This work was supported by NIH HL-10342

REMARQUE: Tous les protocoles d&#39;animaux ont été approuvés par le Comité Université soin et l&#39;utilisation des animaux Johns Hopkins. 1. Préparation des animaux <ol><li> Préparer 6 souris C57BL / 6 de contrôle pour la mesure DFCO, en les anesthésier avec la kétamine et de la xylazine tel que décrit à l&#39;étape 2.3 ci-dessous. </li><li> Préparer tous les autres souris avec les différentes pathologies pulmonaires indiqués dans le tableau 1 en utilisant le même mode opératoire que pour les témoins. Les détails spécifiques nécessaires pour établir chacun de ces modèles se trouvent dans les références pertinentes. Les souris témoins et ceux dans les autres cohortes pathologiques sont tous les 6 à 12 semaines d&#39;âge. </li></ol> 2. Mesure de Diffusion Facteur pour le monoxyde de carbone (DFCO) <ol><li> Mettre en place le module de chromatographie en phase gazeuse fourni avec la machine à mesurer les pics de l&#39;azote, de l&#39;oxygène, le néon, et le monoxyde de carbone. Pour cette utilisation de l&#39;application uniquement les données de néon et de CO. NOTE: Cet instrument utilise un s moléculairesIEVE colonne avec de l&#39;hélium comme gaz porteur, avec un 12,00 um film 320,00 ID um et 10 m de longueur. La colonne de Chromatographie a un volume de 0,8 ml, mais on utilise 2 ml d&#39;assurer la compensation adéquate de la tubulure de liaison avec l&#39;échantillon. </li><li> Au début de chaque journée expérimentale, avant de prendre les mesures des échantillons provenant des souris, prendre un échantillon de 2 ml directement d&#39;un sac de mélange gazeux contenant environ 0,5% Ne, 0,5% de CO, et d&#39;équilibrer l&#39;air, et utiliser cet exemple pour calibrer le chromatographe en phase gazeuse. </li><li> Anesthésier les souris avec de la kétamine (90 mg / kg) et de xylazine (15 mg / kg), et de confirmer l&#39;anesthésie par l&#39;absence de mouvement réflexe. Appliquer une pommade vétérinaire sur les yeux pour prévenir la sécheresse. Tracheostomize les souris avec une canule d&#39;aiguille de stub (18 G chez les adultes ou 20 G chez les très jeunes souris). NOTE: Le DFCO est terminée en moins de 10 min après l&#39;anesthésie et avant toute ventilation mécanique ou d&#39;autres procédures. </li><li> Chez les souris de plus de 6 semaines d&#39;âge, utiliser une seringue de 3 ml to retirer 0,8 ml de gaz dans le sac de mélange de gaz. Connectez la seringue à la canule trachéale et gonfler rapidement le poumon. Utiliser un métronome, comptez 9 sec, puis rapidement retirer les 0,8 ml (air expiré). </li><li> Diluer cette retirés 0,8 ml air expiré à 2 ml avec l&#39;air ambiant, laisser reposer pendant au moins 15 secondes. Puis injecter la totalité de l&#39;échantillon dans le chromatographe en phase gazeuse pour analyse. </li><li> Bien que l&#39;analyse de ce premier échantillon DFCO, gonfler le poumon de souris avec une seconde 0,8 ml dans le sac de mélange de gaz, puis traiter cet échantillon identique au premier échantillon. La moyenne des deux mesures DFCO. NOTE: Pour les mesures dans des souris aussi jeunes que 2 semaines d&#39;âge, utiliser un volume de 0,4 ml, 0,8 ml est depuis un volume trop important de faire des mesures dans les poumons de souris très jeunes. Il est préférable d&#39;utiliser le volume de 0,8 ml pour les souris âgées de 6 semaines, et que si le volume de 0,4 ml est nécessaire pour certaines souris, il devrait être utilisé systématiquement pour toutes les souris de la cohorte à l&#39;étude. </li><li> Calculer DFCOcomme une - (CO 9 / CO c) / (Ne 9 / Ne c), où c et 9 indices se rapportent aux concentrations de gaz d&#39;étalonnage et les gaz injectés retirées après un temps de maintien 9 souffle sec, respectivement. </li><li> Analyser et comparer les différences avec une analyse de variance à une voie et d&#39;évaluer le niveau avec la correction de Tukey pour les comparaisons multiples dans toutes les souris de la cohorte de signification. Considérez p &lt;0,05 de valeur aussi importante. NOTE: Toutes les souris utilisées ici ont fait partie des études expérimentales impliquant plusieurs mesures ultérieures de la ventilation pulmonaire, la mécanique, le lavage du poumon, ou l&#39;histologie, qui ne sont pas signalés ici. En outre, puisque le procédé est le même dans tous les modèles expérimentaux comme l&#39;a fait ci-dessus dans les souris témoins, seuls les résultats des différents modèles pathologiques sont présentés. De plus amples informations sur ces modèles est présentée dans le tableau supplémentaire. </li><li> Euthanasier les animaux par anesthe profondesurdosage tic suivie par dislocation cervicale ou décapitation. Le cas échéant, éliminer les cellules et / ou tissus pulmonaires des souris mortes pour la poursuite ou de la transformation biologique et l&#39;analyse histologique. </li></ol>

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D., 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=Intercellular+adhesion+molecule-1+contributes+to+pulmonary+oxygen+toxicity+in+mice:+role+of+leukocytes+revised.">Intercellular adhesion molecule-1 contributes to pulmonary oxygen toxicity in mice: role of leukocytes revised.</a> Lung. 170 (5), 267-279 (1992).</li><li>Reinhard, 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=Inbred+strain+variation+in+lung+function.">Inbred strain variation in lung function.</a> Mamm Genome. 13 (8), 429-437 (2002).</li><li>Sabo, J. P., Kimmel, E. C., Diamond, L. <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+the+Clara+cell+toxin,+4-ipomeanol,+on+pulmonary+function+in+rats.">Effects of the Clara cell toxin, 4-ipomeanol, on pulmonary function in rats.</a> J Appl Physiol. 54 (2), 337-344 (1983).</li><li>Depledge, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiration+and+lung+function+in+the+mouse,+Mus+musculus+(with+a+note+on+mass+exponents+and+respiratory+variables).">Respiration and lung function in the mouse, Mus musculus (with a note on mass exponents and respiratory variables).</a> Respir Physiol. 60 (1), 83-94 (1985).</li><li>Depledge, M. H., Collis, C. H., Barrett, 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+technique+for+measuring+carbon+monoxide+uptake+in+mice.">A technique for measuring carbon monoxide uptake in mice.</a> Int J Radiat Oncol Biol Phys. 7 (4), 485-489 (1981).</li><li>Fallica, J., Das, S., Horton, M. R., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Carbon+Monoxide+Diffusing+Capacity+in+the+Mouse+Lung.">Application of Carbon Monoxide Diffusing Capacity in the Mouse Lung.</a> J Appl Physiol. 110 (5), 1455-1459 (2011).</li><li>Chaudhary, N., Datta, K., Askin, F. B., Staab, J. F., Marr, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+transmembrane+conductance+regulator+regulates+epithelial+cell+response+to+Aspergillus+and+resultant+pulmonary+inflammation.">Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.</a> Am J Respir Crit Care Med. 185 (3), 301-310 (2012).</li><li>Foster, W. M., Walters, D. M., Longphre, M., Macri, K., Miller, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+the+measurement+of+mucociliary+function+in+the+mouse+by+scintigraphy.">Methodology for the measurement of mucociliary function in the mouse by scintigraphy.</a> J Appl Physiol. 90 (3), 1111-1117 (2001).</li><li>Yildirim, A. O., 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=Palifermin+induces+alveolar+maintenance+programs+in+emphysematous+mice.">Palifermin induces alveolar maintenance programs in emphysematous mice.</a> Am J Respir Crit Care Med. 181 (7), 705-717 (2010).</li><li>Collins, S. L., Chan-Li, Y., Hallowell, R. W., Powell, J. D., Horton, 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=Pulmonary+vaccination+as+a+novel+treatment+for+lung+fibrosis.">Pulmonary vaccination as a novel treatment for lung fibrosis.</a> PLoS One. 7 (2), e31299 (2012).</li><li>Alessio, F. 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J., 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=The+clinical+course+of+patients+with+idiopathic+pulmonary+fibrosis.">The clinical course of patients with idiopathic pulmonary fibrosis.</a> Ann Intern Med. 142 (12 Pt 1), 963-967 (2005).</li><li>Zhou, L., 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=Correction+of+lethal+intestinal+defect+in+a+mouse+model+of+cystic+fibrosis+by+human+CFTR.">Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.</a> Science. 266 (5191), 1705-1708 (1994).</li></ol>

Aucun conflit d&#39;intérêts, et rien à divulguer.

Dans le présent travail, nous avons défini une nouvelle mesure pour quantifier la capacité des poumons de souris de gaz échanger. Cette métrique est analogue à la capacité de diffusion, une mesure clinique courant qui mesure la fonction primaire du poumon, ce est-à sa capacité à échanger des gaz. La capacité de diffusion est la seule mesure fonctionnelle du poumon qui peut être fait facilement et rapidement chez les souris et les humains. Pour la détection de la maladie du poumon chez la souris, un objecti...

La souris est maintenant l&#39;animal primaire utilisé pour modéliser une variété de maladies pulmonaires. Pour étudier les mécanismes qui sous-tendent ces pathologies, les méthodes phénotypiques sont nécessaires qui peuvent quantifier les changements pathologiques il. Bien qu&#39;il existe de nombreuses études sur les souris où la mécanique de ventilation sont mesurés, ces mesures sont généralement sans rapport avec les évaluations standard de la fonction pulmonaire chez l&#39;homme fait normalement. Ce est malheureux, car la capacité d&#39;effectuer des mesures équivalentes dans les souris et les sujets humains peut faciliter la traduction des résultats dans des modèles murins de maladies humaines. Une des mesures les plus communs et facilement faites sur des sujets humains est la capacité de diffusion du monoxyde de carbone (DLCO) 1,2, mais cette mesure n&#39;a que rarement été fait dans les modèles de souris. Dans les études où il a été rapporté 3-7, il ya eu aucune étude de suivi, en partie parce que les procédures sont souvent lourds ou peuvent REQUIRE équipements complexes. Une autre approche consiste à utiliser une méthode de CO réinspiration dans un système de l&#39;état d&#39;équilibre, qui a l&#39;avantage d&#39;être capable de mesurer la diffusion de CO chez des souris conscientes. Cependant cette méthode est très lourd, et les résultats peuvent varier avec le niveau de la ventilation de la souris ainsi que O 2 et CO 2 concentrations 8,9. Ces difficultés semblent avoir empêché l&#39;utilisation systématique de la capacité de diffusion pour détecter des pathologies pulmonaires chez la souris, en dépit de ses nombreux avantages. Pour contourner les problèmes de mesure de la capacité de diffusion chez les souris, les détails d&#39;un moyen simple de mesurer dans des souris ont été récemment rapporté 10. La procédure élimine le difficile problème de prélèvement des gaz alvéolaire non contaminée par échantillonnage rapidement un volume égal à l&#39;ensemble du gaz inspiré. Ce procédé aboutit à une mesure très reproductible, appelée coefficient de diffusion pour le monoxyde de carbone (DFCO), qui est sensible à un hôte de pathologic changements dans le phénotype du poumon. Le DFCO est donc calculé comme 1 - (CO 9 / CO c) / (Ne 9 / Ne c), où le c et 9 indices se rapportent aux concentrations des gaz d&#39;étalonnage injectés et les gaz retirés après un temps d&#39;apnée 9 sec, respectivement. DFCO est une variable sans dimension, qui varie entre 0 et 1, avec une réflexion absorption complète de tous CO, 0 et reflétant pas l&#39;absorption de CO. Dans cette présentation, nous montrons comment faire cette mesure de la capacité de diffusion, et comment il peut être utilisé pour documenter les changements dans presque tous les modèles de maladies pulmonaires de la souris existants, y compris l&#39;emphysème, la fibrose, une lésion pulmonaire aiguë et les infections virales et fongiques.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gas Chromatograph</td>
			<td>Inficon</td>
			<td>Micro GC Model 3000A</td>
			<td>Agilent makes a comparable model</td>
		</tr>
		<tr>
			<td>18 g Luer stub needle</td>
			<td>Becton Dickenson</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr>
			<td>3 mL plastic syringe</td>
			<td>Becton Dickenson</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr>
			<td>Polypropylene gas sample bags</td>
			<td>SKC</td>
			<td>1 or 2 liter capacity works well</td>
			<td>Other gas tight bags will work well</td>
		</tr>
		<tr>
			<td>Gas tank, 0.3% Ne,0.3% CO, balance air; (size ME)</td>
			<td>Airgas, Inc</td>
			<td>Z04 NI785ME3012</td>
			<td>This is the standard mixture used for DLCO in humans</td>
		</tr>
		<tr>
			<td>25 TCID50/mouse of influenza virus A/PR8 diluted in phosphate buffered saline.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine pancreatic elastase</td>
			<td>Elastin Products, Owensville, MO</td>
			<td></td>
			<td>5.4 U</td>
		</tr>
		<tr>
			<td>Bleomycin</td>
			<td>APP Pharmaceuticals, Schaumburg, IL</td>
			<td></td>
			<td>0.25 U</td>
		</tr>
		<tr>
			<td>Escherichia coli LPS8</td>
			<td>Sigma L2880</td>
			<td></td>
			<td>3 &#956;g/g body weight; O55:B5</td>
		</tr>
		<tr>
			<td>Aspergillus fumigatus (isolate Af293) conidia were collected from mature colonies grown on potato dextrose agar.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

phenotyping mouse pulmonary function in vivo with the lung diffusing capacity

La souris est maintenant l&#39;animal primaire utilisé pour modéliser une variété de maladies pulmonaires. Pour étudier les mécanismes qui sous-tendent ces pathologies, les méthodes phénotypiques sont nécessaires qui peuvent quantifier les changements pathologiques. En outre, de fournir la pertinence de translation les modèles de souris, de telles mesures devraient être des tests qui peuvent facilement être faites chez les humains et les souris. Malheureusement, dans la littérature actuelle quelques mesures phénotypiques de la fonction pulmonaire ont une application directe pour l&#39;homme. Une exception est la capacité de diffusion du monoxyde de carbone, qui est une mesure qui se fait couramment chez l&#39;homme. Dans le présent rapport, nous décrivons un moyen de mesurer rapidement et simplement cette capacité de diffusion chez les souris. La procédure implique une brève inflation des poumons avec un gaz témoin dans une souris anesthésiée, suivie d&#39;un temps d&#39;analyse des gaz 1 min. Nous avons testé la capacité de cette méthode pour détecter plusieurs pathologies pulmonaires, y compris l&#39;emphysème, la fibrose, une lésion pulmonaire aiguë et de la grippe etinfections pulmonaires fongiques, ainsi que la surveillance maturation pulmonaire chez les jeunes chiots. Les résultats montrent une diminution significative dans toutes les pathologies du poumon, ainsi que l&#39;augmentation de la capacité de diffusion de la maturation pulmonaire. Cette mesure de la capacité de diffusion du poumon fournit donc un test de la fonction pulmonaire qui a une large application avec sa capacité à détecter des changements structurels phénotypiques avec la plupart des modèles pulmonaires pathologiques existants.

La figure 1 montre les mesures DFCO des souris adultes dans les groupes A, B, C, D, E et F. Il y avait une diminution significative avec les deux infections à Aspergillus et à la grippe, ainsi que des diminutions significatives de la fibrose, emphysème, et aiguë modèles de lésion pulmonaire. La figure 2 montre les changements dans le développement du Groupe G en DFCO au fil du temps que l&#39;âge de la souris 2-6 semaines. Il y avait une augmentation légère mais significative ...

Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Phénotypage Souris fonction pulmonaire<em&gt; In Vivo</em&gt; Avec le Capacité diffusion pulmonaire

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

phenotyping-mouse-pulmonary-function-vivo-with-lung-diffusing

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Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

10.5K vues.  Johns Hopkins University Bloomberg School of Public Health. We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Vidéo: Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Recherche

Biologie

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caractérisation des isolés, ventilés et instrumentés poumons de souris perfusées avec un flux pulsatile

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

L&#39;examen BCL-2 Fonction famille avec de grandes vésicules unilamellaires

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culture de cellules humaines épithéliales nasales à l&#39;interface Air Liquide

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Isolement des mitochondries à partir des quantités minimes de souris muscle squelettique pour les mesures à haut débit microplaques respiratoires

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

La protéine c-FOS détection immunohistologique: Un outil utile comme marqueur des voies centrales impliquées dans les réponses physiologiques spécifiques<em&gt; In Vivo</em&gt; et<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Etude in vivo de Human Interaction Endothelial-péricytes Utilisation du Gel Matrice Plug - test chez la souris

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Mesure de la rigidité de<em&gt; Ex Vivo</em&gt; Souris aortes Utiliser Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

Induction de l'échec ventriculaire droit par constriction pulmonaire d'artère et évaluation de la fonction ventriculaire droite chez les souris

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

Mesure in vivo de la fonction musculaire extensrice du genou chez la souris

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...