Ce travail a été soutenu par le NIH K99 HL09222601 et le S &amp; R Fondation Ryaji Uneo Award décerné à MNH.

1. L&#39;anesthésie <ol><li> Anesthésier les souris avec de l&#39;isoflurane peut faciliter la manipulation des animaux et à la retenue avant d&#39;administrer l&#39;anesthésie intraperitaneally. En utilisant un anesthésique par inhalation peut être ou ne pas être approprié pour une étude particulière: d&#39;abord déterminer l&#39;impact de l&#39;utilisation de l&#39;isoflurane, ou tout autre anesthésique, sur l&#39;étude des poumons proposé. </li><li> Anesthetize animaux à l&#39;isoflurane. La vidéo montre l&#39;utilisation d&#39;une chambre d&#39;isoflurane vaporisateur fixé à 2% d&#39;isoflurane mélangé avec l&#39;oxygène. Si vous n&#39;avez pas une chambre de l&#39;isoflurane, vous pouvez également utiliser des techniques standard de l&#39;exposition ouverte à goutte. Joindre des rats dans une cloche de verre (ou d&#39;autres non poreux contenant) avec un grillage monté sur le fond. Pour un tampon de gaze placée sous le treillis métallique, appliquez un mélange de 30% v / v isoflurane dans du propylène glycol. L&#39;utilisation de la maille assure que l&#39;animal ne vienne pas en contact avec le tampon imbibé d&#39;isoflurane, ce qui pourrait causer des irritations de la peau et le surdosage potentiel. Après courte exposition à l&#39;isoflurane, l&#39;animal doit être anesthésié et ne répondra pas à un réflexe de redressement lorsque le pot est incliné et ne répondra pas, après pincement de l&#39;orteil. </li><li> Souris plus calmes avec une injection intra-péritonéale d&#39;un mélange de xylazine et de kétamine. (Il est de bonne pratique lors de la réalisation des injections intrapéritonéales de reculer pour s&#39;assurer que des portions de l&#39;intestin ou d&#39;autres organes vitaux n&#39;ont pas été pénétrés). Mélanger 150 uL xylazine (100 mg / mL) avec 1000 kétamine uL (100 mg / mL) en 8850 uL de phosphate stérile à 0,9% saline tamponnée (PBS). Pour les souris, la dose sera d&#39;environ 80 pi par 10 g de poids corporel. Les concentrations finales sera de 10 mg / kg de xylazine et de 100 mg / kg de kétamine. Les animaux doivent être gardés au sec et isolés pour prévenir la perte excessive de chaleur corporelle. Pommade ophtalmique peut être appliqué sur les yeux pour éviter le dessèchement de la cornée. </li></ol> 2. Préparation de la zone chirurgicale <ol><li> Rasage de la zone où l&#39;incision chirurgicale sera faite empêche la contamination de la non-préparée domaines. Un rasoir électrique peut se raser de près la fourrure de l&#39;animal. La zone rasée doit être plus grand que le site d&#39;incision. </li><li> L&#39;application ultérieure de crème dépilatoire, comme Nair, va supprimer toutes les fourrures restantes. Appliquer une couche épaisse de crème d&#39;épilation à la zone chirurgicale et laisser agir pendant 3 minutes. Essuyer la fourrure et la crème d&#39;épilation avec une serviette humide. Ne laissez pas la crème épilation au-delà de 10 minutes. </li><li> Utilisez de l&#39;alcool à 70%, ou une solution gommage alternative, pour préparer la zone aseptique chirurgicale. </li></ol> 3. Procédure chirurgicale <ol><li> Position de l&#39;animal anesthésié sur une planche inclinée chirurgicale, ou un support de retenue angulaires. </li><li> Faire une petite incision près de la face antérieure du cou (région de la gorge). </li><li> Disséquer loin le peaucier et antérieure muscles trachéaux, afin de visualiser et accéder aux anneaux de la trachée. </li><li> Typiquement, les volumes d&#39;injection intratrachéale de 3 grammes ul par des poids (environ 30 à 75 volumes uL final). Administrer la quantité appropriée de l&#39;aide d&#39;une instillation sous-cutanée de 1 ml (sous-Q) seringue avec un calibre 30, l&#39;aiguille 5/16-inch. Tenez le côté conique de seringues et parallèle à la trachée, et injecter le volume complet de instillation dans la trachée. </li><li> L&#39;animal peut réagir en haletant après l&#39;instillation de soluté dans les poumons. Présentation de l&#39;air à travers la seringue et en observant si les poumons se dilatent confirmera également les techniques de l&#39;intubation. </li><li> Retirez la seringue de la trachée. </li></ol> 4. Sutures. <ol><li> Saisir sertissage de l&#39;aiguille (partie la plus épaisse de l&#39;aiguille où le matériel de suture est ci-jointe) avec un porte-aiguille. </li><li> Pénètrent dans la peau avec la pointe de l&#39;aiguille à 90 ° environ 1-3 mm Angle loin du bord de la plaie, en fonction de l&#39;épaisseur de la peau. Saisir l&#39;aiguille de quitter après un passage à travers le tissu. Points de suture simple, nouées individuellement, seront efficacement fermer la plaie. </li></ol> 5. Récupération des animaux <ol><li> Les animaux devraient se remettre complètement de l&#39;anesthésie et l&#39;intervention chirurgicale, après 3-24 heures. </li></ol> 6. Les résultats représentatifs Instillation intratrachéale de composé fluorescent, Cy5.5 conjugué à dextrane, montre l&#39;instillation réussie de composé fluorescent dans B6 poumons de souris (figure 1). Le colorant fluorescent est principalement ciblée sur les poumons et est uniformément répartie. Signal fluorescent dans le tractus gastro-intestinal, immédiatement après la procédure indique que l&#39;œsophage a été intubé par inadvertance lors de la procédure (figure 2). Les enquêteurs peuvent également vérifier la livraison ciblée de composés dans le poumon, et de vérifier la distribution de l&#39;instillation, en suivant la procédure en gonflant les poumons et la préparation des tranches de tissu pulmonaire (2, 3) ou fixes histologique des échantillons. <img src="/files/ftp_upload/1941/1941fig1.jpg" alt="Figure 1" /> Figure 1. Fluorescent Cy5.5 signal de co-enregistrées avec des rayons X dans les poumons de souris image de succès inculqué. 1,4 mg Cy5.5 Dextran (à une concentration de 10 ug / uL) a été instillée dans la trachée des poumons de souris B6. Cy5.5 signal n&#39;a été détecté en utilisant un filtre d&#39;excitation 675 nm et 695 nm d&#39;un filtre d&#39;émission. <img src="/files/ftp_upload/1941/1941fig2.jpg" alt="Figure 2" /> Figure 2. Co-enregistrement de signal fluorescent montré avec X-ray superposition de souris mal inculquée. Intubation inapproprié de l&#39;oesophage au cours trachéale des résultats du protocole d&#39;instillation dans Cy5.5 livraison à l&#39;intestin.

<ol>
	<li>Driscoll, K. E., Costa, D. L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H., Schlesinger, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratracheal+instillation+as+an+exposure+technique+for+the+evaluation+of+respiratory+tract+toxicity:+uses+and+limitations.">Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations.</a> Toxicol Sci. 55, 24-35 (2000).</li><li>Helms, M. N., Jain, L., Self, J. L., Eaton, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+regulation+of+epithelial+sodium+channels+examined+in+alveolar+type+1+and+2+cells+patch-clamped+in+lung+slice+tissue.">Redox regulation of epithelial sodium channels examined in alveolar type 1 and 2 cells patch-clamped in lung slice tissue.</a> J Biol Chem. 283, 22875-22883 (2008).</li><li>Helms, M. N., Self, J., Bao, H. F., Job, L. C., Jain, L., Eaton, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+activates+amiloride-sensitive+sodium+channels+in+alveolar+type+I+cells+in+lung+slice+preparations.">Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations.</a> Am J Physiol Lung Cell Mol Physiol. 291, L610-L618 (2006).</li><li>Iyer, S. S., Ramirez, A. M., Ritzenthaler, J. D., Torres-Gonzalez, E., Roser-Page, S., Mora, A. L., Brigham, K. L., Jones, D. P., Roman, J., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidation+of+extracellular+cysteine/cystine+redox+state+in+bleomycin-induced+lung+fibrosis.">Oxidation of extracellular cysteine/cystine redox state in bleomycin-induced lung fibrosis.</a> Am J Physiol Lung Cell Mol Physiol. 296, L37-L45 (2009).</li><li>Li, T., Folkesson, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+alpha-ENaC+inhibits+rat+lung+fluid+absorption+in+vivo.">RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo.</a> Am J Physiol Lung Cell Mol Physiol. 290, L649-L660 (2006).</li><li>Sager, T., Porter, D., Robinson, V., Lindsley, W., Schwegler-Berry, D., Castranova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+to+disperse+nanoparticles+for+in+vitro+and+in+vivo+investigation+of+toxicity.">An improved method to disperse nanoparticles for in vitro and in vivo investigation of toxicity.</a> Nanotoxicol. 1, 118-129 (2007).</li><li>Xu, J., Gonzalez, E. T., Iyer, S. S., Mac, V., Mora, A. L., Sutliff, R. L., Reed, A., Brigham, K. L., Kelly, P., Rojas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+senescence-accelerated+mouse+model+in+bleomycin-induced+lung+injury+suggests+that+bone+marrow-derived+cells+can+alter+the+outcome+of+lung+injury+in+aged+mice.">Use of senescence-accelerated mouse model in bleomycin-induced lung injury suggests that bone marrow-derived cells can alter the outcome of lung injury in aged mice.</a> J Gerontol A Biol Sci Med Sci. 64, 731-739 (2009).</li><li>Yeh, T. F., Lin, H. C., Chang, C. H., Wu, T. S., Su, B. H., Li, T. C., Pyati, S., Tsai, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+intratracheal+instillation+of+budesonide+using+surfactant+as+a+vehicle+to+prevent+chronic+lung+disease+in+preterm+infants:+a+pilot+study.">Early intratracheal instillation of budesonide using surfactant as a vehicle to prevent chronic lung disease in preterm infants: a pilot study.</a> Pediatrics. 121, e1310-e1318 (2008).</li></ol>

Instillations intra-trachéale ont été utilisées dans plusieurs études différentes pour évaluer la toxicité des composés d&#39;essai (6), induisent des lésions pulmonaires alvéolaires (4, 7), remplacer les tensioactifs (8), ainsi que de modifier l&#39;expression des gènes via la livraison de petits oligonucleotides directement dans le poumon (5). Nous explorons actuellement l&#39;utilisation d&#39;instiller 1) fluorescentes redox matériau sensible dans les poumons afin de mesurer le stress oxydatif in vivo d...

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Isoflurane	</td>
<td>&nbsp;</td>
<td>Webster Veterinary Sterling , MA</td>
<td>14043-220-05</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Lloyd Laboratories Shenandoah, IA</td>
<td>&nbsp;</td>
<td>New Animal Drug Application #139-236</td>
</tr>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Bioniche Pharma Lake Forest, IL</td>
<td>67457-001-10</td>
<td>&nbsp;</td>
<tr>
<td>Nair</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Available at drug stores</td>
</tr>
<tr>
<td>1mL SubQ Syringe</td>
<td>&nbsp;</td>
<td>BD Syringe Franklin Lakes, NJ</td>
<td>309597</td>
<td>26 5/8 G</td>
</tr>
<tr>
<td>4-0 Nylon Suture</td>
<td>&nbsp;</td>
<td>Ethicon San Lorenzo, Puerto Rico</td>
<td>1894G.S30</td>
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direct tracheal instillation of solutes into mouse lung

Instillations intratrachéale offrir solutés directement dans les poumons. Cette procédure vise la livraison de l&#39;instillation dans les régions distales du poumon, et est donc souvent incorporées dans des études visant à étudier les alvéoles. Nous fournissons un protocole de survie détaillées pour effectuer des instillations intra-trachéale chez les souris. En utilisant cette approche, on peut cibler la livraison des solutés de test ou des solides (comme les traitements du poumon, tensioactifs, les virus et les oligonucléotides de petite taille) dans le poumon distal. Instillations trachéales peut être la méthode privilégiée, sur les protocoles d&#39;inhalation qui peut ciblent principalement les voies respiratoires supérieures et peut exposer le chercheur à des substances potentiellement dangereuses. En outre, en utilisant le protocole d&#39;instillation trachéale, les animaux peuvent se remettre complètement de la procédure non-invasive. Cela permet de faire suite des mesures physiologiques sur les animaux d&#39;essai, ou à la réinstallation en utilisant le même animal. Le montant de l&#39;instillation introduit dans les poumons doit être soigneusement déterminé et osmotiquement équilibrée pour assurer la récupération des animaux. Typiquement, le volume de 30 à 75 uL instillation peuvent être introduits dans des poumons de souris.

Watch this Scientific Journal Video about Direct Tracheal Instillation of Solutes into Mouse Lung at JoVE.com

Direct Tracheal Instillation of Solutes into Mouse Lung

Instillations intratrachéale offrir solutés directement dans les poumons. Cette procédure vise la livraison de l&#39;instillation dans les régions distales du poumon, et est donc souvent incorporées dans des études visant à étudier les alvéoles. Nous fournissons un protocole de survie détaillées pour effectuer des instillations intra-trachéale chez les souris.

L&#39;instillation trachéale directe des solutés dans les poumons de souris

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

direct-tracheal-instillation-of-solutes-into-mouse-lung

Research

JoVE Journal

Biology

29.0K vues.  Emory University. Instillations intratrachéale offrir solutés directement dans les poumons. Cette procédure vise la livraison de l'instillation dans les régions distales du poumon, et est donc souvent incorporées dans des études visant à étudier les alvéoles. Nous fournissons un protocole de survie détaillées pour effectuer des instillations intra-trachéale chez les souris.

Vidéo: Direct Tracheal Instillation of Solutes into Mouse Lung

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Transfert nucléaire dans des ovocytes de la souris

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

Recherche

Biologie

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

Préparation standardisée des suspensions unicellulaires provenant de tissus pulmonaires de la souris en utilisant les dissociateur gentleMACS

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Culture poumon embryonnaire de la souris, un système pour évaluer les mécanismes moléculaires des Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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 ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Livraison directe de morpholinos MIF Into the otocyste Zebrafish par injection et électroporation affecte le développement oreille interne

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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 ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolement et caractérisation de Hoechst Faible CD45 Négatifs Souris Cellules souches mésenchymateuses du poumon

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Différenciation efficace des cellules souches embryonnaires de souris dans les neurones moteurs

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Génération de souris dérivées de cellules souches pluripotentes induites

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Protein Transfection of Mouse Lung

Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes1. In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice2,3 or viral or non-viral vectors that elevate protein levels via increased gene expression4. Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model5. While conditional transgenics avert problems associated with chronic gene expression6, the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement7. As with transgenics, the use of viral and non-viral vectors is expensive8 and can provoke dose-dependent inflammatory responses that confound results9 and hinder expression10. Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector11,12. Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung13.
Using &beta;-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 &mu;l of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice14. The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity15. Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27&plusmn;4 control vs. 31&plusmn;5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.

Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.

Protéines transfection de poumon de souris

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...