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1。手続きのための動物と機器を準備<ol><li>手術を開始する前に、手術部位や動物を準備します。 PE50のチューブ12の一端 &quot;に23ゲージのブラント針を挿入することにより、頸動脈にカテーテルを作る。カテーテルの反対側の端では約45°の斜面をカットします。前の手順と機器を滅菌する。ヘアボンネット、外科用マスクを着用する、無菌手袋。 </li><li>手術が行われる表面に加熱パッドを配置します。それをアクティブにし、それが温まることができます。吸収ベンチパッドを使用して領域をカバーしています。 </li><li>あなたが結晶せずに溶液中で25％のマンニトールを持っていることを確認します。結晶が存在する場合は、80℃の温水浴でバイアルを置き、定期的に攪拌することにより、それらを溶かす。 </li><li>動物の質量を記録する。 </li><li> 0.1ミリリットル/分の速度で0.8ミリグラム/ kg /分で配達のためのプロポフォール希釈を作る。 </li><li> 3分間インダクションボックスにラットを配置します。設定：5％イソフルラン、2L /分人工呼吸器の流量。 </li><li>使い捨てタオルの上に、首、両内側の太ももを剃る。 </li></ol> 2。ラットを挿管<ol><li> 3分間インダクションボックスの動物の背中を置きます。 </li><li> IVカテーテルを使用して気管内チューブを準備します。このサイズとラットの歪みのために、私たちは18 GAのカテーテルを使用しています。チューブが損傷を引き起こすことなく、十分な通気を確保できるように十分な大きさですが、気管内に収まるように十分に小さくなければならない。プラスチック製のカテーテルの針を引き出し、慎重に針約を破る。その鋭い先端から1cm。 bunted針はあなたのスタイラスになります。カテーテルに針をしなさい。 </li><li>傾斜面にその歯によってラットを掛けます。 </li><li>動物の首に直接光を当てると耳の間に並んでいた。 </li><li>小さなへら約を挿入します。中咽頭への3センチメートル。そっと口から舌を引き出しながら上方に持ち上げます。 </li><li>気管の開口部を視覚化します。あなたは、そういうものでしょうEA小さな穴が開き、各息を閉じます。 </li><li>そっと穴にカテーテルを挿入します。あなたは時々プラスチックは気管の軟骨を入力感じることができます。気管と小文字が区別されます。カテーテルを無理に押し込まないでください。 </li><li> 1 L /分、60拍と2％イソフルレンに人工呼吸器を設定します。 </li><li>操作面への人工呼吸器とテープチューブにカテーテルを接続します。 </li><li>動物がつま先のピンチを行うことにより、麻酔であることを確認します。 </li></ol> 3。プロポフォール静脈麻酔を確立<ol><li>操作面に四肢と尾をテーピングすることによりラットを固定します。 </li><li>クロルヘキシジン石鹸で剃って、首と脚の付け根の部位3回こすります。クロルヘキシジンが3回すすぎ次いでスクラブ。 </li><li> IVの拡張セットにプロポフォールの注射器を取り付けます。輸液ポンプにシリンジをマウントします。 </li><li>剃ら太ももの皮膚を切ってメスを使用している場合。筋肉を切ることは避けてください。 </li><li>力で切開を開くpsの大腿静脈を可視化する。 </li><li>血管を露出させるために動脈を介して組織を引き離します。 </li><li>静脈内に26 GA monoject獣医静脈カテーテルを挿入します。針が静脈の壁を穿刺するときには、それが押しやすくなって感じることができる。針を撤回し、血管内にカテーテルを押してください。 </li><li> 0.1ミリリットル/分でプロポフォール点滴を開始します。 </li><li> 3-5分プロポフォール注入を開始した後、50％O 2、50％N 2 Oの混合物に麻酔ガスを切り替えて、イソフルランを遮断してください。 </li></ol> 4。総頸動脈分岐部を露出<ol><li>メスの刃で首に正中切開を行います。 </li><li>ハサミやメスを持つ2つの大唾液腺の間で切断。 </li><li>頸動脈を露出させるために正中線に対して横方向に組織を離れて解剖する。組織の層を開くためにピンセットを使用しています。あなたは鉗子で公開している部分を削除し、minimallあることを決定するためにハサミを使用することができますyの血管。必要に応じてガーゼ、綿棒、焼灼で出血をコントロール。 </li><li>中等度から重度の出血があった場合には3ミリリットル生理食塩水IPを注入します。 </li><li>頸動脈から組織を引き離しトラクタを使用しています。分岐を探す。 </li><li>分岐のすぐ遠位後頭動脈を公開します。鉗子の間それを焼灼し、電気メスを使って区切ります。 </li><li>完全に頸動脈分岐部、できるだけECAの限りを公開するためにピンセットを使用しています。 </li><li>後頭動脈の遠位にECAを分岐上甲状腺動脈を公開します。鉗子の間それを焼灼し、電気メスを使って区切ります。 </li></ol> 5。外頸動脈にカテーテルを挿入する<ol><li> ECAの下4/0シルクの2の長さを置く。 </li><li>あなたの止血と縫合糸の長さは1をつかんで、（ラットの足に向かって）背尾側にECAを引っ込める。 </li><li>限り頭側（ラットの口に向かって）可能なワットなどの他の糸とネクタイを取るi番目の外科医の結び目。これにより、ECAを犠牲にする恒久的な合字を形成している。 </li><li>第一止血（尾側張力をかけたもの）を削除し、他の縫合（ECAの周りに結ばれているもの）を取得し、止血と頭側で穏やかな緊張を適用します。 </li><li>分岐から遠位および永久結紮の近位 - 疎（かろうじて結ばまま固定しないでください）​​別の外科医の結び目を作る。 </li><li> 1％は動脈をリラックスさせるリドカイン洗浄してください。 </li><li>ヘパリン加生理食塩水を使用してカテーテルを埋める。 </li><li>分岐点のできるだけ近くに一時的な血管クリップを配置します。カテーテル挿入のためのスペースを最大限にするための角度で置き血管クリップ。クリップに向かって緩い結び目をスライドさせます。 </li><li> microscissorsを使用してリガチャーの直近のECAの小さな切開（カット）を行う。クリップとリガチャーが安全である場合も出血があってはなりません。 </li><li> ECAの壁を制御するためにピンセットを用いて、血管内にカテーテルの面取り端を養う。 </lI&gt; <li>緩い縫合糸の結び目でカテーテルを固定します。カテーテルの周りの動脈を縛る。 </li><li>クリップを外して、綿棒でその領域をきれいにしてください。 </li><li>分岐部の遠位に約1mmにカテーテルを進める。容器が固定化されていることを確認するために結び目の近位に鉗子で動脈を開催しています。 </li></ol> 6。マンニトール投与<ol><li>気泡を作成せずにマンニトールを持つ8 &quot;IVの拡張セットと6 mlの注射器を埋める37℃インキュベーターでこれを℃で保存注：マンニトールで迅速に仕事 - それは容易に室温で溶液から析出されます。 </li><li> BBBD可視化するためには、エバンスブルー染料を投与することができる、それがバリアが破壊されている脳の領域を入力見守る。これを行うには、プロポフォール注入に反対脚の、新しいIVサイトを通じて動物を2％エバンスブルーの2ミリリットル/ kgを与える。 </li><li> 5μmのフィルターに設定拡張子を取り付け、エアーバブを削除するにはマンニトールでそれを埋めるBLES。その後、泡を作成せずにコックにこれを添付してください。 </li><li> 0.09 25のml /秒秒でマンニトールを管理します。 ICAの直近時点での総頸動脈の流れに平衡に達するのにマンニトールを監視します。これは、ラットの心拍数を持つパルスに見えるはずです。マンニトールは、単に心臓に向かって流下する場合は、ラットの血圧を克服している。これは、生命を脅かす合併症と貧しいBBBDを引き起こす可能性があります。 </li></ol>

<ol>
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特別な利害関係は宣言されません。

BBBDの効果を最大化するいくつかの手段があります。カットダウンフェーズ中に、出血を最小限に抑えることが重要です。血圧と心拍数はかなりの出血の影響を受ける可能性があり、これらの要因はBBBD 13の程度に影響を及ぼすことが知られている。出血は分割しなければならないような優れた甲状腺および後頭部動脈などの主要な血管を、結紮する縫合糸を使用することで軽減する?...

<table border="1"><tbody><tr><td> 材料名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ロングエバンスラット</td><td>ハーランラボラトリーズ</td><td></td><td> 210〜250グラム、男性</td></tr><tr><td> PE50チュービング</td><td>ベクトン·ディッキンソン</td><td></td><td></td></tr><tr><td> 18ゲージ×2.5 &quot;IVカテーテル</td><td>テルモ</td><td></td><td> ETチューブ用</td></tr><tr><td> 30 &quot;IVの拡張セット</td><td>アボット</td><td></td><td></td></tr><tr><td> 26ゲージ獣医IVカテーテル</td><td> Monoject </td><td></td><td></td></tr><tr><td>エバンスブルー染料</td><td>シグマ</td><td> E2129 </td><td></td></tr><tr><td>バイポーラ</td><td> Codman </td><td></td><td></td></tr><tr><td>フィルタは5μm </td><td>ブラウン</td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

基底膜とアストロサイトの端の足と一緒に、タイトジャンクションと内皮細胞が血液脳関門1を形成する脳血管を囲んでいます。障壁は、選択的に血液とそのサイズと電荷に基づいて脳間交雑種の分子を排除する。この関数は、神経疾患の治療薬の送達を妨げることができます。化学療法薬の数は、例えば、効果的に腫瘍細胞2に到達するために血液脳関門を越えることはありません。従って、血液脳関門を横切る薬物の送達を改善することは興味のある分野である。 脳への薬物の送達を増強するための最も一般的な方法は、直接脳注入および血液脳関門の崩壊3アール。直接脳内注入は、治療が脳に到達することを保証しますが、この方法は、薬4を分散する能力が制限されています。血液脳関門の破壊（BBBD）アル循環系から直接流れる安値薬 脳になるため、より効果的に分散した腫瘍細胞に到達する。バリアの破壊には3つの方法が浸透バリアの破壊、薬理学的なバリアの破壊、そしてマイクロバブルによる集束超音波を含む。 Neuweltによって開拓浸透破壊は、彼らのタイトジャンクショ​​ンを縮小し、混乱させる原因となって、血液脳関門の細胞を脱水25％マンニトールの高張液を使用しています。バリアの破壊はまた、ヒスタミン及びブラジキニン5 6などの血管作動性化合物を用いて薬理学的に行うことができます。しかし、この方法は、主に脳の腫瘍関門7用に選択的である。さらに、RMP-7、ブラジキニンペプチドのアナログは、25％マンニトール8と浸透BBBDと頭に頭を比較した場合、劣ることが判明した。もう一つの方法は、マイクロバブル超音波造影剤と共に集束超音波（FUS）も示されている可逆的に血液脳関門9を開きます。 FUSのと比較して、しかし、25％のマンニトールはトランスレーショナル·リサーチ10月12日のための実証済みのツールとなって、人間の患者における安全性の長い歴史を持っています。 BBBDを達成するために、マンニトールは、直接脳の動脈循環に高いレートで配信される必要があります。ヒトでは、血管内カテーテルは迅速、直接の流れを達成することができる脳へと導かれる。このプロトコルモデル人間はできるだけ密接BBBD。総頸動脈分岐部にカットダウンに続いて、カテーテルにECAに逆行性に挿入し、内頸動脈（ICA）の循環に直接マンニトールを配信するために使用されています。プロポフォールとN 2 O麻酔はバリアの破壊13の効果を最大限に発揮する能力のために使用されます。適切に実行された場合、この手順は安全に、効果的に、かつ可逆的に血液脳関門と即興を開く機能を持っている通常の電子頭脳8,13,14に達していない薬剤の送達。

<img alt="図1" src="/files/ftp_upload/50019/50019fig1.jpg" /> 図1。エバンスブルー色素の血管外漏出を介して血液脳関門の崩壊を可視化する。エバンスブルー色素は、アルブミンに結合して、生理的条件下で脳内に溢出されていない顔料である。非中断半球が変わらないまま、脳の片側の血液脳関門の破壊は、エバンスブルーが中断半球青を入力して染色する?...

Watch this Scientific Journal Video about Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics at JoVE.com

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

血液脳関門の乱れは、脳にある種の薬物の送達を助ける。動脈配信マンニトールは、物理的障壁を破壊するために、血管周囲の細胞を縮小します。

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 ...

rat-model-blood-brain-barrier-disruption-to-allow-targeted

Research

JoVE Journal

Medicine

19.3K ビュー.  Vanderbilt University School of Medicine. 血液脳関門の乱れは、脳にある種の薬物の送達を助ける。動脈配信マンニトールは、物理的障壁を破壊するために、血管周囲の細胞を縮小します。

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

ラットモデルでの経頭蓋集束超音波を用いた血液脳関門のMRI誘導破壊

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

医学

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

として脳微小血管内皮細胞（CEND）でストレッチ<em&gt;インビトロ</em血液脳関門の&gt;外傷性脳損傷モデル

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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 ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

などの酸素グルコース欠乏および再酸素化<em&gt;インビトロ</em&gt;虚血再灌流傷害モデル血液脳関門の機能障害を研究するための

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

ラット頸動脈バルーン損傷モデルは、抗血管リモデリングの治療薬をテストします

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

唾液機能低下のラットモデルにおけるRetroductal顎下腺点滴とローカライズされた分割照射

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

血液脳関門メッシュ様血管形成および崩壊を評価するためのインビトロアッセイ

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.

Methods Article

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