This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

Todo el trabajo con animales en este protocolo se realiza con estricto apego a la Universidad de Newcastle Comité de Ética Animal procedimientos aprobados. 1. Preparación de los instrumentos, medios de cultivo y Platos <ol><li> Pre-caliente de medios 199 (TC199) o de Eagle modificado por Dulbecco (DMEM) medios de comunicación a 37 DO. Pre-oxigenar el medio por burbujeo con 95% de O2 / 5% de CO 2. Comprobar que el medio tiene un pH final de 7,3. </li><li> Prepara sutura cortando dos secciones 5 cm para cada salida. Bucle de las suturas en un nudo sin cerrar. </li></ol> 2. La disección y preparación del Tracto Gastrointestinal <ol><li> Retirar el alimento sólido de 12 horas antes de la eutanasia. Si se desea, colocar los animales sobre los suplementos de gel de nutrientes durante este tiempo. </li><li> La eutanasia a los ratones mediante una sobredosis de pentobarbital sódico ([200 mg / Kg], inyección intraperitoneal), seguido por dislocación cervical de acuerdo con la ética proto institucionalcols y rocían 70% de etanol en el abdomen y el tórax. </li><li> Usando unas tijeras, hacer una incisión horizontal en el centro del abdomen y exponer el peritoneo. </li><li> Proceder para separar y eliminar el tracto gastrointestinal por el corte de la parte superior del intestino desde el estómago en el esfínter pilórico y cortar el intestino grueso en el margen anal. Utilice unas pinzas para eliminar cuidadosamente el mesenterio. Coloque el tracto intestinal en pre-calentado, medio oxigenado. </li><li> Identificar la sección del intestino que deben evaluarse para la permeabilidad (Figura 1) y se corta esta sección libre del resto del tracto intestinal. <ol><li> Con el fin de mantener la coherencia entre los animales, medir secciones de duodeno y el yeyuno en relación con el estómago, y medir secciones del colon y el íleon en relación con el ciego. </li><li> Al seleccionar segmentos de tejido, tenga en cuenta la presencia de los tejidos linfoides asociados a mucosas, tales como placas de Peyer. Estos pueden ser identificados como pequeño guiñoEGLAS en el lado serosal de la luz. </li><li> Usando una jeringa de 1 ml, lavar suavemente el contenido luminal del segmento intestinal en una placa Petri con PBS pre-calentado (37 ° C). Estos contenidos fecales pueden ser descartados o se almacenaron a -80 ° C para el análisis futuro si lo deseas. </li></ol></li></ol> 3. Preparación de Intestinal Sacs <ol><li> Preparar una jeringa de 1 ml con un volumen de 300 l de compuesto de ensayo o molécula. Por integridad de la mucosa, una solución de 1 mg / ml de FITC-dextrano M.Wt. 4400 puede ser utilizado. Las sondas que van de 4,400-70,000 Da de tamaño se pueden usar para una mayor sensibilidad. encajar de forma segura un pequeño catéter vascular de los animales en la jeringa. </li><li> Medida 5 cm de la apertura del segmento intestinal y atar el segmento de seguridad cierran con una sutura de lazo en este punto. Con cuidado, coloque un pre-atado de sutura de lazo alrededor de la abertura del intestino e inserta el catéter despuntada. Tirar de la soga cerrado, así como para asegurar el segmento intestinaly liberar el volumen de 300 l de la jeringa en el intestino, asegurando que se inyecta toda la solución. </li><li> Retire con cuidado el catéter accionando al mismo tiempo el lazo de sutura para asegurar el cierre del saco intestinal. Cortar el saco intestinal suelto en el intestino y colocar en un tubo cónico de 50 ml lleno con 20 ml de medio oxigenado, precalentado a 37 a DO. </li></ol> 4. Medición de la Permeabilidad <ol><li> Colocar los tubos cónicos que contienen los sacos intestinales en un baño de agua de calefacción ajustada a 37 DO. A 0, 30, 60, 90 y 120 puntos de tiempo min, tomar una muestra de 100 l desde el tubo cónico y la transferencia a una placa de 96 pocillos, en sustitución del volumen con 100 l de medio fresco en cada caso. </li><li> Después de que se haya tomado la muestra final, cortar sacos abiertos en el punto de sutura y abajo de la longitud del segmento, la exposición de la superficie de la mucosa. </li><li> Medir la longitud y la anchura de cada segmento intestinal. Si desirojo, broche congelar los segmentos y almacenar a -80 ° C para la proteína o el análisis bioquímico, o, alternativamente, almacenar en solución de estabilización de ARN para análisis moleculares. </li><li> Construir una curva estándar de diluciones de registro de moléculas marcadas con FITC van de 1 a 1 x 10 -6. </li><li> medir las muestras y estándares para FITC en un lector de placas de fluorescencia, FITC excitación / emisión: 495 nm / 519 nm. </li></ol> 5. Cálculo de la permeabilidad aparente para cada individuo intestinal Sac <ol><li> Convertir unidades de tiempo de segundos. </li><li> Para cada punto de tiempo, calcular la concentración acumulativa, Q Q t = (C * t V r) + (Q t suma * V s), Donde: Q t = concentración acumulativa en el tiempo t C t = concentración en el tiempo t V r = Volumen en el lado del receptor Q t = suma Suma de todos los Q anterior t = V svolumen muestreado </li><li> Parcela Q en función del tiempo (T) y calcular la pendiente: δQ / dt </li><li> Se calcula la permeabilidad aparente (P app) P = aplicación (δQ / dt) / (A * Co), donde: A = área de tejido C 0 = concentración inicial </li></ol>

<ol>
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Los autores declaran que no tienen intereses financieros en competencia.

Aquí, hemos detallado el aislamiento y la preparación de los sacos intestinales para evaluar la función barrera de la mucosa ex vivo. preparaciones saco intestinal principalmente se han utilizado en la investigación farmacéutica, el examen de la absorción de los fármacos candidatos a través del intestino. Sin embargo, este ensayo es igualmente bien adecuada para el estudio de la enfermedad intestinal. la permeabilidad intestinal puede variar considerablemente según la región y la evaluación específi...

La barrera epitelial intestinal del tracto gastrointestinal es un área de superficie de la mucosa se ​​estima en 400 m 2 en el adulto humano. En consecuencia, se expone constantemente a desafiar a partir de microbios, fármacos ingeridos, nutrientes y toxinas bacterianas. El anfitrión no sólo debe distinguir entre las bacterias comensales tolerables y patógenos potenciales, pero debe evitar que estas especies y sus moléculas secretadas de atravesar la barrera epitelial, mientras que al mismo tiempo que permite la absorción de nutrientes. Por lo tanto, el papel del epitelio intestinal es actuar como una barrera selectiva a los contenidos luminales 1. Esto se consigue, en parte, por el sistema de defensa innata epitelial en la mucosa, que actúa a través de un sistema biológico sensible que consiste en mecanismos constitutivos e inducibles 2. La pérdida de la función de la barrera epitelial es una patología que es característica de un número de enfermedades gastrointestinales. In vivoexamen de la función de barrera epitelial puede ser evaluada a través de una sonda nasogástrica de una molécula del marcador, y su posterior análisis del suero 3. Sin embargo, esta técnica no ofrece ninguna indicación en cuanto al sitio de la disfunción de la barrera. In vitro y ex vivo evaluación de la resistencia transepitelial utilizando sistemas Transwell 3 y cámaras Ussing 4,5 respectivamente, se emplean comúnmente como marcadores indirectos de la función de la barrera epitelial, pero carecen de la fisiología de la enfermedad contribuyendo de modelos animales 6. En este protocolo se describe un modelo de preparación de tejido ex vivo que permite la evaluación directa y localizada de la integridad intestinal y que puede utilizarse para evaluar la función barrera de la mucosa en un número de niveles. Es importante destacar que esta técnica puede aplicarse a modelos animales de enfermedad, o puede ser manipulado para permitir farmacológicamente en interrogatorio profundidad de disfunción de la barrera de la mucosa.

<table border="0" cellpadding="0" cellspacing="0" width="904">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dekantel&#160; Non-absorbable Silk suture</td>
			<td style="width:227px;">Braintree Scientific</td>
			<td style="width:227px;">SUT-S 116</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Media 199 (TC199)&#160;</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">11043-023</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:227px;">Life Technologies</td>
			<td style="width:227px;">21063-045</td>
			<td style="width:227px;">No phenol red as this interferes with fluorescence</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">N-acetylcysteine</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;"></td>
			<td style="width:227px;">Use at 10mM in media</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:227px;">Small animal vascular cathether: Physiocath</td>
			<td style="width:227px;">Data Sciences International</td>
			<td style="width:227px;">277-1-002</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 4400 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-4</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 20,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-20</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:227px;">FITC-Dextran 70,000 MW</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:227px;">FD-70</td>
			<td style="width:227px;"></td>
		</tr>
	</tbody>
</table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

La barrera epitelial es la primera defensa innata del tracto gastrointestinal y regula selectivamente el transporte desde el lumen a los compartimentos de tejido subyacente, lo que restringe el transporte de moléculas más pequeñas a través del epitelio y casi prohíbe completamente el transporte macromolecular epitelial. Esta selectividad se determina por la capa de gel mucoso, lo que limita el transporte de moléculas lipófilas y tanto los receptores apicales y apretado complejos de proteínas de unión del epitelio. Modelos in vitro de cultivo de células del epitelio son convenientes, pero como modelo, que carecen de la complejidad de las interacciones entre la microbiota, mucosa-gel, el epitelio y el sistema inmunológico. Por otra parte, la evaluación in vivo de la absorción intestinal o la permeabilidad se puede realizar, pero estos ensayos miden la absorción gastrointestinal en general, sin ninguna indicación de especificidad de sitio. Ex vivo usando ensayos de paso de &quot;sacos intestinales&quot;; son un método rápido y sensible de la medición de ya sea la integridad intestinal en general o el transporte comparativo de una molécula específica, con la ventaja adicional de especificidad de sitio intestinal. Aquí se describe la preparación de sacos intestinales para estudios de permeabilidad y el cálculo de la aparente permeabilidad (app P) de una molécula a través de la barrera intestinal. Esta técnica se puede utilizar como un método de evaluación de la absorción del fármaco, o para el examen disfunción de la barrera epitelial regional en modelos animales de la enfermedad gastrointestinal.

Este protocolo puede ser usado para examinar los cambios regionales en función de la barrera intestinal en modelos animales de enfermedad gastrointestinal. Al medir el flujo de una sonda paracelular a través de la superficie de la mucosa a diferentes áreas del tracto gastrointestinal 7, la integridad de las uniones estrechas epiteliales se puede evaluar. Además, mediante la variación de la naturaleza de la sonda paracelular por tamaño (Figura 2) o hidrof...

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Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo intestinal Sacs para evaluar la permeabilidad de la mucosa en modelos de enfermedad gastrointestinal

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

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Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

20.9K vistas.  University of Newcastle. This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Video: Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

Un<em&gt; In vivo</em&gt; Ensayo de prueba de permeabilidad Vasos Sanguíneos

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Investigación

Medicina

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Modelos De Interacción Gen-Ambiente Para Desenmascarar Los Mecanismos De Susceptibilidad En La Enfermedad De Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

Adaptación videofluoroscópicos Swallow Métodos de Estudio Humanos para detectar y caracterizar disfagia en modelos murinos de enfermedades

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

En vivo y ex vivo para estudiar Cáncer de ovario metastásico colonización de Estructuras Vía lugar en Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

El uso de ultra-alta MRI Field en Modelos pequeño roedor de la enfermedad renal poliquística para<em&gt; En Vivo</em&gt; Fenotipificación de Drogas y Vigilancia

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

El uso de una temperatura píldora ingerible telemétrico para evaluar la temperatura gastrointestinal durante el ejercicio

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Evaluación de la función renal en modelos de ratón de la enfermedad Glomerular

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

En Vivo Photolabeling de las células en el Colon para evaluar potencial migratorio de las células hematopoyéticas en ratones neonatales

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Ex Vivo intestinal Sacs para evaluar la permeabilidad de la mucosa en modelos de enfermedad gastrointestinal

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