CIBC Gemeinschaft (JA) und NSERC Zuschuss (RZ). Wir sind dankbar, dass Roch M. Comeau (Brainsight) für seine Hilfe über die Infrarot-Kamera, die MR-kompatiblen Trackern und anderen Hardware-Unterstützung. Wir sind auch dankbar, dass Brian Hynes (Hybex Innovations Inc.), die Mehrgelenkarm für Spulenhalter konzipiert und wird einige der Figuren in dem Video angezeigt. Und einen besonderen Dank an alle MR Techniker und M. Ferreira aus dem McConnell Brain Imaging Center des Montreal Neurological Institute, die uns die Optimierung der Konstruktion des Experimentes geholfen.

Das Protokoll ist in einem Zwei-Tage-Sitzung (nicht unbedingt aufeinanderfolgenden) unterteilt. Der erste Tag besteht aus einem fMRI Localizer mit einer anatomischen und einer funktionellen MR-Scans, um für jeden Teilnehmer die Bereiche mit TMS gezielt auf definieren zusammen. Der zweite Tag besteht in den fMRI-Sitzungen Pre-und Post-TMS, wo TMS Inneren des Scanners mit einem speziellen MR-kompatible TMS Spule (Magstim Ltd, Wales, UK) und eine rahmenlose Stereotaxie System (Brainsight) angewendet wird. Letzteres wird auf die Position in Echtzeit die TMS-Spule auf die kortikale Bereiche in Bezug auf jedes Teilnehmers anatomischen und funktionellen Daten verwendet. Ein. Localizer Session <ul><li> Starten Sie mit dem Erwerb einer hochauflösenden anatomischen Bild des Teilnehmers. </li><li> Dann erwirbt funktionelle Bilder unter Verwendung eines Gradientenecho EPI Puls und Sparse Probenahme Paradigma um jegliche BOLD Effekt oder auditiven Maskierung aufgrund MRI Abtasten Rauschen 14,15 minimieren. In unserem Fall wird fMRI aus d durchgeführtährend eine Melodie Aufgabe, in der Teilnehmer zu ermitteln, ob zwei aufeinander folgende 5-Noten-Melodien die gleich oder verschieden sind 2,16. A Nichtdiskriminierung auditiven Regelaufgabe ist ebenfalls enthalten, in denen die Probanden hören zwei gleich langen Mustern aus fünf Tönen, die alle in der gleichen Tonhöhe von C5 und angewiesen werden, die linke Taste nach dem zweiten Reiz klicken. Perioden der Stille auch zufällig unter den Task Versuchen in jedem Lauf eingeschoben. Insgesamt werden 72 Studien in zufälliger Reihenfolge präsentiert: 24 Studien der Melodie Diskriminierung, 24 auditive kontrollierte Studien und 24 Perioden des Schweigens, für eine Gesamtdauer von 12 min 16 sek. </li><li> Definieren Sie die Stimulation Website mit anatomischen und / oder funktionellen Landmarken. Man muss beachten, dass TMS begrenzt ist in Bezug auf die Tiefe der Stimulationsstelle wegen der Dämpfung von der elektrischen Feldstärke in der Tiefe, und kann nicht erwarten, dass Bereiche tiefer als 3 cm 6,17 erreichen. Ein entscheidender Schritt ist, um ähnlichen Orientierungspunkten für jeden Teil verwendenicipant, was schwierig sein könnte, weil die Unterschiede in der Anatomie und Funktion zwischen den Teilnehmern. Hier streben wir Heschl der Gyrus in jedem Teilnehmer, sich mit beiden anatomischen und funktionellen Landmarken. Wir verwenden Masken Heschl der Gyrus durch die Harvard-Oxford strukturelle Atlanten (vorausgesetzt <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> ) und der TMS Ziel wird einzeln von der Spitze des definierten Aktivierung innerhalb der Heschl der Gyrus 2. Darüber hinaus definieren wir auch die Scheitelpunkt-Position, die als Kontrolle vor Ort verwendet werden soll, um für unspezifische Effekte von TMS wie akustische und somatosensorischen Artefakte zu steuern. Der Scheitelpunkt ist anatomisch als ein Punkt in der Mitte zwischen dem Inion und der Brücke der Nase definiert, und in gleichem Abstand von den rechten und linken intertragal Kerben. Die Reihenfolge der Website der Stimulation (dh Heschl der Gyrus oder Vertex) über aufgewogenEinzelpersonen. </li></ul> 2. Pre-und Post-TMS fMRI Experiment Pre-TMS fMRI-Sitzung <ul><li> Bereiten Sie die Teilnehmer direkt in den Scanner. Dies schließt das Entfernen von Metall-und Befüllen der TMS-und MR-Screening Form. </li><li> Starten Sie den MR Akquisition mit einer anatomischen und einer funktionellen Scans (identisch mit dem in der Localizer-Sitzung durchgeführt, siehe Abschnitt 1). </li></ul> Rahmenlose Stereotaxie und TMS in der MRT-Umgebung Die rahmenlose Stereotaxie-System besteht aus einer Infrarot-Kamera (Polaris Spectra), einige Werkzeuge und trackers (Brainsight) für das Registrierungsverfahren und einem Computer verwendet komponiert. Der Computer außerhalb des Scanners Raum befindet sich aber am Eingang positioniert des Scanners und der Scanner Raum geöffnet wird während der TMS-Anwendung aufbewahrt. Die Werkzeuge und Tracker sind MR-kompatibel, ebenso wie das Stativ (hausgemachte), die die Infrarot-Kamera und sind therefore verwendet das Innere des Scanners Zimmer. Die Infrarot-Kamera ist nicht MR-kompatibel ist, und wird daher im Inneren des Scanners Raum nahe dem Filmkammertür bei etwa zwei Meter von der Scanner-Bett (siehe Diskussion für Sicherheitsverfahren) positioniert. Die TMS-Stimulator ist in einem Raum neben dem Magnetresonanztomographen Raum befinden. Wir verwenden ein MRI kompatiblen TMS Spule im Inneren des Scanners Raum angeordnet und mit dem TMS-System über eine 7-m Kabel durch einen HF-Filterrohr. <ul><li> Laden Sie Ihre Teilnehmer anatomische und funktionelle Bilder und die Stimulation Ziele in der stereotaktischen Software-Paket (zB Brainsight). Hier werden wir gezielt den richtigen Heschl der Gyrus. </li><li> Nach dem Pre-TMS fMRI Akquisition, entfernen Sie die obere MR Kopfspule Teil des 32-Kanal-Kopfspule (bei Verwendung des Siemens 3T Scanner und die 32-Kanal-Kopfspule Konfiguration). </li><li> Dann schieben Sie die Teilnehmer auf das Scanner-Bett. </li><li> Befestigen Sie das Kopfband und tracker Satz auf dem participant Kopf. </li><li> Montieren Sie die Mehrgelenkarm um das Scanner-Bett und befestigen Sie den MR-kompatible TMS Spule auf den Arm. </li><li> Stellen Sie sicher, dass alle die Tracker und die Spule in das Blickfeld der Kamera sind. Hier wird die Kamera geringfügig nach rechts verschoben des Teilnehmers, um ein leichteres Nachführung der Spule Verschiebungen ermöglichen, wenn gezielt die rechte Hemisphäre. </li><li> Kalibrieren Ihres Motivs Kopf mit den Stereotaxie Werkzeuge (dh Zeiger-Tool). Dies wird durch coregistering mehrere Orientierungspunkte an der Kopfgröße des Teilnehmers (zB in unserem Fall die Spitze der Nase, der Nasion und dem Tragus beider Ohren) mit den gleichen Landmarken auf den anatomischen Daten durchgeführt. In diesem Verfahren werden zwei Experimentatoren Bedarf ein, die der Teilnehmer den Kopf zu schließen, um den Zeiger-Werkzeug auf der Kopfgröße des Teilnehmers zu positionieren, und das andere Experimentator am Eingang des Scanner-Raum, um die Registrierung auf dem Computer ausführen. </li><li> Positionieren Sie den MR-kompatible TMS-Spule tangential zu ter Kopfhaut, und die Spule Tracker auf den Infrarot-Kamera gerichtet. Die Spule orientiert ist mit der Spule Griff nach hinten und parallel zu der Mittellinie 2. Fix die Spule Position mit den Schrauben an der Mehrgelenkarm. </li><li> Im Raum neben der MRI-Scanner, auf dem TMS System einschalten und beginnen Stimulation. TMS ist nach einer strukturierten Protokoll, dh, kontinuierliche theta Burst Stimulation (CTBS), bestehend aus 3 Impulsen bei 50Hz, bei 5Hz für 40 wiederholt angewendet. Wir verwenden eine feste Stimulationsintensität (41%) durch den Stimulator Ausgang 18,19 definiert. Wir entschieden uns für dieses Protokoll, da es sich gezeigt hat zu modulieren kortikale Plastizität für eine Dauer von bis zu 30 min nach Stimulation Aufhören in gesunden Populationen 20, (siehe Diskussion im Abschnitt Sicherheitsverfahren). </li></ul> Post-TMS fMRI-Sitzung <ul><li> Sobald Stimulation beendet ist, ist es wichtig, den Gegenstand in den Scanner so schnell wie möglich zu erhalten. Entfernen Sie die TMS Spule von der Scanner-Raum, und entfernen Sie die Mehrgelenkarm. Schieben Sie den Teilnehmer den Kopf in den MR-Kopf Spule. Sichern Sie sich Ihren Scanner ist vorbereitet und bereit zu gehen. Unser Rat ist, den Körper-Plattform während der gesamten TMS-Sitzung angehoben zu halten, und verringern Sie die Anzahl und die Dauer der Localizer Scans auf ein Minimum. </li><li> Da die Wirkungen von rTMS vergänglich sind, sollte die endgültige Scan-Sitzung mit dem funktionellen Scanvorgang zu starten. Auch führten wir fMRI während einer 12-min Lauf der Melodie Aufgabe. </li><li> Nach der endgültigen Scan abgeschlossen ist, mit einer anatomischen Scan zu beenden. </li></ul> 3. Repräsentative Ergebnisse Analysen von fMRI Daten werden getrennt sowohl für die Pre-und Post-TMS fMRI-Sitzung durchgeführt. Für jeden fMRI-Sitzung (dh Pre-und Post-TMS), zeigt der Kontrast zwischen den Melodien und der auditiven Regelaufgabe aufgabenbezogene Aktivität in der linken und rechten Heschl die Gyri temporalis superior Gyri, inferioren frontalen Gyrus und precentrale Gyri (Abbildung 1 A, B). Um Unterschiede zwischen Pre-und Post-TMS fMRI-Sitzungen auszuwerten, führen wir eine random-Wirkungs-Analyse unter Verwendung des Student gepaarten t-Test. Bedeutung wird mittels Cluster az&gt; 2 Schwelle und einer korrigierten Cluster Schwelle von p = 0,05 ermittelt. Abbildung 1 C stellt den Kontrast post-minus pre-CTBS für einen einzelnen Teilnehmer. Die Daten deuten darauf hin, dass CTBS Targeting das Recht Heschl der Gyrus (schwarzer Kreis) eine Erhöhung der fMRI Antwort in der kontralateralen (links) auditorischen Kortex, einschließlich des linken Heschl der Gyrus induziert. Änderungen im fMRI Antwort auch in der linken Gyrus postcentralis gefunden, links Insula, und in der lateralen okzipitalen Kortex bilateral. Es wird jedoch keine signifikante Veränderung in fMRI Reaktion unter der Spule gesehen. Zusätzlich wird ähnlich kombinierten TMS-fMRI Protokoll wiederholt, um den Scheitel (Steuerstelle) zu stimulieren. Vergleich von Pre-und Post-fMRI-Sessions mit CTBS über den Scheitelpunkt angewendet zeigten keine significant-Effekt (Daten nicht gezeigt). <img src="/files/ftp_upload/3985/3985fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Analyse der einzelnen Vor-TMS fMRI-Daten (A), post-TMS fMRI-Daten (B) und Post-minus pre-TMS fMRI-Daten (C). A. Ergebnisse der Kontrast Melodie Diskriminierung minus auditiven kontrollierte Studien für einen einzelnen Teilnehmer in der Pre-TMS fMRI-Sitzung (A) und in der post-TMS fMRI-Sitzung (B). Von links nach rechts: axiale, koronale und sagittale Ansichten. In beiden (A) und (B), das TMS-Spule wird die richtige Ausrichtung der Heschl Gyrus (schwarzer Kreis), der an x ​​= 54, y = -13 und z = 1 (MNI152 Standardleerzeichen). Bei beiden Pre-und Post-TMS fMRI Sitzungen werden Koordinaten an x = -54 angezeigt, y = -13 und z = 1 (MNI152 Standardleerzeichen), um Änderungen in der linken Hemisphäre an der Stelle der Stimulation (dh rechts Heschl den Gyrus zeigen ). C. Ergebnisse des Kontrastes post-minus pre-TMS fMRI Sitzungen mit Student gepaarten t-Test.

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Wir beschreiben ein Protokoll kombiniert offline TMS und fMRT, um die funktionelle Organisation des auditorischen Kortex zu untersuchen. In den nächsten Abschnitten werden wir diskutieren, die methodischen Faktoren zu berücksichtigen, wenn die Durchführung solcher Ansatz. Erwerb und Timing für post-TMS fMRI-Sitzung Order of Scans Erwerb und Gegengewicht der Pre-und Post-TMS fMRI-Sitzungen Es ist entscheidend, um ein MR-anato...

<table border="1"><tbody><tr><td> Material Name </td><td> Typ </td><td> Firma </td></tr><tr><td> Transkranielle Magnetstimulation </td><td> Magstim Super Rapid2 Stimulator, Rapid-2 Plus One Module </td><td> Magstim Ltd, Wales, UK </td></tr><tr><td> Spule für Magnetstimulation </td><td> MRI-kompatiblen 70 mm figure-of-eight-coil </td><td> Magstim Ltd, Wales, UK </td></tr><tr><td> Kernspintomographie </td><td> 3-T Siemens Trio Scanner, 32-Kanal-Kopfspule </td><td> Siemens, Inc., Deutschland </td></tr><tr><td> Rahmenlose Stereotaxie </td><td> Brainsight </td><td> Rogue Research Inc., Montreal, Kanada </td></tr><tr><td> Optisches Messsystem </td><td> Polaris Spectra </td><td> Northern Digital Inc., Ontario, Kanada </td></tr><tr><td> Mehrgelenkarm für Spulenhalter </td><td> Standard </td><td> Hybex InnovatIonen Inc., Anjou, Kanada </td></tr><tr><td> MRI-kompatiblen Einsteckhörern </td><td> Sensimetrics, Modell 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

Auditive Verarbeitungs ist die Grundlage von Sprache und Musik-bezogene Verarbeitung. Transkranielle Magnetstimulation (TMS) wurde erfolgreich auf kognitive, sensorische und motorische Systeme zu studieren verwendet, aber selten angewendet worden, um vorzuspielen. Hier untersuchten wir TMS mit funktioneller Magnetresonanztomographie kombiniert, um die funktionelle Organisation des auditorischen Kortex zu verstehen.

Kartierung der Nachwirkungen der Theta Burst Stimulation auf das menschliche Gehör Cortex mit 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 ...

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

Research

JoVE Journal

Biology

13.7K Aufrufe.  McGill University. Auditive Verarbeitungs ist die Grundlage von Sprache und Musik-bezogene Verarbeitung. Transkranielle Magnetstimulation (TMS) wurde erfolgreich auf kognitive, sensorische und motorische Systeme zu studieren verwendet, aber selten angewendet worden, um vorzuspielen. Hier untersuchten wir TMS mit funktioneller Magnetresonanztomographie kombiniert, um die funktionelle Organisation des auditorischen Kortex zu verstehen.

Serie: 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.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

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

Forschung

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.

Labeling Stammzellen mit fluoreszierenden Farbstoffen zur nicht-invasiven Detektion mit 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.

Funktionelle Bildgebung mit Verstärkung, Eyetracking und Physiologische Überwachung

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.

Elektronen-Spin-Resonanz-Micro-Imaging an lebenden Spezies, bei Oxygen Mapping

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.

Ein Allel-spezifische Gene Expression Assay zur Funktionsanalyse Grundlage genetischer Verbände testen

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.

Funktionelle Bildgebung Brown Fat in Mäusen mit 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.

Verfahren zur Beurteilung der Auswirkungen einer Reihe von Wellenlängen und Intensitäten von Rot / Nah-Infrarot-Licht-Therapie auf oxidativen Stress<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.

Interzelluläre Gap Junction-Kommunikation: Eine funktionelle Biomarker zur Beurteilung Unerwünschte Auswirkungen von Schadstoffen und Toxinen und gesundheitlichen Vorteile von Natural Products

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.

SA--Galactosidase-basierter Screening-Test zur Identifizierung von Senotherapeutischen Arzneimitteln

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.

Anwendung der passiven Kopfbewegung zur Erzeugung definierter Beschleunigungen an den Köpfen von Nagetieren

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