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., Alison, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Cancer+Handbook."> Cancer Handbook.</a> , 243-262 (2007).</li><li>Ward, J. M., Treuting, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+intestinal+epithelial+carcinogenesis:+pathology+and+preclinical+models.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+mouse+pathologists:+ten+years+of+workshops+on+the+Pathology+of+Mouse+Models+of+Human+Disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+fat+influences+on+polyp+phenotype+in+multiple+intestinal+neoplasia+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+device+to+rapidly+prepare+whole+mounts+of+murine+intestine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+best+measure+cell+proliferation+in+samples+from+the+intestine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucin-depleted+foci+(MDF)+in+the+colon+of+rats+treated+with+azoxymethane+(AOM)+are+useful+biomarkers+for+colon+carcinogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+vascular+endothelial+growth+factor+receptor-2+signaling+reduces+tumor+burden+in+the+ApcMin/++mouse+model+of+early+intestinal+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Delrin</td>
			<td>Ryan Plastics, Earls Barton, UK</td>
			<td>N/A</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>N/A</td>
			<td>Aluminium allow dating back to 1909 so alterative suppliers are available&#160;</td>
		</tr>
		<tr>
			<td>Finished device(s) ready for use</td>
			<td>Contact Dr Goodlad&#160; r.goodlad@imperial.ac.uk</td>
			<td>N/A</td>
			<td>Contact&#160; r.goodlad@imperial.ac.uk&#160; 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...

Watch this Scientific Journal Video about A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine at JoVE.com

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 ...

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

Research

JoVE Journal

Medicine

19.5K 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

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

La medición precisa y simple de la citoquina pro-inflamatoria IL-1β mediante un análisis de sangre entera estimulación

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Investigación

Medicina

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La medición y tratamiento de la represión en la ambliopía

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Un sencillo método de intubación ratón de pulmón

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

El aislamiento y uso de las Secciones de bovino mesentérica arteria y la vena como un bioensayo para la Prueba de Vasoactividad en el intestino delgado

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Un procedimiento mecánico simple de crear deficiencia de células madre limbares en ratón

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Guiado por ecografía intracardiaca inyección de células madre mesenquimales humanas para aumentar autoguiado hacia el blanco en el intestino para su uso en modelos murinos de enfermedades de intestino inflamatorias experimentales

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

Sobreacumulación de Ileectomy inducida por bilis en intestino de ratón

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

Mecanismos subyacentes de la secreción de la hormona del intestino utilizando los aislados perfusión intestino de rata

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

Un método simple modificado para la inducción de infarto de miocardio en ratones

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Aplicación de la tomografía de coherencia óptica a un modelo de retinopatía en ratones

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...