著者は、この作業を支援する Leduq 財団によって資金を供給 Sphingonet コンソーシアムを認めたいと思います。この作品も「血管分化と改造」共同研究センターによって支えられた (CRC/Transregio23、プロジェクト C1) と 7。FP、COFUND、ゲーテ国際ポスドク プログラム移動で、291776 号の資金。我々 はさらに処理および遺伝子型解析マウスと彼女のテクニカル サポートのキャサリン ・ ソマーを認めます。

すべての動物は、処理中に痛みや不快感を最小限に抑え最大限大切に取り扱われました。この手順では、当院の動物医療のガイドラインに従って、(承認番号 FK/1044 Regierungspraesidium ダルムシュタット) のローカル委員会によって承認されています。
マウスにおける透過試金の生体内でのための作業手順の概略を図 1に示します。各手順の詳細は次のとおりです。
1. 動物取扱
<ol><li>
準備およびトレーサーと麻酔薬の投与
	<ol>
<li>透磁率測定前日の埋め込み、少なくとも組織のラベルの付いたチューブと金型を準備します。きれいな発煙のフードの下で働いて、80% エタノールで洗浄ツールを使用して、プロトコルの中で無菌状態を維持します。現在のプロトコルのための 3-6 ヶ月の成熟した大人の年齢層の男性または女性の野生型 (WT) またはアンジオポエチン 2 (Ang-2) 関数の利得 (GOF) の CD1 マウスを使用します。10を前述のようにマウスのジェノタイピングを実行します。 注: 80% アルコール滅菌器具にはなりませんが、試料の汚染を最小限に抑えられます。</li>
		<li>2 mM 株に滅菌 PBS ですべてのトレーサーを希釈し、-20 ° C で光から保護因数を格納</li>
		<li>(テトラメチル ローダミン) TMR などのトレーサーを選択し、(かに) 個別励起/蛍光スペクトルとの FITC は、蛍光顕微鏡による色素間の干渉を最小限に修正可能なリジン (TMR を使用してまたはFITC フィルターそれぞれ)、蛍光測定透水性測定用のプレート リーダー。 注: TMR デキストラン 3 kD FITC デキストラン 3 kD 研究に使用修正可能な両方のリジンおよびそれ故にされる可能性がありますもとアルデヒドの免疫組織化学的解析ポスト固定の地域差を調査するために。</li>
		<li>すべての動物を介護施設ガイドラインに従い細心の注意をもって処理します。1:1 の混合10,13各トレーサーの 100 μ L を使用して追加のトレーサーを組み合わせた場合、200 μ L まで増やすことができます 100 μ L トレーサー ソリューションで各マウス腹腔内を挿入します。トレーサー蛍光バック グラウンド減算の偽コントロールとしてではなく単独で PBS で少なくとも 1 匹の動物を注入します。</li>
		<li>トレーサー注入後 5 分麻酔のケタミン ・ キシラジン i.p 注入と動物 (100 mg と 5-10 mg 0.9% で本体重量 kg あたり生理食塩水重量、それぞれ 25 g マウスあたりカクテル 150 μ L)。 注: 獣医軟膏適用されませんでした目のプロシージャの間に動物の麻酔と灌流/犠牲間の期間は短かったと (10 分) が観察場所目の任意の乾燥はなかった。</li>
	</ol>
</li>
	<li>
血のコレクションおよび心臓の血流
	<ol>
<li>心筋灌流 10 分局所麻酔薬投与後の動物を準備します。動物の麻酔外科平面に達したことを確認するために足けいれん反応がないことを確認してください。</li>
		<li>自分の背中に動物を置き、80% エタノールを腹部の皮膚に適用されます。小さなハサミを使用して、胸郭のすぐ下に小さな切開 (2 cm) で腹壁を開きます。横隔膜から肝臓を分離し、ゆっくりと横隔膜の胸腔内15の公開を切り取ります。</li>
		<li>両側に胸郭をカットし、左心室を公開するためにカットの胸骨を修正します。左心室の後部の蠕動灌流システムに接続されている 21 ゲージ蝶針を挿入します。</li>
		<li>右心房を穴をあけるとすぐに (10 s) 血清採血管に血液 (と最後カットオフ) 1 mL ピペット チップを使用して胸腔内に放出の 200-300 μ L を収集して氷の上保存します。 注: この灌流プロシージャは非生存です。</li>
		<li>血液を収集すると、すぐに灌流システム (10-12 rpm, 5 mL/分) に切り替えるし、暖かい (RT) 1x PBS で 3 分のための動物を灌流 (Ca の無料2 +/Mg2 +イオン)。 注: PBS 含んでいる Ca2 +/Mg2 +イオンはより良い心臓の活動の灌流中に利用できます。灌流に使用される PBS の合計量は 15 〜 20 mL の範囲内です。</li>
		<li>肝臓の色に注意して灌流品質を評価する腎臓灌流後ホワイト/ペールに表示されます。 注: 腎臓や肝灌流必ずしも大動脈から脳が特に心房穿刺中に手順 3 で準備中に破裂を得る動脈 (頸動脈・椎骨) 到達として脳血流。</li>
	</ol>
</li>
</ol>2. ティッシュの処理
<ol><li>オルガン コレクションとストレージ
	<ol>
<li>灌流末、頚部転位と収穫脳と腎臓によって動物の死を確認します。脳血流を脳 (髄膜の血管で目に見える血) の色によって灌流の品質が良い場合、透磁率の解析から動物を除外するを確認します。メス (図 1) を使用して 2 hemibrains に脳を分けます。</li>
		<li>メスの 1 つ hemibrain の嗅葉と小脳の無料で解剖し、大脳半球を 2 mL チューブに転送します。小脳の透水性評価に必要な場合、その他のチューブでヘミ小脳を格納します。</li>
		<li>1 つの腎臓を別の 2 mL チューブに転送します。ドライアイスにサンプルをすぐに保存します。</li>
		<li>組織-tek 社最適切削温度 (O.C.T) ドライアイスの化合物で残りの腎臓とヘミ脳 (小脳と嗅葉から成る) をネイティブで埋め込みます。</li>
		<li>最終的には、遠心分離機の 10、000 g、4 ° C で 10 分間で血清採血管で氷の上保存血液サンプル血清培養上清を 1.5 mL チューブに転送し、ドライアイス コンテナーに配置します。</li>
		<li>さらに処理まで-80 ° C の冷凍庫にドライアイスで収集されたすべてのサンプルを転送します。 注:、凍結融解後の均質化効率の増加として均質化の手順に進む前にサンプルを凍結することが重要です。</li>
	</ol></li>
	<li>均質化および遠心分離
	<ol>
<li>氷-80 ° C でダウン凍結ヘミ大脳と腎臓のサンプルを解凍し、臓器を含むチューブの重量を量る。これらの重みから組織重量を得るためのいくつかの空管 (約 20) の平均値を減算します。追加 300 μ L、200 μ L 冷 1 の腎臓とヘミ-大脳をそれぞれ含むチューブ X PBS。 注: (ヘミ小脳、100 mg 以下) のような組織の量を減らすため個々 のチューブを計量することをお勧めします。</li>
		<li>約 15 ストロークを実行することによって電気のオーバーヘッド攪拌 (1, 000 rpm) に接続されているポリテトラフルオロ エチレン (PTFE) 杵と元エッペン チューブ内の各サンプルを均質化 (1 ストロークを 1 と 1 を =)。サンプルの間に PBS で杵を洗い、次のサンプルに進む前に乾燥拭いてください。 注: 螢光を潜在的な干渉により均質化時に洗剤の使用を避けた。ただし、組織を徹底的に均質化、1 つのラウンドの凍結融解後、どちらがより効率的に細胞内のトレーサーがこのプロトコルで検出されたも。</li>
		<li>光と卓上遠心分離機で 4 ° C、20 分 15,000 g で最後にすべて一緒に遠心から保護されている氷の上の均質化のサンプルを格納します。即時蛍光または後で解析する-80 ° C で氷の上清を新しい 1.5 mL チューブに転送します。</li>
	</ol></li>
	<li>蛍光測定と定量化
	<ol>
<li>前の手順の最後に固定されている場合は、氷の上箔と光から守り-80 ° C で保存組織培養上清、血清サンプルを解凍します。</li>
		<li>黒 384 ウェル プレートに希釈血清 (30 μ L 1 × PBS + 20 μ L の血清) または組織培養上清 (として) の 50 μ L をピペットします。適切な背景差分を確保するため血清および組織培養上清の偽動物から少なくとも 1 つのサンプルは、この組織ホモジネートの背景は通常希釈剤として使用される PBS のそれより高い。 メモ: 3 偽動物の平均は偽蛍光値 (データは示されていない) に非常に小さな変動が観察されたが、自発に使用できます。また脳サンプルなど低蛍光サンプルの信号対雑音比を高めることができる PBS で希釈して使用される血清の量を減らせます。40 μ L の PBS で希釈し最大 10 μ L の血清を調べた。泡が井戸に存在しないことを確認します。</li>
		<li>384 ウェル ブラック プレートをプレート リーダーに挿入し、蛍光測定を選択する新しいスクリプトを開きます。</li>
		<li>ゲインを最適に設定し、それぞれ TMR や FITC dyee の 550/580 または 490/520 の励起/蛍光 (nm) の値に使用生蛍光ユニット (RFUs) を取得する測定を開始します。</li>
		<li>プレート リーダーから未加工の蛍光ユニット (RFUs) を使用して、対応する偽値を差し引いた透磁率指数 (PI) を計算します。<ol>
<li>
* 透水性インデックス(mL/g)= (組織 RFUs/g 組織重量)/(血清血清 RFUs/mL)
</li>
		</ol>
</li>
		<li>例の計算脳透磁率指数 (PI) 動物 GOF1 (表 1):<ol>
<li>GOF1 脳 RFUs - 偽脳 RFUs 154 22.5 を = = 131.5</li>
			<li>GOF1 血清 RFUs - 偽血清 RFUs 38305 27 を = = 38278</li>
			<li>脳の重量 (g) = 0.195</li>
			<li>血清量 (ml) = 0.02</li>
			<li>脳の透過性インデックス (10-3 mL/g) = (131.5/0.195)/(38278/0.02) = 0.352 注: 透磁率指数の計算は、絶対と同等の実験と組織型との間にある値を得られます。これらはしかし 2 グループ12間の比較の容易さのための比率として表示できます。1 まだ維持間の動物変化対照群をコントロール グループ平均を運転、制御グループの平均 PI と両方のグループで各動物の PI を割ることによってこれを実現できます。この変換は、1 に設定されて、コントロール グループを基準にして実験的グループ (またはグループ) の値を提供します。</li>
		</ol>
</li>
	</ol></li>
	<li>トレーサーの蛍光可視化
	<ol>
<li>クライオスタットのセクションは-20 ° C に設定し、室温で置かれたスライドをセクションを転送 10 μ m に組織-tek 社化合物 (O.C.T) (ステップ 2.1) でネイティブに埋め込まれた腎臓/ヘミ-脳ブロックをカットします。セクション乾燥 (約 30 分)、一度転送は使用するまで-80 ° C のフリーザーにスライドします。</li>
		<li>染色の日に 10 分、37 ° C でスライドを解凍し、PBS でクイック洗濯後室温で 10 分間 4 %paraformaladehyde (PFA) のセクションを修正します。</li>
		<li>室温で 1 時間 0.5% トリトン X-100 pH 7.5 1 %bsa を含む滅菌 PBS の透過/ブロック バッファーのセクションを孵化させなさい。</li>
		<li>CD31 一次抗体と室温で 1.5 h のスライドを孵化させなさい (0.5 mg/ml、クローン メック 13.3)、トリトン X-100 (pH 7.2) PBS の 3 つの 5 分洗浄後滅菌 PBS、0.5% BSA 0.25% を含むバッファーの 1: 100 で希釈しました。</li>
		<li>蛍光標識抗体希釈 1: 500 (2 mg/mL) の種特異的に室温で 1 時間上のバッファーの二次抗体の孵化を実行します。CD31、1: 500 の希釈でヤギ抗ラット 568 アレクサやアレクサ 488 を使用できます。DAPI を含める (300 μ M) 二次抗体で、核を染色する (1:1、ストックから 000 希釈) をミックスします。</li>
		<li>アクア polymount とステンド グラスのセクションをマウントし、暗闇の中で常温で重合のために一晩置いておきます。</li>
		<li>スペクトル イメージング共焦点レーザー スキャン顕微鏡システムを使用して画像を取得します。NIS 要素ソフトウェア (バージョン 4.3) によってイメージを分析します。モンタージュの数字の生成のため、Photoshop などのソフトウェアを使用できます。</li>
	</ol></li>
</ol>

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(65), e3564 (2012).</li><li>Hoffmann, A., Bredno, J., Wendland, M., Derugin, N., Ohara, P., Wintermark, 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+and+Low+Molecular+Weight+Fluorescein+Isothiocyanate+(FITC)-Dextrans+to+Assess+Blood-Brain+Barrier+Disruption:+Technical+Considerations.">High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations.</a> Translational stroke research. 2 (1), 106-111 (2011).</li><li>Armulik, A., Genov&#233;, G., 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=Pericytes+regulate+the+blood-brain+barrier.">Pericytes regulate the blood-brain barrier.</a> Nature. 468 (7323), 557-561 (2010).</li><li>Daneman, R., Zhou, L., Kebede, A. A., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+are+required+for+blood-brain+barrier+integrity+during+embryogenesis.">Pericytes are required for blood-brain barrier integrity during embryogenesis.</a> Nature. 468 (7323), 562-566 (2010).</li><li>Bell, R. D., Winkler, E. 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=Pericytes+control+key+neurovascular+functions+and+neuronal+phenotype+in+the+adult+brain+and+during+brain+aging.">Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.</a> Neuron. 68 (3), 409-427 (2010).</li><li>Banks, W. A., Gray, A. M., 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=Lipopolysaccharide-induced+blood-brain+barrier+disruption:+roles+of+cyclooxygenase,+oxidative+stress,+neuroinflammation,+and+elements+of+the+neurovascular+unit.">Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit.</a> Journal of Neuroinflammation. 12, 223 (2015).</li><li>Krause, G., Winkler, L., Mueller, S. L., Haseloff, R. F., Piontek, J., Blasig, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+claudins.">Structure and function of claudins.</a> Biochimica et biophysica acta. 1778 (3), 631-645 (2008).</li><li>Johansson, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-Brain+Barrier:+Role+of+Brain+Endothelial+Surface+Charge+and+Glycocalyx.">Blood-Brain Barrier: Role of Brain Endothelial Surface Charge and Glycocalyx.</a> Ischemic Blood Flow in the Brain. , 33-38 (2001).</li><li>Fu, B. M., Li, G., Yuan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+effects+of+the+blood-brain+barrier+on+the+transport+of+charged+molecules.">Charge effects of the blood-brain barrier on the transport of charged molecules.</a> The FASEB Journal. 22 (1 Supplement), (2008).</li><li>Goebl, N. A., Babbey, C. M., Datta-Mannan, A., Witcher, D. R., Wroblewski, V. J., Dunn, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Fc+receptor+mediates+internalization+of+Fc+in+transfected+human+endothelial+cells.">Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells.</a> Molecular biology of the cell. 19 (12), 5490-5505 (2008).</li><li>Lopez-Quintero, S. V., Ji, X. -. Y., Antonetti, D. A., Tarbell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-pore+model+describes+transport+properties+of+bovine+retinal+endothelial+cells+in+normal+and+elevated+glucose.">A three-pore model describes transport properties of bovine retinal endothelial cells in normal and elevated glucose.</a> Investigative ophthalmology &amp; visual science. 52 (2), 1171-1180 (2011).</li><li>Hallmann, R., Mayer, D. N., Berg, E. L., Broermann, R., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+mouse+endothelial+cell+surface+marker+is+suppressed+during+differentiation+of+the+blood+brain+barrier.">Novel mouse endothelial cell surface marker is suppressed during differentiation of the blood brain barrier.</a> Developmental dynamics: an official publication of the American Association of Anatomists. 202 (4), 325-332 (1995).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

血液脳関門の機能障害は、プライマリとセカンダリの脳腫瘍や脳卒中を含む神経疾患の数に関連付けられます。BBB 破壊はしばしば命にかかわる中枢神経浮腫を関連付けです。開始の分子機構の解明や BBB の閉鎖したがって神経疾患一般の治療的意義の調査の研究者。ただし、調査方法は蛍光画像17,18の面倒で主観的な定量化に依存する技術的な問題...

血液脳関門 (BBB) は、基底膜の鞘は、密接に関連するペリサイト (Pc)、および自分の終わり足の1 で基底膜を包むアストロ サイト (ACs) でサポートされている微小血管内皮細胞 (Ec) で構成されています ,2。ECs は、いくつかの細胞の種類をサポートし、主に ACs と Pc、バリア機能を調節すると対話も神経細胞とミクログリア、すべての一緒にユニットを形成、神経血管 (NVU)。NVU は、血液媒介性毒素や脳を入力してから病原体の輸送を制限する BBB の機能にとって重要です。この関数は、ECs ともトランスポーター p 糖タンパク質 (P ・ gp) その流出内皮を入力分子に戻すなどの操作のために存在するクローディン 5, オクルーディン, 精巣毛細血管-1, などタイト結合分子の結果1,ルーメン32,船。ただし、BBB は EC 膜1,2,3で表現される特定のトランスポーターで栄養 (ブドウ糖、鉄、アミノ酸) など重要な分子の輸送のためできます。EC 層は内腔 (血側) と特定とベクトル輸送機能報4,5 ように abluminal (脳向け膜) の間様々 なトランスポーターの分布に関して高偏極します。.BBB は中枢神経系の環境をしっかりと規制に関して保護、機能 BBB とパーキンソン病などの疾患に CNS ドラッグデリバリーのための大きな課題です。BBB 機能障害神経疾患でもバリア機能障害はアルツハイマー病 (AD) のように、例えば特定のトランスポーター ターゲットへの損傷を含めることができます、特に脳薬物送達が増加することを想定できません。広告で LRP1、怒り、P gp などいくつかのアミロイド β のトランスポーターが調節不全として知られている、無駄な6,7、8をかもしれないそれ故にこれらの運送者を対象とします。BBB は、ストローク、広告、髄膜炎、多発性硬化症と脳腫瘍9,10,11などのいくつかの神経疾患の障害です。治療戦略の重要な部分は、バリア機能を回復させる、従ってその評価は重要です。
この仕事については客観的かつ我々 正しく適用されているマウスのいくつかの行に両方実験的な遺伝子疾患モデル10,12,13 齧歯動物で透水性の試金のための量的なプロトコル ,14。メソッドは、血管コンパートメントからトレーサーを削除するマウスの灌流が続く蛍光トレーサーの単純な腹腔内注射に基づいています。脳や他の臓器が収集した記事の灌流および透水性の客観的評価と蛍光プレート リーダーで組織ホモジネートの測定に基づく絶対透磁率インデックス。組織ホモジネートまたは任意のトレースを受け取りません偽動物からの血清を使用して背景のすべての未加工の蛍光値が修正されます。十分な正規化は、血清量、血清蛍光組織、従って絶対と同等の実験と組織型との間にある透過性インデックスを降伏の重量に含まれます。グループ間の比較を容易にするため絶対浸透率のインデックス値容易に変換できる比率と我々 は以前に12を実行していた。同時に、ストアド ヘミ脳や腎臓に利用できる可視化トレーサー蛍光顕微鏡10。古典的な蛍光顕微鏡とはいえ面倒な切片と半定量分析のためのイメージの主観的な選択のために透磁率の地域差を取得する価値があります。プロトコルの詳細な手順が表示されます、適切な場所にノートが追加されます。これは、他の小動物にスケーリングすることができますマウスの体内透過性アッセイを正常に実行するために必要な情報を提供します。アッセイは、トレーサーの多くの種類に適用できる特異的な蛍光スペクトルとトレーサーの組み合わせによって透過性評価に基づいた料金とサイズを可能にします。

<table>
	<tbody>
		<tr>
			<td>Tetramethyl Rhodamine (TMR) dextran 3kD</td>
			<td>Thermosfisher</td>
			<td>D3308</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate (FITC) dextran 3kD</td>
			<td>Thermosfisher</td>
			<td>D3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine (Ketavet)</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Saline</td>
			<td>Fresenius Kabi Deutschland GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-tek O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldhehyde solution</td>
			<td>Sigma</td>
			<td>252549-1L</td>
			<td>prepare a 4% solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, fraction V</td>
			<td>Roth</td>
			<td>8076.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>rat anti CD31 antibody, clone MEC 13.3</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rat alexa 568</td>
			<td>Molecular Probes</td>
			<td>A-11077</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rat alexa 488</td>
			<td>Molecular Probes</td>
			<td>A-11006</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua polymount</td>
			<td>Polyscience Inc</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge butterfly needle</td>
			<td>BD</td>
			<td>387455</td>
			<td></td>
		</tr>
		<tr>
			<td>serum collection tube</td>
			<td>Sarstedt</td>
			<td>41.1500.005</td>
			<td></td>
		</tr>
		<tr>
			<td>2mL eppendorf tubes</td>
			<td>Sarstedt</td>
			<td>72.695.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech precision wipes tissue wipers</td>
			<td>Kimberley-Clark Professional</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>384-well black plate</td>
			<td>Greiner</td>
			<td>781086</td>
			<td></td>
		</tr>
		<tr>
			<td>slides superfrost plus</td>
			<td>Thermoscientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE pestle</td>
			<td>Wheaton</td>
			<td>358029</td>
			<td></td>
		</tr>
		<tr>
			<td>electric overhead stirrer</td>
			<td>VWR</td>
			<td>VWR VOS 14</td>
			<td></td>
		</tr>
		<tr>
			<td>plate reader</td>
			<td>Tecan</td>
			<td>Infinite M200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Microm GmbH</td>
			<td>HM 550</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon C1 Spectral Imaging confocal Laser Scanning Microscope System</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic perfusion system</td>
			<td>BVK Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microcentrifuge</td>
			<td>eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo blood-brain barrier permeability assay in mice using fluorescently labeled tracers

血液脳関門 (BBB) は、毒素や病原体循環から脳微小環境を保護し、脳の恒常性を維持する特殊なバリアです。バリアの主な場所はタイトジャンクショ細胞間と細胞膜に発現排出トランスポーター起因するバリア機能が脳の毛細血管の内皮細胞です。この関数は、ペリサイトおよび単位 (NVU) を形成しているアストロ サイトによって規制されています。脳卒中、アルツハイマー病 (AD) などいくつかの神経疾患脳腫瘍は BBB 機能障害に関連付けられます。BBB 透過性の評価は神経疾患の重症度と治療戦略を採用の成功の評価に重要なため。
いくつかのマウスに正常に適用されている堅牢な透磁率測定モデル遺伝的および実験両方まだここで単純なを提示します。メソッドは、定量性は一般的に適用される顕微鏡によるトレーサー蛍光分析と比較して客観的にです。このメソッドは、マウスはマウスを麻酔続いて水性の不活性蛍光トレーサーのミックスと腹腔内注入されます。動物の心臓の血流は、脳、腎臓や他の臓器を収穫する前に実行されます。臓器の均質化し、遠心上清からの蛍光測定が続きます。直前に灌流心臓穿刺から採血血管コンパートメントに正規化の目的提供しています。組織蛍光が量的に入手するぬれた重量および血清蛍光に正規化されたトレーサー透磁率インデックス。追加の確認のため蛍光可視化用トレーサー対側の半脳免疫組織化学のために保存を利用できます。

我々 は最近、アンジオポエチン 2 (Ang-2) 関数の利得 (GOF) マウスではある健康の条件10の高いコントロール マウスより脳血管透過性を示しています。脳卒中誘発マウス、GOF マウスが大きな梗塞サイズは、コントロールの同腹子より大きな透磁率を持っていたことを示していますがだった。これらの結果は、BBB で透磁率にアン 2 の重要な役割を表示し?...

Watch this Scientific Journal Video about An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers at JoVE.com

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

生体内で血液脳関門透過性アッセイを用いたマウス蛍光トレーサーに分類

血液脳関門の機能障害の動物モデルに適用可能な灌流が続く蛍光トレーサーの腹腔内注入によるマウス脳血管透過性アッセイをご紹介します。1 逸見脳は、透水性の定量的評価と可視化トレーサー/染色の他に使用されます。10 匹のマウスに 5-6 時間を要します。

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

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Neuroscience

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

24.3K ビュー.  Goethe University Hospital. 血液脳関門の機能障害の動物モデルに適用可能な灌流が続く蛍光トレーサーの腹腔内注入によるマウス脳血管透過性アッセイをご紹介します。1 逸見脳は、透水性の定量的評価と可視化トレーサー/染色の他に使用されます。10 匹のマウスに 5-6 時間を要します。

ビデオ: An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

として不死化マウス脳微小血管内皮細胞株の生成<em&gt; in vitroで</em&gt;血液脳関門モデル

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

神経科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

超音波血液脳関門の破壊とマンガン増強MRIを用いた脳機能イメージングの試み

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6.
In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.
To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2).
This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

We describe a live whole animal quantitative measurement for permeability of the embryonic zebrafish brain. The technique analyzes the ability to retain cerebrospinal fluid and molecules of different molecular weights within the neural tube lumen and quantifies their movement out of the ventricles. This method is useful for determining differences in epithelial permeability and maturation during development and disease.

ゼブラフィッシュ胚性神経上皮の透水性に関するアッセイ

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

マウスにおける低温感受性と適応を決定するためのシンプルで安価な方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同時<em&gt; ex vivoで</emによる二つの網膜の&gt;機能テスト<em&gt; in vivoで</em&gt;電図システム

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

神経変性疾患のマウスモデルで FITC 標識アルブミンの点滴静注による血液脳関門透過性の評価

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

虚血性脳卒中のモデルラットの血液脳関門の破壊のIn Vivo評価

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

ラットモデルにおける集中超音波を用いた位置特異的血液脳関門開口へのベンチトップアプローチ

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

げっ歯類における血液脳関門開口のためのハイスループット画像誘導定型神経ナビゲーションと集中超音波システム

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...