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.

Les différentes procédures expérimentales et l&#39;euthanasie sont approuvés par le comité d&#39;éthique institutions d&#39;accueil et par les organismes de réglementation appropriés. L&#39;euthanasie est normalement effectuée en utilisant CO 2 asphyxie suivie par dislocation cervicale. 1. Dispositif <ol><li> Machine de la partie principale de la base de l&#39;appareil à partir d&#39;un bloc solide de résine acétyl-haute densité, tel que décrit dans ce manuscrit. A chaque extrémité de créer 3 mm de haut-élevé bandes, et une partie machine hors de ces 2,5 mm bosquets de prendre les tiges (voir la figure 2 et la figure 3). </li><li> Assurez-barres de l&#39;acier inoxydable d&#39;un diamètre de 2,4 mm avec des extrémités arrondies. REMARQUE: Le cadre principal du couvercle a été usiné sur plaque de Duralumin. Un nombre limité de dispositifs finis sont disponibles à partir de Dr. Goodlad. </li></ol> 2. Utilisation du dispositif <ol><li> Disséquer l&#39;intestin grêle et le côlon à partir de la cavité abdominale à l&#39;aide de ciseauxet doigts gantés. Retirez le mésentère en le tenant avec des pinces courbes et en tirant doucement à l&#39;écart de l&#39;intestin. </li><li> Rincer les intestins avec du phosphate froide saline tamponnée (PBS, pH 7,4) à l&#39;aide d&#39;une pipette de type Gilson, qui avait été coupé à son extrémité la plus large de sorte qu&#39;il puisse tenir sur un raccord seringue en plastique de 10 ou 20 ml luer. </li><li> Disposez l&#39;intestin grêle (SB) sur une feuille de papier absorbant et diviser en trois parties égales en longueur (proximale, moyenne et distale - SB1, SB2 et SB3) en utilisant des ciseaux. Retirer le caecum et de jeter le côlon. </li><li> Faire plusieurs très petites coupures dans les segments intestinaux avec des ciseaux pour permettre au fluide d&#39;échapper et de placer une feuille de serviette en papier sur les intestins et doucement courir un doigt sur les préparatifs pour faire sortir le fluide et le sécher. </li><li> Soulever les sections et insérer les tiges en acier inoxydable qui ont été précédemment mouillés par trempage dans un bêcher de solution saline tamponnée au phosphate. NOTE: L&#39;intestin peut être enconverti si désiré (tourné à l&#39;envers). </li><li> Étiqueter un morceau de carton ou de papier filtre coupe à la taille pour tenir dans la base de l&#39;appareil. Crayon est la meilleure, car elle est affectée par la quasi-totalité des solutions. Date de l&#39;autopsie, code expérimental et le numéro d&#39;identification des animaux sont suggérées. </li><li> Placez la carte ou papier filtre dans la base de l&#39;appareil. Insérer les tiges et les intestins dans les fentes de la base de l&#39;appareil. Assurez-vous que l&#39;extrémité proximale des segments est positionné de manière standardisée. Les extrémités proximales devraient être près les étiquettes de cartes. </li><li> Placez la partie supérieure de l&#39;appareil sur la base. Utilisez les barres obliques pour guider une lame de scalpel pour couper longitudinalement les sections. NOTE: Il aide si le tissu est doucement et soigneusement tenue avec un doigt pour veiller à ce qu&#39;il ne se déplace pas avec le couteau. </li><li> Retirer la partie supérieure de l&#39;appareil. Retirez délicatement le papier et tissus filtre (toujours sur leurs tiges). REMARQUE: Un morceau de carton rigide placés sous le filpapier ter aidera levage. </li><li> Légèrement humide (avec un doigt ganté trempé dans PBS) les segments. Rouler la tige, côté à l&#39;autre d&#39;ouvrir l&#39;intestin et l&#39;étaler à plat. Le tissu sera ensuite adhérer au papier filtre. </li><li> Examiner visuellement les préparatifs pour des lésions macroscopiques. </li><li> Transférer le tissu adhéré au papier filtre pour un bain peu profond (ou boîte à sandwich) contenant fixateur. NOTE: Après le tissu adhère à la papier filtre ou à la carte, il restera fermement attaché. Une large gamme de fixateurs peut être utilisé pour conserver la préparation. Il est recommandé que le liquide de Carnoy doit être utilisé pour l&#39;étude des proliférations cellulaires intestinales 6, ce qui est idéal pour la fixation des meilleures méthodes de prolifération cellulaire; cependant la formaline ou d&#39;autres fixateurs peuvent également être utilisés si d&#39;autres paramètres sont nécessaires. </li><li> Après fixation (généralement 3 h), transférer les préparatifs dans 70% d&#39;éthanol. Les échantillons peuvent ensuite être stockés jusqu&#39;à utilisation. Les préparations cun être étudié en visage et / ou utilisés pour la préparation des échantillons histologiques pour H &amp; E ou d&#39;autres techniques de coloration histologique, immunohistochimique et / ou hybridation in situ. REMARQUE: fixation prolongée peut réduire la réactivité immunohistochimique et / ou l&#39;hybridation in situ réactivité. Boîtes à sandwich polypropylène sont idéales pour stocker un grand nombre de préparations. </li><li> Si nécessaire les tissus fixés à partir des différents segments intestinaux peut être transformé en «roulés» et colorées avec toute la gamme des méthodes histologiques 6, y compris ceux décrits ci-dessus. Pour faire un &#39;roll suisse »les segments intestinaux sont retirés du papier filtre, enroulé autour d&#39;un bâtonnet à cocktail ou un cure-dent, fixé avec une broche entomologique et traitée pour la cire intégrant 6. </li><li> Si nécessaire, tacher le tissu en vrac avec du bleu de méthylène pour visualiser foyers de cryptes aberrantes 7. </li><li> Note les préparations fixées auloisirs. </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.

Le dispositif accélère grandement le processus de sorte que deux opérateurs peuvent autopsie complète d&#39;une souris dans 6 min. Cela inclura un contrôle visuel pour les lésions macroscopiques évidentes et de pesage et fixant les principaux organes. Il permettra également aux opérateurs de rincer, Blot et pèsent l&#39;estomac, le caecum, l&#39;intestin grêle et du côlon et de préparer l&#39;ensemble des supports. Le tissu aplati peut être fixé très rapidement et ainsi éviter des artefacts de dégradat...

La souris et la souris génétiquement modifiée 1-3 sont un modèle inestimable pour l&#39;étude de diverses pathologies cancéreuses en particulier et de sa relation à l&#39;inflammation dans le tractus gastro-intestinal. Cela nécessite souvent la préparation de supports entiers de l&#39;intestin grêle et / ou du côlon. La préparation des montures entières peut être fait avec une paire de ciseaux de décalage de précision, mais il peut prendre environ 20 minutes par souris 4. Ce ne sont pas très pratique pour des études comparatives, qui nécessitent une quantité importante de souris (disons 10 par groupe soumis à une analyse statistique). En outre, la longueur de temps nécessaire augmente le risque de dégradation tissulaire. Une tentative pour atténuer ce problème a conduit à l&#39;essai de diverses aides à la préparation de coupe. Le premier a été basé sur une série de plaques de métal, avec des bosquets de coupe, mais les plaques occulté les tissus et la coupe était donc difficile à contrôler. Un conventional crayon à six faces finalement fourni l&#39;inspiration pour le dispositif de coupe, comme nous pourrions alors voir comment une forme de demi-crayon ferait un bon guide pour un scalpel (Figure 1). Cela a abouti à la conception d&#39;un cadre avec quatre guides de coupe 5. L&#39;efficacité de ce dispositif a été spectaculaire que les préparatifs de la figure 4 démontrent la différence entre la préparation de ciseaux et de la préparation de l&#39;appareil. Les sommets des dispositifs original a été construit à partir de plusieurs morceaux de métal, mais ceux plus tard, et le modèle actuel a été usiné sur un morceau solide de duralumin. La base a été usinée en résine haute densité plus haute qualité. Le dispositif est avéré être d&#39;une grande utilité pour la préparation de supports entiers de intestin de la souris car il accélère grandement et améliore la qualité et la cohérence des préparatifs, qui ont un large éventail d&#39;applications.

<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

Préparation montures entières de l&#39;intestin grêle et le côlon de la souris pour l&#39;analyse ou la quantification ultérieure peut être longue et difficile. Nous décrivons l&#39;utilisation d&#39;un dispositif simple de couper et les intestins &#39;roll&#39; de la souris pour préparer rapidement les préparatifs entiers de montage de qualité supérieure et uniforme à ce qui peut être réalisé à la main. Le dispositif comprend une base qui contient 4 tiges en acier inoxydable et une partie supérieure, qui sert de guide de coupe. Les tiges sont insérés dans le lumen de l&#39;intestin grêle [divisé en tiers] et le côlon. Les tiges et les échantillons sont ensuite placés sur un morceau de papier ou de carton filtre dans les fentes de retenue dans la base de l&#39;appareil. La partie supérieure du dispositif est alors positionné et sert de guide de coupe. Les deux sections inclinées dans le centre de la pièce supérieure sont utilisés pour guider un couteau ou un scalpel et les intestins coupés longitudinalement sur la partie supérieure des tiges. Une fois les sections intestinales ont été coupés, le supérieur est retiré et la carte, tissue et les tiges délicatement retiré de l&#39;appareil et placé sur le banc. Les tiges sont ensuite roulées délicatement sur le côté pour aplatir et coller les segments intestinaux sur la pièce sous-jacente de papier ou de carton filtre. La préparation finale peut ensuite être examinée et fixée ou stockée pour une analyse ultérieure. Les préparatifs sont très précieux pour l&#39;étude des changements dans les modèles intestinales normales ou génétiquement modifiées souris. Les préparations ont été utilisées pour l&#39;étude et la quantification des effets de l&#39;inflammation (colite), des dommages, des lésions pré-cancéreuses (foyers de cryptes aberrantes (ACF) et mucine foyers (MDF) appauvri) et des polypes ou des tumeurs.

Les polypes sont faciles à identifier sous un stéréomicroscope avec une source de lumière froide à l&#39;autre illuminer le tissu (Figure 4). Il est utile de marquer la carte avec les marques de crayon quand un polype est observée afin que la notation peut être discuté plus tard et différents opérateurs peut parvenir à un accord. Le tissu peut également être teinté vrac [au bleu de méthylène] pour identifier les foyers de cryptes aberrantes 8 ou mucine foyers appauvri (MDF) <s...

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 dispositif simple d&#39;élaborer rapidement entiers Monts de l&#39;intestin de souris

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

19.4K vues.  National Institutes of Health. The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

Vidéo: 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&amp;#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.

Mesure précise et simple de la cytokine pro-inflammatoire IL-1β en utilisant un dosage de sang total Stimulation

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

Recherche

Médecine

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.

Une méthode simple d&#39;intubation poumon de souris

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.

L&#39;isolement et l&#39;aide de sections d&#39;encéphalopathie mésentérique artère et la veine comme les essais biologiques pour tester vasoactivité dans l&#39;intestin grêle

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 Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Une simple critique de taille fémorale défauts modèle chez la souris

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

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.

Une procédure mécanique simple pour créer Limbal Carence de cellules souches dans la souris

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.

Guidée par échographie intracardiaque Injection des cellules souches mésenchymateuses d’augmenter le retour vers l’intestin pour une utilisation dans des modèles murins de maladies intestinales inflammatoires expérimentale

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.

Suraccumulation induite par le Ileectomy de Bile dans l’intestin de souris

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.

Mécanismes sous-jacents Gut sécrétion de l’Hormone à l’aide de l’isolé perfusé intestin grêle de Rat

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.

Méthode simple modifiée pour l’induction d’un infarctus du myocarde chez la souris

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.

Application de la tomographie par cohérence optique à un modèle murin de rétinopathie

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