Los autores desean dar las gracias a Benjamin J Weigler, D.V.M., pH.d. y Walt Mandeville, D.V.M. (recursos animales y Campus asistir a médico veterinario, Universidad de Nevada, Reno) por su excelente servicio de animal suministrado a los ratones, así como su asesoramiento en procedimientos quirúrgicos.

El siguiente protocolo ha sido aprobado por el institucional cuidado Animal y el Comité uso (IACUC) en la Universidad de Nevada Reno (UNR) los recursos y cumple con todas las éticas con respecto al uso de animales de investigación.
1. los animales.
<ol><li>Obtener ratones C57BL/6 de maduro (4-6 semanas de edad) entre 20-30 g. casa de la colonia de ratones de laboratorio en un centro de animales centralizado en UNR los recursos.</li>
</ol>2. parcial obstrucción cirugía
Nota: Las cirugías se realizan en una sala dedicada a procedimientos quirúrgicos. Todos los instrumentos quirúrgicos son autoclave antes de la cirugía. Guantes y batas quirúrgicas estériles deben llevarse por todo el personal en la sala quirúrgica en todo momento.
<ol><li>Preparación para el ratón quirúrgico<ol>
<li>Compruebe el sistema de anestesia para asegurar el suministro de oxígeno e isoflurano es adecuada para el procedimiento. Encienda el suministro de oxígeno. Encienda el medidor de caudal de gas y ajustar a 500-1000 mL/min lugar el animal en la sala de inducción y la parte superior del sello.</li>
		<li>Encienda el vaporizador de isoflurano al 5% y controlar al animal hasta que se convierte en decúbito. Cambiar el sistema de anestesia para el cono de nariz.</li>
		<li>Limpie la cámara de inducción de cualquier resto de gas con oxígeno, luego abra la línea de la cámara de inducción manteniendo la línea del cono de nariz.</li>
		<li>Retire el animal de la cámara y coloque cuidadosamente la pomada oftálmica en los ojos del animal.</li>
		<li>Coloque el cono de nariz en una almohadilla caliente precalentada cuando gas sigue fluyendo.</li>
		<li>Cambiar el flujo de oxígeno a 100-200 mL/min, con 2-3% de isoflurano. Si el animal empieza a moverse, gentlyrestrain el animal con el cono de nariz en hasta completamente anestesiados otra vez.</li>
		<li>Controlar la respiración y la respuesta a la estimulación durante el procedimiento y ajuste el porcentaje de isoflurano (2-5%) según sea necesario. Nivel de anestesia animal es supervisado por la falta de reflejo de pellizco del dedo del pie antes de realiza la cirugía.</li>
		<li>Inyecte analgésicos (buprenorfina, 1 μg/g de peso corporal) por vía intraperitoneal de la zona de la incisión.</li>
		<li>Aplicar loción de eliminación de pelo en el abdomen utilizando un hisopo de algodón limpio. Que la loción se siente durante 3-5 min en ratón, luego eliminar el vello utilizando hisopos de Gasa y algodón. Repita este paso hasta que se ha eliminado todo el pelo del abdomen del ratón.</li>
		<li>Limpiar la piel con etanol al 70% usando torundas de Gasa y algodón. Aplique solución swabsticks o povidona yodada para limpiar el abdomen.</li>
	</ol>
</li>
	<li>Cirugía de la obstrucción parcial<ol>
<li>Cubra el sitio quirúrgico con papel estéril 25 x 50 cm con 2,5 x 2,5 cm en el centro para el área de cirugía. Asegure el cobertor al animal colocando tiras estériles en los límites de la apertura y la piel.</li>
		<li>Hacer una incisión abdominal de cm ~3.0 longitudinalmente con bisturí hoja nº 15, asegurando que sólo la piel es incidida y evitar el corte en la capa de musculoperitoneal en este momento.</li>
		<li>Utilizando pinzas y tijeras quirúrgicas, cuidadosamente separe la piel de la capa de musculoperitoneal sin causar ninguna incisión a la capa de musculoperitoneal. Después de que las capas se han separado lo suficiente (aproximadamente 1 cm x 4 cm), identificar la linea alba en la capa de musculoperitoneal y corte ~ 2 cm a lo largo de la linea alba para exponer la cavidad intraperitoneal utilizando micro-pinzas y tijeras.</li>
		<li>Cuidadosamente Localice e identifique el ciego. Despacio y con cuidado retire el ciego de la cavidad intraperitoneal con micro-pinzas, trayendo el colon proximal y el ileon con el ciego fuera sobre el cobertor esterilizado. Inmediatamente humedecer el tejido intestinal con una gasa empapada salina estéril 0,9% y mantener tejidos expuestos humedecido en todo momento mientras estén fuera de la cavidad abdominal.</li>
		<li>Localice e identifique el mesenterio entre el íleon y el colon proximal. Hacer una incisión (~ 1 cm) paralela a y justo debajo, el íleo, en el mesentery y evite cortar cualquier vasculatura.</li>
		<li>Toma un anillo de silicona autoclave (6 mm de longitud, 4 mm de diámetro exterior, diámetro interior de 3,5 mm). Cortar longitudinalmente para abrir el tubo y abrir el anillo con micro pinzas.</li>
		<li>Inserte un extremo del anillo abierto a través de la incisión en el tejido del mesenterio. Devolver el anillo a una forma de anillo terminado poniendo un extremo en contacto con otro, con el íleon rodeado por el anillo.</li>
		<li>Asegurar que el anillo de silicona rodea completamente el íleo y cerrar el anillo con la sutura cuidadosamente vuelva a colocar el intestino en la cavidad intraperitoneal.</li>
	</ol>
</li>
	<li>Cierre de la cirugía<ol>
<li>Realizar una simple sutura continua en la capa de musculoperitoneal a lo largo de la linea alba para cerrar el musculoperitoneal la herida con una sutura absorbible. Una vez finalizada la sutura, limpiar cualquier sangrado con una gasa empapada salina estéril 0,9%.</li>
		<li>Para cerrar completamente la herida con una sutura de nylon separadas, realizar una sutura continua simple en la piel.</li>
		<li>Ambas suturas cuando hayan terminado, limpie la herida con nuevo swabstick o povidona yodada.</li>
		<li>Inyectar por vía intraperitoneal antibióticos (gentamicina, 150 μL por ratón basado en el peso corporal de 20-30 g).</li>
		<li>Después de la terminación del procedimiento, apagar el vaporizador de isoflurano y dejar que el animal respirar sólo fluye oxígeno hasta que empezar ganando consciencia.</li>
		<li>Una vez que el animal está despierto, colocar al animal en un área de recuperación separado con soporte térmico hasta que se recuperó completamente.</li>
	</ol>
</li>
</ol>3. posterior a la operación observación.
<ol><li>Después de la terminación de la cirugía, mueva los animales a una incubadora en una sala de recuperación donde se regulación la temperatura y la humedad. Monitorear los animales después de la operación cada 15 minutos durante la primera hora después cada 30 min para la segunda hora mientras los animales están en la incubadora.</li>
	<li>Tan pronto como la observación prescrita esté completa, mueva los animales a su jaula individual y controlarlos diariamente para indicaciones clínicas del dolor18y para asegurar que la herida está sanando correctamente sin signos de complicaciones ( dehiscencia) presente.</li>
</ol>

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<li>Liu, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Voltage+dependent+potassium+channel+remodeling+in+murine+intestinal+smooth+muscle+hypertrophy+induced+by+partial+obstruction%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.</a> PLoS One. 9 (2), e86109(2014).</li>
<li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Down-regulation+of+hydrogen+sulfide+biosynthesis+accompanies+murine+interstitial+cells+of+Cajal+dysfunction+in+partial+ileal+obstruction%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Down-regulation of hydrogen sulfide biosynthesis accompanies murine interstitial cells of Cajal dysfunction in partial ileal obstruction.</a> PLoS One. 7, e48249(2012).</li>
<li>Yang, J., Zhao, J., Chen, P., Nakaguchi, T., Grundy, D., Gregersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interdependency+between+mechanical+parameters+and+afferent+nerve+discharge+in+hypertrophic+intestine+of+rats%5BTitle%5D)+AND+%22Am+J+Physiol-Gastr+L%22%5BJournal%5D)">Interdependency between mechanical parameters and afferent nerve discharge in hypertrophic intestine of rats.</a> Am J Physiol-Gastr L. 310, G376-G386 (2016).</li>
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<li>Bowen, E. J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22129371">Duodenal Brunner's glade adenoma causing chronic small intestinal obstruction in a dog.</a> J Small Anim Pract. 53, 136-139 (2012).</li>
<li>Bettini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypertrophy+of+intestinal+smooth+muscle+in+cats%5BTitle%5D)+AND+%22Res+Vet+Sci%22%5BJournal%5D)">Hypertrophy of intestinal smooth muscle in cats.</a> Res Vet Sci. 75, 43-53 (2003).</li>
<li>Macdonald, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Smooth+muscle+phenotypic+plasticity+in+mechanical+obstruction+of+the+small+intestine%5BTitle%5D)+AND+%22J+Neurogastroenterol+Motil%22%5BJournal%5D)">Smooth muscle phenotypic plasticity in mechanical obstruction of the small intestine.</a> J Neurogastroenterol Motil. 20, 737-740 (2008).</li>
<li>Ha, S. E., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/28806761">Transcriptome analysis of PDGFRα+ Cells identifies T-types Ca2+ channel CACNA1G as a new pathological marker for PDGFRα+ cell hyperplasia.</a> PLoS One. 12, e0182265(2017).</li>
<li>Park, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum+response+factor+is+essential+for+prenatal+gastrointestinal+smooth+muscle+development+and+maintenance+of+differentiated+phenotype%5BTitle%5D)+AND+%22J+Neurogastroenterol+Motil%22%5BJournal%5D)">Serum response factor is essential for prenatal gastrointestinal smooth muscle development and maintenance of differentiated phenotype.</a> J Neurogastroenterol Motil. 21, 589-602 (2015).</li>
<li>Spencer, N. J., Sanders, K. M., Smith, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Migrating+motor+complexes+do+not+require+electrical+slow+waves+in+the+mouse+small+intestine%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Migrating motor complexes do not require electrical slow waves in the mouse small intestine.</a> J Physiol. 553, 881-893 (2003).</li>
<li>Langford, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Coding of facial expressions of pain in the laboratory mouse.</a> Nat Methods. 7, 447-449 (2010).</li>
<li>Terez, S. D., Notari, L., Sun, R., Zhao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+smooth+muscle+responses+to+inflammation%5BTitle%5D)+AND+%22Neurogastroenterol+Motil%22%5BJournal%5D)">Mechanisms of smooth muscle responses to inflammation.</a> Neurogastroenterol Motil. 24, 802-811 (2012).</li>
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<li>Huizinga, J. D., Chen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interstitial+Cells+of+Cajal%3A+Update+on+Basic+and+Clinical+Science%5BTitle%5D)+AND+%22Curr+Gastroenterol+Rep%22%5BJournal%5D)">Interstitial Cells of Cajal: Update on Basic and Clinical Science.</a> Curr Gastroenterol Rep. 16, 363(2014).</li>
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</ol>

Los autores no tienen nada que revelar.

Hemos demostrado que ratones recibiendo la cirugía PO intestinal consistente y reproducible desarrollan hipertrofia del músculo liso intestinal, que imita a la obstrucción intestinal humana. Cirugía de obstrucción intestinal ha sido desarrollado para diferentes animales, incluyendo ratones7,10de ratas, conejillos de Indias11, perro12 y gatos13. El modelo de ratón de la obstrucción intestinal tien...

Obstrucciones intestinales son una obstrucción parcial o completa en el intestino pequeño o grande que impide que alimentos digeridos, líquidos y gases en movimiento a través de los intestinos1. Debido a la obstrucción, la obstrucción induce las paredes intestinales para ser espesada, estrechamiento de la luz2. La obstrucción intestinal puede ocurrir como resultado de cirugías abdominales o pélvicas que causan formación de tejido de adherencias abdominales o de trastornos GI como enfermedades inflamatorias del intestino (enfermedad de Crohn), diverticulitis, hernia, vólvulo, estenosis, invaginación intestinal, estreñimiento, impactación fecal, seudo-obstrucción, tumores y cánceres de3,4,5. Obstrucciones intestinales en estos casos a menudo conducen a la hipertrofia de los muscularis de tunica de los intestino6.
PO de la luz induce distensión intestinal y aumenta el espesor de la capa de músculo liso alrededor de la obstrucción en respuesta a la necesidad de seguir peristaltismo funcional7,8,9,10, 11,12,13. Modelos animales de PO intestinal se han desarrollado para estudiar la hipertrofia del músculo liso en ratones7ratas10, conejillos de Indias11, perros12y gatos13 que constantemente desarrollan hipertrofia similar dentro de la capas del músculo intestinal.
Un modelo de ratón de PO intestinal es la manera más rentable para generar y estudiar obstrucciones intestinales en vivo. Obstrucciones de intestino delgado se llevan a cabo en ratones mediante el uso de un anillo de silicona que se coloca quirúrgicamente alrededor del íleo. El PO los ratones mostraron un incremento temprano en el número de células (hiperplasia) y un aumento en espesor de la capa del músculo (hipertrofia) después PO cirugía8,15. SMC son las principales células plásticas que están creciendo dentro de las capas musculares lisas en respuesta a las condiciones hipertrófica14, sino otras células como los ICC PDGFRα+ células y que están estrechamente asociados con SMC, también se repobló. Hemos divulgado previamente que los ratones PO desarrollan hipertrofia en el intestino delgado, en que SMC son dedifferentiated en células PDGFRα+ que son altamente proliferativas7,15,16. Por el contrario, ICC se degeneró y perdió dentro de las capas de músculo liso hipertrofiado durante el desarrollo de obsruction intestinal7. Otro beneficio importante del modelo PO es su capacidad para inducir cambios en el sistema nervioso entérico y propagación de patrones motores neurogénicos. La importante propagación de patrón motor neurogénica en el intestino de ratón es el migrar motor complejo (MMC), que es neurogénica y no requiere de ICC o de las ondas eléctricas lentas17. El modelo PO puede proporcionar penetraciones claras en cómo MMCs y los nervios entéricos son remodelados por obstrucción parcial.
Aquí, proponemos un protocolo murino para la cirugía PO intestinal usando un anillo de silicona. Ratones que recibieron cirugía PO confiablemente producen hipertrofia en las muscularis de tunica del intestino. Dentro de músculo hipertrófico, SMC, ICC, PDGFRα+y células neuronales son remodeladas dramáticamente.

<table><tbody><tr><td>Surgical drape</td><td>Medical and veterinary supplies</td><td>SMS40</td><td>40&amp;rdquo;X100 yards</td></tr><tr><td>Underpad, econ, pro plus</td><td>Medical and veterinary supplies</td><td>MSC281224</td><td>17x24&amp;rdquo;</td></tr><tr><td>Iris scissors</td><td>Braintree scientific, Inc</td><td>SC-i-130</td><td></td></tr><tr><td>Iris scissors</td><td>Vantage</td><td>V95-304</td><td></td></tr><tr><td>Dumont electronic &amp;amp; jeweler tweezers</td><td>Dumont</td><td>98-180-3</td><td></td></tr><tr><td>Braided absorbable suture</td><td>Covidien polysorb</td><td>SL-5687G</td><td>5-0, polyglactin</td></tr><tr><td>Nylon non-absorbable mono filament</td><td>AD surgical</td><td>S-N618R13</td><td>6-0, nylon</td></tr><tr><td>Surgical blade</td><td>Dynarex</td><td></td><td>No.15</td></tr><tr><td>Needle holder</td><td>Jacobson microvascular</td><td>36-1342TC</td><td>8.5 inch</td></tr><tr><td>Scalpel handle</td><td>Flinn scientific</td><td>AB1049</td><td></td></tr><tr><td>Microsurgical scissor</td><td>WPI</td><td>503305</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Puralube VET</td><td></td><td>3.5 g</td></tr><tr><td>Fluriso (isoflurane)</td><td>Vetone</td><td>V1 502017</td><td>250 ml</td></tr><tr><td>Steri-strip reinforced skin closure</td><td>3M</td><td>R1547</td><td></td></tr><tr><td>Surgical gloves</td><td>Medline</td><td>MSG2270</td><td></td></tr><tr><td>Ear loop face mask</td><td>The safety zone</td><td>RS700</td><td></td></tr><tr><td>Avant gauze non-woven sponges</td><td>Caring</td><td>PRM25444</td><td></td></tr><tr><td>Surgical cup</td><td>Admiral&amp;nbsp; craft OYC-2</td><td>725-A42</td><td>2.5 oz</td></tr><tr><td>Swabstick</td><td>ChloraPrep</td><td>260103</td><td>2% w/v Chlorhexidine&amp;nbsp; Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)</td></tr><tr><td>Cotton tipped applicator</td><td>Puritan</td><td>806-WC</td><td></td></tr><tr><td>Buprenorphine</td><td>Zoo pharm</td><td>BZ8069317</td><td>1 mg/ml</td></tr><tr><td>Gentamycin sulfate</td><td>Vetone</td><td>G-6336-04</td><td>100 mg/ml</td></tr><tr><td>Fast acting gel cream remover</td><td>Veet</td><td>8111002</td><td></td></tr><tr><td>Syringe</td><td>AHS</td><td>AH01T2516</td><td>1 ml with needle</td></tr><tr><td>Silicon ring</td><td>VWR</td><td>60985-720</td><td>6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter</td></tr><tr><td>C57BL/6 mice</td><td>The Jackson Laboratory</td><td></td><td>4-6 weeks old</td></tr></tbody></table>

a mouse model of intestinal partial obstruction

Obstrucciones intestinales, que impiden o bloquean el movimiento peristáltico, pueden ser causadas por adherencias abdominales y más gastrointestinales (GI) enfermedades incluyendo crecimientos tumorales. Sin embargo, el celular mecanismos de remodelación involucrado en, y causada por, obstrucciones intestinales son poco conocidas. Se han desarrollado varios modelos animales de obstrucciones intestinales, pero el modelo de ratón es más coste/tiempo eficaz. El modelo de ratón utiliza la implantación quirúrgica de una obstrucción intestinal parcial (PO) que tiene una alta tasa de mortalidad si no se realiza correctamente. Además, ratones recibiendo cirugía PO no desarrollan hipertrofia si un bloqueo apropiado se utiliza o no correctamente colocado. Aquí, describimos un protocolo detallado para la cirugía PO que produce obstrucciones intestinales fiables y reproducibles con una tasa de mortalidad muy baja. Este protocolo utiliza un anillo de silicona colocado quirúrgicamente que rodea el íleo que bloquea parcialmente la circulación digestiva en el intestino. La obstrucción parcial hace que el intestino se dilatan debido a la interrupción de circulación digestiva. La dilatación del intestino induce la hipertrofia del músculo liso en el lado oral del anillo que desarrolla progresivamente durante 2 semanas hasta que provoca la muerte. El modelo de ratón PO quirúrgico ofrece un modelo en vivo de hipertrófico tejido intestinal útil para el estudio de los cambios patológicos de las células intestinales incluyendo células del músculo liso (SMC), células intersticiales de Cajal (ICC), PDGFRα+, neuronales y células durante el desarrollo de la obstrucción intestinal.

Obstrucción parcial (PO) fue quirúrgicamente inducida en ratones de un mes de edad colocando un anillo de silicona alrededor del íleo cerca el esfínter ileocecal. Este anillo creado una obstrucción parcial en el íleo. Operaciones simuladas (tan) se realizaron sin un anillo de edad/sexo emparejado ratones y estos ratones no mostraron síntomas similares a los encontrados en ratones de PO. Ratones se recuperaron rápidamente de cirugía PO dentro de unas horas. No demostraron ninguna ...

Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

A Mouse Model of Intestinal Partial Obstruction

Obstrucciones intestinales son una obstrucción parcial o completa del intestino que puede causar dolor abdominal severo, náuseas, vómitos y evitando el paso de las heces. Este procedimiento para crear tiene obstrucciones parciales intestinal en ratones es confiable en el estudio de los mecanismos de crecimiento celular patológico y la muerte en el intestino.

Un modelo de ratón de la obstrucción Intestinal parcial

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

a-mouse-model-of-intestinal-partial-obstruction

Research

JoVE Journal

Biology

21.1K vistas.  University of Nevada, Reno School of Medicine. Obstrucciones intestinales son una obstrucción parcial o completa del intestino que puede causar dolor abdominal severo, náuseas, vómitos y evitando el paso de las heces. Este procedimiento para crear tiene obstrucciones parciales intestinal en ratones es confiable en el estudio de los mecanismos de crecimiento celular patológico y la muerte en el intestino.

Video: A Mouse Model of Intestinal Partial Obstruction

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Evaluación funcional de la motilidad intestinal y la inflamación de la pared intestinal en roedores: Análisis de un modelo estandarizado de manipulación intestinal

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Investigación

Medicina

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Modelado Colitis asociada a cáncer con azoximetano (AOM) y Sodio sulfato de dextrano (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Modelo murino de isquemia-reperfusión intestinal Lesiones

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Evaluación Funcional De La Permeabilidad Intestinal Y La Migración Transepitelial Neutrófilo En Ratones Usando Un Modelo De Bucle Intestinal Estandarizado

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Inmunología e Infección

<meta charset="utf8"></head><body>Modelo murino para el estudio del íleo postoperatorio mediante manipulación intestinal

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

<meta charset="utf8"></head><body>Modelo murino de íleo postoperatorio

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Modelo murino de íleo postoperatorio

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Bypass duodeno-ileal de anastomosis única con modelo de gastrectomía en manga en ratones

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Modelo en roedor de lesión por isquemia-reperfusión intestinal por oclusión de la arteria mesentérica superior

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...

Murine Ileal Resection Model: Studying Ileectomy-Induced Bile Metabolism in Murine Model

Source: Zhang, R., et al. <a href="https://www.jove.com/v/55728"> Ileectomy-induced Bile Overaccumulation in Mouse Intestine.</a> J. Vis. Exp. (2017)
This video describes the detailed protocol for intestinal resection in a murine model. This technique provides insights into ileectomy-induced bile malabsorption, overaccumulation, and toxicity in the mouse intestine.

Modelo de resección ileal murina: estudio del metabolismo biliar inducido por ileectomía en modelo murino

Source: Zhang, R., et al.  Ileectomy-induced Bile Overaccumulation in Mouse Intestine. J. Vis. Exp. (2017)
This video describes the detailed protocol for ...

Un modelo de ratón de la obstrucción Intestinal parcial

Resumen

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