Name: an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke
Uploaded: 2018-03-11T14:00:00.000+00:00
Duration: 12 min 19 s
Description: Watch this Scientific Journal Video about An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

著者らは、財政支援のアルダビール大学医療科学 (アイルランド) の研究のための副首相に感謝している (許可 No: 9607)。

動物実験を行い、アルダビール大学医療科学研究評議会のガイドラインに従ってすべてのプロシージャを行った (倫理的な ID 番号: 赤外線ARUMS。REC.1394.08). この可視化の研究で牧草研究所 (イラン ・ テヘラン) から得られる男性の大人の Sprague-dawley ラット (300-350 g) を使用しています。
1. 麻酔および血流計
<ol><li>4% イソフルランを使用して麻酔し、手術の間に亜酸化窒素 (70 %v/v) と酸素 (30 %v/v) の混合物にイソフルラン (1 1.5%) とそれを維持します。</li>
	<li>フィードバック制御暖房毛布にうつ伏せで麻酔動物を置き、この加熱装置に接続された直腸プローブによる 37±0.5 ° C の体温を維持します。</li>
	<li>乾燥を防ぐために両方の目に眼軟膏の少量を適用します。</li>
	<li>頭蓋骨の左側にある外科の領域を電気バリカンで剃る。ガーゼのパッドと肌の消毒に betadine ソリューションを使用します。この地域の 70% のエタノールを含む滅菌パッドを洗い流し、3 サイクル17の両方の手順を繰り返します。</li>
	<li>鎮痛の外科領域17に 0.5% ブピバカイン 0.2 mL を皮下注入します。</li>
	<li>(左耳のベースに左目の眼から延びる) 頭蓋骨 1 cm 長い皮膚切開を行い、頭蓋骨を公開する左の側頭筋を解剖します。作成小さなバリ穴 5 mm 外側 1 mm 後方前18レーザードップラー血流計ペンシル プローブの先端の配置を容易にします。</li>
	<li>仰臥位になりやすいからラットを切り、局所脳血流量 (rCBF) を監視するこれまでにドリル頭蓋骨ブラインド穴にレーザー ペンシル プローブを配置します。</li>
	<li>それぞれの動物のベースライン rCBF を記録し、これを 100% として考慮します。特に、80% 以上の脳血流量の低下が検出された19、20中大脳動脈閉塞 (MCAO) が開始されます。</li>
</ol>2. 焦点煎じつめて虚血の誘導
<ol><li>頸部領域を剃る、betadine ソリューションと 70% エタノールで皮膚を消毒してください。鎮痛17外科サイトで皮下 0.5% ブピバカイン 0.2 mL を注入します。</li>
	<li>左の総頸動脈 (LCCA) にアクセスする首の腹側表面で 2 cm 長い切開を行います。(ECA) 外頚動脈と内頸動脈 (ICA) の分岐にアクセスする近隣の筋膜や迷走神経から LCCA を分離します。</li>
	<li>LCCA または 5-0 絹糸を用いた ECA のいずれか完全に結紮そして翼口蓋動脈のレベルは無料 ICA を解剖します。</li>
	<li>緩く LCCA、周り 5-0 絹糸のネクタイを配置、その後一時的に ICA を血管のマイクロ クリップをクランプします。</li>
	<li>小切開は、以前に配置した緩いネクタイ前 LCCA と ICA の内腔の空間に 4-0 シリコン コーティング ナイロン縫合糸を挿入し、ナイロン縫合糸を保護し、血液の漏れを防ぎます LCCA 周り縫合糸を締めます。</li>
	<li>ICA から血管のマイクロ クリップを削除します。その後、MCA 起源の閉塞を示す rCBF の顕著な減少を観察までシリコン被覆管腔内のフィラメントを進めます。虚血期間 (90 分) の終わりに、腔内縫合21を撤回することによって再灌流を開始します。</li>
</ol>3. 頚静脈穿刺とエバンス ブルー (EB) 注射
<ol><li>正中線の左側に首で 1 cm の縦切開を行うそして、にべもなく左頸静脈 (LGV) の外の枝にアクセスする距離浅筋膜を解剖します。その後、5-0 絹糸と LGV の頭蓋の終わり完全に縛る。緩く、静脈周囲の関係を 2 つを配置し、心臓血管のマイクロ クランプと LGV 末一時的に。</li>
	<li>小切開は、2 つの縫合線の間の LGV と LGV の内腔の容量に満ちてヘパリン血清カテーテルを挿入し事前にそれ約 10 mm。 その後、カテーテルをセキュリティで保護し、出血を防ぐために静脈周囲の合字を締めます。少量の静脈の崩壊を避けるために血清を注入します。</li>
	<li>再灌流期間の初めに、ゆっくりと 5 分にわたって EB 色素 (生理食塩水で 2 %eb 液の 1 mL/kg) を挿入します。その後、0.5 mL 通常生理食塩水の注入カニューレを洗浄し、静脈12,22から注入カニューレを撤回します。</li>
	<li>最後に、首と頭の切開を縫合し、ブプレノルフィン 0.05 mg/kg を腹腔内注入 (IP) 鎮痛手術17再灌流期間中にすべての 6-8 h 注射を繰り返します。</li>
	<li>回復ケージ17水和を提供するために予め温めておいた saline(IP) の 5 mL を注入します。</li>
	<li>温度と空調制御場所特別な回復ケージ内の動物、動物の麻酔からの回復を監視します。</li>
</ol>4. 血液脳関門の透過性の評価
<ol><li>再灌流の 24 h 後深くチオペン タール ナトリウムと動物を麻酔します。胸骨の下に小さな穴を開けることで、胸腔内を開きます。</li>
	<li>右房内の小さな開口部を作るし、生理食塩水 0.9% に予め温めておいた (37 ° C) 左心室心房から普通の生理食塩水の出口になる無色23まで循環から EB を洗って 15 分間を通じて 110 mmHg の圧の 250 mL を注入します。</li>
	<li>すぐに頭蓋骨から脳を取り除くし、脳行列に配置。嗅球や小脳の解剖し、きれいなかみそりの刃を使用して別の脳の右半球正中線に沿って左 (Lesioned) から。</li>
	<li>各半球の重量を量るとリン酸緩衝生理食塩水 2.5 mL で個別にそれらを均質化し、渦のマシンを使用して 2 分の 2.5 ml のトリクロロ酸 (60%) のこれをミックスします。</li>
	<li>1,322 × g で 30 分間脳サンプルを遠心し、10 分の 4 ° C 冷蔵庫でクールダウンしてサンプルを許可します。</li>
	<li>培養上清を使用して、610 nm23で、分光光度計による EB 吸光度を推定します。</li>
	<li>脳組織の μ g/g として標準曲線と結果を表現に対する EB 濃度を計算します。</li>
</ol>5. 理事会の標準曲線の生産
<ol><li>リン酸緩衝生理食塩水 5 mL に EB の 1-10 μ g の濃度と理事会の 10 のサンプル ソリューションを準備します。</li>
	<li>610 の各サンプルの吸光度を測定分光光度計を用いた nm。</li>
	<li>Y 軸 (図 1) のスプレッドシートと X 軸上の現在の EB 濃度と吸光度の値を使用して散布図を作成します。</li>
	<li>その方程式と曲線の線形トレンド ラインを定義します。それを使用し、EB サンプル濃度を取得します。</li>
	<li>脳組織重量の μ g/g として EB 血管外漏出の最終結果を報告します。</li>
</ol>6. 偽操作
<ol><li>Sham のグループ (含む作り切開頸部に EB 注入) のラットと同じ手術のプロセスを実行しますが、MCAO を除外します。</li>
</ol>

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K., Kleinschnitz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke+and+their+application+in+clinical+research.">Animal models of ischemic stroke and their application in clinical research.</a> Drug Des Devel Ther. 9, 3445-3454 (2015).</li><li>Noor, R., Wang, C. X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hyperthermia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats.">Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats.</a> Neurosci Lett. 349 (2), 130-132 (2003).</li><li>Shin, H. K., 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=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Bottiger, B. W., 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=Global+cerebral+ischemia+due+to+cardiocirculatory+arrest+in+mice+causes+neuronal+degeneration+and+early+induction+of+transcription+factor+genes+in+the+hippocampus.">Global cerebral ischemia due to cardiocirculatory arrest in mice causes neuronal degeneration and early induction of transcription factor genes in the hippocampus.</a> Brain Res Mol Brain Res. 65 (2), 135-142 (1999).</li></ol>

著者が明らかに何もありません。

これまで、放射線と放射性トレーサー24,25, 蛍光顕微鏡26,27, EB 血管外漏出テクニック20,の検出などのさまざまな方法23血液脳関門の損傷を評価するために使用されています。EB 色素が強く血清アルブミンと結合することができると血管漏出を検出し、BBB 内訳11,28,29の定量化のトレーサーとして使用されます。承認済みで信頼性の高い方法としては、EB 血管外漏出手法は、虚血性脳卒中を含む脳の別の傷害によって影響を受ける BBB の整合性に直接推定を提供します。
BBB のin vivo評価には、虚血による分子病態脳浮腫の病態生理学的メカニズムを研究し、新しい治療上の介在を見つけるには研究者ことができます。このモデルは、特別な設備を必要としない、実験 (80% 以上)13,20で高い成功率と信頼できる結果を生成することができます。脳組織へ直接アクセスでき、このモデルは BBB の整合性の高精度な評価を有効に、長期的な研究に制限されています。
虚血性脳卒中による BBB の病理学的変化を 3 段階に分けて開発: (時間) 以内に急性の日に亜急性 (時間)、および慢性 (ヶ月日)30,31。明らかに、初期の治療は急性病態期に貴重な保護効果を生成します。EB 投与量と注入の時点は、虚血性の侮辱の後 BBB の動的な性質から信頼性の高い結果を得るための 2 つの重要なパラメーターです。したがって、ゆっくりと再灌流期間の初めの後の適切な投与量 (生理食塩水で 2 %eb 液 1 mg/kg) を使用して静脈カニューレを介して EB 色素の注入の重要な要因は、でき、病態生理学的変化に関する研究ストロークの初期の段階で.
いくつかの実験的方法は、虚血性脳卒中を研究に導入されています。この実験で、人間ストローク21,32と同様の条件を作成する腔内フィラメント法による MCAO を使いました。このテクニックはシンプルで信頼性の高いです。しかし、その実行は、さらに技術のパフォーマンスを向上し、その精度を保証するいくつかの技術的なポイントを考慮する必要があります。体温は、血圧と血液ガス監視対象33,34,35必要がありますしながら、手術中に生理的範囲内で保つ必要があります。レーザーのドップラー流量計と脳血流量の記録と適切な準備を使用して定数シリコーン コーティング フィラメントだけでなく MCAO 成功率を高める、また死亡率を減らします。

血液脳関門 (BBB) 破壊による分子病態脳浮腫には、虚血性脳卒中の重要な合併症と脳卒中患者1,2の生存率の主要な決定要因が残っています。脳毛細血管内皮細胞 (BCECs) によって形成される、異なる神経血管コンポーネント (BCECs、ペリサイト、グリア、および神経細胞3間タイトジャンクショなど) で構成される、血液-脳関門 (BBB) を提供します、中枢神経系 (CNS)、末梢血循環4,5間の専門的かつ動的なインタ フェース。虚血再灌流傷害など侮辱でした BBB の機能の整合性を混乱させるし、最終的に原発性脳腫瘍の怪我や脳の炎症を引き起こす脳実質に白血球の循環のそれに続く侵入につながる6,7. 脳卒中の発生に続く BBB の機能不全の正確な検出には動物モデルが必要です。このようなモデルは、非常に重要な病態生理学的メカニズムの基礎となる、新しい神経保護戦略を導入することを勉強するためです。BBB の細胞培養技術を用いたモデル体外は高い開発し、BBB 生理病理学8,9,10の分子生物学研究に使用されます。それにもかかわらず、生体内で動物モデル、人間の臨床条件に類似の BBB の虚血性損傷を作り出す、この点で非常に価値があるも。エバンス ブルー (EB) の血管外漏出の定量的検出は BBB の整合性と神経変性疾患、虚血性脳卒中11,を含む機能の評価のために使用されているよく受け入れられ、敏感な技術12,13,14します。 この方法は、実験室でコスト効果の高い、可能、再現性のある、完全に該当します。その実装では、放射性トレーサー15や磁気共鳴画像 (MRI)16、他の方法のための前提条件となるなど、高度な機器は必要ありません。この記事で包括的に虚血性脳卒中のモデルラットによる EB 血管外漏出 bbb ランク評価の基本的な技術的なプロセスを示しています。

<table><tbody><tr><td>Isoflurane</td><td>Piramal</td><td>AWN 34041100</td><td>20 - 25 &amp;deg;C</td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride (TTC)</td><td>Molekula</td><td>31216368</td><td>4 years</td></tr><tr><td>Sprague&amp;ndash;Dawley rats&amp;nbsp;</td><td>Pasture Institute (Tehran, Iran)</td><td></td><td>300-350g</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>314-13-6</td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>76-03-9</td><td>2 years</td></tr><tr><td>Bupivacaine HCl (0.5%)</td><td>Delpharm Tours</td><td></td><td>below&amp;nbsp; 25 &amp;deg;C</td></tr><tr><td>Bupernorphine</td><td>Exir (Iran)</td><td></td><td></td></tr><tr><td>Sodium Carbonate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>497-19-8</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>7647-14-5</td><td></td></tr><tr><td>Di- Sodium hydrogen phosphate</td><td>EMD Millipore&amp;nbsp;</td><td>231-448-7</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;7447-40-7</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>64-17-5</td><td></td></tr><tr><td>silicone(Xantopren)</td><td>Heraeus</td><td>EN ISO 4823</td><td></td></tr><tr><td>Activator universal plus</td><td>Heraeus</td><td>66037445</td><td></td></tr><tr><td>Micro-Dissecting forceps</td><td>Stoelting</td><td>52100-41</td><td></td></tr><tr><td>Spring Scisors</td><td>Stoelting</td><td>52130-00</td><td></td></tr><tr><td>Operating&amp;nbsp; Scissors</td><td>Roboz</td><td>52140-70</td><td></td></tr><tr><td>Brain matrix&amp;nbsp;</td><td>Stoelting</td><td>51390</td><td></td></tr><tr><td>Anesthesia Machine for Small Animals |</td><td>&amp;nbsp;Kent Scientific</td><td>SS-01</td><td></td></tr><tr><td>Power Lab system</td><td>AD Instruments</td><td>ML880</td><td></td></tr><tr><td>Laser Doppler flowmeter</td><td>AD Instruments</td><td>ML191</td><td></td></tr><tr><td>Heating feed back system</td><td>Harvard Appratus</td><td>72-7560</td><td></td></tr><tr><td>Vascular micro clamp</td><td>FineScience Tools</td><td>18055-03</td><td></td></tr><tr><td>Silk 5-0 suture thread</td><td>Ethicon</td><td>682G</td><td></td></tr><tr><td>Ethilon 4-0 suture thread&amp;nbsp;</td><td>Ethicon</td><td>EH6740G</td><td></td></tr></tbody></table>

an in vivo assessment of blood-brain barrier disruption in a rat model of ischemic stroke

虚血性脳卒中は、分子病態脳浮腫、血液脳関門 (BBB) の破壊を介するその後原発性脳損傷につながります。誘発虚血性脳卒中ラットは確立され、BBB の機能の整合性を調査するため生体内でモデルとして使用されます。エバンス ブルー (EB) の虚血性損傷脳のサンプルの吸光光度検出は研究と新規治療戦略の開発のための信頼性の高い正当化を提供すること。このメソッドは、再現性のある結果を生成し、特別な装置を必要としない任意の研究室では、適用。ラットにおける虚血性脳卒中の誘導 EB の血管外漏出の検出に関する可視化および技術的なガイドラインを紹介します。

Sham ラットの左半球と右半球の EB レベルに差はありませんでした (1.06 ± 0.1 μ g/g と 1.1 ± 0.09 μ g/g、それぞれ)。図 2A 2B、一過性虚血の誘導に示すように (90 分虚血/24 h 再灌流) 理事会レベルの差の原因 (10.41 ± 0.84 μ g/g, p < 0.001) と比較して、それぞれの虚血ラットの左半球のsham ラットの半球。総称して、これらの調査結果を示し通常の条件下で EB が脳実質内に BBB を容易に越えることはできません、脳虚血性の侮辱 (2A および 2B の数字) BBB の高められた透磁率による EB の血管外漏出を誘発します。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57156/57156fig1.jpg"> 図 1: 標準曲線を使用して吸光度値から EB 濃度を決定します。<a href="/files/ftp_upload/57156/57156fig1large.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>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57156/57156fig2.jpg"> 図 2: EB 血管外漏出虚血性脳卒中後 24 時間で BBB の中断の評価します。偽手術および虚血性動物 (A) の脳の写真。脳組織 (青色) の EB を血管外漏出の強度は破壊の半球の BBB の中断の範囲から発生します。EB (破壊) の左から調製した試料中濃度と sham と虚血性動物 (B) では、脳の半球を右 (n = 6、*p< pシャム群左半球に比べて 0.001 < 0.001 と比較すると同じグループの同側半球)。<a href="/files/ftp_upload/57156/57156fig2large.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 An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke at JoVE.com

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

この手順の全体的な目標は、虚血性脳卒中のモデルラットの血液脳関門の破壊の生体内評価の再現性の高い技術を提供することです。

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

an-vivo-assessment-blood-brain-barrier-disruption-rat-model-ischemic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

11.3K ビュー.  Ardabil University of Medical Sciences. この手順の全体的な目標は、虚血性脳卒中のモデルラットの血液脳関門の破壊の生体内評価の再現性の高い技術を提供することです。

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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

ラット中大脳動脈の血管縫合閉塞による局所脳虚血モデル

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

神経科学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

医学

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

設定アップ<em&gt;体外</emラット血液脳関門の&gt;モデル（BBB）：BBB不透過性および受容体媒介輸送に注力

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

虚血性脳卒中をin vitroで研究するためのヒト血液脳関門のトリプル初代細胞培養モデル

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

生化学

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

マウスにおける反復低酸素プレコンディショニングと過渡中大脳動脈閉塞後に神経血管保護の定量

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

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 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 ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

げっ歯類の脳サンプルの単一のセットで脳卒中後脳浮腫、梗塞ゾーンおよび血液脳関門破壊を測定する

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

虚血性脳卒中のウサギモデルにおける血圧のリアルタイムモニタリングと調節

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

<meta charset="utf8"></head><body>ラットMCAO脳卒中モデルにおける細胞死を研究するための免疫蛍光染色

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.

<meta charset="utf8"></head><body>虚血性脳卒中のラットモデルにおける免疫蛍光法による細胞死シグナル伝達の評価

Rat in vivo models are indispensable tools to investigate the pathological mechanisms and therapeutic targets for ischemic stroke. This work will describe performing immunofluorescence staining in infarcted brain slices following middle cerebral artery occlusion (MCAO) in rats.

虚血性脳卒中のラットモデルにおける免疫蛍光法による細胞死シグナル伝達の評価

Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery ...