CIBC bourse (JA) et subvention du CRSNG (RZ). Nous sommes reconnaissants à M. Roch Comeau (Brainsight) pour son aide en ce qui concerne la caméra infrarouge, les trackers compatibles IRM et le soutien d&#39;autres matériels. Nous sommes également reconnaissants à Brian Hynes (Hybex Innovations Inc) qui a conçu le bras multi-articulé pour porte-bobine et a fourni quelques-uns des chiffres qui s&#39;affichent dans la vidéo. Et un grand merci à tous les techniciens MR et M. Ferreira du Centre d&#39;imagerie cérébrale McConnell de l&#39;Institut neurologique de Montréal qui nous ont aidés optimisation de la conception de l&#39;expérience.

Le protocole est divisé en une session de deux jours (pas nécessairement consécutifs). La première journée se compose d&#39;une piste composée d&#39;une IRMf anatomique et quelques balayages IRM fonctionnelle pour définir pour chaque participant les zones à cibler avec TMS. Le deuxième jour consiste en des sessions IRMf pré-et post-TMS TMS où est appliquée à l&#39;intérieur du scanner à l&#39;aide d&#39;une bobine spéciale MR compatible TMS (Magstim Ltd, Pays de Galles, Royaume-Uni) et un système stéréotaxique sans cadre (Brainsight). Ce dernier est utilisé à la position en temps réel de la bobine TMS sur les aires corticales par rapport aux données anatomiques et fonctionnelles de chaque participant. 1. Session Localizer <ul><li> Commencez par l&#39;acquisition d&#39;une image anatomique haute résolution de votre participant. </li><li> Ensuite, acquérir des images fonctionnelles qui utilisent un écho de gradient EPI impulsion et un paradigme échantillonnage clairsemé afin de minimiser tout effet BOLD ou masquage auditif dû au bruit de numérisation IRM 14,15. Dans notre cas, l&#39;IRMf est effectuée dendant une tâche mélodie dans laquelle les participants doivent déterminer si deux mandats consécutifs de 5-notes mélodies sont l&#39;2,16 identiques ou différents. Une tâche de contrôle non-discrimination auditive est également inclus, dans lesquelles les sujets entendre deux de longueur égale modes de cinq notes, tous à la même hauteur de C5 et demande de cliquer sur le bouton gauche en suivant le second stimulus. Périodes de silence sont également insérés au hasard parmi les essais des tâches dans chaque série. Au total, 72 sont des essais présentés dans un ordre aléatoire: 24 essais de discrimination mélodie, 24 essais de contrôle auditif et 24 périodes de silence, pour une durée totale de 12 min 16 sec. </li><li> Définir le site de stimulation à l&#39;aide anatomiques et / ou fonctionnelles repères. Il faut être conscient que la TMS est limitée en ce qui concerne la profondeur du site de stimulation en raison de l&#39;atténuation de l&#39;intensité du champ électrique en profondeur et ne peut pas s&#39;attendre à atteindre les zones les plus profondes de 3 cm 6,17. Une étape cruciale consiste à utiliser des repères similaires pour chaque partieicipant, ce qui pourrait s&#39;avérer difficile en raison des différences dans l&#39;anatomie et la fonction entre les participants. Ici, nous ciblons gyrus Heschl à chaque participant, situé utilisant deux repères anatomiques et fonctionnelles. Nous utilisons des masques de gyrus Heschl fournies par les atlas Harvard-Oxford structurelles ( <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> ) et la cible TMS est définie individuellement par le pic de activation dans le gyrus l&#39;Heschl 2. En outre, nous avons également définir la position du sommet, qui sera utilisé comme site de contrôle pour contrôler les effets non spécifiques des TMS tels que des artefacts acoustiques et somato-sensoriel. Le sommet est défini comme anatomiquement point milieu de l&#39;inion et l&#39;arête du nez, et à égale distance des encoches droite et gauche intertragal. L&#39;ordre du site de stimulation (gyrus dire Heschl ou un sommet) est contrebalancée dansindividus. </li></ul> 2. Pré-et post-TMS expérience IRMf Pré-session de TMS IRMf <ul><li> Préparer le participant pour aller directement dans le scanner. Cela comprend l&#39;enlèvement de métal et de remplir le formulaire de sélection TMS et MR. </li><li> Lancement de l&#39;acquisition MR avec un anatomiques et fonctionnelles quelques balayages (identique à celle effectuée lors de la session de piste, voir la section 1). </li></ul> Stéréotaxie sans cadre et TMS dans l&#39;environnement IRM Le système de stéréotaxie sans cadre est composé d&#39;une caméra infrarouge (Polaris Spectra), des outils et des pisteurs (Brainsight) utilisés pour la procédure d&#39;enregistrement et un ordinateur. L&#39;ordinateur est situé à l&#39;extérieur de la chambre scanner, mais placée à l&#39;entrée de la chambre et la porte du scanner scanner est maintenu ouvert pendant l&#39;application TMS. Les outils et les trackers sont compatibles MR, ainsi que le trépied (fait maison) supportant la caméra infrarouge et sont eerefore utilisé à l&#39;intérieur de la chambre du scanner. La caméra infrarouge n&#39;est pas compatible IRM, et par conséquent est positionné à l&#39;intérieur de la salle du scanner, près de la porte du scanner à environ deux mètres de la vitre du scanner (voir la discussion de procédure de sécurité). Le système TMS stimulateur est situé dans une pièce adjacente à la salle du scanner IRM. On utilise une bobine MRI compatible TMS situé à l&#39;intérieur de la chambre et du scanner relié au système TMS par un câble 7-m dans un tube filtre RF. <ul><li> Charger des images anatomiques et fonctionnelles votre participant et les objectifs de stimulation dans le logiciel stéréotaxique (Brainsight par exemple). Ici, nous allons cibler le gyrus Heschl de droite. </li><li> Après l&#39;acquisition IRMf pré-TMS, enlever la partie supérieure MR bobine tête de la bobine de tête 32-canal (si vous utilisez le scanner 3T Siemens et la configuration à 32 canaux tête de bobine). </li><li> Ensuite, faites glisser vers le bas le participant sur la vitre du scanner. </li><li> Fixez le serre-tête et jeu de tracker sur la participant la tête. </li><li> Montez le bras à plusieurs articulations à la vitre du scanner et de fixer le MR bobine compatible TMS sur le bras. </li><li> Vérifiez que tous les trackers et de la bobine sont dans le champ de vision de la caméra. Ici, la caméra est légèrement déplacé vers le côté droit du participant pour permettre un suivi plus facile des déplacements de bobine lorsque le ciblage de l&#39;hémisphère droit. </li><li> Calibrer tête de votre sujet avec les outils de stéréotaxie (c.-à-outil pointeur). Cela se fait en plusieurs points de repère coregistering sur la tête du participant (par exemple, dans notre cas, le bout du nez, le nasion et le tragus des deux oreilles) avec les points de repère sur les mêmes données anatomiques. Dans cette procédure, deux expérimentateurs sont nécessaires, l&#39;une près de la tête du participant pour positionner l&#39;outil pointeur sur la tête du participant, et l&#39;expérimentateur autre à l&#39;entrée de la salle du scanner pour effectuer l&#39;enregistrement sur l&#39;ordinateur. </li><li> Placez le MR bobine TMS compatible tangente à til cuir chevelu, et les suiveurs de bobine orientée vers la caméra infrarouge. La bobine est orienté avec la poignée bobine pointant vers l&#39;arrière et parallèle à la ligne médiane 2. Fixer la position de la bobine à l&#39;aide des vis sur le bras à plusieurs articulations. </li><li> Dans la chambre à côté de l&#39;appareil d&#39;IRM, mettez le système TMS et de commencer la stimulation. TMS est appliquée suivant un protocole motifs, c&#39;est à dire, continue la stimulation thêta burst (CTBS) consistant à 3 impulsions à 50 Hz, répétée à 5Hz pour 40s. On utilise une intensité de stimulation fixe (41%), définie par le stimulateur de sortie 18,19. Nous avons choisi ce protocole, il a été montré pour moduler la plasticité corticale pour une durée allant jusqu&#39;à 30 minutes après l&#39;arrêt de la stimulation à la santé des populations 20, (voir la section de discussion pour les procédure de sécurité). </li></ul> Session de l&#39;IRMf post-TMS <ul><li> Une fois que la stimulation est terminée, il est important d&#39;obtenir le sujet dans le scanner le plus tôt possible. Retirez le TMS bobine de la salle du scanner et retirez le bras à plusieurs articulations. Faire glisser la tête du participant dans la bobine tête MR. Assurez votre scanner est préparé et prêt à partir. Notre conseil est de garder la plate-forme corporelle élevée pendant toute la session de TMS, et de réduire le nombre et la durée de l&#39;analyse d&#39;alignement de piste à un minimum. </li><li> Parce que les effets de la SMTr sont transitoires, la session de numérisation définitive devrait commencer par l&#39;analyse fonctionnelle. Encore une fois, nous avons procédé à l&#39;IRMf pendant un cycle de 12 min de la tâche mélodie. </li><li> Après la numérisation finale est terminée, terminez par une analyse anatomique. </li></ul> 3. Les résultats représentatifs L&#39;analyse des données d&#39;IRMf sont effectuées séparément pour la session IRMf pré-et post-TMS. Pour chaque session IRMf (ie, pré et post-TMS), le contraste entre les mélodies et la tâche de contrôle auditif montre les tâches liées à l&#39;activité de la gauche et la droite gyri Heschl, supérieur gyri temporal, frontal inférieur gyri et precentrale gyri (figure 1 A, B). Pour évaluer les différences entre les sessions IRMf pré-et post-TMS, nous effectuons une analyse aléatoire effet à l&#39;aide de Student t-test apparié. L&#39;importance est déterminée en utilisant les clusters identifiés par az&gt; seuil de 2 et un seuil de cluster corrigée de p = 0,05. Figure 1 C représente le contraste post-moins pré-CTBS pour un seul participant. Les données suggèrent que CTBS ciblant le droit gyrus Heschl (cercle noir) induit une augmentation de la réponse IRMf dans le cortex (à gauche) auditive, y compris le gyrus gauche Heschl. Changements dans la réponse IRMf se trouvent également dans le gyrus post-central gauche, gauche insula et le cortex occipital latéral bilatéral. Toutefois, aucune modification significative de la réponse IRMf est vu sous la bobine. En outre, même combinée TMS-IRMf protocole est répété pour stimuler l&#39;(site témoin) sommets. Comparaison des séances de pré-et post-IRMf avec CTBS appliquées sur le sommet n&#39;a montré aucune significaeffet nt (données non présentées). <img src="/files/ftp_upload/3985/3985fig1.jpg" alt="Figure 1"/> Figure 1. Analyse des données individuelles IRMf pré-TMS (A), post-TMS IRMf données (B) et post-moins pré-TMS données IRMf (C). A. Résultats de la discrimination mélodie revanche moins les essais contrôlés auditifs pour un seul participant à la session IRMf pré-TMS (A) et à la session de l&#39;après-TMS IRMf (B). De gauche à droite: coupes axiales, coronales et sagittales. Dans les deux cas (A) et (B), la bobine TMS vise le droit gyrus Heschl (cercle noir) situé à x = 54, y = -13, z = 1 (MNI152 espace standard). Pour les deux sessions de pré-et post-TMS IRMf, les coordonnées sont affichées en x = -54, y = -13, z = 1 (MNI152 espace standard) pour montrer les changements dans l&#39;hémisphère gauche sur le site de stimulation (gyrus droit soit Heschl ). C. Résultats de l&#39;opposition post-moins sessions IRMf pré-TMS à l&#39;aide de Student t-test apparié.

Aucun conflit d&#39;intérêt déclaré.

Nous décrivons un protocole combinant TMS hors ligne et l&#39;IRMf pour étudier l&#39;organisation fonctionnelle du cortex auditif. Dans les sections suivantes, nous allons étudier les facteurs méthodologiques à considérer lors de la réalisation de ces approches. Acquisition et le calendrier de l&#39;après-TMS session de l&#39;IRMf Afin d&#39;acquisition et de contrebalancer les analyses de sessions IRMf pré-et post-TMS ...

<table border="1"><tbody><tr><td> Nom du produit </td><td> Type </td><td> Entreprise </td></tr><tr><td> La stimulation magnétique transcrânienne </td><td> Magstim super-Rapid2 stimulateur, Rapid-2 Plus One Module </td><td> Magstim Ltd, Pays de Galles, Royaume-Uni </td></tr><tr><td> Bobine de stimulation magnétique </td><td> Compatible avec l&#39;IRM 70 mm figure en huit-bobine </td><td> Magstim Ltd, Pays de Galles, Royaume-Uni </td></tr><tr><td> Imagerie par résonance magnétique </td><td> 3-T scanner Trio Siemens, Bobine Head à 32 canaux </td><td> Siemens, Inc, Allemagne </td></tr><tr><td> La stéréotaxie sans cadre </td><td> Brainsight </td><td> Rogue Research Inc, Montréal, Canada </td></tr><tr><td> Système de mesure optique </td><td> Polaris Spectra </td><td> Northern Digital Inc, Ontario, Canada </td></tr><tr><td> Multi-bras articulé pour porte-bobine </td><td> Standard </td><td> Hybex Innovations Inc, Anjou, Canada </td></tr><tr><td> Écouteurs Insérer compatible avec l&#39;IRM </td><td> Sensimetrics, modèle S14 </td><td> Sensimetrics Corporation, MA, USA </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

Traitement auditif est à la base de la parole et de la musique liés transformation. Stimulation magnétique transcrânienne (TMS) a été utilisée avec succès pour étudier cognitifs, systèmes sensoriels et moteurs, mais a rarement été appliquée à l&#39;audition. Ici, nous avons étudié TMS associés à l&#39;imagerie par résonance magnétique fonctionnelle pour comprendre l&#39;organisation fonctionnelle du cortex auditif.

Cartographie des séquelles de Theta Burst Stimulation sur le cortex auditif humain à l&#39;imagerie fonctionnelle

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

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Biology

13.7K vues.  McGill University. Traitement auditif est à la base de la parole et de la musique liés transformation. Stimulation magnétique transcrânienne (TMS) a été utilisée avec succès pour étudier cognitifs, systèmes sensoriels et moteurs, mais a rarement été appliquée à l'audition. Ici, nous avons étudié TMS associés à l'imagerie par résonance magnétique fonctionnelle pour comprendre l'organisation fonctionnelle du cortex auditif.

Vidéo: 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.

Étiquetage CSEh et hMSCs d&#39;oxyde de fer nanoparticules pour les non-invasives dans le suivi in ​​vivo avec IRM

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

Recherche

Biologie

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.

Étiquetage des cellules souches avec des colorants fluorescents pour les non-invasive de détection avec l&#39;imagerie optique

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.

Imagerie fonctionnelle avec des renforts, Eyetracking, et en surveillance physiologique

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.

Résonance de spin électronique Micro-imagerie d&#39;espèces vivantes pour la cartographie d&#39;oxygène

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 allèle spécifique Assay expression du gène pour l&#39;épreuve la base fonctionnelle des associations génétiques

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.

Imagerie fonctionnelle de la graisse brune chez les souris avec 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.

Méthode d&#39;évaluation des effets d&#39;une gamme de longueurs d&#39;onde et des intensités de Luminothérapie Rouge / proche infrarouge sur le stress oxydatif<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.

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

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

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.

Essai de dépistage basé sur sA-MD-Galactosidase pour l'identification des médicaments sérothérapeutiques

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.

Application du mouvement passif de la tête pour générer des accélérations définies à la tête des rongeurs

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