L&#39;induction de l&#39;inflammation expérimentale: <ol><li> Un coup de soleil au premier degré est induite chez les non-tanné la peau d&#39;une cuisse avec l&#39;aide d&#39;un étalonnage, source de rayons ultraviolets B (Saalmann Multitester CFF LT 499, Saalmann GmbH, Hereford, Allemagne). Le coup de soleil est induite 24 heures avant de commencer une expérience microdialyse en appliquant trois fois la quantité de rayons UVB nécessaire pour causer des rougeurs complète de la peau exposée, ou trois fois la dose minimale érythémateuse (DME). Le MED est déterminé dans chaque sujet avant le lancement du projet. Livraison précise d&#39;énergie est assurée avec un UV-mètre (12 RM, le Dr Grobel UV-Electronic GmbH, Ettlingen, Allemagne). </li></ol> Cathéter et la collecte de l&#39;échantillon: <ol><li> Préparer une aire de repos avec des gants stériles, 1 porte-aiguille stérile, une seringue 1cc embout Luer avec une jauge de 23 (G) l&#39;aiguille, une seringue avec une aiguille 1cc 30G, 1% de lidocaïne, tampons d&#39;alcool, 2x2 compresses de gaze stériles, cathéters microdialyse attachée à une fin à une aiguille 23G 4cm (dialyse cutanée, Erlangen, Allemagne), et des tubes de 1,5 ml de collecte Microcent (E &amp; K Scientific, Santa Clara, CA.) avec une solution inhibiteur de protéase 4μl par échantillon 100 ul sur glace sèche. solution inhibiteur de la protéase est faite par instruction du fabricant en utilisant des comprimés inhibiteur de protéase cocktail (Roche Diagnostics GmbH, Mannheim, Allemagne). </li><li> Préparer une solution à 1% d&#39;albumine humaine dans la solution de Ringer lactate et de l&#39;attirer dans la seringue embout Luer 1cc, en veillant à l&#39;absence de bulles d&#39;air restant dans la seringue. </li><li> Charger la seringue avec le 1% d&#39;albumine humaine enrichie solution de Ringer lactate dans le berceau (microdialyse multi-seringue en rack, Harvard Apparatus, South Natick, MA) monté sur une pompe à perfusion de haute précision (pompe 22, Harvard Apparatus, South Natick, MA ). Assurez-vous que la barre de la pompe à perfusion de conduite de la seringue est en plein contact avec le piston de la seringue. Réglez le débit de la pompe après avoir vérifié que le diamètre de seringue a été correctement entré dans la pompe. Taux de perfusion couramment utilisés varient entre 2 à 4μl/min. </li><li> Définir la zone que vous prévoyez pour recueillir votre échantillon à partir. Lors de l&#39;échantillonnage d&#39;une zone créée artificiellement enflammés, tels que les coups de soleil décrit ci-dessus, le plan sur l&#39;insertion du cathéter au bord de l&#39;insolation. </li><li> Nettoyez une petite zone sur les bords opposés de la lésion, ou sur le site vous avez l&#39;intention de recueillir votre échantillon à partir, avec l&#39;alcool. Ces zones seront nettoyées à l&#39;insertion prévu et les points de sortie pour le cathéter. Injecter juste assez lidocaïne intra-cutanée (en témoigne blanchissement de la peau à l&#39;injection) sur ces sites afin de créer une bulle très petite taille (maximum de 2 mm). L&#39;injection de quantité limitée tels des échanges intra-cutanée de lidocaïne à l&#39;entrée et de sortie du cathéter de microdialyse ne devrait pas modifier les résultats de l&#39;essai biologique, mais ne font l&#39;insertion du cathéter significativement moins douloureuse pour les sujets de l&#39;étude. </li><li> Utiliser des techniques aseptiques, insérer le cathéter de microdialyse avec l&#39;aide d&#39;une aiguille 23G attachés sur toute la longueur de la zone de la peau explorées. La profondeur de l&#39;insertion est à l&#39;interface entre derme et l&#39;hypoderme. Insertion à une profondeur suffisante peut être vérifié optiquement. Comme l&#39;aiguille et le cathéter attachés sont déplacés vers l&#39;avant, l&#39;aiguille doit provoquer un peu de place-plissement de la peau ainsi que quelques blanchissement de la peau. Aucun saignement devrait se produire. Si le saignement ne se produit, cela indique que l&#39;aiguille se trouve plus profond que prévu et la profondeur d&#39;insertion doit être ajustée. Une fois que l&#39;aiguille a été poussé à travers le point de sortie de la peau, rincer les cathéters avec 1% d&#39;albumine enrichi solution de Ringer lactate jusqu&#39;à ce que vous voyez gouttelettes le long de la longueur du cathéter. Ceci est fait en prenant soin d&#39;insérer la fin aiguille d&#39;une seringue (aiguille 23G) dans l&#39;extrémité distale du cathéter de microdialyse et injectant lentement solution. </li><li> Après le rinçage du cathéter tirez doucement sur l&#39;aiguille pour nourrir le cathéter fixé par la peau. Une manipulation délicate est nécessaire parce que les cathéters sont de petit diamètre, et par conséquent fragile. Tirez le cathéter jusqu&#39;à ce qu&#39;il soit complètement à travers la peau, c&#39;est à dire la base du tube en plastique intégrant plus de l&#39;extrémité distale du cathéter touche la peau au point d&#39;entrée. Comme mentionné ci-dessus, peu ou pas de saignements à la fin de la procédure confirme le positionnement du cathéter optimal. </li><li> Fixez l&#39;extrémité distale du cathéter à la pointe de l&#39;aiguille de la seringue 23G de repos dans le rack de la pompe à perfusion. Les soins sont nécessaires pour éviter la perforation de l&#39;aiguille à travers la paroi du cathéter, ce qui causerait des fuites potentielles. </li><li> Couper l&#39;aiguille à l&#39;extrémité proximale du cathéter. </li><li> En utilisant ¼ de pouce ruban rose, fixez le tube de 1,5 ml de collecte préparés Microcent à la peau à proximité du site de sortie du cathéter. Placez l&#39;extrémité du cathéter dans le tube de collecte pour permettre la collecte des microdialysat. Démarrer la pompe de perfusion à r prédéterminésmangé et par intermittence vérifier que le volume livré et recueillies correspondent. Dans le cas où les volumes ne correspondent pas, la fuite du site d&#39;entrée de peau doit être exclu (peut parfois être résolu en tirant sur la partie du cathéter qui est incorporé par les tubes en plastique plus étroitement sur le site d&#39;entrée de peau). Alternativement, un cathéter peut être nécessaire de remplacer si les volumes livrés et recueillis ne correspondent pas. </li><li> Une fois l&#39;échantillonnage terminé, les échantillons sont placés sur la glace sèche jusqu&#39;à ce stockage à -80 ° C. </li></ol>

<ol>
	<li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, 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=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation and administration of a COX-inhibitor: A microdialysis study.</a> Pain. 139, 15-27 (2008).</li></ol>

The authors have nothing to disclose.

L&#39;utilisation combinée des techniques de microdialyse et la technologie immuno multiplex est un précieux temps réel, in vivo bioessai humaine offrant un aperçu des événements biochimiques dans les tissus d&#39;intérêt. Combiné avec des tests comportementaux tels que tests de douleur, la méthode permet d&#39;étudier les interactions complexes des produits biochimiques médiation douleur et l&#39;inflammation. La méthode est prometteuse pour 1) l&#39;identification de biomarqueurs spécifiques de tissu médiation douleur et l&...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Microdialysis catheter</td>
<td>Tool</td>
<td>Dermal Dialysis</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Pump 22</td>
<td>Tool</td>
<td>Harvard Apparatus</td>
<td>552219</td>
<td>&nbsp;</td>
<tr>
<td>Saalmann Multitester SBB LT 400</td>
<td>Tool</td>
<td>Saalmann GmbH</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>RM-12 IP-65 Radiometer</td>
<td>Tool</td>
<td>Dr. Grobel UV Elektronik GmbH</td>
<td>82120000/81112100</td>
<td>&nbsp;</td>
<tr>
<td>Complete Proteinase Inhibitor</td>
<td>Reagent</td>
<td>Roche Bioscience</td>
<td>11836145001</td>
<td>&nbsp;</td>
<tr>
<td>Microcent tube</td>
<td>Tool</td>
<td>E&K Scientific</td>
<td>380150-X</td>
<td>&nbsp;</td>
<tr>
<td>Albumin 5%</td>
<td>Reagent</td>
<td>ZLB Behring</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Lactated Ringer</td>
<td>Reagent</td>
<td>Hospira Inc</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Xylocaine-MPF</td>
<td>Other</td>
<td>Astra Zeneca</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Syringe 1mL</td>
<td>Tool</td>
<td>Becton Dickinson Co</td>
<td>309602</td>
<td>&nbsp;</td>		</tbody>
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human in-vivo bioassay for the tissue-specific measurement of nociceptive and inflammatory mediators

Ce bio-essai in vivo humaine peut être utilisée pour étudier des volontaires humains et des patients. Les échantillons sont prélevés à partir de sites pertinents, tels que les tissus de la peau via des cathéters insérés microdialyse aseptique (dialyse cutanée, Erlangen, Allemagne). Illustré dans cet exemple est la collection de liquide interstitiel de la peau enflammée expérimentalement chez des volontaires humains. Prélèvement d&#39;échantillon peut être combiné avec d&#39;autres tests expérimentaux. Par exemple, l&#39;évaluation simultanée de produits biochimiques libérés localement et de la sensibilité subjective à un stimulus douloureux de la peau enflammée expérimentalement fournit l&#39;essentiel biochimique et comportementale lien d&#39;identifier des biomarqueurs de la douleur et l&#39;inflammation. Présenté test dans l&#39;organisme humain vivant permet de comprendre mécanistes dans les processus spécifiques de tissus sous-jacents des douleurs et / ou l&#39;inflammation. La méthode est également bien adapté pour examiner l&#39;efficacité des interventions existantes ou nouvelles - telles que de nouveaux candidats médicaments - ciblant le traitement des affections douloureuses et / ou inflammatoire. Cet article propose une description détaillée sur l&#39;utilisation des techniques de microdialyse pour recueillir le liquide interstitiel lésion cutanée expérimentale enflammées des sujets de l&#39;étude humaine. Les échantillons de liquide interstitiel sont généralement traitées avec l&#39;aide de tests immunologiques multiplexe array billes permettant titrant jusqu&#39;à 100 analytes dans des échantillons de faible volume de 50 microlitres.

Watch this Scientific Journal Video about Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators at JoVE.com

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

Une technique est présentée pour la collection in-vivo d&#39;échantillons de liquide interstitiel des tissus à partir de sites pertinents (peau ici, expérimentalement enflammés) pour la mesure de médiation biochimiques douleur et l&#39;inflammation.

L&#39;homme in vivo des essais biologiques pour la mesure de tissu-spécifique des médiateurs inflammatoires et nociceptifs

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

human-vivo-bioassay-for-tissue-specific-measurement-nociceptive

Research

JoVE Journal

Biology

10.2K vues.  Stanford University School of Medicine. Une technique est présentée pour la collection in-vivo d'échantillons de liquide interstitiel des tissus à partir de sites pertinents (peau ici, expérimentalement enflammés) pour la mesure de médiation biochimiques douleur et l'inflammation.

Vidéo: Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In vivo la micro-circulation de mesure dans le muscle squelettique par voie intra-vitale microscopie

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Recherche

Biologie

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

Recueillir et mesurer les médiateurs nociceptifs et inflammatoires dans les plaies chirurgicales

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Mesure du pH vacuolaire et cytosolique<em&gt; In Vivo</em&gt; En cellule de levure Suspensions

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo modélisation du génome humain morbide utilisant Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Un bioessai simple pour l&#39;évaluation des facteurs de croissance endothélial vasculaire

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap Junctional intercellulaire Communication: Un biomarqueur fonctionnelle pour évaluer les effets néfastes des substances toxiques et les toxines, et bienfaits pour la santé des produits naturels

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Blessure à la gorge aortique murine: une efficacité<em&gt; In vivo</em&gt; Modèle de prolifération des cellules musculaires lisses et fonction endothéliale

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Utilisation de Caenorhabditis elegans pour dépister les interactions tissulaires spécifiques des chaperons

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

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 ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Fixation et isolement rapides in vivo de complexes translationnels à partir de cellules eucaryotes

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...