CIBC comunione (JA) e NSERC sovvenzione (RZ). Siamo grati a Roch M. Comeau (Brainsight) per il suo aiuto per quanto riguarda la macchina fotografica a raggi infrarossi, gli inseguitori MR compatibili e di supporto hardware. Siamo anche grati a Brian Hynes (Hybex Innovations Inc.) che ha progettato il multi-articolato braccio porta bobine e fornito alcune delle figure mostrate nel video. E un grazie speciale a tutti i tecnici e MR M. Ferreira del cervello Centro McConnell Imaging del Montreal Neurological Institute, che ci ha aiutato a ottimizzare la progettazione dell&#39;esperimento.

Il protocollo è suddiviso in una sessione di due giorni (non necessariamente consecutivi). Il primo giorno consiste in un localizzatore fMRI composto con un anatomica e funzionale scansioni MR definire per ogni partecipante le zone da mirata con TMS. Il secondo giorno consiste in sessioni fMRI pre-e post-TMS in cui si applica TMS all&#39;interno dello scanner con una speciale bobina MR compatibile TMS (Magstim Ltd., Galles, Regno Unito) e un sistema stereotassico frameless (Brainsight). Quest&#39;ultimo è utilizzato in posizione in tempo reale la bobina TMS su aree corticali relativi a dati anatomici e funzionali di ogni partecipante. 1. Localizer sessione <ul><li> Inizia con l&#39;acquisizione di una immagine ad alta risoluzione anatomica del partecipante. </li><li> Poi, acquisire immagini funzionali utilizzando un gradiente echo EPI impulso e un paradigma campionamento ridotto, al fine di ridurre gli effetti di BOLD o mascheramento uditivo a causa di rumore scansione MRI 14,15. Nel nostro caso, fMRI viene effettuata durante un compito melodia in cui i partecipanti devono determinare se due consecutivi 5 note melodie sono uguali o diversi 2,16. Un compito non discriminazione uditiva di controllo è anche incluso, in cui i soggetti sentono due di uguale lunghezza modelli di cinque note, tutte alla stessa altezza di C5 e viene richiesto di fare clic sul pulsante a sinistra in seguito il secondo stimolo. I periodi di silenzio, sono anche inseriti in modo casuale tra le prove delle attività in ogni seduta. In totale, le prove 72 sono presentati in uno studio randomizzato in ordine: 24 prove di discriminazione melodia, 24 prove di controllo uditivo e 24 periodi di silenzio, per una durata totale di 12 min 16 sec. </li><li> Definire il sito di stimolazione con anatomica e / o punti di riferimento funzionali. Si deve essere consapevoli che TMS è limitato per quanto riguarda la profondità del sito di stimolazione per l&#39;attenuazione del campo elettrico in profondità, e non può aspettarsi di raggiungere le zone più profonde di 3 cm 6,17. Un passo fondamentale è quello di utilizzare punti di riferimento simili per ogni parteicipant, che potrebbe essere difficile a causa delle differenze di anatomia e la funzione tra i partecipanti. Qui, puntiamo giro Heschl a ciascun partecipante, che si trova con entrambi i punti di riferimento anatomici e funzionali. Usiamo le maschere di giro di Heschl fornite dalle università di Harvard e Oxford atlanti strutturali ( <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> ) e il target TMS è definito individualmente dal picco di attivazione entro il giro di Heschl 2. Inoltre, si definisce anche la posizione del vertice, che sarà utilizzato come sito di controllo per controllare per effetti non specifici di TMS come artefatti acustici e somatosensoriale. Il vertice è definito anatomicamente come un punto a metà strada tra il inion e il ponte del naso, ed equidistante dalle tacche destro e sinistro intertragal. L&#39;ordine del sito di stimolazione (cioè Heschl di giro o vertice) è controbilanciato attraversoindividui. </li></ul> 2. Pre-e post-TMS esperimento fMRI Pre-TMS fMRI sessione <ul><li> Preparare il partecipante di andare direttamente nello scanner. Ciò include la rimozione di metallo e compilando il TMS e la forma di screening MR. </li><li> Avviare l&#39;acquisizione MR con un anatomico e un scansioni funzionali (identico a quello effettuato nella sessione localizzatore, vedere sezione 1). </li></ul> Stereotassia Frameless e TMS nell&#39;ambiente MRI Il sistema di stereotassi senza telaio è composto da una telecamera a infrarossi (Polaris Spectra), alcuni strumenti e inseguitori (Brainsight) utilizzati per la procedura di registrazione e un computer. Il computer si trova fuori della camera scanner, ma posizionato all&#39;ingresso della camera e la porta scanner scanner viene mantenuta aperta durante l&#39;applicazione TMS. Gli strumenti e gli inseguitori sono MR compatibili, così come il treppiede (fatti in casa) a sostenere la telecamera a infrarossi e sono esimoutilizzato erefore all&#39;interno della stanza dello scanner. La telecamera a infrarossi non è MR-compatibile, e quindi è posizionato all&#39;interno della stanza dello scanner, vicino alla porta dello scanner a circa due metri dal piano dello scanner (vedi la discussione per la procedura di sicurezza). Il sistema TMS stimolatore si trova in una stanza adiacente alla sala scanner MRI. Usiamo un MRI coil compatibile TMS situato all&#39;interno della camera scanner e collegato al sistema TMS tramite un 7-m cavo attraverso un tubo filtro RF. <ul><li> Caricare immagini anatomiche e funzionali vostro partecipante e gli obiettivi di stimolazione nel pacchetto software stereotassica (Brainsight ad esempio). Qui, ci sarà il giro di mira Heschl di destra. </li><li> Dopo il pre-TMS acquisizione fMRI, rimuovere la parte superiore testa MR bobina del 32 canali bobina testa (se si utilizza lo scanner 3T Siemens e il 32-canale di configurazione della bobina testa). </li><li> Quindi, far scorrere verso il basso il partecipante sul piano dello scanner. </li><li> Fissare la fascia e set inseguitore sulla parteciptesta di formica. </li><li> Montare il multi-articolato braccio al piano di scansione e fissare la compatibilità MR bobina TMS sul braccio. </li><li> Verificare che tutti gli inseguitori e la bobina sono nel campo visivo della telecamera. Qui, la fotocamera è leggermente spostata verso il lato destro del partecipante per consentire un facile monitoraggio degli spostamenti bobina per orientare l&#39;emisfero destro. </li><li> Calibrare la testa del soggetto con gli strumenti stereotassia strumento puntatore (ad esempio). Questo viene fatto coregistering più punti di riferimento sulla testa del partecipante (ad esempio, nel nostro caso la punta del naso, il nasion e il trago di entrambe le orecchie) con i punti di riferimento stessi sui dati anatomici. In questa procedura, sono necessarie due sperimentatori, uno vicino alla testa del partecipante per posizionare l&#39;utensile puntatore sulla testa del partecipante, e l&#39;altra sperimentatore all&#39;ingresso della camera scanner per eseguire la registrazione sul computer. </li><li> Posizionare la bobina TMS compatibile MR tangente al tlui cuoio capelluto, e gli inseguitori bobina rivolta verso la telecamera a raggi infrarossi. La bobina è orientato con la maniglia bobina rivolta all&#39;indietro e parallelamente alla linea mediana 2. Fissare la posizione della bobina con le viti sul multi braccio snodabile. </li><li> Nella stanza adiacente allo scanner MRI, accendere il sistema TMS e iniziare la stimolazione. TMS viene applicata seguendo un protocollo fantasia, vale a dire, la stimolazione continua raffica theta (CTBs) composta da 3 impulsi a 50Hz, ripetuto a 5Hz per 40. Usiamo una intensità di stimolazione fissa (41%) definita dalla uscita stimolatore 18,19. Abbiamo scelto questo protocollo, come è stato dimostrato di modulare la plasticità corticale per un periodo fino a 30 minuti dopo la cessazione stimolazione in popolazioni sane 20, (vedere sezione discussione per procedura di sicurezza). </li></ul> Post-TMS fMRI sessione <ul><li> Una volta che la stimolazione è completa, è importante per ottenere il ritorno soggetto nello scanner appena possibile. Rimuovere il TBobina MS dalla stanza dello scanner e rimuovere il multi braccio snodabile. Spingere indietro la testa del partecipante nella bobina testa MR. Assicurarsi che lo scanner è preparato e pronto ad andare. Il consiglio è di mantenere il corpo piattaforma sollevata durante l&#39;intera sessione TMS, e ridurre il numero e la durata delle scansioni localizzatore al minimo. </li><li> Poiché gli effetti della rTMS sono transitori, la sessione di scansione finale dovrebbe iniziare con l&#39;analisi funzionale. Ancora una volta, abbiamo condotto fMRI nel corso di 12 minuti di esecuzione dell&#39;operazione melodia. </li><li> Dopo la scansione finale è completo, finire con una scansione anatomica. </li></ul> 3. Risultati rappresentativi L&#39;analisi dei dati fMRI sono condotte separatamente sia per la sessione di fMRI pre-e post-TMS. Per ogni sessione di fMRI (vale a dire, pre e post-TMS), il contrasto tra le melodie e il compito di controllo uditivo mostra compito attività legate a sinistra ea destra di circonvoluzioni Heschl, circonvoluzioni temporali superiore, frontale inferiore e circonvoluzioni precentrale gyri (Figura 1 A, B). Per valutare le differenze tra le sessioni fMRI pre-e post-TMS, eseguiamo un casuale effetto analisi utilizzando Student t-test accoppiato. Importanza è determinato utilizzando i cluster individuati dalla az&gt; 2 soglia e una soglia di cluster corretto di p = 0.05. Figura 1 C rappresenta i post-meno contrasto pre-CTBs per un solo partecipante. I dati suggeriscono che il diritto di targeting CTBs Heschl di giro (cerchio nero) induce un aumento della risposta fMRI nel controlaterale (a sinistra) corteccia uditiva, tra cui giro la sinistra di Heschl. I cambiamenti in risposta fMRI si trovano anche nel giro sinistra postcentrale, insula sinistra, e nella corteccia laterale occipitale bilateralmente. Tuttavia, nessun cambiamento significativo della risposta fMRI è vista sotto la bobina. Inoltre, analogamente combinato TMS-fMRI protocollo viene ripetuto per stimolare il vertice (sito controllo). Confronto di sessioni di pre-e post-fMRI con CTBs applicato sopra il vertice non ha mostrato alcun significant effetto (dati non mostrati). <img src="/files/ftp_upload/3985/3985fig1.jpg" alt="Figura 1"/> Figura 1. Analisi dei singoli pre-TMS dati fMRI (A), post-TMS fMRI dati (B) e post-meno pre-TMS dati fMRI (C). A. Risultati della discriminazione melodia contrasto meno prove di controllo uditivi per un singolo partecipante nel pre-TMS sessione di fMRI (A) e nel post-TMS sessione di fMRI (B). Da sinistra a destra: vista assiale, coronale e sagittale. In entrambi (A) e (B), la bobina TMS si rivolge destra Heschl di giro (cerchio nero) situato in x = 54, y = -13, z = 1 (MNI152 spazio standard). Per entrambe le sessioni pre-e post-TMS fMRI, coordinate vengono visualizzate in x = -54, y = -13, z = 1 (MNI152 spazio standard) per mostrare i cambiamenti nell&#39;emisfero sinistro nel sito di stimolazione (cioè a destra giro di Heschl ). C. Risultati del contrasto post-meno pre-TMS sessioni fMRI utilizzando Student t-test accoppiato.

Nessun conflitto di interessi dichiarati.

Descriviamo un protocollo che unisce TMS offline e fMRI per studiare l&#39;organizzazione funzionale della corteccia uditiva. Nelle prossime sezioni, discuteremo i fattori metodologici da considerare per lo svolgimento di tale approccio. Acquisizione e tempi per il post-TMS sessione fMRI Ordine di acquisizione scansioni e di controbilanciamento delle sessioni fMRI pre-e post-TMS È fondamentale acquisire una scansione anatomica ...

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Tipo </td><td> Azienda </td></tr><tr><td> La stimolazione magnetica transcranica </td><td> Magstim Super Rapid2 stimolatore, Rapid-2 Plus One Module </td><td> Magstim Ltd., Galles, Regno Unito </td></tr><tr><td> Bobina per la stimolazione magnetica </td><td> MRI-compatibile 70 millimetri figura di otto-coil </td><td> Magstim Ltd., Galles, Regno Unito </td></tr><tr><td> La risonanza magnetica </td><td> 3-T Trio Siemens scanner a 32 canali Testa Coil </td><td> Siemens, Inc., Germania </td></tr><tr><td> Stereotassi Frameless </td><td> Brainsight </td><td> Rogue Research Inc., Montreal, Canada </td></tr><tr><td> Sistema di misurazione ottica </td><td> Polaris Spectra </td><td> Northern Digital Inc., Ontario, Canada </td></tr><tr><td> Multi-braccio snodato porta bobine </td><td> Standard </td><td> Hybex Innovationi Inc., Anjou, Canada </td></tr><tr><td> RM-compatibili Inserite gli auricolari </td><td> Sensimetrics, modello S14 </td><td> Sensimetrics Corporation, MA, Stati Uniti d&#39;America </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

Elaborazione uditiva è la base di parlato e musica legati elaborazione. Stimolazione Magnetica Transcranica (TMS) è stato usato con successo per lo studio dei sistemi cognitivi, sensoriali e motori, ma raramente è stata applicata per un provino. Qui abbiamo studiato TMS combinati con risonanza magnetica funzionale per comprendere l&#39;organizzazione funzionale della corteccia uditiva.

Mappatura delle postumi di Theta Burst stimolazione sulla corteccia uditiva umana con imaging funzionale

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 Views.  McGill University. Elaborazione uditiva è la base di parlato e musica legati elaborazione. Stimolazione Magnetica Transcranica (TMS) è stato usato con successo per lo studio dei sistemi cognitivi, sensoriali e motori, ma raramente è stata applicata per un provino. Qui abbiamo studiato TMS combinati con risonanza magnetica funzionale per comprendere l'organizzazione funzionale della corteccia uditiva.

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.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

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

Ricerca

Biologia

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.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con 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 ...

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.

Imaging funzionale con rinforzo, eyetracking e monitoraggio fisiologico

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.

Electron Spin Resonance micro-imaging delle specie dal vivo per la mappatura di ossigeno

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.

Una allele-specifica espressione genica saggio per testare la base funzionale delle Associazioni di genetica

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.

Imaging funzionale del grasso bruno nei topi 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.

Metodo per la valutazione degli effetti di un intervallo di lunghezze d&#39;onda e intensità di / vicino-infrarosso di terapia della luce rossa sullo stress ossidativo<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.

Spazi di giunzione intercellulare di comunicazione: un biomarker funzionale per valutare gli effetti avversi dei tossici e tossine, e benefici per la salute di prodotti naturali

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.

Saggio di screening basato su SA-Galactosidase per l'identificazione dei farmaci senoterapici

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.

Applicazione del movimento passivo della testa per generare accelerazioni definite alle teste dei roditori

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