CIBCフェローシップ（ja）およびNSERC助成（RZ）。我々は、赤外線カメラに関する彼の助けのためロッシュM.コモ（Brainsight）、MRコンパチブルトラッカーや他のハードウェアのサポートに感謝しています。我々はまた、コイルホルダ用の多関節アームを設計し、ビデオで表示される数値のいくつかを提供するブライアン·ハインズ（Hybexイノベーション株式会社）に感謝しています。そして、私たちは、実験の設計を最適化する助けモントリオール神経学研究所のマクコーネル脳イメージングセンターからすべてのMR技術とM.フェレイラ氏に感謝します。

プロトコルは2日間のセッション（必ずしも連続しない）に分かれています。初日は領域は、TMSと対象とするために、各参加者のために定義するために解剖学的および機能的MRスキャンで構成fMRIのローカライザーで構成されています。二日目は、fMRIのセッションで構成されています事前とTMSは特別なMRコンパチブルTMSコイル（Magstim株式会社、ウェールズ、英国）とフレームレス定位システム（Brainsight）を使用して、スキャナの内部に適用されるポストTMS。後者は、各参加者の解剖学的および機能的なデータに相対皮質領域でリアルタイムTMSコイルの位置に使用されます。 1。ローカライザーセッション<ul><li>あなたの参加者の高解像度の解剖学的画像を取得して起動します。 </li><li>次に、任意のBOLD効果やMRIスキャンノイズ14,15に起因する聴覚マスキングを最小限に抑えるために、勾配エコーEPIパルスおよびスパースサンプリングのパラダイムを用いて機能画像を取得する。我々のケースでは、fMRIはdを行っている参加者が2つの連続した5分音符のメロディーは2,16、同一または異なっているかどうかを判断する必要がありますているメロディータスクをuring。被験者は5ノートの等しい2つの長さのパターン、C5の同じピッチですべてを聞いて、第2刺激後の左ボタンをクリックするように指示されている無差別の聴覚制御タスクも、含まれています。沈黙の期間はまた、各実行中のタスクの試験間でランダムに挿入されます。 12分16秒の合計持続時間のために、メロディの弁別、24聴覚制御試験と沈黙の24周期の24試験：合計で72試験は無作為化された順に記載しています。 </li><li>解剖学的および/または機能的なランドマークを用いた刺激部位を定義します。一つは、TMSがあるため深さにおける電界強度の減衰の刺激部位の深さに関する制限されていることを認識する必要があり、3センチメートル6,17よりも深い領域に達することを期待することはできません。重要なステップは、パートごとに類似したランドマークを使用することですなぜなら参加者間の構造と機能に違いがあるため難しいかもしれicipant、。ここでは、解剖学的および機能的なランドマークの両方を使用して配置された各参加者でHeschlの回を、ターゲットにしています。我々は、ハーバード·オックスフォード構造アトラス（が提供するHeschlの回のマスク使用<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> ）とTMSターゲットがピークによって個別に定義されていますHeschlの回2内活性化。加えて、我々はまた、このような音響と体性感覚の成果物としてのTMSの非特異的効果を制御するためのコントロールサイトとして使用される頂点の位置を定義します。頂点はイニオンと鼻筋、左右intertragalノッチから等距離の点の間に中間として解剖学的に定義されています。刺激部位（ すなわち Heschlの回または頂点）の順序は全体で相殺される個人。 </li></ul> 2。事前·事後のTMSのfMRI実験プリTMS fMRIのセッション<ul><li>スキャナに直接移動する参加者を準備します。これは、金属の除去を含むとTMSとMRスクリーニングフォームを充填。 </li><li>解剖学的および機能的なスキャン（ローカライザーのセッションで行われたものと同じ、第1節を参照）で、MRの取得を開始します。 </li></ul> MRI環境におけるフレームレス定位とTMS フレームレス定位システムは、赤外線カメラ（ポラリススペクトル）、登録手順とコンピュータのために使用するいくつかのツールと​​トラッカー（Brainsight）から構成される。コンピュータがスキャナ室の外側に位置するが、スキャナ室の入口に位置し、スキャナのドアは、TMSの適用中に開いたままです。ツールやトラッカーは、MR互換性があるだけでなく、赤外線カメラを支える三脚（自家製）とthアールereforeはスキャナ室の内部で使用。赤外線カメラは、MR-互換性がありませんので、スキャナーベッドから約2メートルで、スキャナのドアの近くスキャナ室、（安全手順についての説明を参照してください）​​の内側に配置されています。 TMSの刺激システムは、MRIスキャナ室に隣接する部屋に位置しています。我々は、MRI適合性のTMSコイルスキャナ室の内側に位置しており、RFフィルターチューブを通って、7メートルケーブルを介して、TMSシステムに接続を使用しています。 <ul><li>定位ソフトウェアパッケージ（ 例えば Brainsight）にご参加者の解剖学的および機能画像と刺激ターゲットをロードします。ここでは、右Heschlの回をターゲットにされます。 </li><li>プリTMS fMRIの買収後、32チャネル·ヘッド·コイル（シーメンス3Tスキャナと32チャンネルヘッドコイルの構成を使用している場合）の上部MRヘッドのコイル部分を削除します。 </li><li>次に、スキャナのベッドの上で参加者を下にスライドさせます。 </li><li> participで設定ヘッドバンドとトラッカーを修正アリの頭部。 </li><li>スキャナベッドに多関節アームを取り付け、アームにMRコンパチブルTMSコイルを固定してください。 </li><li>すべてのトラッカーとコイルはカメラの視野にあることを確認します。ここでは、カメラが若干右半球をターゲットとした場合、コイルの変位の簡単追跡を有効にする参加者の右側に移動されます。 </li><li>定位ツール（ つまり 、ポインタツール）を使用して被検者の頭部をキャリブレーションします。これは、解剖学的データで同じランドマークの参加者の頭（私たちの例では、例えば鼻の先、[後]と両耳の耳珠）でいくつかのランドマークをcoregisteringすることによって行われます。この手順では、2つの実験は、一人の参加者の頭の上にポインタツールを配置するため、参加者の頭の近くにして、コンピュータの登録を行うスキャナ室の入り口に他の実験を必要とされている。 </li><li>トンに対する接線のMRコンパチブルのTMSコイルを置き彼頭皮、および赤外線カメラ向けコイルトラッカー。コイルは正中2に後方と平行ポインティングコイルハンドル付き配向されている。多関節アームにネジを使用して、コイルの位置を修正します。 </li><li> MRIスキャナに隣接する部屋では、TMSのシステムの電源をオンにし、刺激を開始します。 TMSは、40代のための5Hzので繰り返し50Hzで3パルスで構成されるパターン化されたプロトコル、 すなわち 、連続シータバースト刺激（CTBS）、続いて適用されます。我々は、刺激出力18,19によって定義された固定の刺激強度（41％）を使用します。それは（安全手順については議論のセクションを参照）、健康な集団20の刺激中止後30分間の持続時間のために皮質可塑性を調節することが示されているように我々は、このプロトコルを選択しました。 </li></ul>ポストTMSのfMRIのセッション<ul><li>刺激が完了したら、それはできるだけ早くスキャナに対象を取り戻すことが重要です。 Tを削除するMSがスキャナ室からコイル、及び多関節アームを取り外します。 MRヘッドのコイルに、参加者の頭を後ろにスライドさせます。お使いのスキャナが準備済みで行く準備ができていることを確認します。我々のアドバイスは、全体のTMSのセッション中に発生した身体のプラットフォームを維持し、最小限にローカライザースキャンの回数と期間を削減することです。 </li><li> rTMSの効果が一時的なものなので、最終的なスキャンセッションは、機能スキャンで始まらなければなりません。再び、我々はメロディタスクの12分間実行中にfMRIを実施しました。 </li><li>最後のスキャンが完了したら、解剖スキャンで仕上げてください。 </li></ul> 3。代表的な結果 fMRIデータの解析は、事前·事後のTMS fMRIのセッションの両方に対して個別に行われている。各fMRIのセッションのために（ すなわち 、事前と事後TMS）は、メロディーと聴覚制御タスクとのコントラストが左右Heschlの脳回、上側頭脳回、下前頭脳回とprecenでタスク関連活性を示す最大スペクトル脳回（ 図1のB）。事前·事後のTMS fMRIのセッションとの違いを評価するために、我々はスチューデントのt検定を用いて、ランダム効果分析を行う。意義は、z&gt; 2のしきい値とpの補正されたクラスタのしきい値= 0.05で識別されるクラスタを使用して決定されます。 図1 Cは単一の参加者のためのコントラストポストマイナスプレCTBSを表しています。データは右Heschlの回（黒丸）を標的CTBSは左Heschlの回を含む反対（左）聴覚野におけるfMRIの応答の増加を誘導することを示唆している。 fMRIの応答の変化はまた、二国間で左中心後回、左の島にと横後頭葉皮質で発見されています。しかし、fMRIの応答に有意な変化は、コイルの下に見られません。また、同様の結合されたTMS-fMRIのプロトコルは、頂点（コントロール·サイト）を刺激するために繰り返されます。頂点の上に適用さCTBSと事前·事後fMRIのセッションの比較は、どのsignificaを示さなかったntの効果（データは示さず）。 <img src="/files/ftp_upload/3985/3985fig1.jpg" alt="図1"/> 図1：個々のプレTMS fMRIのデータ（）、ポストTMS fMRIデータ（B）とポストマイナスプレTMS fMRIのデータ（C）の分析。プリTMS fMRIのセッション（A）中とポストTMS fMRIのセッション（B）の単一の参加者のためのコントラストメロディ差別マイナス聴覚制御試験のAの結果。左から右へ：軸冠状および矢状ビュー。 （a）も（b）、TMSのコイルは、次の場所に右Heschlの回（黒丸）を標的としたx = 54、Y = -13、Z = 1（MNI152標準スペース）。事前·事後のTMS fMRIのセッションの両方の場合、座標値は、x = -54、Y = -13、Z = 1（MNI152標準スペース）刺激部位（ すなわち右Heschlの回で左半球の変化を示すことで表示されます）。スチューデントのt検定を用いたコントラストポストマイナスプレTMS fMRIのセッションの後は、結果。

特別な利害関係は宣言されません。

我々は、聴覚野の機能的な組織を調査するためにオフラインTMSおよびfMRIを組み合わせるプロトコルを記述します。次のセクションでは、我々はそのようなアプローチを実施する際に考慮すべき方法論的な要因を説明します。 ポストTMS fMRIのセッションの取得とタイミング 事前·事後のTMS fMRIのセッションのスキャンの取得と相殺の順序 <p class=...

<table border="1"><tbody><tr><td> 材料名 </td><td> タイプ </td><td> 会社 </td></tr><tr><td>経頭蓋磁気刺激</td><td> MagstimスーパーRapid2刺激すると、Rapid-2プラスワンモジュール</td><td> Magstim株式会社、ウェールズ、イギリス</td></tr><tr><td>磁気刺激用コイル</td><td> MRI対応70ミリメートル8の字コイル</td><td> Magstim株式会社、ウェールズ、イギリス</td></tr><tr><td>磁気共鳴画像</td><td> 3-Tシーメンストリオスキャナ、32チャネル·ヘッドコイル</td><td>シーメンス社、ドイツ</td></tr><tr><td>フレームレス定位脳手術</td><td> Brainsight </td><td>ローグリサーチ社、モントリオール、カナダ</td></tr><tr><td>光学測定システム</td><td>ポラリススペクトル</td><td>ノーザン·デジタル社は、オンタリオ州、カナダ</td></tr><tr><td>コイルホルダ用の多関節アーム</td><td>標準</td><td> Hybex Innovatイオン（株）、アンジュ、カナダ</td></tr><tr><td> MRI対応インサートイヤホン</td><td> Sensimetrics、モデルS14 </td><td> Sensimetricsコーポレーション、米国マサチューセッツ州</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

聴覚処理は、音声や音楽関連の処理の基礎となっています。経頭蓋磁気刺激（TMS）を認知、感覚と運動のシステムを研究するために正常に使用されているが、まれにオーディションを受けるために適用されていません。ここでは、聴覚皮質の機能的な組織を理解することは機能的磁気共鳴画像法と組み合わせるTMSを検討した。

機能イメージングによるヒト聴覚皮質上のシータバースト刺激の後遺症のマッピング

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 ビュー.  McGill University. 聴覚処理は、音声や音楽関連の処理の基礎となっています。経頭蓋磁気刺激（TMS）を認知、感覚と運動のシステムを研究するために正常に使用されているが、まれにオーディションを受けるために適用されていません。ここでは、聴覚皮質の機能的な組織を理解することは機能的磁気共鳴画像法と組み合わせるTMSを検討した。

ビデオ: 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.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

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

生物学

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.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

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.

強化、Eyetracking、および生体モニタリングによる機能イメージング

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.

電子スピン共鳴酸素マッピングのためのライブ種のマイクロイメージング

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.

遺伝的関連の機能的基準をテストするための対立遺伝子特異的遺伝子発現アッセイ

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.

FDG micro-PET/CTマウスにおける褐色脂肪の機能イメージング

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.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</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.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

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.

セノ治療薬同定のためのSA-β-ガラクトーシダーゼベーススクリーニングアッセイ

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.

げっ歯類の頭部に定義された加速度を生成するための受動的頭部運動の適用

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