilita el engraftment aumento y proliferación de las células trasplantadas por neovascularización aceleración y migración de células hacia el hígado. Aquí, describimos un protocolo simple para realizar 30% hepatectomía y trasplante de células en el bazo de un no obeso diabético grave había combinado NOD inmunodeficiente. SCID (CABECEO. CB17-Prkdcscid/j) ratón.
En este procedimiento, se realizan dos pequeñas incisiones. La primera incisión es para exponer y resecar el lóbulo izquierdo del hígado, y otra pequeña incisión para exponer el bazo para el intrasplenic trasplante de células. Este procedimiento no requiere ninguna habilidad quirúrgica especializada y puede realizarse en 5-7 minutos con menos estrés y dolor, una recuperación más rápida y mejor supervivencia. Hemos demostrado el trasplante de hepatocitos aislados de una proteína fluorescente verde (GFP) expresando su ratón (transgénicos C57BL/6-Tg (UBC-GFP) 30Scha/J), así como los hepatocitos como células de origen humano (NeoHep) en hepatectomized parcialmente NOD. Ratones de SCID.

Proliferación de hepatocitos después de hepatectomy parcial 30%: La proliferación de hepatocitos en el resto de hígado después de hepatectomy 30% fue examinada por inmunohistoquímica (IHC) para un marcador de proliferación celular, Ki-67. Un día post hepatectomía, los ratones fueron sacrificados, los lóbulos de hígado restantes fueron suprimidos y se obtuvieron secciones de la parafina. Las secciones fueron teñidas con anticuerpos Ki-67, seguido por etiquetado con rábano peroxidasa (HRP) con...

Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Hemos descrito un protocolo para la realización de hepatectomía parcial (PHx) y trasplante de la célula vía bazo en cabeceo. SCID (CABECEO. CB17-Prkdcscid/j) ratones. En este protocolo, se realiza una incisión para exponer y resecar el lóbulo izquierdo del hígado, seguido por otra incisión para el intrasplenic trasplante de células.

Intrasplenic trasplante de hepatocitos después de Hepatectomy parcial en cabeceo. Ratones de SCID

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

intrasplenic-transplantation-hepatocytes-after-partial-hepatectomy

Research

JoVE Journal

Medicine

13.9K vistas.  National Institute of Immunology. Hemos descrito un protocolo para la realización de hepatectomía parcial (PHx) y trasplante de la célula vía bazo en cabeceo. SCID (CABECEO. CB17-Prkdcscid/j) ratones. En este protocolo, se realiza una incisión para exponer y resecar el lóbulo izquierdo del hígado, seguido por otra incisión para el intrasplenic trasplante de células.

Video: Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

El trasplante de células del estroma mamario y células de carcinoma en los ratones C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Investigación

Medicina

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

El trasplante ortotópico aórtica en los ratones para el Estudio de la Enfermedad Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Procedimientos Quirúrgicos para un modelo de rata de trasplante hepático ortotópico parcial con reconstrucción arterial hepática

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotópico cervical trasplante cardiaco en ratones

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

La visualización del parénquima vascular y la regeneración después del 70% hepatectomía parcial en ratones normales

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Volver a arterializada rata parcial trasplante del hígado con una en vivo orientada al barco 70% hepatectomía

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

Trasplante de células madre derivadas de tejido adiposo hoja para reducir fugas después de colectomía parcial en un modelo de rata

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Terapia del Gene mediada por vectores lentivirales de hepatocitos Ex Vivo para trasplante autólogo en cerdos

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Generación de un modelo de rata de insuficiencia hepática aguda mediante la combinación de 70% de hepatectomía parcial y paracetamol

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

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 ...