Este trabajo fue apoyado por la Fundación Rusa de Ciencias, subvención no. 14-50-00060 y LLC RUSCHEMBIO. Este trabajo también fue apoyado por la Fundación Crohn`s y Colitis de América conceder 294.083 (a AVT), y por el NIH subvención RO1 DK47700 (CJ).

Se llevaron a cabo estudios con animales de acuerdo con el Instituto Nacional de Salud directrices y fueron aprobados por el Cuidado de Animales institucional y el uso comité del Instituto Trudeau. 8-12 semanas de edad C57BL / 6 ratones se utilizaron para el estudio. 1. Preparación para la Cirugía <ol><li> Preparar y esterilizar instrumentos quirúrgicos. </li><li> Preparar sistema de anestesia basada en isoflurano con el cono de la nariz y la almohadilla caliente. Asegúrese de que la almohadilla caliente no se ha recalentado (&lt;39 ºC). </li><li> Asegúrese de que el isoflurano recipiente de recogida de gases está colocado correctamente para asegurar que los orificios de escape en la parte inferior de la lata no se bloquean o ocluido de ninguna manera. Pesar recipiente de recogida de gases antes del procedimiento y el peso del documento en el cartucho. Deseche el cartucho cuando el peso del recipiente es superior a 50 g de peso acumulado (~ 12 h). </li></ol> 2. anestesia <ol><li> Anestesiar al ratón con 3% de isoflurano en una cámara de inducción (1 L / minO 2). <ol><li> Evaluar la profundidad anestésica por una incapacidad para permanecer en posición vertical, pérdida de movimiento intencional voluntaria, pérdida del reflejo de parpadeo, la relajación muscular y la pérdida de respuesta a la estimulación reflex (dedo del pie o pellizco de la cola con una presión firme). </li><li> Evaluar la frecuencia respiratoria y el patrón mediante la observación de los movimientos de la pared torácica y abdominal. Bajo anestesia óptima, la tasa de respiración debe ser ~ 55-65 respiraciones por minuto. </li><li> Retire ratón desde la cámara de inducción y afeitar rápidamente el área del abdomen del ratón. </li></ol></li><li> Para evitar la desecación de la córnea, colocar pomada oftálmica sosa en los ojos. </li><li> Coloca el ratón sobre la almohadilla caliente y conectarlo a través de cono de la nariz con el sistema de anestesia. Asegúrese de que la membrana cono de la nariz de látex se ajusta firmemente sobre la cabeza del ratón y no hay fugas de isoflurano. </li><li> Reducir la tasa de isoflurano al 1,5%, e inyectar buprenorfina (0,1 mg / kg) y ketamina (10 mg / kg) por vía subcutánea para prevenir wind-up del dolor en cascada. </li><li> limpie THe la piel de la zona de operación con un hisopo de algodón estéril empapada con una solución de Betadine quirúrgico seguido de etanol al 70%. </li></ol> 3. Cirugía <ol><li> Hacer una laparotomía cm de la línea media de 3-5 con tijeras de funcionamiento. Área de actividad de la cubierta con el cojín no adherente estéril humedecido con solución salina. Aislar ciego y el íleon y exponer la arteria mesentérica superior usando hisopos de algodón humedecidos en solución salina. </li><li> Para facilitar la aplicación de clips, hacer pequeñas muescas en el mesenterio que rodea la arteria mesentérica superior usando iris finas scissors.To ello, levante suavemente el intestino con una pinza vestidores y cortar mesenterio a ambos lados de la arteria mesentérica superior en la posición de corte deseada (Figura 1A). A continuación, añadir algunas gotas de solución salina estéril a la zona de la posición de clip deseado antes de aplicar clips. Nota: Para realizar la cirugía simulada, siga el procedimiento quirúrgico hasta el paso 3.2. No aplicar clips. En su lugar, mantener el tejido húmedo por el agregado s tibiaaline como se describe en 3.6 durante 1 h. Después, continúe en el paso 4.1, </li><li> Ocluir las primeras ramas de la orden de la arteria mesentérica superior con clips microvasculares (70 g de fuerza) utilizando un aplicador de clips para crear una región de 5-7 cm del íleon isquémica adyacente al ciego (Figura 1B). Aunque la posición de los vasos es conservadora, puede haber ligeras variaciones entre los ratones (ver ejemplos en la Figura 1). Por lo tanto, se requieren generalmente 2 o 3 clips (ver la situación de los clips en la Figura 1A, D, E, flechas negras). Nota: Utilice grapas de vaso de alta calidad. clips de alta presión puede dañar los vasos y evitar la regeneración mientras que los clips de baja presión (&lt;30 g) no puede bloquear completamente el flujo sanguíneo. </li><li> Bloquear el flujo sanguíneo colateral a través del intestino usando dos clips microvasculares través de los vasos (40 g fuerza), demarcación de la región del intestino isquémico (Figura 1). Se requiere la oclusión de vasos colaterales aimpide el suministro de sangre de los vasos sanguíneos adyacentes (ver la situación de los clips en la figura 1A, D, E, flechas verdes). </li><li> Opcional: Añadir solución de heparina (6 USP unidades / ml), para prevenir la coagulación de la sangre. Gota a gota añadir 0,5 ml de solución de heparina al intestino aislado. </li><li> Húmedos almohadilla no adherente delicados toallitas estériles con solución salina precalentada a 37 ° C y se aplican a la zona quirúrgica. Asegúrese de que las toallitas húmedas se mantiene durante todo el procedimiento. </li><li> Mantener la isquemia durante 60 minutos usando 1-1,5% a lo largo de la anestesia con isoflurano. Si la isquemia procedimiento se realiza correctamente, la región isquémica cambiará a rojo vino de color en aproximadamente 30 minutos. Tenga en cuenta que los vasos sanguíneos distales a la posición del clip se agrandan durante la isquemia (Figura 1, paneles de la derecha) que indica la oclusión exitosa. </li><li> Vigilar de cerca el ratón durante la etapa de la isquemia. Seguir aplicando una solución salina para la almohadilla no adherente que cubre el sitio de la cirugía. </li><li> marcalos bordes de la zona isquémica mediante pipeteo 20 l de Gill`s 3 hematoxilina sobre el tejido para facilitar la cosecha el tejido isquémico y el tejido sano adyacente de la misma para la comparación de ratón (Figura 1E, panel derecho). </li></ol> 4. Etapa de reperfusión <ol><li> Al final de la isquemia añadir unas gotas de solución salina en el área de clip y retire suavemente clips microvasculares con aplicador de clips. A continuación, empuje suavemente el intestino de nuevo a la cavidad abdominal con solución salina humedecido puntas de algodón. Retire la almohadilla no adherente y cierre de la pared abdominal y la piel utilizando acero inoxidable grapas para heridas 9 mm. Si la reperfusión se lleva a cabo durante más de 3 horas, utilice una sutura absorbible vicryl para cerrar la pared abdominal antes de la aplicación de grapas de heridas en la piel. </li><li> Mantener los ratones en una jaula limpia calentó durante cantidad deseada de tiempo (30 min, 60 min, 120 min, o 180 min) para la fase de reperfusión. </li><li> Compruebe los animales al menos cada 30 minutos para asegurar la estabilidad. </li></ol><pclass = &quot;jove_title&quot;&gt; 5. La necropsia y recolección de intestino delgado <ol><li> La eutanasia a los ratones por CO 2 sobredosis seguido por dislocación cervical en el momento deseado después de la reperfusión. </li><li> Abra cavidad abdominal y recoger el tejido intestinal isquémica para su posterior análisis. Cosecha tejido normal sano adyacente al tejido lesionado como un control interno para dar cuenta de cualquier reacción sistémica a la lesión. Nota: Este control es más apropiada que la operación simulada ratones de control porque los ratones con operación simulada no se someten a una reacción sistémica a la lesión inducida por IR. </li><li> Lave el contenido intestinal mediante jeringa de 30 ml con aguja de sonda unida llena con solución salina y luego se corta el intestino longitudinalmente. Si se requiere una muestra de intestino para el análisis de la expresión génica, cortar un fragmento de 1,5 mm en sentido longitudinal, y el uso de la pieza restante para el análisis histológico. </li><li> Para el análisis histológico, preparar un rollo suizo usando un par de pinzas para hacer rodar la intestine. </li><li> Para mantener la forma enrollada, colocar las piezas de intestino entre almohadillas de espuma de biopsia en cassettes de tejido (Figura 2). Coloque los cassettes en 10% de formalina tamponada. </li><li> Fijar el tejido en formalina durante al menos 24 h. Reemplazar formol con un 70% de etanol durante 24 horas más. tejido de la tienda en 70% de etanol indefinidamente a temperatura ambiente. </li><li> Incrustar en parafina, se cortaron secciones de 5 micras y se tiñen con hematoxilina y eosina utilizando un protocolo estándar (Figura 3). </li></ol> 6. Scoring <ol><li> Puntuación de la lesión por isquemia-reperfusión murino tal como se resume en la Tabla 2. Elija un método de puntuación apropiada. </li><li> Opcional: Se divide el campo de visión en cuatro secciones ya que la gravedad de la lesión varía a lo largo de la sección. </li><li> Calcular la nota media de cada sección de las puntuaciones obtenidas a ciegas. </li><li> Comparar el grado del tejido lesionado entre los casos y de control, así como a the ileso tejido utilizando una prueba de Kruskal-Wallis, seguido de la prueba de comparaciones múltiples de un Dunn. </li></ol>

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<li>Stallion, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ischemia%2Freperfusion%3A+a+clinically+relevant+model+of+intestinal+injury+yielding+systemic+inflammation%5BTitle%5D)+AND+%22J+Pediatr+Surg%22%5BJournal%5D)">Ischemia/reperfusion: a clinically relevant model of intestinal injury yielding systemic inflammation.</a> J Pediatr Surg. 40, 470-477 (2005).</li>
<li>Blikslager, A. T., Roberts, M. C., Rhoads, J. M., Argenzio, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+reperfusion+injury+an+important+cause+of+mucosal+damage+after+porcine+intestinal+ischemia%3F%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">Is reperfusion injury an important cause of mucosal damage after porcine intestinal ischemia?</a> Surgery. 121, 526-534 (1997).</li>
<li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li>
<li>Victoni, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+and+remote+tissue+injury+upon+intestinal+ischemia+and+reperfusion+depends+on+the+TLR%2FMyD88+signaling+pathway%5BTitle%5D)+AND+%22Med+Microbiol+Immunol%22%5BJournal%5D)">Local and remote tissue injury upon intestinal ischemia and reperfusion depends on the TLR/MyD88 signaling pathway.</a> Med Microbiol Immunol. 199, 35-42 (2010).</li>
<li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-like+receptor+2+mediates+ischemia-reperfusion+injury+of+the+small+intestine+in+adult+mice%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Toll-like receptor 2 mediates ischemia-reperfusion injury of the small intestine in adult mice.</a> PloS one. 9, e110441(2014).</li>
<li>Pope, M. R., Fleming, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TLR2+modulates+antibodies+required+for+intestinal+ischemia%2Freperfusion-induced+damage+and+inflammation%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">TLR2 modulates antibodies required for intestinal ischemia/reperfusion-induced damage and inflammation.</a> J. Immunol. 194, 1190-1198 (2015).</li>
<li>Leung, F. W., Su, K. C., Passaro, E. Jr, Guth, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regional+differences+in+gut+blood+flow+and+mucosal+damage+in+response+to+ischemia+and+reperfusion%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Regional differences in gut blood flow and mucosal damage in response to ischemia and reperfusion.</a> Am J Physiol. 263, G301-G305 (1992).</li>
<li>Chiu, C. J., McArdle, A. H., Brown, R., Scott, H. J., Gurd, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+mucosal+lesion+in+low-flow+states.+I.+A+morphological%2C+hemodynamic%2C+and+metabolic+reappraisal%5BTitle%5D)+AND+%22Arch+Surg%22%5BJournal%5D)">Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal.</a> Arch Surg. 101, 478-483 (1970).</li>
<li>Quaedackers, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+evaluation+of+methods+for+grading+histologic+injury+following+ischemia%2Freperfusion+of+the+small+bowel%5BTitle%5D)+AND+%22Transplant+Proc%22%5BJournal%5D)">An evaluation of methods for grading histologic injury following ischemia/reperfusion of the small bowel.</a> Transplant Proc. 32, 1307-1310 (2000).</li>
<li>Bianciardi, P., Scorza, R., Ghilardi, G., Samaja, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xanthine+oxido-reductase+activity+in+ischemic+human+and+rat+intestine%5BTitle%5D)+AND+%22Free+Radic+Res%22%5BJournal%5D)">Xanthine oxido-reductase activity in ischemic human and rat intestine.</a> Free Radic Res. 38, 919-925 (2004).</li>
<li>Yandza, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pig+as+a+preclinical+model+for+intestinal+ischemia-reperfusion+and+transplantation+studies%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">The pig as a preclinical model for intestinal ischemia-reperfusion and transplantation studies.</a> J Surg Res. 178, 807-819 (2012).</li>
</ol>

The authors declare no conflict of interest

El desarrollo de modelos de ratón de IR lesión intestinal han mejorado en gran medida la comprensión de los mecanismos de lesión tisular y ayudado en el desarrollo de estrategias terapéuticas potenciales para minimizar los daños 7,9,11,34. Los pasos críticos de este protocolo son la colocación apropiada de las grapas microvasculares, la coordinación correcta de la evaluación histológica isquemia y adecuada de la lesión IR. La duración de la isquemia es crítica para d...

El intestino es muy sensible a la interrupción del flujo de sangre que provoca isquemia y daño epitelial. Reperfusión después de la isquemia proporciona re-oxigenación del tejido, y puede promover aún más la patología. Por lo tanto, la lesión por isquemia y reperfusión intestinal se asocia con una amplia gama de patologías, incluyendo enterocolitis necrotizante, rechazo de aloinjertos en el trasplante de intestino delgado, las complicaciones de la cirugía de aneurisma de aorta abdominal, derivación cardiopulmonar, y 1,2 enfermedad inflamatoria intestinal. IR lesión intestinal, isquemia mesentérica aguda sobre todo, es una condición que amenaza la vida como resultado de la morbilidad y la mortalidad 3. Aunque poco entendido, se cree que intestinal por isquemia-reperfusión (IR) daño a estar asociado con cambios en la microflora intestinal, así como la producción de especies reactivas del oxígeno y citoquinas inflamatorias y quimiocinas 1,4-6. Esto conduce a la activación de ambos enNate y mecanismos inmunes adaptativas, que promueven la inflamación y el tejido 1,7,8 lesión. Los modelos animales son fundamentales para la comprensión de los mecanismos de la lesión por IR, ya que permiten una fácil ganancia y la pérdida de experimentos genéticos de la función. Varios modelos animales de IR se han desarrollado, que incluyen la oclusión vascular completa, bajo la isquemia de flujo, y segmentada de oclusión vascular (que se resumen en una revisión exhaustiva reciente 9). La isquemia intestinal causado por la oclusión vascular completa de la arteria mesentérica superior (SMA) es un modelo sencillo y de uso común de IR en animales grandes y roedores 9-11. Sin embargo, las diferentes áreas del intestino tienen diferente susceptibilidad a la lesión. Además, la diversa gama de anestésicos, analgésicos, técnicas de oclusión de la arteria, así como la inconsistencia en la duración de la lesión isquémica y resultado de recuperación en grados variables de lesión de confusión nuestra comprensión de la biología de IR a través de múltiples studies. La Tabla 1 muestra estas inconsistencias en los estudios de IR murino. El mayor inconveniente del uso de los tiempos de isquemia más cortos (30-45 min) se dirige a la ventana de la recuperación sobre la cual se pueden observar diferencias discernibles entre los casos y controles. lesión leve en el epitelio puede ser resuelto de una hora después de la reperfusión, por lo tanto, se especializó métricas patológicos pueden ser necesarios para encontrar las diferencias en la restitución epitelial. Por el contrario, un daño excesivo, como se ve por 100 min de la lesión isquémica puede resultar en la denudement completa del epitelio, cuando la restitución ya no es posible, el aumento de la tasa de mortalidad y el tiempo de recuperación. Por lo tanto, aquí se describe el procedimiento detallado de la RI intestinal en ratones que produce lesiones reproducibles sin mortalidad para fomentar la estandarización de esta técnica a través de nuestro campo. Este modelo de lesión intestinal IR se puede utilizar para estudiar los mecanismos celulares y moleculares de la lesión y regeneración.

<table><tbody><tr><td>Heated Pad</td><td>Sunbeam</td><td>E12107-819</td><td>Alternative: Braintree Scientific heated pad</td></tr><tr><td>Table top research anesthesia Machine</td><td>Vasco</td><td>UCAP 0001-0000171</td><td>Alternative: Parkland Scientific, V3000PS</td></tr><tr><td>Nose Cone</td><td>Parkland Scientific</td><td>ARES500</td><td></td></tr><tr><td>Scavenger canister and replacement cartridge</td><td>Parkland Scientific</td><td>80000, 80120</td><td></td></tr><tr><td>Induction Chamber</td><td>Surgivet</td><td>V711802</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare</td><td>NDC 66794-013-10</td><td>Controlled substance, contact IACUC</td></tr><tr><td>Animal clipper&amp;nbsp;</td><td>Oster&amp;nbsp;</td><td>Oster Golden A5 078005-050-003</td><td></td></tr><tr><td>Ophthalmic ointment</td><td>Webster</td><td>8804604</td><td></td></tr><tr><td>Buprenorphine</td><td>McKesson</td><td>562766</td><td>Controlled substance,contact IACUC</td></tr><tr><td>Ketaset (Ketamine HCl)</td><td>Pfizer</td><td>NADA 45-290</td><td>Controlled substance, contact IACUC</td></tr><tr><td>Cotton tips</td><td>Puritan medical products</td><td>806-WC</td><td>Autoclave before use</td></tr><tr><td>Betadine</td><td>Purdue Products</td><td>67618-150-17</td><td>10% Povidone-Iodine</td></tr><tr><td>Sterile saline solution</td><td>Aspen</td><td>46066-807-60</td><td>Adjust to room temperature before use</td></tr><tr><td>IR rodent thermometer</td><td>BIOSEB</td><td>BIO-IRB153</td><td></td></tr><tr><td>Micro vascular clips, 70 g</td><td>Roboz Surgical&amp;nbsp;</td><td>RS5424, RS5435</td><td>Alternative: WPI 14121, for SMA occlusion</td></tr><tr><td>Micro vascular clips, 40 g</td><td>Roboz Surgical&amp;nbsp;</td><td>RS6472</td><td>Alternative:WPI 14120, for collateral vessels occlusion</td></tr><tr><td>Clip applying forceps</td><td>World Precision Instruments</td><td>14189</td><td>Alternative: Roboz #RS-5410 or&amp;nbsp; #RS-5440</td></tr><tr><td>Gill&#039;s 3 hematoxylin</td><td>Thermo Scientific</td><td>14-390-17</td><td></td></tr><tr><td>Surgical staples, Reflex 9 mm</td><td>Cell Point Scientific</td><td>201-1000</td><td></td></tr><tr><td>Autoclip applier</td><td>Beckton Dickinson</td><td>427630</td><td></td></tr><tr><td>Byopsy foam pad</td><td>Simport</td><td>M476-1</td><td></td></tr><tr><td>Tissue cassette</td><td>Fisher Healthcare</td><td>15182701A</td><td>Histosette II combination lid and base</td></tr><tr><td>10% buffered formalin</td><td>Fisher Scientific</td><td>245-684</td><td></td></tr><tr><td>Surgical iris scissors</td><td>World Precision Instruments</td><td>501263-G SC</td><td>Alternative: Roboz RS6816</td></tr><tr><td>Operating scissors</td><td>World Precision Instruments</td><td>501219-G</td><td>Alternative: Roboz RS6814</td></tr><tr><td>Dressing forceps</td><td>Roboz Surgical&amp;nbsp;</td><td>RS-5228, RS-8122</td><td>Alternative: World Precision Instruments 1519-G</td></tr><tr><td>Heparin, endotoxin free, 300 USP units/vial, 50 mg</td><td>Sigma</td><td>2106</td><td></td></tr><tr><td>Reflex wound clip removing forceps</td><td>Roboz Surgical&amp;nbsp;</td><td>RS-9263</td><td>Alternative: World Precision Instruments: 500347</td></tr><tr><td>Mice C57BL/6J mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>Stock No 0664</td><td></td></tr><tr><td>Telfa non-adherent dressings, 3 x 4, sterile</td><td>Coviden</td><td>1050</td><td></td></tr><tr><td>Fisherbrand transfer pipets</td><td>Fischer Scientific</td><td>13-711-5AM</td><td>Use pipets to dropwise add saline</td></tr></tbody></table>

murine model of intestinal ischemia-reperfusion injury

La isquemia intestinal es una afección potencialmente mortal asociado con una amplia gama de condiciones clínicas que incluyen la aterosclerosis, trombosis, hipotensión, enterocolitis necrotizante, trasplante de intestino, el trauma y la inflamación crónica. lesión intestinal por isquemia-reperfusión (IR) es una consecuencia de isquemia mesentérica aguda, causada por el flujo inadecuado de sangre a través de los vasos mesentéricos, lo que resulta en el daño intestinal. Reperfusión después de la isquemia puede exacerbar aún más el daño del intestino. Los mecanismos de la lesión IR son complejos y poco conocidos. Por lo tanto, los modelos experimentales de animales pequeños son fundamentales para la comprensión de la fisiopatología de la lesión IR y el desarrollo de nuevas terapias. Aquí se describe un modelo de ratón de lesión IR intestinal aguda que proporciona lesión reproducible del intestino delgado sin mortalidad. Esto se consigue por la isquemia inducir en la región del íleon distal por temporalmente ocludinag las ramas colaterales periféricos y terminales de la arteria mesentérica superior durante 60 minutos con clips microvasculares. Reperfusión durante 1 hora, o 2 horas después de la lesión resulta en lesión reproducible del intestino examinado por análisis histológico. La correcta posición de las pinzas microvasculares es crítica para el procedimiento. Por lo tanto el clip de vídeo proporciona una detallada descripción visual paso a paso de esta técnica. Este modelo de lesión intestinal IR se puede utilizar para estudiar los mecanismos celulares y moleculares de la lesión y regeneración.

Hemos optimizado el protocolo experimental de la cirugía de infrarrojos para obtener la lesión inducida reproducible IR del íleon en ratones. Los resultados representativos se manifestaron en esta sección. La Figura 1 muestra ejemplos de posición clips microvasculares para inducir isquemia del íleon. Las flechas negras indican la posición de los principales clips que ocluyen las ramas de primer orden de...

Watch this Scientific Journal Video about Murine Model of Intestinal Ischemia-reperfusion Injury at JoVE.com

Murine Model of Intestinal Ischemia-reperfusion Injury

Modelo murino de isquemia-reperfusión intestinal Lesiones

A continuación se describe el procedimiento detallado de isquemia-reperfusión intestinal en ratones que produce lesiones reproducibles sin mortalidad para fomentar la estandarización de esta técnica a través del campo. Este modelo de lesión de isquemia-reperfusión intestinal puede ser utilizado para estudiar los mecanismos celulares y moleculares de la lesión y regeneración.

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

murine-model-of-intestinal-ischemia-reperfusion-injury

Research

JoVE Journal

Medicine

19.5K vistas.  Trudeau Institute.  A continuación se describe el procedimiento detallado de isquemia-reperfusión intestinal en ratones que produce lesiones reproducibles sin mortalidad para fomentar la estandarización de esta técnica a través del campo. Este modelo de lesión de isquemia-reperfusión intestinal puede ser utilizado para estudiar los mecanismos celulares y moleculares de la lesión y regeneración. 

Video: Murine Model of Intestinal Ischemia-reperfusion Injury

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Un murino tórax cerrado modelo de isquemia y reperfusión miocárdica

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Investigación

Medicina

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Modelo murino de tumores hepáticos metastásicos en el escenario de la lesión por reperfusión de isquemia

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Investigación sobre el cáncer

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 ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Aislamiento de células madre intestinales y de la cultura en un modelo porcino de isquemia Intestinal pequeño segmentaria

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

Oclusión de los vasos dos ratón modelo de isquemia-reperfusión Cerebral

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modelo de infarto de miocardio murino usando ligadura permanente de la arteria coronaria descendente anterior izquierda

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

An Effective Mouse Model of Unilateral Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models are the only available resources that mimic the complexities of the IRI-associated damage encountered in vivo. This paper describes an effective mouse model of unilateral renal IRI that delivers highly reproducible data. Ischemia is induced by occluding the right renal pedicle for 30 min followed by reperfusion. In addition to the surgical procedure, a sequential overview of the expected physiological and histopathological changes following renal IRI will be provided by comparing data from seven different reperfusion times (4 h, 8 h, 16 h, 1 day, 2 days, 4 days, and 7 days). Critical data for planning experiments ahead, such as mean surgical time, average anesthetic consumption, and body weight changes over time, will be shared. This work will help researchers implement a reliable renal IRI model and select the appropriate reperfusion time that aligns with their intended investigative goals.

Renal ischemia-reperfusion injury is associated with high morbidity and mortality in hospitalized patients. Here, we present a simple and effective mouse model of unilateral renal ischemia-reperfusion injury and provide a sequential overview of representative pathological changes observed in the kidney.

Un modelo de ratón eficaz de lesión unilateral por isquemia-reperfusión renal

Ischemia-reperfusion injury (IRI) is the leading cause of acute renal failure and is a significant contributor to delayed graft function. Animal models ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Inducción de infarto de miocardio y lesión por isquemia-reperfusión miocárdica en ratones

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

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, ...

Modelo murino para el estudio de la lesión por isquemia-reperfusión pulmonar

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows for&#160;detailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

Autor destacado: Perspectivas sobre el efecto de la reperfusión por isquemia en el trasplante de pulmón

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse models.