著者らは、リーグ・フランセーズcontre L&#39;épilepsie（MA-Lに）、（FJ）に研究所ナショナル・デ・ラ・サンテら・デ・ラ・RECHERCHEMédicaleグラントAVENIR R12087FSは、モンペリエの大学からの助成金（FJ）と補助金を承認したいと思います連盟から（NM）にラRECHERCHEシュルルCerveauを注ぎます。私たちは、モンペリエのin vivoイメージングのコアプラットフォームの施設内IPAMでChrystelラフォンの技術支援を認めます。また、原稿を校正するためのメアリーVernov（ワイルコーネル医科大学を）感謝します。

マウスは、食物、水を自由に摂取へのアクセスを許可し、12時間の明暗サイクルで維持されています。実験動物に関わるすべての手順は、国家と欧州の法律に準拠し、教育や科学研究（CEEA-LR-00651から01）のためのフランスの省によって承認されました。 6トラ​​ンスジェニック5xFADと4同腹の野生型（WT）対照マウスの合計は、この手順のために使用されました。 1.術前の準備<ol><li>腹腔内（IP）メトキシX04（10mg / kgの）Aβ沈着物11を標識する手術前48時間を注入。 </li><li>無菌の人工脳脊髄液（aCSFの）（120 mMのNaClを、26.2 mMの炭酸水素ナトリウム、2.5 mMの塩化カリウムを、準備1 mMののNaH 2 PO 4、1.3のMgCl 2、10 mMグルコース、95％O 2/5％CO 2の前とバブル2.5 mMのCaCl 2を）加えます。 </li><li>無菌の前にオートクレーブまたはホットビーズ滅菌器を使用して、手術道具（はさみ、ピンセット、カミソリの刃とドリルビット）を滅菌します手術。 </li><li> 70％エタノールで手術用ベンチや顕微鏡プレートを清掃し、清潔な吸収性の布でカバーしています。 </li><li>ケタミン/キシラジン（75 mgの/ kgおよび10mg / kg、それぞれ）と鎮痛剤ケトプロフェン（2.5ミリグラム/キログラム）IPを注入します。 </li><li>足や尾のピンチで麻酔の適切なレベルを確認してください。 </li><li>目に無菌眼軟膏を適用し、ウィスカを避けながら毛皮（耳、鼻）を剃ります。脱毛クリームであれば、それは慎重に皮膚刺激を防止するために、水ですすぎが続いているとして使用することができます。かみそりの刃で、残りの毛を剃ります。ポビドンヨード消毒液で皮膚を滅菌します。 </li><li>双眼顕微鏡下でマウスを置き、鼻に耳から皮膚切開を行います。頭蓋骨を露出するように横向きに肌を引き出します。骨膜を切開し、繰り返し可能な出血を止めるために、滅菌aCSFので頭蓋骨をすすぎます。 </li></ol> 2.血管系標識および間伐スカルウィンドウの準備（40分） <ol><li>綿棒で眼軟膏を削除します。眼科用局所麻酔剤（テトラ1％）の低下を適用し、優しく突起を達成するために、眼窩の周りの皮膚に圧力をかけます。 </li><li>ヤーデニと同僚12によって記載された手順以下の眼窩に、デキストラン（70 kDaの）（皮下インスリン注射針を100mg / kgまたは50 mg / mlで溶液50μl）と結合したフルオレセインイソチオシアネート（FITC）を注入します。目に無菌眼軟膏を適用します。 </li><li>皮膚が後退し、頭蓋骨が選択された撮像領域の周り清潔で乾燥していることを確認してください。デバイスをマウントヘッドの窓周りのシアノアクリル接着剤の少量を適用し、数秒10のための穏やかな圧力を適用し、ターゲット領域の周囲、及び接着剤（積み重ねかみそり刃からなる、 図1を参照）。 </li><li>領域があることを確認すること、デバイスをマウントヘッドの窓の縁に皮膚の端を引いてドライ。皮膚および獣医グレードのシアノアクリル接着剤の少量のウィンドウの端を固定し、乾燥した（ 図1）になるまで待ちます。 </li><li>ヘッドマウント装置を使用しての段階で動物を固定します。滅菌aCSFのですすぎ、よく皮膚やヘッドマウントデバイスとの間が完全に密閉されていることを確認してください。正常体温を維持するために、生存毛布にマウスを包みます。 </li><li>以前に10を説明したようにaCSFの溶液中に頭蓋骨の間伐を行います。頭皮が完全に除去されないことに注意してください。定期的に骨の破片をクリアすると、組織の過熱を回避するために、aCSFのを変更します。残りの骨の破片が短い空気パフを用いて除去することができます。 aCSFの溶液は、穿孔によって作成された振動を減衰させます。 注：マウスの頭蓋骨の厚さは、動物の年齢と関心領域に応じて80〜150ミクロンの間で変化します。 </li><li> （中程度の速度でと0.7ミリメートルバリ）歯科用ドリルを使用すると、エリア1に頭蓋骨の表面を薄く開始します。頭蓋骨への定期的な上下動パラレルを使用して削除直径0ミリメートル海綿骨の大部分（最初の40から70ミクロン）。 aCSFのごとに20〜30秒を交換し、3-4秒に中断されない掘削を制限することを忘れないでください。縫合糸の近くに骨が高度に血管であることに注意してください。 aCSFのが最終的な出血を止めるためにゲルフォームを予め浸し適用します。 </li><li>過度の圧力がかからないように注意しながら、頭蓋骨の間伐を続行するには鋭利な使い捨ての眼科顕微ブレードを使用してください。 35ミクロン - 最後の領域は直径20の厚さと、約0.5mmです。 注：これにより、骨の再成長および瘢痕化のために、それは、各撮像セッションでウィンドウをさらに薄くする必要があります。 </li></ol> 3.二光子顕微鏡（45分） <ol><li>必要に応じて、適切な麻酔を維持するために19ミリグラム/ kgのケタミン注射します。 </li><li>二光子レーザー顕微鏡（headmountステージに固定された）マウスを転送します。 20X水浸OBJを使用して、、1.0の開口数ectiveエピ蛍光ランプを用いた光フィールドの中央に薄く頭蓋窓を探します。目的は常にaCSFの中に浸漬されていることを確認してください。 </li><li>モードロックパルスレーザーチタン（Ti-サファイア680-1040nm）でレーザスキャンを開始します。それぞれ青と緑のチャンネルにメトキシXO4およびFITCデキストランの放出された蛍光を検出するために、750 nmで励起波長を設定します。 注：顕微鏡は、506 nmおよび555 nmで2ビームスプリッタを持っています。青色光は、470±12 nmのフィルターを備えたNDD検出器によって捕捉されます。緑色光は、525±25 nmのフィルターを備えた高感度のGaAsP検出器によって捕捉されます。目的から放出された最大のレーザパワーは、レーザ誘起組織の損傷を避けるために、10ミリワットを超えてはなりません。 </li><li> ROIの正確な再局在化のための3Dマップを作成するために、0.7X数値ズームで、低倍率のスタック（2ミクロンステップ500ミクロン×500ミクロン、512×512ピクセル）を取得後の時点で。 </li><li> 2X数値ズーム（;1μmのステップは200μm×200μmで、512×512ピクセル）で4高いデジタル倍率画像のモザイクを取得します。イメージングソフトウェアを使用すると、すべての領域をキャプチャするために200μm以下の手順でウィンドウを移動します。スタックの深さは、モザイクの各画像に軟膜表面から出発し、一般的には250μmです。 </li><li>顕微鏡からマウスを削除する前に、写真を撮るか、イメージング分野の将来の再局在化のための軟膜血管系の手描きの2Dマップを作ります。 </li></ol> 4.リカバリと再イメージング<ol><li>下に頭蓋骨を保持しながらトラクションを適用することにより、ヘッドマウントデバイスを削除します。任意の接着剤は、頭蓋骨や皮膚に付着したままの場合、細かい鉗子で慎重にそれを削り取ります。頭皮を縫合し、ホームケージに戻す前に、麻酔からの回復を監視するために加熱されたケージにマウスを置きます。私は、縫合糸への局所抗生物質のクリームを適用し、ケトプロフェン（2.5ミリグラム/キログラム）を注入しますPは2日後。 </li><li>繰り返しは、繰り返しイメージングセッションのために1.1〜2.5を繰り返します。必要であれば、画像の最適な品質を確保するために顕微ブレードと骨を剃ります。 注：イメージングセッションの間の間隔は1ヶ月より長い場合、歯科用ドリルで追加の薄型化が必要になることがあります。 </li><li>前回のセッション（ステップ3.6）から取られた軟膜血管系の2Dマップに従って落射蛍光光を用いて顕微鏡下にマウスを置きます。 </li><li>二光子レーザー走査を開始し、ステップ3.4で得られた3次元地図を用いたマイクロメートルスケールでのアラインメントを達成するために顕微鏡のステージを調整します。ステップ3.5で説明したように、詳細な撮影に進みます。 </li></ol> 5.買収後三次元再構成と画像解析<ol><li>例えばIMARIS（バージョン8.0及び7.7.2を使用）などの3D画像解析ソフトウェアを用いて脈管構造およびアミロイド沈着の三次元再構成を得ます。 </li&gt; <li>画像スタックの深さ全体で2つのチャネルの理想的なコントラストを得るために、画像処理メニューやコントラスト変化サブメニューのノーマライズレイヤプラグインを使用します。 </li><li> 同時に全ての時点で同じ領域の画像を開き、常に異なる時点を比較するために並行して処理します 。 </li><li>フィラメントトレーサーモジュールを使用してトレース容器。 <ol><li> 、新しいフィラメントを作成するために、フィラメントのアイコンをクリックして自動トレーサーをスキップして、自動パス方法を選択します。 </li><li>選択されたチャンネルは、血管画像に対応していることを確認してくださいとポインタの選択機能を使用して、開始点を決定するために、シフト+コントロールキーを押しながらイメージングセッション間で保存されている血管分岐部でクリックします。ポインタの選択機能を使用して、フィラメントのエンドポイントを選択するには、Shiftキーを押しながらクリックします。 注：obtaiの比較NEDスケルトンは、血管系の構造変化が表示されます。 </li></ol></li><li>新しいプラークを追跡するために、平面分析による平面を行います。 Aβプラークの成長を追跡するための青チャンネルに表面モジュールを適用します。新しいサーフェスを作成し、自動化された表面の作成に進むには、表面のアイコンをクリックしてください。バックグラウンド信号を設定し、減算するしきい値メソッドを使用します。統計メニューは、個々のアミロイド沈着物の表面に関するデータを提供します。 </li></ol>

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著者には、競合する金融利害関係を持っていません。

in vivoでの 2光子顕微鏡のためのオープン頭蓋骨技術は大きな撮像視野13,14の無制限のイメージングセッションの利点を提供しています。しかし、この技術は、互換性のない、多くの場合、関心14の領域における炎症を生成または神経血管リードアウト15に影響を与えます。逆に、薄くなった頭蓋骨の頭蓋技術は、脳血管の構造およびプラーク蓄積10,14...

脳血管系は、解剖学的および機能的ニューロンに結合されている多細胞構造です。血管の動的なリモデリングは、脳の発達全体および中枢神経系（CNS）1,2の病状の進行中に発生します。広く脳損傷が、てんかん、アルツハイマー病（AD）、外傷性脳損傷および脳炎3,4を含むいくつかのCNS疾患の顕著な特徴であることが認められています。発症から慢性期に、CNS疾患をモデル化するときしたがって、in vivoでの脳血管の変化を追跡することは重要になります。脳血管の修飾は、しばしば、神経損傷または可塑性と同時に起こるように、神経血管系の​​画像化は、CNS疾患の病態生理学を解読するためのキーのエントリポイントを表します。 このプロトコルは、マウスモデルにおいて脳血管系の再構築を追跡するために、長手方向の二光子ベースの手順を説明しますAD、アミロイド形成性プラーク沈着5-7による大と小口径血管に脳血管の欠陥によってマークプログレッシブ病理。この手順では、病気の経過を通して神経血管リモデリングに対するアミロイド沈着の可視化とその位置と成長の追跡を可能にします。バイタル蛍光色素は、トランスジェニックADマウス8における脳血管およびアミロイド斑の可視化のための各撮像セッションの前に注入されます。薄くなった頭蓋骨の頭蓋窓を通してROIのイメージングセッションを非侵襲的であり、生きているマウスの脳2,5,9,10に神経血管リモデリングを評価するための選択の方法を繰り返します。 以下の手順は、外科プロトコル、画像取得および処理の概要を説明します。主に大軟膜と貫通細動脈における脳アミロイド血管症（CAA）の早期の進行を特徴としています。

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">methoxy-X04</td>
			<td style="width:125px;">tocris</td>
			<td align="right" style="width:119px;">4920</td>
			<td style="width:167px;">use 10 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-Dextran 70Kda</td>
			<td style="width:125px;">sigma</td>
			<td align="right" style="width:119px;">46945</td>
			<td>use 100 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gelfoam/Bloxang</td>
			<td style="width:125px;">Bausch and Lomb</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micorsurgical blade</td>
			<td style="width:125px;">surgistar</td>
			<td align="right" style="width:119px;">6900</td>
			<td>must be sharp and not dented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">povidone-iodine</td>
			<td style="width:125px;">betadine</td>
			<td style="width:119px;"></td>
			<td>antisceptic solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">binocular stereomicroscope</td>
			<td style="width:125px;">olympus</td>
			<td style="width:119px;">SX10</td>
			<td>optimal image contrast is crucial for this procedure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-photon microscope</td>
			<td style="width:125px;">zeiss</td>
			<td style="width:119px;"></td>
			<td>Zeiss LSM 710mp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine scissors-toughcut</td>
			<td style="width:125px;">Fine science tools</td>
			<td>14058-09</td>
			<td>this scissors are optimized for cutting skin and soft tissue</td>
		</tr>
	</tbody>
</table>

longitudinal in vivo imaging of the cerebrovasculature: relevance to cns diseases

脳血管系のリモデリングは、脳の病理の共通の特徴です。 in vivoでのイメージング技術は、脳血管可塑性または損傷が残業を発生し、神経活動や血液の流れに関連して検出することが基本です。 生体内では 2光子顕微鏡は、生きている脳内の大規模な細胞単位の構造的および機能的可塑性の研究を可能にします。具体的には、間引き頭蓋骨ウィンドウの準備は重要な脳の炎症を誘発することなく、関心領域（ROI）の皮質領域の可視化を可能にします。皮質ROIの反復的なイメージングセッションは、多数のCNS疾患の進行中に、時間をかけて病気の特徴の特徴付けを提供する、実現可能です。脳の250μmの範囲内軟膜構造にアクセスするこの技術は、遺伝子細胞マーカーおよび/または生体染色色素によってコードされた蛍光プローブの検出に依存しています。後者（ 例えば、蛍光デキストラン）はLUMINをマッピングするために使用されています脳血管構造のアルコンパートメント。本明細書に記載のプロトコルに密接には、アルツハイマー病（AD）の進行を評価するためのアミロイド沈着のインビボマーカー、メトキシO4の使用です。我々はまた、血管の変化およびアミロイド沈着を追跡するために使用されるポスト取込み画像処理を説明します。 ADのモデルに現在着目して、記載されているプロトコルは、病理学的な脳血管変更が発生し、他のCNS障害に関連しています。

このプロトコルは、残業脳血管アミロイド沈着物を可視化するための方法を説明します。蛍光染料は、アミロイド沈着（メトキシXO4）11を標識することおよび脳血管内腔（FITC-デキストラン）1を埋めるために注入しました。 3D画像分析ソフトウェアモジュールは、連続した時点で撮像視野一定の視野の3D画像を作成するために使用されました。 5XFADマ?...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases at JoVE.com

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

この原稿が縦2光子顕微鏡を用いたin vivoでのアミロイド斑の蓄積の間に脳血管系のリモデリングを追跡するための手順を説明します。間引き頭蓋骨製剤は、アルツハイマー病のマウスモデルにおける脳損傷の進行を評価するために、蛍光色素の可視化を可能にします。

縦<em&gt;インビボ</em&gt;脳血管系のイメージング：中枢神経系疾患との関連

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

longitudinal-vivo-imaging-cerebrovasculature-relevance-to-cns

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

7.0K ビュー.  Institute of Functional Genomics. この原稿が縦2光子顕微鏡を用いたin vivoでのアミロイド斑の蓄積の間に脳血管系のリモデリングを追跡するための手順を説明します。間引き頭蓋骨製剤は、アルツハイマー病のマウスモデルにおける脳損傷の進行を評価するために、蛍光色素の可視化を可能にします。 

ビデオ: Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

医学

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

前臨床研究における薬理学的チャレンジのfMRIの利用：5-HTシステムへの応用

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

アウェイクマウスに対する中枢神経系治療薬の経鼻投与

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

敗血症性関節炎の経時的評価のためにMRターゲット造影剤を用いたin vivoイメージングのマクロファージ

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls. 	
DTI data analysis is performed in a variate fashion, i.e. voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e. differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.	
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.

Diffusion tensor imaging (DTI) basically serves as an MRI-based tool to identify in vivo the microstructure of the brain and pathological processes due to neurological disorders within the cerebral white matter. DTI-based analyses allow for application to brain diseases both at the group level and in single subject data.

神経変性疾患の分析における拡散テンソル磁気共鳴画像

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

縦用の眼の前房に移植、非侵襲<emインビボ&gt;</emリアルタイムでのシングルセル分解能&gt;イメージング

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

縦二光子イメージングに続いてマウスの急性脳外傷

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...