Le présent travail a été soutenu par le Programme de l&#39;Union européen IST (Projet &quot;INTREPID - une réalité virtuelle multi-capteur intelligent système portable pour le traitement des phobies&quot; - IST-2002-507464).

Le protocole clinique pour le traitement du trouble anxieux généralisé (TAG) est programmé simultanément à l&#39;intérieur et à l&#39;extérieur du bureau du thérapeute. Programme de relaxation est une combinaison de «Formation relaxation musculaire progressive» (PMRT), la procédure classique décrite par Jacobsen, qui consiste à tendre et de détendre les groupes musculaires différents tout en prenant note des sensations contrastées, et &quot;L&#39;imagerie guidée» du programme. Dans notre imagerie de formation est remplacé par le repère visuel d&#39;un environnement de détente virtuel. Dans le bureau du thérapeute. Les patients participent à 8 séances de formation avec le thérapeute, répartis en quatre semaines (2 séances par semaine). Chaque séance commence et s&#39;arrête avec une évaluation clinique qui donne la mesure de l&#39;anxiété du patient. Plan de séances dans le bureau du thérapeute: <ol><li> Évaluation clinique initiale de l&#39;état du patient; </li><li> Le patient est relié à des biocapteurs qui enregistrent son / ses paramètres physiologiques (conductance de la peau, la fréquence cardiaque, la respiration). Une mesure de base de ces paramètres est enregistré pendant 3 minutes; </li><li> Le patient porte un écran monté sur la tête relié à un PC et les poignées d&#39;un joystick; </li><li> Le patient commence à explorer l&#39;environnement virtuel: une belle île tropicale face à l&#39;océan. Le patient, après le récit enregistré par le thérapeute, atteint l&#39;île en bateau et de l&#39;explorer. Suite à un sentier qui guide lui / elle à travers l&#39;île, le patient arrive au point de départ, où différents panneaux indiquent les directions dans les zones cibles différentes. Dans chacun de ces domaines, un exercice de relaxation est fourni; au cours de cette formation, en suivant les indications données par le guide vocal, le patient essaie de se détendre lui / elle-même tandis que certains éléments de l&#39;environnement virtuel sont modifiés directement par son / ses paramètres physiologiques enregistrés en temps réel. Ainsi, le patient reçoit une rétroaction immédiate de son / son niveau d&#39;activation (comme dans les techniques de biofeedback traditionnelle), mais avec la valeur supplémentaire donnée par l&#39;environnement virtuel qu&#39;il / elle est immersive à explorer. Les zones cibles sont liés à: <ul><li> Campfire: paramètres physiologiques de contrôle de l&#39;intensité du feu, de sorte que la réduction des résultats du patient activation physiologique sur une réduction du feu jusqu&#39;à ce qu&#39;il s&#39;éteigne; </li><li> Vagues: paramètres physiologiques de contrôle des vagues jusqu&#39;à l&#39;océan devient complètement calme; </li><li> Cascade: la réduction de l&#39;excitation physiologique correspond à une réduction de l&#39;intensité de courant jusqu&#39;à ce que l&#39;eau cesse de tomber. </li><li> Gazebo: différemment des autres, cet exercice est personnalisable par le patient, qui peut choisir des mots ou des images liées à lui / elle personnelle des événements stressants. Cette technique sert un programme d&#39;inoculation du stress, et le patient peut éprouver de la réduction de la taille-fading-flottante de l&#39;élément choisi - selon son / sa capacité à se détendre lui / elle-même; </li></ul></li><li> Une fois terminé la séance de la réalité virtuelle, les paramètres physiologiques sont enregistrés à nouveau en état de repos; </li><li> Final d&#39;évaluation clinique de l&#39;état du patient. </li></ol> BUREAU À L&#39;EXTÉRIEUR DE Thérapeute: En vue d&#39;améliorer l&#39;efficacité de la formation et à augmenter les effets de la relaxation, les patients auront la pratique des techniques de relaxation devant le bureau du thérapeute en éprouvant un scénario à domicile. Ceci est réalisé en présentant le même environnement virtuel expérimentés au cours de la thérapie sur un appareil mobile. L&#39;appareil mobile est connecté via Bluetooth à un ensemble de biocapteurs qui comprend la fréquence cardiaque et la conductance de la peau. Cet équipement est assez petit et facile à utiliser pour être manipulé de manière autonome par le patient (voir fig. 1). Le patient est nécessaire pour former les capacités de relaxation au moins une fois par jour pendant toute la durée du traitement (un mois), suivant le plan de traitement prévu par le thérapeute. Les résultats représentatifs Parce que les deux biofeedback et la réalité virtuelle sont connus pour être efficaces pour induire la relaxation, le principal résultat attendu est que d&#39;une combinaison de ces deux méthodes est plus efficace dans la réduction de l&#39;anxiété chez les patients affectés par le trouble d&#39;anxiété généralisée, que les deux techniques utilisées individuellement. <img src="/files/ftp_upload/1554/Fig1.jpg" alt="Figure 1" /> Figure 1: GSR / HR module de biocapteurs.

<ol>
	<li>Arntz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+therapy+versus+applied+relaxation+as+treatment+of+generalized+anxiety+disorder.">Cognitive therapy versus applied relaxation as treatment of generalized anxiety disorder.</a> Behav Res Ther. 41 (6), 633-633 (2003).</li><li>Siev, J., Chambless, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+treatment+effects:+cognitive+therapy+and+relaxation+for+generalized+anxiety+and+panic+disorders.">Specificity of treatment effects: cognitive therapy and relaxation for generalized anxiety and panic disorders.</a> Journal of consulting and clinical psychology. 75 (4), 513-513 (2007).</li><li>Jacobsen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progressive relaxation. , (1938).</li><li>Villani, D., Riva, F., Riva, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+technologies+for+relaxation:+the+role+of+presence.">New technologies for relaxation: the role of presence.</a> International journal of Stress Management. 14 (3), 260-260 (2007).</li></ol>

The authors have nothing to disclose.

Notre hypothèse est que la combinaison de la réalité virtuelle et le biofeedback qui modifie directement l&#39;environnement virtuel, par rapport à la réalité virtuelle seul, se traduira par un assouplissement plus vite et mieux apprendre. Si oui, ce nouvel instrument créé pour traiter les troubles anxieux pourraient être appliquées et testées, même dans d&#39;autres pathologies liées à l&#39;anxiété.

the use of biofeedback in clinical virtual reality: the intrepid project

Trouble anxieux généralisé (TAG) est un trouble psychiatrique caractérisé par une anxiété constante et non spécifiques qui interfère avec la vie quotidienne des activités. Sa prévalence est élevée dans la population générale et les limites sévères qu&#39;elle cause, soulignent la nécessité de trouver de nouvelles stratégies efficaces pour la traiter. Ensemble, avec les traitements cognitivo-comportementale, la relaxation représente une approche utile pour le traitement du TAG, mais il a la limitation qu&#39;il est difficile d&#39;être appris. Le projet vise INTREPID à mettre en œuvre un nouvel instrument pour traiter les troubles anxieux et de tester son efficacité clinique dans la réduction de l&#39;anxiété symptômes liés. L&#39;innovation de cette approche est la combinaison de la réalité virtuelle et le biofeedback, de sorte que le premier est directement modifiée par la sortie de la seconde. De cette façon, le patient est mis au courant de ses réactions à travers la modification de certaines caractéristiques de l&#39;environnement VR en temps réel. En utilisant des exercices mentaux du patient apprend à contrôler ces paramètres physiologiques et l&#39;utilisation de la rétroaction fournie par l&#39;environnement virtuel est capable de mesurer sa réussite. L&#39;utilisation supplémentaire d&#39;appareils portables, tels que PDA ou téléphones intelligents, permet au patient d&#39;effectuer à la maison, individuellement et de manière autonome, les mêmes exercices expérimentés dans le bureau du thérapeute. L&#39;objectif est d&#39;ancrer le protocole a appris dans un contexte de vie réelle, afin de renforcer la capacité des patients à traiter leurs symptômes. Le résultat attendu est une meilleure et plus rapide d&#39;apprentissage des techniques de relaxation, et donc une efficacité accrue du traitement si l&#39;on compare avec les traditionnels protocoles cliniques.

Watch this Scientific Journal Video about The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project at JoVE.com

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Le Projet sur la Réalité Virtuelle intelligente multi-capteur du système pour le traitement des troubles anxieux (Intrepid) vise à développer une multi-capteurs sensibles au contexte du système de réalité virtuelle pour le traitement des troubles anxieux.

L&#39;utilisation du biofeedback dans la Réalité Virtuelle clinique: Le projet INTREPID

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

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15.1K vues.  Istituto Auxologico Italiano. Le Projet sur la Réalité Virtuelle intelligente multi-capteur du système pour le traitement des troubles anxieux (Intrepid) vise à développer une multi-capteurs sensibles au contexte du système de réalité virtuelle pour le traitement des troubles anxieux.

Vidéo: The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

Évaluation du stress psychophysiologiques utilisant le biofeedback

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Recherche

Biologie

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification de la M. magneticum Strain AMB-1 Protein magnétosome Associated MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

L&#39;utilisation de chémostats dans microbienne biologie des systèmes

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

PCR en temps réel basée sur une sonde Approches de mesure quantitative des microARN

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

Utilisation des Biocapteurs pour quantifier enzymatique endogène ATP ou H<sub&gt; 2</sub&gt; O<sub&gt; 2</sub&gt; Dans le rein

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Haute résolution Time-lapse d&#39;imagerie et d&#39;analyse automatisée des microtubules Dynamics Living ombilicale humaine Cellules endothéliales de veine

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...