著者らは、この手法の開発に有益なコメントや技術的なサポートのためにローラ·ディアス、ケヴィン·フィリップス、ウィラA. Hsueh、王で李に感謝したいと思います。

1。動物の準備とコールドトリートメント<ol><li>実験用マウスを収容するための承認されている4℃の低温室を探して、点検してください。 </li><li>寒い部屋の中で一晩プレチル動物ケージ。ケージは、飼料及び敷料なしであるが、水のボトルと組み立てられる。 </li><li>実験当日の午前中に、場所のマウスの30分間隔で予冷ケージのそれぞれに一つずつ。それはイメージングラボに輸送される前に、各単独ケージマウスは、ほぼ4時間寒い部屋に滞在する必要があります。マウスは断食をしているが、水へのアクセスを確認します。 </li><li>イメージングラボ一度に4時間後に低温処理輸送で一匹は30分毎。これは、氷と発泡スチロールの容器に充填し、ボックス内の氷の上に予め冷却住宅ケージを配置することによって実現できます。緩く発泡スチロールの箱にカバーを置きます。 </li></ol> 2。セットアップMicro-PET/CTイメージングワークフロー<p class=&quot;jove_content&quot;&gt;このプロトコルmicro-PET/CTイメージングにおいては、Siemens Inveon専用PET（dPET）システムとドッキングモードでInveonのモダリティ（MM）システム（CT / SPECT）を用いて達成される。動物は、MMの入り口から最初に配置された解剖学的な参照のためのCTでスキャンして、スタティックF18 PETの取得のためにdPETの中央にスライドさせる。ホストコンピュータが自動的にこれらのシーケンシャルタスクを遂行するために有効にするためには、以下の &quot;ワークフロー&quot;は前に実際イメージングセッションへInveon新株予約職場（IAW）ソフトウェアでプログラムされます。 <ol><li> CTの取得 ：全身CTスキャンについては、500 UA、80kVの電圧が、200ミリ秒の露光時間、240°の回転のための240のステップで電流を設定します。 X線検出器については、78ミリメートル軸イメージング分野とシングルベッドモードで &quot;低システム倍率&quot;で解像度を選択します。そのため、 &quot;共通のコーンビーム再構成&quot;メソッドを使って &quot;リアルタイム再構築&quot;を選択してホストPCの協議タスクを開始するために、専用のリアルタイム再構成コンピュータ（コブラ）。 </li><li> PETのエミッション取得 ： &quot;時間によって獲得する&quot;オプションで&quot;固定スキャン時間&quot;で設定した600秒（10分）。 &quot;研究同位体&quot;としてF-18を選択して、 &quot;エネルギーレベル&quot;として350から650 keVのを使用しています。 </li><li> PETの排出ヒストグラム ：静的スキャンを実現するための全体の持続期間のための1フレームのようにデータを処理するために&quot;黒&quot;に設定&quot;ダイナミックフレーム&quot;。ヒストグラムのタイプとして &quot;3D&quot;を選択し、 &quot;いいえ散乱補正&quot;を選択してください。 </li><li> PETの再建 ：再構成アルゴリズムとして2D順序サブセット期待値最大化（OSEM2D）を使用します。 </li></ol> 3。 FDGの注射<ol><li> 〜30分予定され、最初の注射の前にイメージングラボへの到着のための地域のベンダーから、18 F-FDG（10 MCI）の臨床的なパッケージをご注文ください。放射性マテリアを含むパッケージを受信し、調査する研究所の安全手順に従ってくださいLS（RAM）。 </li><li> Lブロックテーブルトップシールド、アリコートFDGと滅菌生理食塩水で希釈することによって提供される保護を持つ。 FDGの希釈された放射能濃度は、それぞれの注射用200から300μCi/100ULで利用可能であるべきです。 26G 1/2インチ針で1mlシリンジにFDGのソリューションを描画し、線量校正器と注射器全体の放射能を測定します。 </li><li>ちょうど腹腔内（ip）経路を介してのFDG溶液100μlと（ステップ1.4参照）寒い部屋から運ばれている動物を注入します。噴射時間を記録します。ドーズキャリブレータで再び注射器の残留放射能を測定します。 </li><li>氷で維持発泡スチロールのクーラー内部冷たいケージに戻って動物を置く。 FDGの取り込みのために1時間寒い（〜4℃）で動物をインキュベートする。 </li><li>次式により、各マウスのために注入FDGの活動を計算します。 注射前に注射器で注入された活動（μCiの）=活動- 注入後、シリンジ内の活動</li></ol> 4。 Micro-PET/CTイメージング<ol><li> micro-PET/CTイメージングは​​FDGの注射または低温処理後5時間後に1時間を開始します。酸素でイソフルラン3％麻酔導入室に動物を入れてください。 </li><li>一度麻酔が誘発され、動物は2L /分の流量で連続的にイソフルラン提供コーンフェイスマスク（2％）以内で休んで、その頭を持つマイクロCTペレット（動物用ベッド）の上に移動されます。電気加熱パッド（BioVet、M2Mイメージング株式会社）は体温を維持するために動物の下に配置されます。 </li><li> MMのスキャナの入り口に動物をスライドさせ、ツールバーから &quot;レーザー&quot;アイコンをアクティブにして、動物の胸が水平と垂直のレーザーラインのクロスになるようにベッドを移動するタッチパッドのコントロールを使用します。 &quot;レーザー整列&quot;ウィンドウでOPTIOとして &quot;PET収集、ワークフローに含まれている&quot; CTスキャンのように &quot;最初のスキャンの種類&quot;を選択し、nである。 </li><li> &quot;スカウトビュー&quot;ウィンドウを開き、X線レントゲン写真スカウトビューを取得します。 CTのビューの中心分野はマウス本体（肝臓）の中心部に位置していますように、動物のベッドの位置を調整するIAWを使用しています。必要な場合は、この手順を繰り返します。 </li><li>ステップ2で確立された &quot;ワークフロー&quot;を起動します。オプションがポップアップするとき、CT再構成に使用する適切な3D PET-CT変換行列ファイルを選択して、無減衰補正のPET再構成のために最近作成された正規のファイルを選択します。 IAWその後、CTやPETスキャン順次プログラムどおりに開始されます。 </li><li>ワークフロー全体が完了した後、20〜25分かかることがあり、簡単にASIPro VM、マイクロPET解析ソフトウェアIAWと共インストールされて取得されたCTとPET画像の品質を評価する。アーカイブデータストレージデバイスにイメージングデータまたはさらに詳細な分析のためのポスト画像解析ワークステーション（ステップ5を参照）に、ネットワークを介してデータを転送します。 </li><li> ReleaSEは、イメージングシステムから動物、そして室温で、その回復のための正常な食糧と水の供給とクリーンな住宅ケージに入れてください。システムは現在、キュー内の次の動物のための準備が整いました。 &quot;RAMを注入した実験動物の取り扱い&quot;に関する研究所の規制に従うべきであるイメージング後の動物の管理と取り扱いに注意してください。また、研究所の関連するRAMの廃棄物処理規制に従って針/注射器、吸収性パッド、手袋、およびすべての寝具を使用し、糞便が、放射性廃棄物として考慮されるべきであると扱われることに注意してください。 </li></ol> 5。ポストのイメージング解析<ol><li>オープンInveonリサーチ職場（IRW）ソフトウェア（シーメンス）、手動でCTとPETの両方のデータセットをインポートします。共同レジスタCTやPET画像 &quot;一般的な分析&quot;機能をツールを使用して &quot;登録&quot;ウィンドウで、 &quot;レビュー&quot;ウィンドウの下にあるすべての3次元的にCTとPETの画像間の確認に最適なアライメントを行う。 </li><li>&quot;関心領域（ROI）を定量化&quot;ウィンドウ、共同登録CT画像により提供される参照を持つ、首の肩甲骨間の領域では、BATを識別し、成体マウスの中で最も優勢な低温誘導性BATから、関心のあるボリュームを描く（VOI）PETデータセット上のBATの。 &quot;定量化の種類&quot;として &quot;ボクセル強度&quot;を選択し、ベクレル/ mlとしてVOI内の平均放射能を記録します。ベクレル/ mlまでカウント/秒に変換する校正係数は、以前から知​​られている放射能を持つファントムをスキャンすることによって確立されています。 </li><li> BAT内のFDG集積は、減衰補正を持つグラム組織当たりの割合注入量（％ID / g）として定量化される。注入された用量は、ステップ3.5の結果である、しかし、ベクレル（Bq）のユニット（1μCiの= 37,000ベクレル）に変換し、我々は組織の1ミリリットルが1グラムに等しいと仮定しています。 </li></ol>

<ol>
	<li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+importance+of+brown+adipose+tissue+in+energy+homeostasis.">The role and importance of brown adipose tissue in energy homeostasis.</a> Curr. Opin. Pediatr. 22, 478-484 (2010).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiol. Rev. 84, 277-359 (2004).</li><li>Collins, S., Surwit, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-adrenergic+receptors+and+the+control+of+adipose+tissue+metabolism+and+thermogenesis.">The beta-adrenergic receptors and the control of adipose tissue metabolism and thermogenesis.</a> Recent Prog. Horm. Res. 56, 309-328 (2001).</li><li>Ouellet, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outdoor+temperature,+age,+sex,+body+mass+index,+and+diabetic+status+determine+the+prevalence,+mass,+and+glucose-uptake+activity+of+18F-FDG-detected+BAT+in+humans.">Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-detected BAT in humans.</a> J. Clin. Endocrinol. Metab. 96, 1115-1125 (2011).</li><li>Cypess, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> N. Engl. J. Med. 360, 1509-1517 (2009).</li><li>Fruhbeck, G., Becerril, S., Sainz, N., Garrastachu, P., Garcia-Velloso, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAT:+a+new+target+for+human+obesity.">BAT: a new target for human obesity.</a> Trends Pharmacol. Sci. 30, 387-396 (2009).</li><li>Cypess, A. M., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+fat+as+a+therapy+for+obesity+and+diabetes.">Brown fat as a therapy for obesity and diabetes.</a> Curr. Opin. Endocrinol. Diabetes Obes. 17, 143-149 (2010).</li><li>Virtanen, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> N. Engl. J. Med. 360, 1518-1525 (2009).</li><li>van Marken Lichtenbelt, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> N. Engl. J. Med. 360, 1500-1508 (2009).</li><li>Cohade, C., Mourtzikos, K. A., Wahl, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="USA-Fat":+prevalence+is+related+to+ambient+outdoor+temperature-evaluation+with+18F-FDG+PET/CT.">"USA-Fat": prevalence is related to ambient outdoor temperature-evaluation with 18F-FDG PET/CT.</a> J. Nucl. Med. 44, 1267-1270 (2003).</li><li>Garcia, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+brown+fat+2-deoxy-2-[F-18]fluoro-D-glucose+uptake+by+controlling+environmental+temperature+prior+to+positron+emission+tomography+scan.">Reduction of brown fat 2-deoxy-2-[F-18]fluoro-D-glucose uptake by controlling environmental temperature prior to positron emission tomography scan.</a> Mol. Imaging Biol. 8, 24-29 (2006).</li><li>Saito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58, 1526-1531 (2009).</li><li>Agrawal, A., Nair, N., Baghel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+for+reduction+of+brown+fat+uptake+on+FDG+PET.">A novel approach for reduction of brown fat uptake on FDG PET.</a> Br. J. Radiol. 82, 626-631 (2009).</li><li>Soderlund, V., Larsson, S. A., Jacobsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+FDG+uptake+in+brown+adipose+tissue+in+clinical+patients+by+a+single+dose+of+propranolol.">Reduction of FDG uptake in brown adipose tissue in clinical patients by a single dose of propranolol.</a> Eur. J. Nucl. Med. Mol. Imaging. 34, 1018-1022 (2007).</li><li>Zhao, J., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenesis+is+beta3-+but+not+beta1-adrenergically+mediated+in+rat+brown+fat+cells,+even+after+cold+acclimation.">Thermogenesis is beta3- but not beta1-adrenergically mediated in rat brown fat cells, even after cold acclimation.</a> Am. J. Physiol. 275, 2002-2011 (1998).</li><li>Rowland, D. J., Garbow, J. R., Laforest, R., Snyder, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Registration+of+[18F]FDG+microPET+and+small-animal+MRI.">Registration of [18F]FDG microPET and small-animal MRI.</a> Nucl. Med. Biol. 32, 567-5672 (2005).</li></ol>

特別な利害関係は宣言されません。

本研究ではmicro-PET/CT-basedイメージング法は、単に風邪治療や市販の18 F-FDGの1注入を必要とする成体マウスにおけるBATの活動を検出するために開発されました。全体の手順は、すべての動物が扱われ、画像化されるまで、30分ごとに開始され、治療と撮像シーケンス後に1日で行うことができます。概説した実験条件下で、10匹のマウス（または5匹の2群）の合計は、単一の撮像システム?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> Micro-PET/CTイメージングシステム</td><td>シーメンスメディカルソリューションズUSA社</td><td> Inveon専用のPETシステムとInveonモダリティCT / SPECTシステム（ドッキング） </td><td></td></tr><tr><td>プロプラノロール</td><td>シグマ</td><td> P0884 </td><td></td></tr><tr><td> BRL 37344 </td><td>シグマ</td><td> B169 </td><td></td></tr><tr><td> 18 F-FDG </td><td> Cyclotope株式会社</td><td></td><td></td></tr><tr><td> C57BL/6J雄マウス</td><td>ジャクソン研究所</td><td> 000664 </td><td> 3から4ヶ月</td></tr></tbody></table>

functional imaging of brown fat in mice with 18f-fdg micro-pet/ct

褐色脂肪組織（BAT）はその離散場所と豊富な血管新生およびミトコンドリアの高密度化のために赤褐色の色によって白色脂肪組織（WAT）とは異なります。 BATは、エネルギー消費と非ふるえ熱産新生児哺乳類においてだけでなく、大人1において主要な役割を果たす。 BATの媒介熱発生は非常にβアドレナリン受容体2、3を介して主に、交感神経系によって調節されている。最近の研究では、人間の成人におけるBATの活動が否定的にボディ·マス·インデックス（BMI）と他の糖尿病パラメータ4月6日と相関していることが示されている。 BATはこうしてエネルギーバランス6-8の変調に着目しanti-obesity/anti-diabetes療法のための潜在的なターゲットとして提案されている。いくつかの冷たいチャレンジベースの陽電子放射断層撮影（PET）の方法は、人間のBAT 9-13を検出するために確立されている間、イメージングおよびquantifのための標準化されたプロトコルは、基本的にありませんマウスなどの小動物モデルにおけるBATのication。ここでは、マウスでのBATの機能評価のための堅牢なPET / CTイメージング法を説明します。彼らは、18 F-フルオロデオキシグルコース（FDG）のいずれかの用量を受信する前に簡単に説明すると、成人のC57BL/6Jマウスは、4時間の持続時間のために絶食条件下で処理冷たかった。マウスは、さらに1時間のポストFDGの注射のために寒さの中に残っており、その後、小動物専用micro-PET/CTシステムをスキャンした。取得したPET画像は、解剖学的参照用のCT画像と共に登録とBATの活動を提示するために肩甲骨間BAT領域にFDGの取り込みについて分析した。この標準化された冷治療とイメージングプロトコルは14プロプラノロールのβ-アドレナリン受容体拮抗薬の治療によって抑制BATの活性化、15、または強化されたBATの活性化は、β3アゴニストBRL37344 16により、例えば、薬理学的介入の間にテストBATの活動を通じて検証されています。方法デここcribedはBATの活性を調節する薬剤/化合物をスクリーニングするために適用することができ、または様々な前臨床および基礎研究のBAT開発や調節に関与している遺伝子/経路を特定することができます。

マウスBATのmicro-PET/CTイメージングの例を図1に示します。 CT撮影は解剖学的情報を提供していますが、PETイメージングは全身の18 F-FDG集積の分布と量をエンコードします。これらの画像データは、（1Aおよび1B）別に見（1C）の融合した、またはそのような最大値投影（MIP、1D）などの3D機能を使用して実証することができます。ここに3Dイメージングツール、関心体積（VOI）...

Watch this Scientific Journal Video about Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT at JoVE.com

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

マウス褐色脂肪組織（BAT）の機能イメージングの方法は、BAT内の18F-フルオロデオキシグルコース（FDG）の冷刺激取り込みが非侵襲的に標準化されたmicro-PET/CTプロトコルと評価されるで説明されています。この方法では、マウスモデルで、BATの活動の違いを検出する堅牢性と小文字が区別されます。

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 ...

functional-imaging-of-brown-fat-in-mice-with-18f-fdg-micro-petct

Research

JoVE Journal

Biology

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

19.0K ビュー.  The Methodist Hospital Research Institute, Houston. マウス褐色脂肪組織（BAT）の機能イメージングの方法は、BAT内の18F-フルオロデオキシグルコース（FDG）の冷刺激取り込みが非侵襲的に標準化されたmicro-PET/CTプロトコルと評価されるで説明されています。この方法では、マウスモデルで、BATの活動の違いを検出する堅牢性と小文字が区別されます。

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

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

マウス用ダーク/ライト遷移テスト

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

生物学

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 ...

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, ...

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

マウス用高架式十字迷路

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

アセンブリ、読み込み、および分析用超遠心器のサンプルセルの整列

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

マウスにおける伝統的な米航空宇宙局（NASA）後肢アンロードモデルへの交代方法

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

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.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

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 ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

非トランスジェニック無脊椎動物における単一ニューロン分解能を用いたネットワークダイナミクスを記録するためのフォトダイオードベースの光学イメージング

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.