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.

Tutti i lavori degli animali in questo protocollo viene eseguita con la stretta aderenza alle Università di Newcastle comitato etico degli animali procedure approvate. 1. Preparazione degli strumenti, Cultura Media e Piatti <ol><li> Pre-caldo media 199 (TC199) o Dulbecco Modified Eagle Medium (DMEM) supporti a 37 ° C. Pre-ossigenare il supporto facendo gorgogliare con il 95% O 2/5% di CO 2. Verificare che il mezzo ha un pH finale di 7,3. </li><li> Preparare sutura tagliando due sezioni 5 cm per ogni uscita. Loop i punti di sutura in un nodo non chiusa. </li></ol> 2. dissezione e preparazione del tratto gastrointestinale <ol><li> Ritirare solido cibo 12 ore prima di eutanasia. Se lo si desidera, inserire gli animali sugli integratori gel nutrienti durante questo periodo. </li><li> Euthanize topi per overdose pentobarbitone sodio ([200 mg / Kg], iniezione intraperitoneale) seguita da dislocazione cervicale in conformità con l&#39;etica proto istituzionaleCols e spruzzare il 70% di etanolo sul addome e torace. </li><li> Usando una forbice, fare una incisione orizzontale nel mezzo dell&#39;addome ed esporre il peritoneo. </li><li> Procedere per separare e rimuovere il tratto gastrointestinale tagliando il piccolo intestino superiore dallo stomaco al sfintere piloro e taglio crasso al margine anale. Utilizzare una pinza per rimuovere delicatamente il mesentere. Posizionare il tratto intestinale in pre-riscaldato, medio ossigenato. </li><li> Identificare la sezione dell&#39;intestino da valutare per permeabilità (Figura 1) e tagliare questa sezione libera dal resto del tratto intestinale. <ol><li> Al fine di mantenere la coerenza tra animali, misurare le sezioni di duodeno e nel digiuno rispetto allo stomaco, e misurare le sezioni di colon e dell&#39;ileo rispetto al cieco. </li><li> Quando si seleziona segmenti di tessuto, notare la presenza di tessuti linfoidi mucosa-associati, come le placche di Peyer. Questi possono essere identificati come piccolo cenno del capoORME sul lato sierose del lume. </li><li> Usando una siringa da 1 ml, lavare delicatamente il contenuto luminale del segmento intestinale in una capsula di Petri con PBS preriscaldato (37 ° C). Questi contenuti fecali possono essere scartati o conservati a -80 ° C per le analisi future, se lo desideri. </li></ol></li></ol> 3. Preparazione intestinale Sacs <ol><li> Preparare una siringa da 1 ml con un volume di 300 microlitri del composto di prova o molecola. Per l&#39;integrità della mucosa, un 1 mg / ml soluzione di FITC-destrano M.Wt. 4.400 possono essere utilizzati. Le sonde che vanno da 4,400-70,000 Da dimensioni possono essere utilizzati per una maggiore sensibilità. Trovare e montare un piccolo catetere vascolare animali sulla siringa. </li><li> Misura 5 cm dall&#39;apertura del segmento intestinale e legare il segmento completamente chiuse con una sutura-loop a questo punto. Posizionare delicatamente una sutura-loop pre-legata intorno all&#39;apertura dell&#39;intestino e inserire il catetere smussata. Tirare il cappio chiuso in modo da assicurare il segmento intestinalee rilasciare il volume 300 microlitri dalla siringa nell&#39;intestino, assicurando che tutta la soluzione viene iniettata. </li><li> Rimuovere delicatamente il catetere e contemporaneamente tirare il cappio di sutura per fissare la chiusura del sacco intestinale. Tagliare il sacco intestinale sciolto dall&#39;intestino e posto in un tubo da 50 ml riempito con 20 ml di terreno ossigenato, preriscaldata a 37 ° C. </li></ol> 4. la misura della permeabilità <ol><li> Luogo provette coniche contenenti le sacche intestinali in un bagno d&#39;acqua riscaldata impostati su 37 ° C. A 0, 30, 60, 90 e 120 punti min tempo, prelevare un campione di 100 microlitri dalla provetta conica e trasferimento ad una piastra da 96 pozzetti, sostituendo il volume con 100 ml di mezzi freschi in ciascun caso. </li><li> Dopo aver prelevato il campione finale, tagliare sacche aperte al punto di sutura e la lunghezza del segmento, esponendo la superficie mucosa. </li><li> Misurare la lunghezza e la larghezza di ogni segmento intestinale. Se Desirosso, scatto congelare i segmenti e conservare a -80 ° C per le proteine ​​o l&#39;analisi biochimica, o, in alternativa, conservare in soluzione di stabilizzazione RNA per saggi molecolari. </li><li> Costruire una curva standard di diluizioni di registro per le molecole FITC-etichettati da 1 a 1 x 10 -6. </li><li> Misure di campioni e standard per FITC su un lettore di piastre a fluorescenza, FITC di eccitazione / emissione: 495 nm / 519 nm. </li></ol> 5. Calcolo di permeabilità apparente per ogni intestinale individuale Sac <ol><li> Convertire unità di tempo di sec. </li><li> Per ogni punto di tempo, calcolare la concentrazione cumulativa, Q Q t = (C t * V r) + (Q t somma * V s), dove: Q t = concentrazione cumulativa al tempo t C t = concentrazione al tempo t V r = Volume sul lato ricevente Q t somma = Somma di tutte le precedenti Q t V s =volume campionato </li><li> Trama Q in funzione del tempo (T) e calcolare la pendenza: AQ / DT </li><li> Calcolare la permeabilità apparente (P app) P app = (AQ / T) / (A * Co), dove: A = area di tessuto C 0 = concentrazione iniziale </li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari concorrenti.

Qui, abbiamo descritto l&#39;isolamento e la preparazione di sacche intestinali per valutare la funzione barriera della mucosa ex vivo. preparazioni sac intestinali sono principalmente utilizzati nella ricerca farmaceutica, esaminando l&#39;assorbimento di farmaci candidati attraverso l&#39;intestino. Tuttavia, questo test è ugualmente adatta per lo studio della malattia intestinale. La permeabilità intestinale può variare notevolmente da regione a regione e la valutazione specifica del sito di permeabilità...

La barriera epiteliale intestinale del tratto gastrointestinale è una superficie mucosa stimato a 400 m 2 nel umano adulto. Di conseguenza, si è costantemente esposto a sfidare da microbi, farmaci ingeriti, nutrienti e tossine batteriche. L&#39;host non solo deve distinguere tra batteri commensali tollerabili e potenziali patogeni, ma deve impedire queste specie e loro molecole secrete di attraversare la barriera epiteliale, mentre allo stesso tempo permette l&#39;assorbimento dei nutrienti. Pertanto, il ruolo dell&#39;epitelio intestinale è di agire come barriera selettiva al contenuto luminale 1. Questo risultato è ottenuto, in parte, dal sistema di difesa epiteliale innato alla mucosa, che agisce attraverso un sistema biologico reattivo costituito da costitutive e inducibili meccanismi 2. La perdita della funzione barriera epiteliale è una patologia che è caratteristica di un certo numero di malattie gastrointestinali. In vivoesame della funzione di barriera epiteliale può essere valutata attraverso sonda gastrica di una molecola tracciante e la successiva analisi del siero 3. Tuttavia, questa tecnica non offre alcuna indicazione sul sito di disfunzione barriera. In vitro e ex vivo valutazione della resistenza transepiteliale utilizzando sistemi Transwell 3 e ussing rispettivamente camere di 4,5, sono comunemente impiegati come indicatori surrogati di funzione di barriera epiteliale, ma manca la contribuendo malattia fisiologia dei modelli animali 6. In questo protocollo si descrive un modello di preparazione del tessuto ex vivo che permette una valutazione diretta e localizzata dell&#39;integrità intestinale e che può essere utilizzato per valutare la funzione barriera della mucosa a diversi livelli. È importante sottolineare che questa tecnica può essere applicata a modelli animali di malattia, o può essere farmacologicamente manipolato per permettere a fondo interrogatori di disfunzione barriera 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 barriera epiteliale è la prima difesa innata del tratto gastrointestinale e regola selettivamente trasporto dal lume ai compartimenti sottostanti, limitare il trasporto di molecole più piccole attraverso l&#39;epitelio e quasi completamente vieta epiteliale trasporto macromolecolare. Questa selettività è determinata dallo strato di gel mucoso, che limita il trasporto di molecole lipofile ed entrambi i recettori apicali e stretti complessi proteici giunzionali dell&#39;epitelio. Modelli in vitro su colture di cellule dell&#39;epitelio sono convenienti, ma come un modello, non hanno la complessità delle interazioni tra microbiota, muco-gel, epitelio e il sistema immunitario. D&#39;altra parte, la valutazione in vivo dell&#39;assorbimento intestinale o permeabilità può essere eseguita, ma questi test misurare il grado di assorbimento gastrointestinale, senza alcuna indicazione di specificità del sito. Ex vivo saggi permeabilità utilizzando &quot;sacche intestinali&quot;; sono un metodo rapido e sensibile per misurare sia l&#39;integrità intestinale globale o il trasporto comparativa di una molecola specifica, con l&#39;ulteriore vantaggio di specificità del sito intestinale. Qui si descrive la preparazione di sacche intestinali per gli studi di permeabilità e il calcolo della permeabilità apparente (P app) di una molecola attraverso la barriera intestinale. Questa tecnica può essere utilizzata come un metodo di valutazione assorbimento del farmaco, o per esaminare disfunzione barriera epiteliale regionale in modelli animali di malattia gastrointestinale.

Questo protocollo può essere utilizzato per esaminare i cambiamenti regionali in funzione di barriera intestinale in modelli animali di malattie gastrointestinali. Misurando il flusso di una sonda paracellular sulla superficie mucosa in diverse aree del tratto gastrointestinale 7, l&#39;integrità delle giunzioni strette epiteliali può essere valutata. Inoltre, variando la natura della sonda paracellular per dimensione (Figura 2) o idrofobicità (Fig...

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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 intestinale Sacs per valutare mucosa Permeabilità in modelli di malattia gastrointestinale

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 Views.  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; Test per la verifica della nave Permeabilità Sangue

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 ...

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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.

Modelli di interazione gene-ambiente per smascherare i meccanismi di suscettibilità nel morbo di 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.

Adattamento umani videofluoroscopia Rondine metodi di studio per individuare e caratterizzare disfagia in modelli di malattia Murine

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.

Un<em&gt; Ex vivo</em&gt; Modello per studiare Hormone azione in seno umano

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.

In vivo ed ex vivo approcci per studiare il cancro ovarico metastatico colonizzazione delle strutture Lattea Spot in peritoneale 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.

L&#39;uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per<em&gt; In Vivo</em&gt; Fenotipizzazione 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 ...

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.

L&#39;utilizzo di un ingestible telemetrico Temperatura pillola per valutare Gastrointestinale temperatura durante l&#39;allenamento

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.

L&#39;esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

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.

Valutazione della funzione del rene in modelli murini di malattia glomerulare

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.

In Vivo Photolabeling delle cellule del colon per valutare il potenziale migratorio delle cellule ematopoietiche in topi neonatali

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

Ex Vivo intestinale Sacs per valutare mucosa Permeabilità in modelli di malattia gastrointestinale

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Modelli di interazione gene-ambiente per smascherare i meccanismi di suscettibilità nel morbo di Parkinson

Adattamento umani videofluoroscopia Rondine metodi di studio per individuare e caratterizzare disfagia in modelli di malattia Murine

Un

In vivo ed ex vivo approcci per studiare il cancro ovarico metastatico colonizzazione delle strutture Lattea Spot in peritoneale adiposo

L'uso di ultra-alto campo MRI in piccolo roditore modelli di malattia del rene policistico per

L'utilizzo di un ingestible telemetrico Temperatura pillola per valutare Gastrointestinale temperatura durante l'allenamento

L'esaurimento delle cellule di topo da xenotrapianti tumorali umani migliora in modo significativo Analisi A valle delle cellule bersaglio

Valutazione della funzione del rene in modelli murini di malattia glomerulare

In Vivo Photolabeling delle cellule del colon per valutare il potenziale migratorio delle cellule ematopoietiche in topi neonatali