CIBC comunión (JA) y NSERC beca (RZ). Damos las gracias a M. Roch Comeau (Brainsight) por su ayuda con respecto a la cámara de infrarrojos, los seguidores de RM compatibles y soporte hardware. También damos las gracias a Brian Hynes (Hybex Innovations Inc.) que diseñó el brazo multi-articulado para que el titular de la bobina y proporcionó algunas de las cifras que aparecen en el video. Y un agradecimiento especial a todos los técnicos de RM y M. Ferreira del Cerebro Centro McConnell Imágenes del Instituto Neurológico de Montreal que nos ayudó a optimizar el diseño del experimento.

El protocolo se divide en un período de dos días (no necesariamente consecutivos). El día primero consiste en un localizador fMRI compuesto con un anatómicas y funcionales a exploraciones por RM para definir para cada participante las áreas objetivo con TMS. El segundo día consiste en las sesiones de resonancia magnética funcional pre-y post-TMS TMS donde se aplica en el interior del escáner con una bobina especial MR TMS compatible (Magstim Ltd., País de Gales, Reino Unido) y un sistema de estereotaxia sin marco (Brainsight). Este último se utiliza para la posición en tiempo real de la bobina de EMT en áreas corticales relativos a los datos anatómicos y funcionales de cada participante. 1. Localizador Sesión <ul><li> Comience con la adquisición de una imagen anatómica de alta resolución de la participante. </li><li> Entonces, adquirir imágenes funcionales utilizando un gradiente de eco EPI pulso y un paradigma de muestreo escaso en el fin de minimizar cualquier efecto de negrita o enmascaramiento auditivo debido al ruido de escaneo MRI 14,15. En nuestro caso, se lleva a cabo fMRI durante una tarea melodía en la que los participantes tienen que determinar si dos consecutivos Nota 5-melodías son iguales o diferentes 2,16. A la no discriminación auditiva tarea de control también se incluye, en el que los sujetos escuchar dos de igual longitud patrones de cinco notas, todos a la misma altura de C5 y pide que pulse el botón izquierdo tras el segundo estímulo. Los períodos de silencio también se insertan aleatoriamente entre los ensayos de tarea en cada carrera. En total, 72 ensayos se presentan en un orden aleatorio: 24 ensayos de discriminación melodía, 24 ensayos de control auditivo y 24 periodos de silencio, con una duración total de 12 min 16 seg. </li><li> Definir el lugar de estimulación mediante anatómicas y / o funcionales de puntos de referencia. Uno tiene que ser consciente de que TMS está limitado en cuanto a la profundidad de la zona de estimulación debido a la atenuación de la intensidad de campo eléctrico en profundidad, y no se puede esperar a llegar a las zonas más profundas de 3 cm 6,17. Un paso fundamental es el uso de marcas similares para cada parteicipant, que podría ser difícil debido a las diferencias en la anatomía y función entre los participantes. Aquí, nos dirigimos circunvolución Heschl en cada participante, que se encuentra usando ambos puntos de referencia anatómicos y funcionales. Nosotros usamos máscaras de giro Heschl proporcionada por los atlas Harvard-Oxford estructurales ( <a href="http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html" target="_blank">http://www.fmrib.ox.ac.uk/fsl/data/atlas-descriptions.html</a> ) y el objetivo de TMS se define individualmente por el pico de activación en la circunvolución del Heschl 2. Además, también definen la posición de vértice, que se utiliza como punto de control para controlar para efectos no específicos de TMS como artefactos acústicos y somatosensorial. El vértice se define anatómicamente como un punto a medio camino entre el inión y el puente de la nariz, y equidistante de las muescas intertragal derecho e izquierdo. El orden del lugar de estimulación (es decir giro Heschl o vértice) se compensa a través deindividuos. </li></ul> 2. Pre-y Post-TMS-fMRI experimento Pre-TMS fMRI sesión <ul><li> Preparar al participante para ir directamente en el escáner. Esto incluye la remoción de metal y llenar el formulario de TMS y MR de selección. </li><li> Inicie la adquisición de RM con una anatomía y una exploraciones funcionales (idéntica a la realizada en la sesión del localizador, véase la sección 1). </li></ul> Estereotaxia sin marco y TMS en el entorno MRI El sistema de estereotaxia sin marco se compone de una cámara de infrarrojos (Polaris Spectra), algunas herramientas y rastreadores (Brainsight) empleados para el procedimiento de registro y un ordenador. El ordenador se encuentra fuera de la sala del escáner, pero situada en la entrada de la sala del escáner y la puerta del escáner se mantiene abierto durante la aplicación de TMS. Las herramientas y los rastreadores son MR compatible, así como el trípode (hecho en casa) que soporta la cámara de infrarrojos y son therefore utilizado dentro de la sala del escáner. La cámara de infrarrojos no es compatible con MR, y por lo tanto se coloca dentro de la sala del escáner, cerca de la puerta del escáner en aproximadamente dos metros de la superficie del escáner (véase la discusión de procedimiento de seguridad). El sistema TMS estimulador se encuentra en una habitación contigua a la sala del escáner de resonancia magnética. Usamos una bobina de MRI TMS compatible ubicado dentro de la sala del escáner y conectado al sistema de TMS a través de un cable de 7 m por medio de un tubo de filtro de RF. <ul><li> Cargar imágenes anatómicas y funcionales de su participante y los objetivos de estimulación en el paquete de software estereotáctica (Brainsight por ejemplo). Aquí, vamos a tener como objetivo la circunvolución Heschl derecho. </li><li> Después de la adquisición fMRI pre-TMS, retire el cabezal superior MR parte bobina de la bobina de la cabeza de 32 canales (si se utiliza el escáner 3T Siemens y la configuración de la bobina de 32 canales cabeza). </li><li> A continuación, deslice hacia abajo el participante en la superficie del escáner. </li><li> Fije la banda para la cabeza y un conjunto de seguimiento sobre la participcabeza de hormiga. </li><li> Monte el brazo multi-articulado a la superficie del escáner y fijar la bobina TMS MR compatible en los brazos. </li><li> Verificar que todos los rastreadores y la bobina están en el campo de visión de la cámara. Aquí, la cámara está ligeramente desplazada hacia el lado derecho del participante para permitir un seguimiento más fácil de los desplazamientos de la bobina cuando la orientación del hemisferio derecho. </li><li> Calibrar la cabeza del sujeto con las herramientas de estereotaxia (es decir, instrumento de puntero). Esto se hace por coregistering varias marcas en la cabeza del participante (por ejemplo, en nuestro caso la punta de la nariz, el nasión y el trago de ambos oídos) con los mismos puntos de referencia sobre los datos anatómicos. En este procedimiento, se necesitan dos experimentadores, uno cerca de la cabeza del participante para posicionar la herramienta de puntero en la cabeza del participante, y el experimentador otra a la entrada de la sala del escáner para realizar el registro en el ordenador. </li><li> Coloque la bobina MR compatible TMS tangencial a tél cuero cabelludo, y los rastreadores bobina dirigida hacia la cámara de infrarrojos. La bobina está orientado con el mango bobina apuntando hacia atrás y paralelamente a la línea 2. Fijar la posición de la bobina con los tornillos en el brazo multi-articulado. </li><li> En la sala adyacente al escáner MRI, encienda el sistema TMS y comenzar la estimulación. TMS se aplica siguiendo un protocolo patrón, es decir, la estimulación continua explosión theta (CTBS) que consiste en 3 pulsos a 50 Hz, 5 Hz para repetirse a los 40 años. Usamos una intensidad de estimulación fijo (41%) definido por la salida del estimulador 18,19. Elegimos este protocolo, ya que se ha demostrado que modulan la plasticidad cortical para una duración de hasta 30 minutos después de cesar la estimulación en poblaciones sanas 20, (véase la sección de discusión de procedimiento de seguridad). </li></ul> Post-TMS fMRI sesión <ul><li> Una vez que se haya completado la estimulación, es importante para obtener el tema de nuevo en el escáner, tan pronto como sea posible. Retire el TMS bobina de la sala del escáner y retire el brazo multi-articulado. Deslice hacia atrás la cabeza del participante en la bobina de la cabeza MR. Asegúrese de que el escáner está preparado y listo para funcionar. Nuestro consejo es mantener la plataforma corporal elevada durante toda la sesión de TMS, y reducir el número y duración de las exploraciones del localizador a un mínimo. </li><li> Debido a que los efectos de la EMTr son transitorios, la sesión de exploración final debe comenzar con el análisis funcional. Una vez más, se realizó fMRI durante una ejecución de 12-min de la tarea melodía. </li><li> Después de la exploración final se ha completado, termine con una exploración anatómica. </li></ul> 3. Los resultados representativos Los análisis de los datos de la fMRI se llevan a cabo por separado tanto para la sesión de fMRI pre-y post-TMS. En cada sesión de fMRI (es decir, antes y después del TMS-), el contraste entre las melodías y la tarea de control muestra auditiva relacionada con las tareas en la actividad de la izquierda y la derecha Heschl giros, circunvoluciones temporal superior, inferior y frontal gyrus precentral circunvoluciones (Figura 1 A, B). Para evaluar las diferencias entre las sesiones de resonancia magnética funcional pre y post-TMS, llevamos a cabo un análisis de efectos aleatorios de Student para datos apareados t-test. La significación se determinó usando grupos identificados por az&gt; 2 umbral y un umbral de clúster corregido de p = 0,05. Figura 1 C representa el contraste minus post-pre-CTBS para un único participante. Los datos sugieren que CTBS dirigidas a la derecha Heschl giro (círculo negro) induce un aumento de la respuesta fMRI en la corteza contralateral (izquierda) auditivos, incluyendo el giro a la izquierda Heschl. Los cambios en la respuesta fMRI también se encuentran en la circunvolución postcentral izquierda, de izquierda ínsula, y bilateralmente en la corteza occipital lateral. Sin embargo, ningún cambio significativo en la respuesta fMRI se ve bajo la bobina. Además, similar combinado TMS-fMRI protocolo se repite para estimular el vértice (sitio de control). Comparación de las sesiones previas y posteriores fMRI con CTBS aplicados sobre el vértice no mostró significant efecto (datos no mostrados). <img src="/files/ftp_upload/3985/3985fig1.jpg" alt="Figura 1"/> Figura 1. Análisis de las distintas pre-TMS datos de la fMRI (A), post-TMS fMRI datos (B) y post-menos pre-TMS datos fMRI (C). A. Resultados de la discriminación melodía contraste menos auditivas ensayos de control para un único participante en la sesión de fMRI pre-TMS (A) y en el período de post-TMS fMRI (B). De izquierda a derecha: vista axial, coronal y sagital. En ambos (A) y (B), la bobina TMS está apuntando a la derecha Heschl giro (círculo negro) situado en x = 54, y = -13, z = 1 (MNI152 espacio estándar). Para ambas sesiones fMRI pre-y post-TMS, se mostrarán las coordenadas en x = -54, y = -13, z = 1 (MNI152 espacio estándar) para mostrar los cambios en el hemisferio izquierdo en el lugar de estimulación (es decir, el giro a la derecha Heschl ). C. Resultados de la post-contraste minus pre-TMS sesiones utilizando fMRI de Student prueba de t pareada.

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Se describe un protocolo que combina TMS sin conexión y resonancia magnética funcional para estudiar la organización funcional de la corteza auditiva. En las secciones siguientes, vamos a discutir los aspectos metodológicos a tener en cuenta al llevar a cabo este enfoque. Adquisición y calendario de post-TMS sesión de fMRI Orden de adquisición exploraciones y contrapeso de las sesiones de resonancia magnética funcional antes y después de TMS- <...

<table border="1"><tbody><tr><td> Nombre del Material </td><td> Tipo </td><td> Empresa </td></tr><tr><td> La estimulación magnética transcraneal </td><td> Magstim súper Rapid2 estimulador, Rapid-2 Plus Módulo Uno </td><td> Magstim Ltd., País de Gales, Reino Unido </td></tr><tr><td> Bobina para la estimulación magnética </td><td> MRI-compatible 70 mm figura-de-ocho-coil </td><td> Magstim Ltd., País de Gales, Reino Unido </td></tr><tr><td> La resonancia magnética </td><td> 3-T escáner Siemens Trio, 32-canal Head Coil </td><td> Siemens, Inc., Alemania </td></tr><tr><td> Estereotaxia sin marco </td><td> Brainsight </td><td> Rogue Research Inc., Montreal, Canadá </td></tr><tr><td> Sistema de medición óptico </td><td> Polaris Spectra </td><td> Northern Digital Inc, Ontario, Canadá </td></tr><tr><td> Multi-articulado brazo de sujeción de bobina </td><td> Estándar </td><td> Hybex innovaiones Inc., Anjou, Canadá </td></tr><tr><td> MRI-Compatible con auriculares de inserción </td><td> Sensimetrics, Modelo S14 </td><td> Sensimetrics Corporation, MA, EE.UU. </td></tr></tbody></table>

mapping the after-effects of theta burst stimulation on the human auditory cortex with functional imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Watch this Scientific Journal Video about Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging at JoVE.com

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

El procesamiento auditivo es la base de la expresión y el procesamiento relacionado con la música. Estimulación Magnética Transcraneal (TMS) se ha utilizado con éxito para estudiar los sistemas cognitivos, sensoriales y motoras, pero rara vez se ha aplicado a la audición. Aquí investigamos TMS combinado con resonancia magnética funcional para comprender la organización funcional de la corteza auditiva.

Mapeo de las secuelas de la theta ráfaga de estimulación sobre la corteza auditiva humana con imágenes funcionales

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

mapping-after-effects-theta-burst-stimulation-on-human-auditory

Research

JoVE Journal

Biology

13.7K vistas.  McGill University. El procesamiento auditivo es la base de la expresión y el procesamiento relacionado con la música. Estimulación Magnética Transcraneal (TMS) se ha utilizado con éxito para estudiar los sistemas cognitivos, sensoriales y motoras, pero rara vez se ha aplicado a la audición. Aquí investigamos TMS combinado con resonancia magnética funcional para comprender la organización funcional de la corteza auditiva.

Video: Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etiquetado y hESCs hMSCs con nanopartículas de óxido de hierro para no invasivo en el seguimiento in vivo mediante RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Investigación

Biología

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etiquetado de las células madre con tintes fluorescentes para la detección no invasiva con imágenes ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

Imágenes funcionales con Refuerzo, Eyetracking y Monitoreo Fisiológico

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

Resonancia de spin electrónico micro imágenes en vivo de las especies para la asignación de oxígeno

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

Un alelo-específicas Gene ensayo de expresión para probar la base funcional de las asociaciones genéticas

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagen funcional de grasa marrón en ratones con FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método para la Evaluación de los efectos de un rango de longitudes de onda e intensidades de / Terapia de luz del infrarrojo cercano Red sobre el estrés oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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.

Brecha ocasiones intercelular de comunicación: un biomarcador funcional para evaluar los efectos adversos de las sustancias tóxicas y toxinas, y de Salud Beneficios de Productos Naturales

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

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

Ensayo de cribado basado en sA-Galactosidasa para la identificación de medicamentos senoterapéuticos

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Aplicación del movimiento pasivo de la cabeza para generar aceleraciones definidas en las cabezas de roedores

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...