El presente trabajo fue apoyado por el programa IST de la Unión Europea (Proyecto &quot;INTREPID - Una Realidad Virtual Inteligente Multi-sensor del sistema usable para el tratamiento de fobias&quot; - IST-2002 hasta 507.464).

El protocolo clínico para el tratamiento del Trastorno de Ansiedad Generalizada (TAG) se ha programado al mismo tiempo dentro y fuera de la oficina del terapeuta. Programa de relajación es una combinación de &quot;Entrenamiento de relajación muscular progresiva&quot; (PMRT), el procedimiento clásico descrito por Jacobsen, que consiste en tensar y relajar los diferentes grupos musculares al mismo tiempo que toma nota de las sensaciones de contraste, y el programa de &quot;imágenes guiadas&quot;. En nuestro imaginario de formación se sustituye por la señal visual de un entorno virtual de relax. Dentro de la oficina del terapeuta. Los pacientes participan en ocho sesiones de entrenamiento con el terapeuta, distribuidos en cuatro semanas (2 sesiones por semana). Cada sesión comienza y termina con una evaluación clínica que da la medida de la ansiedad del paciente. Plan de sesiones en el consultorio del terapeuta: <ol><li> La evaluación clínica inicial del estado del paciente; </li><li> El paciente es conectado con biosensores que registrar su / sus parámetros fisiológicos (conductancia de la piel, la frecuencia cardíaca, la respiración). Una medida habitual de estos parámetros se registra durante 3 minutos; </li><li> El paciente lleva una pantalla montada en la cabeza conectado con un PC y se ocupa de una palanca de mando; </li><li> El paciente comienza a explorar el entorno virtual: una hermosa isla tropical frente al océano. El paciente, después de la narración grabada por el terapeuta, llega a la isla en barco y explorar. Después de un camino que guía a él / ella a través de la isla, el paciente llega al punto de partida, donde los diferentes paneles indican la dirección a las zonas de destino diferente. En cada una de estas áreas un ejercicio de relajación es siempre, durante este entrenamiento, siguiendo las indicaciones dadas por la voz guía, el paciente trata de relajar él / ella misma, mientras que algunos elementos del entorno virtual son directamente modificado por él / ella registró los parámetros fisiológicos en tiempo real. Por lo tanto, el paciente recibe una retroalimentación inmediata de su / su nivel de activación (como en las técnicas de biofeedback tradicional), pero con el valor adicional dado por el entorno virtual que él / ella es immersively explorar. Las áreas objetivo están obligados a: <ul><li> Campfire: los parámetros fisiológicos de control de la intensidad del fuego, por lo que la reducción de los resultados del paciente activación fisiológica en una reducción del fuego hasta que se apague; </li><li> Olas: los parámetros fisiológicos de control de las olas hasta que el océano se convierte en completa calma; </li><li> Cascada: la reducción de la activación fisiológica corresponde a una reducción de la intensidad de corriente hasta que el agua deja de caer. </li><li> Gazebo: diferente a los otros, este ejercicio es personalizable por parte del paciente, que pueden elegir las palabras o imágenes relacionadas con él / ella los acontecimientos estresantes personal. Esta técnica sirve como un programa de inoculación de estrés, y el paciente puede experimentar la reducción en el tamaño de la decoloración de flotación del elemento seleccionado - en función de su / su capacidad de relajar él / ella; </li></ul></li><li> Una vez completada la sesión de realidad virtual, los parámetros fisiológicos se registran nuevamente en condición de reposo; </li><li> Evaluación final del estado clínico de la paciente. </li></ol> OFICINA terapeuta fuera: en el fin de mejorar la eficacia de la formación y aumentar los efectos de la relajación, los pacientes a practicar técnicas de relajación frente a la oficina del terapeuta, disfrutando de un escenario de Atención Domiciliaria. Esto se realiza mediante la presentación del mismo entorno virtual experimentada durante el tratamiento en un dispositivo móvil. El dispositivo móvil se conecta mediante Bluetooth a una serie de biosensores, que incluye el ritmo cardíaco y la conductancia de la piel. Este equipo es lo suficientemente pequeño y fácil de usar que se manejan de manera autónoma por parte del paciente (ver fig. 1). El paciente tiene la obligación de entrenar las habilidades de relajación al menos una vez al día durante toda la duración del tratamiento (un mes), siguiendo el plan de tratamiento proporcionado por el terapeuta. Resultados representante Debido a que tanto el biofeedback y la realidad virtual se sabe que son efectivos para inducir la relajación, el principal resultado esperado es que una combinación de estos dos métodos es más efectivo en la reducción de la ansiedad en los pacientes afectados por el trastorno de ansiedad generalizada, que las dos técnicas utilizadas de forma individual. <img src="/files/ftp_upload/1554/Fig1.jpg" alt="Figura 1" /> Figura 1: GSR / HR módulo de biosensores.

<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.

Nuestra hipótesis es que la combinación de realidad virtual y biofeedback que modifica directamente el entorno virtual, en comparación con la realidad virtual solo, se traducirá en una mejor relajación y un aprendizaje más rápido. Si es así, este nuevo instrumento creado para tratar los trastornos de ansiedad pueden ser aplicados y probados, incluso en otras patologías relacionadas con la ansiedad.

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

Trastorno de ansiedad generalizada (TAG) es un trastorno psiquiátrico caracterizado por una constante ansiedad y no específico que interfiere con las actividades de la vida diaria. Su alta prevalencia en la población general y las graves limitaciones que provoca, señalan la necesidad de encontrar nuevas estrategias eficaces para su tratamiento. Junto con los tratamientos cognitivo-conductual, relajación representa un enfoque útil para el tratamiento del TAG, pero tiene la limitación de que es difícil que aprender. El proyecto está dirigido INTREPID para poner en práctica un nuevo instrumento para el tratamiento de trastornos relacionados con la ansiedad y para probar su eficacia clínica en la reducción de la ansiedad relacionada con los síntomas. La novedad de este enfoque es la combinación de la realidad virtual y el biofeedback, de modo que la primera es directamente modificado por la salida de la segunda. De esta manera, el paciente es consciente de sus reacciones a través de la modificación de algunas de las características del entorno de realidad virtual en tiempo real. El uso de ejercicios mentales que el paciente aprende a controlar estos parámetros fisiológicos y el uso de la información proporcionada por el entorno virtual es capaz de medir su éxito. El uso complementario de los dispositivos portátiles, como PDA o teléfonos inteligentes-, permite que el paciente realice en casa, en forma individual y autónoma, los mismos ejercicios con experiencia en la oficina del terapeuta. El objetivo es fijar el protocolo de aprender en un contexto de vida real, por lo que mejora la capacidad del paciente para hacer frente a sus síntomas. El resultado esperado es una mejor y más rápido aprendizaje de técnicas de relajación, y por lo tanto un aumento de la eficacia del tratamiento en comparación con los protocolos tradicionales de clínicos.

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

El Proyecto de la Realidad Virtual Inteligente Multi-sensor del sistema para el tratamiento de la ansiedad relacionada con los trastornos (INTREPID) tiene por objeto el desarrollo de un multi-sensor sensible al contexto del sistema de realidad virtual para el tratamiento de los trastornos relacionados con la ansiedad.

El uso de la biorretroalimentación en Realidad Virtual Clínica: El Proyecto de INTREPID

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

the-use-biofeedback-clinical-virtual-reality-intrepid

Research

JoVE Journal

Biology

15.1K vistas.  Istituto Auxologico Italiano. El Proyecto de la Realidad Virtual Inteligente Multi-sensor del sistema para el tratamiento de la ansiedad relacionada con los trastornos (INTREPID) tiene por objeto el desarrollo de un multi-sensor sensible al contexto del sistema de realidad virtual para el tratamiento de los trastornos relacionados con la ansiedad.

Video: 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.

Evaluación psicofisiológica de estrés uso de 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 ...

Investigación

Biología

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.

El uso de SC1 (Pluripotin) para apoyar MESC auto-renovación en la ausencia de LIF

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.

La purificación de la M. magneticum Tensión AMB-1 magnetosomas proteína asociada 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.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

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.

El uso de quimiostatos en Microbiana Biología de Sistemas

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.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

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 tiempo real basada en sondeos Enfoques para la medición cuantitativa de 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 ...

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.

El uso de biosensores para cuantificar enzimática endógena ATP o H<sub&gt; 2</sub&gt; O<sub&gt; 2</sub&gt; En el riñón

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.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

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