著者らは、動物のケアとの助けをShawnaリドー·グロスとアレクサンドラガルセスに感謝し、彼女の技術支援のためのPing呉だろう。この作業のサポートは、助成金なしで、米国立衛生研究所によって提供されました。 EB003268、カナダ研究チェアプログラム。

1。超音波やMRIのセットアップ MRI互換性のある3軸集束超音波システム（FUS·インスツルメンツ社、トロント、オンタリオ、カナダ）本研究で使用されています。二つの異なる超音波トランスデューサが使用されています。統合されたとの社内構築551.5 kHzの球状に集束されたトランスデューサーは（曲率半径= 60ミリメートル、外径= 75ミリメートル、内径= 20 mm）、および1.503 MHzで、8セクタの配列単一の要素球状に焦点を当てたトランスデューサー（= 0.8、絞り値= 10センチFN）として駆動PZTハイドロホン（Imasonic株式会社VorayシュルL&#39;Orgnon、フランス）。 MRI互換性PVDF受信機8は、551.5 kHzの振動子を用いた場合アコースティックエミッションを記録するために使用されていました。別の機器が使用されている場合は、次のことが示唆されています。 <ol><li>範囲0.25 MHzの-1.5 MHzの中​​心周波数と1以下のfナンバー（曲/開口部の半径）と適切なキャリブレーション超音波トランスデューサを選択します。 </li><li>水浴FIを配置1.5 Tまたは3 T MRIのベッドの上で暖かく、脱気、脱イオン水でlled。水浴は、動物を保持することができ、トッププレートを持っている必要があります。水面に面しており、MRI互換性のある3軸位置決めステージやシステムの9日にタンク内の超音波トランスデューサをマウントします。 </li><li>浸透パネルを介して超音波駆動装置に変換器ケーブルを実行します。 </li><li>ファンクション·ジェネレータとパワーアンプを使用してトランスデューサの駆動信号を生成します。反映され電力を最小限に抑えるために、外部整合回路を使用しています。 </li><li>トランスデューサの焦点距離を知って、水面でのトランスデューサの焦点を置き、目に見える噴水を生成するために水の表面を超音波処理してください。ステップ1.6および1.7は、生成された噴水を使用してフォーカスを登録する方法について説明します。結果として温度上昇は、MRIで焦点に熱吸収ゲルとイメージングを超音波処理が含まれ、フォーカスを検索するためのより正確な方法は、9に記載されています&lt;/&gt;のsup。 </li><li> MR画像上に表示され、マーカーを使用してフォーカスの位置をマークします。これは、フォーカス上に配置されたときに水でいっぱいになり、中央の穴とプレートを使用して行うことができます。変換器の軸方向の座標が水面から決定することができますが水で満たされた穴は、MRI上に表示されますと2面での焦点の座標を提供します。 </li><li> 3面のローカライザーシーケンスを使用して画像の焦点マーカーとMRI座標系でのフォーカスの位置を記録します。 </li><li>アコースティックエミッションを監視するには、トランスデューサの焦点に向け水浴中で受動的なキャビテーション検出器（PCD）として使用されているMR-互換性のあるハイドロ8をマウントするか、統合されたハイドロフォンのトランスデューサーを使用しています。 </li><li>最大2 Hzの（例えば、ATS460、AlazarTech、ポワントクレア、ケベック州、カナダ）へのPRFが10ミリ秒までのバーストを捕捉するのに十分高速であるスコープのカードにPCDケーブルを実行します。ケーブルはPENEに接地する必要がありますtrationパネルまたはRFシールドは、ノイズを最小限に抑えます。 </li></ol> 2。動物の準備<ol><li>イソフルランガスを使用して動物を麻酔。イソフルランは、BBBの破壊10の効果を持っているので、動物実験の開始前にガスは10分をオフに注意する必要があります。乾燥や他の傷害から角膜を保護するために、麻酔の開始時に各目の場所の眼潤滑剤軟膏。 </li><li>動物の頭と首の上から毛を剃る電気かみそりを使用して、その後脱毛クリーム（例えばNairさんは、教会＆ドワイト社、プリンストン、ニュージャージー州、米国）を使用して、残りの毛を除去し、穏やかな石鹸で頭皮をすすぎ、水。 </li><li> bridine洗浄に続いてbetadineスキンスクラブと尾を準備します。尾静脈を可視化するために、アルコールとの最終wipedownを実行します。 </li><li>尾静脈に3方活栓と22ゲージのカテーテルを挿入し、血栓形成を防止するために、ヘパリン/食塩水混合物（33 U / mL）でフラッシュカテーテルインチ</li><li>筋肉内注射により、注射麻酔薬（40〜50 mg / kgのケタミン、10 mg / kgのキシラジン）を提供し、イソフルランから動物を削除します。 </li><li>トッププレート（図1）の穴を通して水浴の接触の頭の上で超音波測位システムにおける麻酔動物を仰臥位に置きます。超音波ゲルの少量多分トラップairbubblesの可能性を最小限に抑えるために動物の頭の上に適用されます。 </li><li>位置決めシステムへのテープの足を。頭のいずれか一口バーを使用して、場所で開催された、利用可能な場合、またはテープがあご全体にしっかりと置かれるかもしれません。 </li><li>タオルで動物をカバーし、それを暖かく保つために水の毛布を循環させる。 </li></ol> 3。標的選択<ol><li>ベースラインの軸方向のT 2-Tと重み脳の1加重MR画像を取得します。 1.5 T MRIと専用のヘッドサイズのRF-受信表面コイル、適切なSCを使用している場合パラメータは次のようになります。 加重T 2：FSE、TE = 60ミリ秒、TR = 2000ミリ秒 加重T 1：FSE、TE = 10ミリ秒、TR = 500ミリ秒</li><li>心室、脳の正中線を避けて、中脳の深さを選択し、T 2-加重スキャンからターゲットを選択します。 </li><li>ターゲットの場所にトランスデューサのフォーカスを移動します。 </li></ol> 4。マイクロバブルの準備 DEFINITYマイクロバブル（Lantheusメディカルイメージング、MA、米国）がBBBD 2,5,7を誘発したマイクロバブル媒介FUSのために、いくつかのグループによって使用されます。他のマイクロバブルの種類の適切な投与量は、文献11,12に記載されています。 <ol><li>のDefinityマイクロバブルを活性化し、徐々に18ゲージ針を用いて1 mLシリンジに小さな音量を上げて描画します。 </li><li>軽く前後にプランジャーを移動することによってシリンジから閉じ込められた空気を削除します。これは気泡を破ることができるように注射器をタップしないでください。 </li><li>正常な内のDEFINITYを希釈<html>徐々に生理食塩水のシリンジにDEFINITYの必要量を注​​入することによりDefinty〜10:1生理食塩水の割合で生理食塩水。注入配信が使用されている場合、DEFINITYは、50:1または100:1、さらに希釈することができる。 </li><li>優しくても外観が達成されるまで、徹底的にのDefinityと生理食塩水を混ぜて注射器を反転させます。気泡が懸濁液からフロートを開始した場合、直ちに注入前にシリンジの緩やかな反転が必要になることもあります。 </li><li>のDefinityの0.02 ml / kgの、または溶液（10:1希釈）0.2 ml / kgの用量に基づいて、必要な量を計算します。 </li></ol> 5。超音波配信<ol><li>低デューティ·サイクルのバーストではなく、連続波sonicationsを使用して、超音波処理パラメータを設定します。 0.5 MHzで、適切な超音波処理パラメータは、 その場の圧力0.23 MPaで、1 HzのPRF、2分間で10ミリ秒のバーストです。合計超音波処理時間。 その場の圧力は 1.5 MHzでの適切な0.45から0.5 MPaの周りに分類されます。 </li></ol>_content &quot;は&gt;異なる周波数で適切な圧力は、0.46 13の機械的なインデックスを使用して推定することができます。 <ol start="2"><li>動物の頭はまだ水に結合されていることを確認してください。 </li><li>尾静脈カテーテルにゆっくりとマイクロバブルを注入し、0.5 mLの生理食塩水で洗い流してください。注入開始と同時に超音波処理を開始します。 </li></ol> 6。治療成績のMRI評価<ol><li>超音波処理後、0.5 mLの生理食塩水フラッシュに続いて、尾静脈カテーテルを介して（例えば0.2ミリリットル/ kgのOmniscan、GEヘルスケア）MRI造影剤を注入します。 </li><li>コントラストのピークが経過するまで、T 1強調画像を実行します。首尾よく破壊された超音波のサイトが周囲の組織よりも大きい増強が表示されます。 </li><li>浮腫を確認するためにT 2強調画像を実行します。超音波処理現場での高信号は、浮腫の指標である。 </li></ol> 7。代表者結果 MRI造影剤は正常に集束超音波とマイクロバブル循環を使用して、BBBを介して配信することができます。図2は、典型的なプリおよびポストFUS T 1-Wの画像を示しています。図2Bは、4つの超音波処理した場所で異なった焦点距離の開口部とは対照的に強化された（CE）T 1-Wイメージを示しています。超音波位置1と2は特に明るい強化を示しています。図の位置1と2にも浮腫を示し、T 2-W高信号に対応するように見ることができます。 T 2-wの浮腫の程度は、時には矢状スライスに、より簡単に視覚化することができます。図4は、CE-T 1-wとT 2は、-w矢状スライス超音波位置1〜3を示しています。浮腫は位置1ではなく位置3で表示されます。 キャプチャされたアコースティックエミッションデータ（図5）のスペクトル解析では、高調波の排出量および/または安定したキャビテーションが発生している超/サブハーモニックの排出量が表示される場合があります。ハーモンsubとultraharmonic排出量はバブルのアクティビティ14の結果としてのみ発生することができますがICはまた、組織の非直線性から生じる可能性があります。より高い圧力で慣性キャビテーションを示す広帯域の排出量も検出することができます。しかし、これらは、慣性キャビテーション11なしsonicationsより赤血球extravasationsと微小損傷の大きい量に関連付けられている。 小さな焦点スポットサイズに起因する多くのローカライズされた開口部に高い超音波周波数を使用した結果。図6は、高い周波数は、開口部の小さな領域を作成するために使用できることを示しています。これは、より少ない近い頭蓋骨の影響と効果半ば脳の調査を可能にします。 <img alt="図1。" src="/files/ftp_upload/3555/3555fig1.jpg" /> 図1実験セットアップ。 <spクラス= &quot;pdflinebreak&quot;&gt; <img alt="図2。" src="/files/ftp_upload/3555/3555fig2.jpg" /> 図2。前（左）と後（右）の治療法T 1-W 4超音波の位置で強化を示すラットの脳の画像。 <img alt="図3。" src="/files/ftp_upload/3555/3555fig3.jpg" /> （3）前（左）と後（右）の治療法T 2-Wラットの脳の画像（図2と同じ動物）は、超音波位置1と2で、T 2は、-w浮腫を示す図 。 <img alt="図4。" src="/files/ftp_upload/3555/3555fig4.jpg" /> 図4後処理矢T 1-W（左）とT 2-W（右）同じラットの脳からの画像は、図。 2と3。位置1の開口部（左）T 2-wの浮腫（右）と相関しています。場所3は、開口部（左）がないT 2は、-w浮腫を示しています。 </P&gt; <img alt="図5。" src="/files/ftp_upload/3555/3555fig5.jpg" /> 図5：551.5 kHzで1つの10ミリ秒のバースト中に捕捉したデータから周波数スペクトル。基本周波数（f 0）と同様に高調波（2 F 0）とultraharmonics /サブ（0.5 F 0、1.5 F 0）が表示されます。 <img alt="図6。" src="/files/ftp_upload/3555/3555fig6.jpg" /> 図6治療後CE-T 1-W軸（左）と1.503 MHzで4箇所に超音波処理したラットの脳から矢（右）の画像。この周波数でのBBBの開口部は、よりローカライズされたように見られている。

<ol>
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K. HynynenとR.チョプラがFUS·インスツルメンツの共同設立者であり、株式会社R.チョプラ、A. WaspeとK. HynynenはFUSインスツルメンツでは株主である株式会社K. HynynenはFUSの楽器、（株）からの研究支援を受け

動物とマイクロバブルの準備は、この手順の中で最も重要な側面です。動物の頭の毛は完全に超音波ビームの減衰を避けるために削除する必要があります。 BBBは、イソフルラン麻酔下で中断される可能性があります、しかし、それは一貫性のある開口部を達成するために難しくなります。 マイクロバブルは、常にそれらを壊すことを避けるために、策定時に大径の針を?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>小さな動物集束超音波システム</td><td> FUS·インスツルメンツ株式会社</td><td> RK-100 </td></tr><tr><td> DEFINITY </td><td> Lantheusメディカルイメージング</td><td></td></tr><tr><td> Omniscan </td><td> GEヘルスケア</td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

血液脳関門（BBB）の集束超音波（FUS）の中断は、BBB 1-5を回避するためのますます調査手法である。それは小さいサイズ6で約500 Daより分子に脳組織に血管系からの分子の通過を制限するようにBBBは、脳疾患の医薬治療への重要な障害である。 FUS誘導BBBの中断（BBBD）は一時的かつ可逆的4であり、高度にローカライズされていることBBBD誘導の化学的手段利点があります。 FUS誘導BBBDそれ以外の場合は十分な濃度の組織への成果物ではありません脳、上の治療薬の広い範囲の影響を調査するための手段を提供します。超音波パラメータの広い範囲がBBB 2,5,7中断することで成功を収めているが、正確なターゲティングに成功した混乱を確保するための実験手順で、いくつかの重要なステップがあります。この機能実装オールアウトラインはどのように重要な動物の準備と実験のマイクロバブル処理の手順に焦点を当てて、ラットモデルでBBBD誘導されるMRIガイド下FUSを実現しています。

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

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

血液脳関門（BBB）のマイクロバブル媒介集束超音波の乱れは、脳の非侵襲的な標的薬物送達のための有望な技術です。<sup&gt; 1月3日</sup&gt;。このプロトコルは、ラットモデルにおけるMRI誘導頭蓋BBBの中断のための実験手順を説明します。

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 ...

mri-guided-disruption-blood-brain-barrier-using-transcranial-focused

Research

JoVE Journal

Medicine

17.8K ビュー.  Sunnybrook Research Institute. 血液脳関門（BBB）のマイクロバブル媒介集束超音波の乱れは、脳の非侵襲的な標的薬物送達のための有望な技術です。 1月3日。このプロトコルは、ラットモデルにおけるMRI誘導頭蓋BBBの中断のための実験手順を説明します。

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

医学

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 ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

ラットにおける慢性栄養注入のモデル

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

昇順大動脈剛性の測定<em&gt;インビボ</em&gt;超音波​​を使用したマウスにおける

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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 ...

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) ...

MRI-guided Focused Ultrasound Thalamotomy for Patients with Medically-refractory Essential Tremor

Essential tremor (ET) is the most common type of tremor in adults. While ET does not result in decreased life expectancy, the disabilities associated with ET can have a significant impact on quality of life, mood, functional activities, and socialization. Patients suffering from ET not sufficiently treated with first line medications may be eligible for alternative strategies such as deep brain stimulation, radiofrequency ablation, and MRI guided focused ultrasound (MRgFUS). High-intensity MRgFUS is an emerging modality to treat ET, its attraction for patients being that it is noninvasive and associated with short recovery time, as patients are home the day after treatment. While MRgFUS centers are still limited, it will become important for clinicians to consider MRgFUS as a treatment alternative, particularly in the case of a patient for whom open surgery is contraindicated. This article outlines the steps of patient selection, equipment setup, sonication, and post-treatment follow-up, as well as critical steps to be aware of when performing a MRgFUS procedure.

High-intensity MRI guided focused ultrasound is an emerging noninvasive technique to precisely ablate brain tissue. It has been shown to be safe and effective in treating medically-refractory essential tremor. This article describes the protocol for thalamotomy from patient selection to equipment setup to post-treatment follow-up.

医学的に難治性本態性振戦患者に対する MRI ガイド下集束超音波視床

Essential tremor (ET) is the most common type of tremor in adults. While ET does not result in decreased life expectancy, the disabilities associated with ...