この作品が"Fondazione Neuromed"でサポートされている、健康参加する「Ricerca グラーヴェ」イタリア省の資金

動物のすべてのプロシージャは IRCCS Neuromed 動物介護審査会によって、承認された &#34; 会館 Istituto スペリオーレ ディ Sanità &#34; (許可番号: 1163/2015-PR) され、2010 年の EU 指令のガイドラインに従って/63/EU 動物実験。
 1 頸静脈に挿入される FITC-アルブミン溶液の調製 注: すべての実験が実施をマニフェスト (週齢 11) HD R6/2 マウス、時代、性別一致の野生型 (WT) コントロール。同腹子。
<ol><li>ディゾルブ FITC-アルブミン パウダー (材料表 参照) リン酸緩衝生理食塩水 (PBS) で 10 mg/ml で 。
	注: 最適な結果を得るには、このため準備されるべき新鮮な各ラベルの反応</li>。
</ol> 2。手術中に使用する機器の準備 <ol><li>以前オートクレーブの手術器具 (材料の表 を参照) をレイアウトします</li>。
	<li>はクリーン アルコール (70% エタノール溶液) で手術のベンチです。手術滅菌使い捨てタオル ドレープで外科領域 (45 cm × 45 cm) を準備し、営業の実体顕微鏡を設定します</li>。
</ol> 3。頸静脈へ FITC-アルブミン注入術 <ol><li>麻酔マウスの体重を記録します</li>。
	<li>麻酔マウス腹腔内注射 (i.p) ケタミン (100 mg/kg) - キシラジン (10 mg/kg) ソリューションの適切な投与量と</li>。
	穏やかなつま先ピンチによる <li>モニター動物鎮静反射を撤回は、Walantus ら に示すよう 11, 12.</li> <li>フェイス アップ仰臥位で麻酔下のマウスを置き、4 本の脚と尾を手術ワークベンチに粘着テープで固定します</li>。
	<li>電気カミソリで首に適切な外科的マージンを慎重に剃るマウスを確保した後 (材料の表 を参照) し、ポビドン ヨード ソリューションと 70% エタノールで消毒します</li>。
	<li>綿棒で露出剃毛表面を乾燥します</li>。
	<li>優しく剃毛の皮膚を引き上げ、1.5 cm 長い縦切開を行う、メス、鎖骨の中央で線胸部上中間の開始と首の下だけを拡張する.</li>
	<li>鉗子外頸静脈を公開する顕微鏡観察 (5 倍) の下で慎重に離れて結合組織を伸ばす</li>。
	<li>右側に、FITC-アルブミン液の注入用 30 G 針の挿入を容易にするために頸静脈の左側に十分なスペースを確保する</li>。
	<li>動物を静脈内注入 (右頸静脈) ゆっくり (3 分以上) 10 mL/kg FITC-アルブミンの 100 μ L に 30 G の針を使用しています</li>。
	<li>15 分後、斬首でマウスを安楽死させる急速に前述 13 として頭蓋骨から脳を取り除く、中古冷蔵イソペンタンの少なくとも 10 倍脳自体のボリュームで 15 分間浸します</li>。
	<li>中古冷蔵チューブに脳を冷凍イソペンタンを移動し、組織学的断面まで-80 ° C のフリーザーに保存します</li>。
</ol> 4。組織切片の脳を流し込んで FITC-アルブミン <ol><li>埋め込む最適な切削温度 (OCT) で脳を複合冷凍クライオスタットを区分する前に (材料の表 を参照してください).</li>
	<li>直列クライオスタットで 20 μ m 厚いセクションに脳を切る (材料の表 を参照)、顕微鏡用スライド グラスの上にそれらをマウントと</li>。
	<li>特定を用いて FITC-アルブミン/ラミニン共同染色を行う抗ラミニン抗体前述 6 の方法 (材料の表を参照してください).
	<ol><li>簡潔に、一次抗体の抗ラミニン (pAb 1: 1000) でセクションを一晩インキュベート、Cy3 標識適切な二次抗体を続行します</li>。
		<li>Coverslip スライド 4 を含むメディアをマウントを &#39;、6-diamidino-2-phenylindole (DAPI) (吸収ピーク = 360 nm; 発光ピーク = 460 nm (材料の表 を参照してください).</li>
		<li>暗闇の中で 4 ° C で一晩乾燥してスライドのまま</li>。
		<li>蛍光顕微鏡下で染色する蛍光を観察 (材料の表 を参照してください) 両方の FITC 装備 20 倍の倍率で (吸収ピーク = 495 nm; 発光ピーク = 525 nm) および Cy3 フィルター (吸収ピーク = 550 nm; 排出量ピーク = 570 nm)。
		<ol><li>染色後すぐにサンプルを表示するのにはマニキュアを使用してコーナーで coverslip を確保し、(4.3.3) 上記として顕微鏡の下でそれらを検討します</li>。
		</ol></li></ol></li></ol> 5。蛍光顕微鏡を設定とカラー画像集録 <ol><li>蛍光顕微鏡を次のように設定します。
	<ol><li>蛍光ランプをオンに使用する前に約 10 分。顕微鏡、接続されているコンピューターやデジタル カメラをオンにします。画像解析ソフトウェアを実行 (材料の表 を参照してください).</li>
		<li>最適な信号収集および空間分解能の適切な目的を使用し、適切なフィルターを選択します</li>。
	</ol></li> <li>カラー画像の取得 <ol><li>画像解析ソフトウェアの [live] コマンドを選択 (材料の表 を参照してください)、各フィルターの最適な照明のパラメーター設定</li>。
		<li>単一チャネルのカラー画像を取り出してスケールバーを追加 [凍結] コマンドを選択する (&#39; 編集 &#62; を燃やすスケール &#39;)</li>
		<li>内の各イメージを保存、&#39; .tiff &#39; 形式</li>。
		<li>を選択してチャンネルを単一のイメージにマージ &#39; ファイル &#62; マージ チャネル &#39; の保存と、</li>
		<li>tiff &#39; 形式</li>。
		<li>脳スライス (スライドごと 6 セクション) ごと (2200 μ x 3400 μ m) を写真 4 の 24 ビット色の最小値を取得します</li>。
	</ol></li></ol> 6。FITC-アルブミン漏出の評価 <ol><li>自由に利用できる ImageJ 14 , 15 を使用して各単一チャネルの蛍光強度を分析します</li>。
	<li>合計各画像ごとラミニン CY3 蛍光強度による FITC-アルブミンの全蛍光信号強度を正常化し、グリーンの蛍光強度として血管外管外アルブミンを報告します</li>。
</ol>

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著者がある利益相反を開示するには

我々 はここで説明する手法は、主に脳疾患条件下で BBB の漏れを検出するため便利です。BBB の機能不全が多様な神経学的疾患4の共通の特徴として注目されています。我々 は以前 HD6のようなまれな神経変性疾患のマウスモデルで BBB 整合性の初期の異常を説明するのにこのアプローチを使用します。
このメソッドは、相対的な簡易性の利点と正確で信頼性の高い結果を提供するプロシージャの有効性を取ります。それは高感度プレクリニカル ・ リサーチ ツールを表す、スピードと BBB の中断を断面化された脳組織内の蛍光グリーンのシンプルな可視化による質的評価を提供しています即時性のため便利です。ただし、生化学および分子の分析と組み合わせると、テクニックの BBB の相関解析を許可が血管内皮 TJs の構造と機能の整合性についての定量的な情報を提供することに成功したと脳のホメオスタシスを中断。
高分解能電子顕微鏡による16,などの他の方法と比較して安価で時間要求手順 BBB 微細構造についての情報を提供することはできませんそれがここで説明されているプロトコルがあります。17します。 さらに、その簡単なアプリケーションと再現性のため、手順は、神経変性障害前臨床モデルを特徴づける進歩的な脳血管病変を監視するのに役立ちます。
蛍光ベースのアプローチによって BBB 破壊を評価するための偉大な強さある直接リアルタイムでテストして最終的に共通の BBB 機能の微妙な変化の役割を調査するための潜在性および稀な神経疾患の影響BBB-に影響を及ぼす薬物や内因性の分子血液や脳室から適用されます。プロシージャは人間にまだ変換できないが、将来的に臨床練習で使用する新しい脳のトレーサーの開発のための徴候を提供する可能性があります。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author&#39;s names was corrected from:
Maglione Vittorio
to:
Vittorio Maglione

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author's names was corrected.

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

中枢神経系 (CNS) 内の恒常性は、適切なコミュニケーションと神経細胞の機能のための前提条件です。中枢神経系柔密によって封鎖される周辺から、内皮細胞血液脳関門 (BBB)、末梢血流と脳との間のインターフェイスを表し、これらの 2 つの地区の1 間のクロストークで極めて重要な役割を果たしています。 ,2。BBB は複雑な専門マイクロ血管内皮細胞) 細胞複合体 - タイトな接合 (TJs) - によって互いにリンク、囲まれて周を主体とする立体構造物の動的ニューロン終末とアストロ サイトの足プロセス1,2。
生理学的な条件の下でそのまま BBB の非常に低透磁率により、脳の栄養素および他の分子の輸送の厳しい規制とにより中枢神経系の変化からユニークな保護、潜在的な末梢神経活動に影響を与える可能性があります血液の組成は、1,2,3を侮辱します。
BBB の整合性および高められた透磁率の中断長い神経の多くの重要な機能を構成するため知られている、神経変性障害4ただしハンティントンの病気 (HD)5,6, を含む、かどうかこのような機能不全が原因の現象や病気の過程で繁殖イベントはまだ不明です。BBB 破壊のタイミングもとらえどころのないまま、しかし、中断の BBB の整合性が、後半イベント早期のステップ6ではなく、病気の進行で,7を表さないことを示します出現の私達のグループと他の証拠,8、長期的な影響を持っている可能性があります。
これを念頭において、早熟役に立つ病気の進行と脳損傷を予測し、正常に代替しよりターゲットを絞った介入できるを開発する戦略を開発するために神経変性の BBB の内訳を明らかにすることが重要です。このような混乱の臨床結果を軽減します。したがって、BBB 減損の信頼性の高い画像は、脳疾患の診療・研究の主要な重要なのです。
本稿では高分子量フルオレセイン イソチオ シアン酸ラベル-アルブミン (FITC-アルブミン) を使用して、HD マウス モデルの BBB 透過性の評価の成功と単純な手順をについて説明します。通常、脳実質にバリアを越えることはできません FITC-アルブミンの漏出を BBB 漏れの指標として測定しました。この手法は、ラットと cerebrovasculature 障害9,10によって特徴付けられる他の病理学の条件に容易に適応可能であります。

<table>
	<tbody>
		<tr>
			<td>Albumin-fluorescin isothiocyanate conjiugate</td>
			<td>SIGMA</td>
			<td>A9771-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>pAb anti-Laminin</td>
			<td>Novus Biologicals</td>
			<td>NB300-144</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 anti-rabbit made in goat</td>
			<td>MILLIPORE</td>
			<td>AP132</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERFROST PLUS</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips 24 X 50 mm</td>
			<td>Thermo Scientific (DIAPATH)</td>
			<td>61050</td>
			<td></td>
		</tr>
		<tr>
			<td>Kilik Optimal Cutting Temperature (OCT) compound</td>
			<td>Bio Optica</td>
			<td>05-9801</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Media</td>
			<td>VECTOR</td>
			<td>H-1500</td>
			<td>Mounting media with DAPI</td>
		</tr>
		<tr>
			<td>iNSu/Light Insulin Disposible Syringe</td>
			<td>RAYS Health &#38;Safety</td>
			<td>INS1ML26G13</td>
			<td></td>
		</tr>
		<tr>
			<td>30G 1/2&#34;</td>
			<td>BD Microlance</td>
			<td>304000</td>
			<td>Needle for Insulin disposible Syringe</td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>F.S.T.</td>
			<td>91003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#22 Disposable Scalpel blads</td>
			<td>F.S.T.</td>
			<td>10022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris scissors 10.5 cm</td>
			<td>F.S.T.</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5745 45&#176; 0.10 x 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps 10 cm</td>
			<td>F.S.T.</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5 0.1 X 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical patch</td>
			<td>Medicalis</td>
			<td>34788</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable towel drape</td>
			<td>Dispotech</td>
			<td>TVO50VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic Microscope</td>
			<td>NIKON</td>
			<td>SMZ 745 T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic Illuminator LED light (C-FLED2)</td>
			<td>NIKON</td>
			<td>1003167</td>
			<td>Optic Illuminator for Stereoscopic Micrscope</td>
		</tr>
		<tr>
			<td>Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>932162</td>
			<td>Epifluorescence Microscope</td>
		</tr>
		<tr>
			<td>Microscope digital Camera</td>
			<td>Nikon</td>
			<td>DS-Ri2</td>
			<td>Microscope camera</td>
		</tr>
		<tr>
			<td>Intenslight</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Microscope lamp</td>
		</tr>
		<tr>
			<td>NIS-Elements 64 bit</td>
			<td>Nikon</td>
			<td>AR 4.40.00</td>
			<td>Analysis Software</td>
		</tr>
		<tr>
			<td>Electric Razor</td>
			<td>Gemei</td>
			<td>GM-3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease

血液脳関門 (BBB) 整合性の破壊は、異なる神経と神経変性疾患の一般的な機能です。開発と BBB の変化を正確に検出する信頼性の高いプロシージャの検証が重要であるし、便利なツールを表す摂動の BBB の恒常性と脳疾患の病態との間の相互作用は、さらなる調査を必要とするが潜在的疾患を予測するためターゲットを絞った治療戦略を進行と開発。
ここでは、BBB の BBB 透過性の欠陥は明らかに早熟で検出可能なハンチントン病のマウスの前臨床モデルで発生するそのような変性条件で漏れを評価するための簡単で効率的な手順を提案します。病気。具体的には、高分子量かにラベル (FITC)-アルブミン、後者は障害時にのみ、BBB を交差することがあるがマウスの頸静脈と血管や実質地区におけるその分布に鋭く融合その後、蛍光顕微鏡によって決定されます。
脳実質の緑色蛍光アルブミンの蓄積異常 BBB 透過性の指標として機能し、J の画像処理ソフトウェアを使用して量的に表わされるとき緑の蛍光強度として報告されます。

FITC-アルブミン血流から緑色の蛍光トレーサーの BBB は脳 parenchymawhen に血管外漏出に頸静脈結果の適切な注入は、6を妥協しました。生理学的な条件の下で注入蛍光アルブミンの分布は脳の血管の内側に制限と周囲の血管領域や脳実質内に信号が検出できなかった (図 1 a、顕微鏡写真で、トップ)。逆に、BBB 神経病理学的状況下で危険にさらされて FITC-アルブミンの血管周囲スペースに沿うそして脳実質 (図 1 a, 顕微鏡下に) びまん性蛍光パターンが表示されます。血管マーカー染色、ラミニン、はっきり FITC-アルブミン蓄積 (図 1 a) のサイトのローカライズに使用されます。侵害された BBB FITC-アルブミンによって放出される増加緑蛍光画像処理ソフトを使用して評価された (材料の表を参照してください)、緑の蛍光強度として報告 (N = 3; 0.3112 対 0.5765; * * *、p = 0.0004、対 T 検定になっていない) (図 1 b)。上記したように、FITC-アルブミン強度が正常化しました。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56389/56389fig1.jpg"> 
図 1: BBB 損傷のマーカーとして FITC-アルブミン漏出します。(A) 代表的な蛍光顕微鏡下で緑色蛍光アルブミンの差分の分布を示す脳の凍結切片の両方生理学的な (上) と病理学の条件 (下)。赤い染色血管マーカー ラミニンを表します。露出時間: DAPI 20 ms、FITC 500 ms、テキサス州赤 60 さんスケール バー = 100 μ m (マージ モノグラフ)。(B) グリーン蛍光強度として報告は FITC-アルブミンによって放射される蛍光グリーンの数量が表示されます (手順 6.2 を参照)、生理学的な (健康な BBB) の病理学の条件 (侵害された BBB)。N = 3。データは、平均 ± SD を表されます * * * p = 0.0004 (対になっていない T 検定)。<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56389/56389fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

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

本研究では神経変性条件下で血液-脳関門の破壊を評価するための簡単で効率的な手順を提案する.我々 の目的を達成するために我々 は高分子量かにラベル (FITC) を注入-マウス頸静脈にアルブミン静脈および蛍光顕微鏡による脳実質にその漏洩を評価します。

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 ...

assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.8K ビュー.  IRCCS Neuromed. 本研究では神経変性条件下で血液-脳関門の破壊を評価するための簡単で効率的な手順を提案する.我々 の目的を達成するために我々 は高分子量かにラベル (FITC) を注入-マウス頸静脈にアルブミン静脈および蛍光顕微鏡による脳実質にその漏洩を評価します。

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

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

マウスモデルにおける静脈内自己投与方法

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

神経科学

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 ...

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 ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

神経変性疾患のマウスモデルのための嗅覚アッセイ

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

永続かつ広範な神経伝達のための新生児マウス脳の脳室内ウイルス注射

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

神経変性疾患のショウジョウバエモデルにおけるクライミング欠陥の定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

マウス海馬にオリゴマーアミロイドベータの定位注入

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

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

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

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

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

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

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

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 ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

病原体や医薬品との相互作用、脳によって障壁の交差を勉強する人間の血-脳インターフェイス モデル

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...