The evolution of the device started with somewhat different cutting prototype demonstrated at the Imperial Cancer Research Fund (ICRF) mouse club by Bass Hassan, following this there were several discussions with Rob Rudling and Nikki Mandir in the containment facility at ICRF Clare Hall. Jo Kitau of the mechanical workshop at ICRF constructed the initial versions and Andy Lucas designed and perfected the final model in the CRUK workshop in Oxford.

Los diferentes procedimientos experimentales y la eutanasia humanitaria son aprobados por el comité de ética instituciones de acogida y por los órganos de control pertinentes. La eutanasia se logra normalmente mediante asfixia con CO2, seguido por dislocación cervical. 1. Dispositivo <ol><li> Máquina de la porción principal de la base del dispositivo desde fuera de un bloque sólido de resina de acetilo de alta densidad, como se describe en este manuscrito. En cada extremo crear tiras de alto elevado 3 mm, y parte de la máquina fuera de estos campos de 2,5 mm para tomar las varillas (véase la Figura 2 y la Figura 3). </li><li> Hacer varillas de acero inoxidable de 2,4 mm de diámetro con extremos redondeados-off. NOTA: El marco principal de la tapa se mecanizó de placa de duraluminio. Un número limitado de dispositivos acabados están disponibles en Dr. Goodlad. </li></ol> 2. El uso del dispositivo <ol><li> Diseccionar el intestino delgado y el colon de la cavidad abdominal con tijerasy los dedos enguantados. Retire el mesenterio sujetándolo con pinzas curvas y suavemente tirando de él lejos del intestino. </li><li> Enjuagar los intestinos con fosfato fría solución salina tamponada (PBS, pH 7,4) usando una punta de pipeta de tipo Gilson, que había sido cortado hacia abajo en su extremo más ancho de modo que pueda caber en un 10 o 20 ml luer de la jeringa de plástico. </li><li> Coloque el intestino delgado (SB) en una hoja de papel de cocina y se divide en tres secciones iguales en longitud (proximal, medio y distal - SB1, SB2 y SB3) con unas tijeras. Retire el ciego y diseñar el colon. </li><li> Hacer varios cortes muy pequeños en los segmentos intestinales con unas tijeras para permitir que el fluido se escape y coloque una hoja de papel de cocina sobre los intestinos y suavemente ejecutar un dedo sobre los preparativos para exprimir el líquido y secar. </li><li> Levante las secciones e insertar las varillas de acero inoxidable que se humedecieron previamente por inmersión en un vaso de precipitados de solución salina tamponada con fosfato. NOTA: El intestino puede estar enverted si así se desea (vuelto del revés). </li><li> Etiquetar un trozo de cartón o un filtro de corte de papel de tamaño para encajar en la base del dispositivo. Pencil es mejor, ya que no se ve afectada por prácticamente todas las soluciones. Fecha de la autopsia, el código experimental y el número de identificación de los animales se sugieren. </li><li> Coloque la tarjeta o papel de filtro en la base del dispositivo. Inserte las varillas y los intestinos en las ranuras de la base del dispositivo. Asegúrese de que el extremo proximal de los segmentos se posiciona de una manera estandarizada. Los extremos proximales deben estar cerca de las etiquetas de la tarjeta. </li><li> Coloque la pieza superior del dispositivo a través de la base. Utilice las barras en ángulo para guiar una hoja de bisturí para cortar longitudinalmente las secciones. NOTA: Ayuda si el tejido es suave y cuidadosamente celebró con un dedo para garantizar que no se mueve con el cuchillo. </li><li> Retire la pieza superior del dispositivo. Retire con cuidado el papel y tejidos de filtro (todavía en sus barras). NOTA: Un trozo de cartón rígido colocado debajo de la filpapel ter ayudará elevación. </li><li> Ligeramente húmedo (con un dedo enguantado sumergido en PBS) los segmentos. Estirar la varilla, de lado a lado para abrir el intestino y difundirlo plana. El tejido entonces se adhieren al papel de filtro. </li><li> Examinar visualmente los preparativos de las lesiones macroscópicas. </li><li> Transferir el tejido adherido al papel de filtro en un baño poco profundo (o caja de sándwich) que contiene fijador. NOTA: Después de que el tejido se adhiere al papel de filtro o en la tarjeta se mantendrá firmemente unida. Una amplia gama de fijadores se puede utilizar para preservar la preparación. Se recomienda que el fluido de Carnoy se debe utilizar para el estudio de las proliferaciones de células intestinales 6, que es ideal para la fijación de los mejores métodos de proliferación celular; Sin embargo formalina o muchos otros fijadores también se pueden utilizar si se requieren otros criterios de valoración. </li><li> Después de la fijación (por lo general de 3 horas), la transferencia de los preparativos en etanol al 70%. Las muestras se pueden almacenar hasta que sea necesario. Las preparaciones cun ser estudiados en face y / o utilizados para la preparación de muestras histológicas para H &amp; E u otras técnicas de tinción histológica, inmunohistoquímica y / o hibridación in situ. NOTA: La prolongada fijación puede reducir la reactividad inmunohistoquímica y / o en la reactividad hibridación in situ. Cajas de sándwich de polipropileno son ideales para almacenar un gran número de las preparaciones. </li><li> Si es necesario los tejidos fijos de los diversos segmentos de intestino puede convertirse en &#39;brazos de gitano&#39; y teñidas con toda la gama de métodos histológicos 6 incluyendo los descritos anteriormente. Para hacer un &quot;brazo de gitano&quot; los segmentos intestinales se retiran del papel de filtro, enrollado alrededor de un palillo o un palillo de dientes, se sujeta con un pasador entomológica y procesados ​​para la cera de incrustación de 6. </li><li> Si es necesario, manchar el tejido grueso con azul de metileno para visualizar aberrante focos de criptas 7. </li><li> Puntuación de los preparativos fijos enocio. </li></ol>

<ol><li>Goodlad, R. A. Cancer Handbook. Alison, M. R. , John Wiley and Sons, Ltd. 243-262 (2007).</li>
<li>Ward, J. M., Treuting, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+intestinal+epithelial+carcinogenesis%3A+pathology+and+preclinical+models%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Rodent intestinal epithelial carcinogenesis: pathology and preclinical models.</a> Toxicol Pathol. 42, 148-161 (2014).</li>
<li>Sundberg, J. P., Hogenesch, H., Nikitin, A. Y., Treuting, P. M., Ward, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Training+mouse+pathologists%3A+ten+years+of+workshops+on+the+Pathology+of+Mouse+Models+of+Human+Disease%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Training mouse pathologists: ten years of workshops on the Pathology of Mouse Models of Human Disease.</a> Toxicol Pathol. 40, 823-825 (2012).</li>
<li>Wasan, H. S., Novelli, M., Bee, J., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+fat+influences+on+polyp+phenotype+in+multiple+intestinal+neoplasia+mice%5BTitle%5D)+AND+%22Proc+Natl+Acade+Sci+USA%22%5BJournal%5D)">Dietary fat influences on polyp phenotype in multiple intestinal neoplasia mice.</a> Proc Natl Acade Sci USA. 94, 3308-3313 (1997).</li>
<li>Rudling, R., Hassan, A. B., Kitau, J., Mandir, N., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+device+to+rapidly+prepare+whole+mounts+of+murine+intestine%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">A simple device to rapidly prepare whole mounts of murine intestine.</a> Cell Prolif. 39, 415-420 (2006).</li>
<li>Alferez, D., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((To+best+measure+cell+proliferation+in+samples+from+the+intestine%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">To best measure cell proliferation in samples from the intestine.</a> Cell Prolif. 40, 231-240 (2007).</li>
<li>Park, H. S., Goodlad, R. A., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+incidence+of+aberrant+crypt+foci+and+colonic+carcinoma+in+dimethylhydrazine-treated+rats+varies+in+a+site-specific+manner+and+depends+on+tumor+histology%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">The incidence of aberrant crypt foci and colonic carcinoma in dimethylhydrazine-treated rats varies in a site-specific manner and depends on tumor histology.</a> Cancer Res. 57, 4507-4510 (1997).</li>
<li>Park, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+epidermal+growth+factor+and+dimethylhydrazine+on+crypt+size%2C+cell+proliferation%2C+and+crypt+fission+in+the+rat+colon.+Cell+proliferation+and+crypt+fission+are+controlled+independently%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Effects of epidermal growth factor and dimethylhydrazine on crypt size, cell proliferation, and crypt fission in the rat colon. Cell proliferation and crypt fission are controlled independently.</a> Am J Pathol. 151, 843-852 (1997).</li>
<li>Femia, A. P., Dolara, P., Caderni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucin-depleted+foci+%28MDF%29+in+the+colon+of+rats+treated+with+azoxymethane+%28AOM%29+are+useful+biomarkers+for+colon+carcinogenesis%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Mucin-depleted foci (MDF) in the colon of rats treated with azoxymethane (AOM) are useful biomarkers for colon carcinogenesis.</a> Carcinogenesis. 25, 277-281 (2004).</li>
<li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1%5BTitle%5D)+AND+%22Exp+Toxicol+Pathol%22%5BJournal%5D)">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55, 91-106 (2003).</li>
<li>Goodlad, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibiting+vascular+endothelial+growth+factor+receptor-2+signaling+reduces+tumor+burden+in+the+ApcMin%2F%2B+mouse+model+of+early+intestinal+cancer%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer.</a> Carcinogenesis. 27, 2133-2139 (2006).</li>
<li>Mandir, N., Englyst, H., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resistant+carbohydrates+stimulate+cell+proliferation+and+crypt+fission+in+wild-type+mice+and+in+the+Apc%28Min%2F%2B%29+mouse+model+of+intestinal+cancer%2C+association+with+enhanced+polyp+development%5BTitle%5D)+AND+%22Br+J+Nutr%22%5BJournal%5D)">Resistant carbohydrates stimulate cell proliferation and crypt fission in wild-type mice and in the Apc(Min/+) mouse model of intestinal cancer, association with enhanced polyp development.</a> Br J Nutr. 100, 711-721 (2008).</li>
</ol>

The authors have nothing to disclose.

El dispositivo acelera en gran medida el proceso para que dos operadores pueden autopsia plenamente un ratón dentro de 6 min. Esto incluirá una comprobación visual para las lesiones macroscópicas evidentes y el pesaje y la fijación de los órganos principales. También permitirá a los operadores para enjuagar, secar y pesar el estómago, intestino ciego, intestino delgado y el colon y se preparan todo el monturas. El tejido aplanado puede fijarse muy rápidamente y por lo tanto evitar los artefactos de degradació...

El ratón y el ratón modificado genéticamente 1-3 son un modelo de gran valor para el estudio de diversas patologías, especialmente de cáncer y su relación con la inflamación en el tracto gastrointestinal. Esto a menudo requiere la preparación de preparaciones completas del intestino delgado y / o colon. La preparación de los montajes enteros se puede hacer con un par de tijeras de precisión de desplazamiento, pero puede tomar alrededor de 20 minutos por ratón 4. Esto no es muy práctico para estudios comparativos, que requieren una cantidad significativa de los ratones (10 por grupo decir sujetas a análisis estadístico). Además, la longitud de tiempo que se tarda aumenta el riesgo de la degradación del tejido. Un intento de aliviar este problema llevó al juicio de varias ayudas de preparación de corte. El primero se basa en una serie de placas de metal, con arboledas de corte, pero las placas oscurecido el tejido y el corte fue por lo tanto difícil de controlar. Un conventional lápiz de seis lados, finalmente sirvió de inspiración para el dispositivo de corte, ya que entonces podríamos ver como una forma de media lápiz haría una buena guía para un bisturí (Figura 1). Esto dio lugar a un diseño para un marco con cuatro guías de corte 5. La eficacia de este dispositivo era dramático como las preparaciones en la Figura 4 demuestran la diferencia entre la preparación de tijera y la preparación dispositivo. Las copas de los dispositivos originales se construyen a partir de varias piezas de metal, pero los posteriores, y el modelo actual se mecanizan de una pieza sólida de duraluminio. La base se mecanizó en resina de alta densidad más alta calidad. El dispositivo ha demostrado ser de gran utilidad para la preparación de los montajes enteros de intestino del ratón, ya que acelera en gran medida y mejora la calidad y la consistencia de los preparados, que tienen una amplia gama de aplicaciones.

<table><tbody><tr><td>Delrin</td><td>Ryan Plastics, Earls Barton, UK</td><td></td><td>High-density acetal resin similar material would suffice</td></tr><tr><td>Duralumin plate</td><td>Metal Supplies Ltd, Park Road,Dukinfield. SK16 5LP UK</td><td></td><td>Aluminium allow dating back to 1909 so alterative suppliers are available&amp;nbsp;</td></tr><tr><td>Finished device(s) ready for use</td><td>Contact Dr Goodlad&amp;nbsp; r.goodlad@imperial.ac.uk</td><td></td><td>Contact&amp;nbsp; r.goodlad@imperial.ac.uk for supply details</td></tr></tbody></table>

a simple device to rapidly prepare whole mounts of the mouse intestine

Preparación de los montajes enteros del intestino delgado y el colon de ratón para el análisis o cuantificación posterior puede ser lento y difícil. Se describe el uso de un dispositivo fácil de cortar y los intestinos &#39;roll&#39; ratón para preparar rápidamente los preparativos completos de montaje de calidad superior y uniforme a lo que se puede lograr con la mano. El dispositivo comprende una base que tiene 4 varillas de acero inoxidable y una parte superior, que actúa una guía de corte. Las varillas se insertan en el lumen del intestino delgado [dividido en tercios] y el colon. Las barras y las muestras se colocan entonces sobre un trozo de papel de filtro o de la tarjeta en las ranuras de retención en la base del dispositivo. La parte superior del dispositivo se coloca a continuación y sirve como una guía de corte. Las dos secciones en ángulo en el centro de la pieza superior se utilizan para guiar un cuchillo o bisturí y cortar los intestinos longitudinalmente en la parte superior de las barras. Una vez que las secciones intestinales han sido cortadas, la parte superior se elimina y la tarjeta, TISSue y varillas retira suavemente desde el dispositivo y se coloca en el banco. Las barras son entonces rodó suavemente hacia los lados para aplanar y se adhieren los segmentos intestinales en la pieza fundamental de un papel de filtro o de la tarjeta. La preparación final puede entonces ser examinado o fija y se almacena para su posterior análisis. Los preparativos son de gran valor para el estudio de los cambios intestinales en los modelos normales o modificados genéticamente ratón. Las preparaciones se han utilizado para el estudio y cuantificación de los efectos de la inflamación (colitis), daños, lesiones pre-cancerosas (focos de criptas aberrantes (FCA) y mucina focos (MDF) empobrecido) y los pólipos o tumores.

Los pólipos son fáciles de identificar en un estereomicroscopio con una fuente de luz fría a lado iluminar el tejido (Figura 4). Es útil para marcar la tarjeta con marcas de lápiz cuando se observa un pólipo con el fin de que la puntuación se puede discutir más tarde y diferentes operadores pueden llegar a un acuerdo. El tejido también puede ser mayor manchado [con azul de metileno] para identificar focos de criptas aberrantes 8 o mucina focos empobrecido (MDF) 9 por la il...

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A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Un dispositivo sencillo de preparar rápidamente enteros Montes del Intestino Ratón

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

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Research

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Medicine

19.6K vistas.  National Institutes of Health. The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

Video: A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Los protocolos para analizar el papel de las células de Paneth en Regeneración del Murino Intestino utilizando condicional<em&gt; Cre-lox</em&gt; Ratón Modelos

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Investigación

Biología del Desarrollo

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 ...

Medicina

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

Inmuno-fluorescente etiquetado de microtúbulos y proteínas centrosómicas en tejido Intestinal Ex Vivo y 3D In Vitro Intestinal organitas

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

los<em&gt; Ex Vivo</em&gt; Cultura y Reconocimiento de Patrones receptor de estimulación de ratón intestinal organoides

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Inmunología e Infección

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

La visualización de las células intersticiales de Cajal (ICC) de red en ratones

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Biología

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Sistema de Cultura ratón fetal Whole Intestino para<em&gt; Ex Vivo</em&gt; Manipulación de vías de señalización y tridimensional de imágenes en vivo del desarrollo de vellosidades

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

<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 ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Mejora Técnica Swiss-laminación para la preparación del tejido intestinal para inmunohistoquímica y análisis de inmunofluorescencia

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Un dispositivo sencillo de preparar rápidamente enteros Montes del Intestino Ratón

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Mejora Técnica Swiss-laminación para la preparación del tejido intestinal para inmunohistoquímica y análisis de inmunofluorescencia