The authors acknowledge all colleagues who made this work possible; Ann Demarr&#233;, Veerle Laurysens, Jan De Jonge and Erik Quartier for technical assistance. Financial support was from the VUB Research Council, the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), the Beta Cell Biology Consortium (BCBC), the Fund for Scientific Research Flanders (FWO), Diabetes Onderzoek Nederland (DON) and Interuniversity Attraction Pole (IAP).

Todas las manipulaciones siguen las directrices emitidas por el Convenio Europeo para la Protección de los animales vertebrados utilizados para experimentación y otros fines científicos (ETS 123 y y 2010/63 / UE). 1. Preparación del área de trabajo <ol><li> Proporcionar un área de preparación dedicado, un área quirúrgica y un área de recuperación. </li><li> Llevar a cabo todo el procedimiento quirúrgico en una cabina de flujo laminar para reducir al mínimo los contaminantes ambientales. </li><li> Montar los suministros (como se indica en Materiales y Métodos) necesarios en el área de preparación, quirúrgica y la recuperación, utilizando una técnica aséptica adecuada. </li><li> Asegúrese de que los instrumentos quirúrgicos se esterilizan en autoclave antes de la cirugía. </li><li> Proporcionar una almohadilla de calefacción de agua recirculante a una temperatura de 38 ° C para estabilización de la temperatura durante la cirugía. Cubra la almohada eléctrica con un protector impermeable estéril. </li><li> Proporcionar un microscopio con un aumento de al menos 6.3x. </li><li> Utilice un instrumento sterilizer, tal como un esterilizador de cuentas caliente, para esterilizar los instrumentos en entre procedimientos quirúrgicos. </li><li> Prepare un área de recuperación que consiste en una gran jaula, bordeado por las camas papel plano </li></ol> 2. Preparación de los animales para la cirugía <ol><li> Para PDL y cirugía simulada, utilizar 8 semanas de edad ratones machos, alojados en jaulas estándar y mantenidos en un 12 horas de luz / 12 h oscuridad ciclo y alimentados con una dieta ad libitum roedores estándar. Nota: Aquí, utilizamos ratones BALB / cJrJ pero también hemos utilizado con éxito otras cepas y varias cepas transgénicas. </li><li> Utilice buprenorfina como analgesia preventiva (desde 0,05 hasta 0,1 mg / kg) 30 min antes de la cirugía. </li><li> Anestesiar a los ratones por inyección intraperitoneal de 100 mg / kg de ketamina y de 5 - 10 mg / kg de xilazina. </li><li> Evaluar anestesia adecuada mediante la observación de pérdida gradual del movimiento voluntario y la relajación muscular. Prueba de la pérdida de los reflejos por pinzamiento toe. </li><li> Aplicar pomada oftálmica de prevsequedad ent de los ojos, mientras que bajo anestesia. </li></ol> 3. Preparación del sitio quirúrgico <ol><li> Desinfectar tórax y el abdomen con una solución de clorhexidina antiséptico. </li><li> Afeitarse un área de 2,5 cm x 1,5 cm del abdomen. </li><li> Desinfectar la zona afeitada con una gasa empapada con una solución de clorhexidina, a continuación, solución de alcohol y una aplicación final de la solución de clorhexidina. </li><li> Coloque el animal en el área quirúrgica para que el sitio quirúrgico preparado esté orientado hacia arriba el cirujano. </li><li> Coloque el ratón usando un paño quirúrgico a prueba de agua con una abertura que deja la región abdominal desinfectada expuesta al tiempo que cubre el resto del cuerpo para crear un campo de trabajo estéril. Monitorear el ratón antes del procedimiento para la profundidad de la anestesia. </li></ol> 4. Conducto pancreático Ligadura <ol><li> La laparotomía <ol><li> Hacer una incisión en la línea media superior en la piel que se extiende desde la apófisis xifoides hasta el ombligo usando un estérilcuchilla quirúrgica. Separar la línea alba subyacente y el peritoneo con unas tijeras estériles con el fin de exponer el cuadrante abdominal superior. </li><li> Prevenir desecación de los órganos internos por aspersión regular con cloruro de sodio estéril al 0,9%. </li><li> El uso de pinzas o hisopo estéril, retraer el estómago superiormente, exponiendo el bazo y el lóbulo esplénica (la región de la cola) del páncreas. </li><li> Retraer suavemente el duodeno y parte de la parte superior del yeyuno al cuadrante abdominal superior derecha para exponer la cabeza, el cuello y la región cuerpo del páncreas cubiertos por el peritoneo visceral. </li></ol></li><li> Ligadura <ol><li> Busque la posición anatómica del conducto pancreático principal en la región del cuello del páncreas. </li><li> Incisión del peritoneo visceral y el ligamento gastrocólico, la concesión del acceso al retroperitoneo, la exposición de la región del cuerpo y la cola del páncreas. Con el fin de exponer la región del cuello, realizar una maniobra de Kocher. Levante el duodeno y el jefe deel páncreas fuera del retroperitoneo elevándolos de la vena cava y la aorta por debajo inferior. </li><li> Coloque cuidadosamente la espátula debajo de la región del cuello. Ligar el conducto pancreático con 6-0 Prolene hilo en el lado izquierdo de la vena porta que separa los lóbulos gastro-duodenal y esplénicas. </li><li> Realizar una segunda ligadura en estrecha proximidad a la primera ligadura para asegurar que los lóbulos están separados adecuadamente. </li><li> Ligar con mucho cuidado a fin de no dañar los vasos sanguíneos subyacentes, a saber, la arteria pancreaticoduodenal superior, la arteria pancreaticoduodenal inferior y la parte de páncreas de la arteria esplénica. </li><li> Coloca los órganos en la cavidad abdominal. </li><li> Cierre la incisión utilizando 4-0 poliglicol hilo filamentoso en un patrón de sutura continuo para la / capa peritoneal muscular y en un patrón de sutura discontinua para la piel. </li></ol></li></ol> 5. Sham Cirugía <ol><li> Realice todos los pasos como dedescribe en los pasos 1 a 4.1.4. Mientras que el páncreas está expuesta, no realice la ligadura del conducto pancreático. </li><li> Al final de la etapa 4.1.4, colocar los órganos internos en la cavidad abdominal. </li><li> Cierre de la incisión como se describe en 4.2.7. </li></ol> Cuidado 6. Post-operativa y Vigilancia <ol><li> Después de la intervención quirúrgica es completa, coloque el ratón en el área de recuperación. Este consiste en una jaula colocada sobre una almohadilla térmica y forrado con ropa de cama de papel plano con el fin de mantener la temperatura corporal normal. </li><li> Brindar apoyo nutricional para evitar la hipoglucemia post-operatorio por el alimento humedecido colocado en la parte inferior de la jaula. Para este propósito, utilizar gránulos de dieta estándar para roedores remojadas en agua hasta que se ablanden. </li><li> Proporcionar apoyo fluido por el alimento humedecido y proporcionar agua a voluntad. </li><li> Utilice buprenorfina como la analgesia (0,05-0,1 mg / kg) dos veces al día durante 2 días después de la cirugía. Durante todo el experimental, el seguimiento, observe los ratones durante la ocurrencia de posibles signos de infección, incluyendo la secreción de líquido o pus de la herida, o por el deterioro físico que se caracteriza por la reducción en el comportamiento de acicalamiento y nivel de actividad, menor apetito y pérdida de peso corporal. </li></ol> 7. Evaluación del éxito PDL y Cosecha de PDL cola y Sham cola de páncreas <ol><li> La eutanasia a los ratones por dislocación cervical. </li><li> Vuelva a afeitarse la región abdominal para evitar el arrastre de la piel. </li><li> Abra la capa de la piel y el músculo abdominal y eliminar una gran área de piel y músculo para obtener un buen acceso al páncreas. </li><li> El uso de pinzas o hisopo estéril, el estómago se retrae superiormente, exponiendo el bazo y el lóbulo esplénica (la región de la cola) del páncreas. </li><li> Retire con cuidado el duodeno y parte del yeyuno superior para exponer el lóbulo gastroduodenal (región de la cabeza) del páncreas. Nota: La porción ligada del páncreas PDL ahora se ha reducido en tamaño y se ha convertido en casitraslúcido para que los islotes son visibles como pequeños puntos blancos. La parte de la cabeza del páncreas es de color rosa opaco y distinta exocrina lobuli se puede observar. </li><li> Usando tijeras estériles, separar la parte de la cola del páncreas se ligó a partir del bazo, cortando a lo largo del bazo. Cortar perder el tejido conectivo que conecta la región de la cola del páncreas a los órganos internos. </li><li> Cortar la región de la cola PDL justo en frente de la ligación, con exclusión de la ligadura y el tejido inmediatamente adyacente a la misma desde el tejido cosechado. </li><li> Para aislar el tejido cola farsa, siga los pasos 7.1 a 7.5. Usando tijeras estériles separan la región de la cola del páncreas farsa del bazo por el corte a lo largo del bazo. Aislar la región de la cola por el corte en la región del cuello del páncreas y el corte soltar el tejido conectivo que conecta el páncreas cola a los órganos internos Nota: Tanto la cola y región de la cabeza del páncreas simulados son de color rosa opaco, para aislar las dos partes por separado,cortar el páncreas en la región del cuello. </li></ol>

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The authors have nothing to disclose.

In the present study, we describe in detail the methodology behind PDL, a mouse injury model to study beta cell neogenesis and proliferation and transdifferentiation of pancreatic non-beta cells. Ambiguity in data on PDL among labs stimulates the need for a standardized protocol for PDL surgery.
Critical steps in the PDL protocol include the selection of healthy, young mice for surgery. Preferably male mice should be used since unpublished data from our lab suggest that estrogen receptor signa...

El aumento de la prevalencia de la diabetes, que afecta a más de 300 millones de personas en todo el mundo 1,2 ha impulsado la búsqueda de nuevas fuentes de células beta productoras de insulina, tanto in vitro como in vivo, para reponer la masa de células beta deficiente. 3 Identificar clave mecanismos y factores que regulan la proliferación de células beta y beta neogénesis de células, es decir, la diferenciación de las células beta a partir de una célula o células progenitoras no beta, pueden proporcionar nuevas dianas para el desarrollo de terapias regenerativas en la diabetes. En el páncreas de roedores en desarrollo, todos los tipos de células endocrinas diferenciar de una población transitoria de células progenitoras endocrino, que expresa el factor de transcripción Neurogenin3 (Ngn3) 4,5. En el páncreas roedor adulto, en condiciones fisiológicas normales, la masa de células beta es se mantiene a un número óptimo para satisfacer las demandas metabólicas. Los cambios en el tamaño de las células beta,apoptosis y la replicación constituyen los principales mecanismos para la expansión de células beta y turn-over. 6-8 Mientras que el potencial de las células beta a proliferar en condiciones fisiológicas normales es homogénea en toda la población, 9 su tasa de proliferación es baja y re-replicación está restringida por un período dinámico de quiescencia o período refractario 6,7, influenciado por la edad y el metabolismo de la glucosa. 10 Dado que las células progenitoras endocrinas hasta ahora no han sido identificados en el páncreas adulto normal, se cree neogénesis de no contribuir al crecimiento normal de las células beta del adulto. 8 Por lo tanto, la identificación de una célula progenitora endocrino facultativa en el páncreas adulto que es expandible y capaz de producir nuevas células beta podría proporcionar una novela, posiblemente fuente ilimitada de las células beta. Parcial ligadura del conducto (PDL) es un modelo de lesión de animal que ha sido descrito para inducir neogenesi célula betas en el páncreas adulto. 11,12 En este modelo, el conducto pancreático principal drenaje de la cola del páncreas se une quirúrgicamente. La obstrucción resultante de drenaje exocrino induce importante remodelación del tejido, acompañado por la inflamación y atrofia acinar distal a la ligadura. 12-14 Dentro de este entorno inflamatorio, re-expresión del marcador progenitor endocrino Ngn3 se induce y el volumen de las células beta aumenta de dos veces . Esta duplicación en beta resultados del volumen celular de la generación de nuevas células beta a partir de un Ngn3 expresan de tipo embrionario de células progenitoras endocrino y de la proliferación de las células beta pre-existentes y recién formados que son propensos a volver a la duplicación sin &quot;período de inactividad&quot;. 11,15 Neogénesis de células beta y replicación en modelos de lesión, tales como la pancreatectomía 6,7,16-19 y la ablación selectiva de las células beta 20 se han descrito ampliamente. Sin embargo, el resultado regenerativa en thesmodelos e está influenciada por la magnitud de los daños causados ​​y se asocia con una masa de células beta inicial disminuido 21. PDL es un modelo quirúrgico en el que no se ve afectada la masa inicial de las células beta y la neogénesis de células beta y la proliferación se activan robusta. De hecho, en el páncreas de ratones que se sometieron a PDL, las células que expresan Ngn3 se identifican cerca del revestimiento epitelial del conducto. Estas células se pueden aislar a partir de los páncreas de ratones transgénicos ligados Ngn3-GFP usando células activadas por fluorescencia (FACS) y son capaces de diferenciarse hacia células beta funcionales siguiente en el injerto y la cultura ex vivo del páncreas de E12.5 Ngn3 - / - . ratones 11 Del mismo modo, en Ngn3 CreERT;. ratones R26 YFP en el que las células que activan el gen Ngn3 están etiquetados de forma permanente después de la inyección tamoxifeno, las células derivadas de células beta Ngn3 etiquetas positivas se detectan después de PDL 15 Por otra parte, las células beta recién formadas diluir pre -existing células beta y preferentemente localizar en pequeños islotes en el que las células beta muestran un alto potencial de proliferación. 15 Ngn3 es importante para la expansión de las células beta después de PDL ya la disminución de la expresión Ngn3 usando específica de la diana de ARN corto horquilla disminuye significativamente la masa de células beta y la proliferación de las células beta después PDL. 11 En particular, la fracción de células derivadas de células beta Ngn3 y la masa de células beta después de PDL depende críticamente del nivel de Ngn3 inducción 15. Esto está de acuerdo con la observación de que el alto nivel de expresión Ngn3 es un paso fundamental para el compromiso endocrina del páncreas progenitores multipotentes durante el desarrollo de páncreas 22 Además, la ablación selectiva de células que expresan Ngn3 por la administración de la difteria-toxina para Ngn3 CreERT;. Ratones R26 IDTR resultados en el contenido de insulina disminuida y la proliferación de células beta reducido, especialmente en pequeños islotes. 15 <pclass = &quot;jove_content&quot;&gt; Aunque la inducción de la expresión Ngn3 en células de los conductos después de PDL ha sido confirmado por muchos 11,15,16,23,24, Ngn3 expresión en células de islotes 24,25 y discrepancias en el resultado de PDL desafió nuestras observaciones iniciales del aumento de la masa de células beta 26,27, aparición de Ngn3 expresando progenitores conducto derivado endocrinas 24,26,28,29, y aumento de la proliferación de las células beta 27 después de PDL. 30 Estos resultados contradictorios podrían, al menos en parte, atribuirse a una combinación de factores, incluyendo variaciones en los puntos de tiempo post-quirúrgicas de análisis, el peso corporal, el sexo y la edad de los ratones, condiciones fisiológicas y ambientales postoperatorias y, lo más importante, diferencias en la técnica quirúrgica. 30 En nuestras manos, la proliferación de células beta, contenido de insulina, el volumen de las células beta y el número de pequeños islotes se incrementan constantemente después de PDL. TambiénNgn3 ARNm aumenta constantemente, pero hay grandes diferencias en la expresión de ARNm Ngn3 entre PDL páncreas cola, para los que no tenemos explicación directa. La hipótesis de que el nivel de ARNm Ngn3 podría correlacionar con el grado de neogénesis de células beta a partir de células no beta 15, pero esto requiere de mayor fundamentación. A pesar de que no elimina todas las variaciones experimentales, un método estandarizado para la realización de la cirugía PDL permite una mejor uniformidad en los resultados y abre nuevas vías en el estudio de la proliferación de células beta y neogénesis.

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			<td>Hoechst</td>
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surgical injury to the mouse pancreas through ligation of the pancreatic duct as a model for endocrine and exocrine reprogramming and proliferation

Expansión de las células beta pancreáticas in vivo o ex vivo, o la generación de las células beta por la diferenciación de una célula madre embrionaria o un adulto, puede proporcionar nuevas fuentes expandibles de las células beta para aliviar la escasez de donantes en el trasplante de islotes humanos como terapia para la diabetes. Aunque los avances recientes se han hecho hacia este objetivo, los mecanismos que regulan la expansión de las células beta y la diferenciación de una célula madre / progenitoras aún no se han caracterizado. Aquí, se describe un protocolo para un modelo de lesión en el páncreas de ratón adulto que puede funcionar como una herramienta para estudiar los mecanismos de la remodelación tisular y proliferación de las células beta y la diferenciación. Parcial ligadura del conducto (PDL) es una lesión inducida experimentalmente del páncreas roedor que implica la ligadura quirúrgica del conducto pancreático principal que resulta en una obstrucción del drenaje de los productos exocrinas fuera de la región de la cola del páncreas. El daño infligido induce atrofia acinar, infilt célula inmuneración y la remodelación tisular grave. Hemos informado anteriormente de la activación de Neurogenina (NGN) 3 que expresan las células progenitoras como endógenos y un aumento en la proliferación de las células beta después de PDL. Por lo tanto, PDL proporciona una base para estudiar señales implicadas en la dinámica de las células beta y las propiedades de un progenitor endocrino en páncreas adulto. Dado que, todavía sigue siendo en gran medida poco clara, que los factores y las vías contribuyen a la neogénesis de células beta y la proliferación en PDL, un protocolo estandarizado para PDL permitirá la comparación entre laboratorios.

PDL induce atrofia acinar y la inflamación, pero no afecta el peso corporal y la glucemia En 8 semanas de edad BALB / c ratones, el conducto de drenaje de las enzimas exocrinas de la cola del páncreas se liga mientras que la cabeza del órgano, situado adyacente al estómago y el duodeno, no se ve afectado. Edad, sexo y machos BALB / c ratones con peso se someten a cirugía simulada recapitular todas las medidas de la cirugía parcial ligadura del conducto, a excepción de...

Watch this Scientific Journal Video about Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation at JoVE.co

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

Lesión quirúrgica del páncreas del ratón a través de la ligadura del conducto pancreático como un modelo para el endocrino y exocrino Reprogramación y proliferación

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

surgical-injury-to-mouse-pancreas-through-ligation-pancreatic-duct-as

Research

JoVE Journal

Medicine

16.0K vistas.  Vrije Universiteit Brussel. This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation. 

Video: Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Un modelo murino de isquemia-reperfusión miocárdica lesión a través de la ligadura de la arteria descendente anterior

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Investigación

Medicina

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

Ligadura del conducto biliar en los ratones: Inducción de la injuria hepática inflamatoria y fibrosis por colestasis obstructiva

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

Infusión del conducto pancreático: un método eficaz y selectivo de administración de fármacos y virus

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Disección del páncreas de ratón para análisis histológico y perfiles metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Pancreaticojejunostomía lateral robótica para la pancreatitis crónica

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Pancreatectomía Izquierda Radical Laparoscópica para Cáncer de Páncreas: Estrategia Quirúrgica y Video Técnica

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Un modelo de trasplante de pulmón de rata de lesión por isquemia/reperfusión caliente: optimizaciones para mejorar los resultados

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Visualización longitudinal estabilizada a nivel celular in vivo del páncreas en un modelo murino con una ventana de imágenes intravitales pancreáticas

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

Establecimiento de un modelo de pancreatitis aguda grave en ratones mediante inyección retrógrada de taurocolato de sodio en el conducto biliopancreático

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Ligadura parcial del nervio ciático: un modelo de ratón de dolor neuropático crónico para estudiar el efecto antinociceptivo de nuevas terapias

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...