我々は、共焦点レーザー顕微鏡を使用するための財政支援と医療の統合イメージング研究所のテキサスA＆M健康科学センター大学のためにスコットと白病院研究費補助金プログラムを認めます。私たちは、原稿編集のヘルプ氏グレンクライヤーを認めます。

内皮細胞の1播種<ol><li>大人のSDラットからRBMEC年代の初代培養物を得る（または商業的にそれらを得ます）。 </li><li> 100センチメートルフィブロネクチン（50μg/ mlの）ラット脳内皮細胞増殖培地を用いて被覆されたペトリ皿で​​RBMECs育成。密集度に到達するまで、2日ごとに培地を変更します。 </li><li> 80〜90％コンフルエントに達すると、穏やかに旋回を5 mlのリン酸緩衝生理食塩水（PBS）で細胞を洗浄します。次いで、細胞を37℃に平衡化した温かい0.25％トリプシン - エチレンジアミン四酢酸（EDTA）溶液1ml、それらを暴露することによって分離されます。 </li><li>細胞が剥離して分散されるまで2〜5分間、37℃で細胞をインキュベートします。 注：細胞を剥離するために培養皿をタップします。顕微鏡下で見る細胞が皿の表面から細胞を完全に剥離を確認しました。 </li><li>トリプシンを中和するためにペトリ皿に5ミリリットル完全培地を追加します。剥離した細胞を含む培地をピペット15mLの遠心分離管にそれを収集します。 。 </li><li>遠心分離媒体を5分間220×gでの内皮細胞を含みます。 </li><li>上清を吸引した後、自動細胞カウンターまたは血球計数器を用いてカウントされるピペット細胞で軽くそれを混合することにより、およびダウン3〜5ミリリットルの新鮮なラット脳内皮細胞増殖培地中にcells.Suspendにペレットを含むペレットを保存します。 </li><li>ウェルあたり10,000〜15,000細胞の間の範囲の播種密度で8ウェル滅菌チャンバースライドシステム0.7 cm 2で/ウェルをプレコートフィブロネクチンへの細胞懸濁液（50μg/ ml）を転送します。コンフルエンスが達成されるまで37℃で細胞を増殖させます 注：実験によって要求されるようにウェスタンブロットまたは他の実験を行うために、細胞を10cmの細胞培養皿または特殊皿で増殖させることができます。 </li></ol> 2.酸素およびインビトロでのグルコース欠乏、再酸素化モデル注：以下のプロトコルをスルエタらから適応されています。 1997;朱H ら。2012 8,9。 <ol><li> （ 図1参照）でRBMECsのOGD-Rの効果を研究するために、低酸素細胞培養システムを使用してください。セットアップし、製造業者の指示に従って、実験を開始する前に、低酸素細胞培養系を較正します。 </li><li> 37℃のインキュベーターからコンフルエントチャンバースライド（ステップ1.8）を取り外します。 OGD状態を表すために、37℃で2時間、脱酸素とチャンバースライドで完全培地、無グルコース、ダルベッコ改変イーグル培地（DMEM）および95％の低酸素チャンバーに入れ、N 2及び5％CO 2を交換します。 </li><li> 37℃で95％O 2、5％CO 2インキュベーターで逆に細胞を移動させ、新鮮なラット脳内皮細胞の完全な媒体を備え、37℃でさらに1時間インキュベートします。 注：このステップは、再酸素化の状況を表しています。 </li><li>免疫蛍光局在化およびローダミンファロイジン標識のためチャンバースライドを使用します（セクション3を参照）。 </li></ol> 3.免疫ZO-1の局在とローダミンファロイジンを使用したFアクチンラベリング<ol><li> OPTI-MEM /低血清培地/ウェルの1時間の100μlにRBMEC単層を含むチャンバースライドを公開します。リン酸緩衝生理食塩水（PBS、pHが7.0〜7.2）100μlのチャンバースライドを3回洗浄します。 </li><li> 15分間、PBS中の4％パラホルムアルデヒド（pHは7.0〜7.2）の100μLを使用して細胞を固定し、PBSで3回（pHは7.0〜7.2）のためのチャンバースライドを洗浄します。 </li><li>さらに15分間、PBS中0.5％トリトンX-100100μlの、（pHが7.0〜7.2）を用いて細胞を透過性。時間、PBS中の2％ウシ血清アルブミン（BSA）100μlでブロックします。 ZO-1またはF-アクチンのためのこのステップのいずれかで染色/ラベル細胞のいずれかの後に。 <ol><li>免疫STA用ining 1にZO-1に対する抗ウサギ一次抗体で細胞をインキュベートする：150、Oのために/ N 4℃にて2％BSA-PBS中で調製しました。細胞をPBS中で3回、（pHは7.0〜7.2）を洗浄します。フルオレセインイソチオシアネート（FITC）100μlでインキュベートし、室温で1時間、抗ウサギ二次抗体をタグ付き。 </li><li>ローダミンファロイジン標識のために、ブロッキングに続いて、20分間、2％BSA-PBS中で調製し、1：50希釈でローダミンファロイジンの100μlに細胞を公開します。 </li><li> （pHは7.0〜7.2）、PBS中の免疫蛍光染色およびローダミンファロイジン標識から細胞を洗浄します。 DAPIと退色防止試薬を含むマウント培地を用いてチャンバをマウントします。 </li></ol></li><li> 60 Xの水浸レンズを用いて細胞を視覚化し、細胞を共焦点顕微鏡下で単一の光学面に走査されます。 </li></ol>

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The authors have nothing to disclose.

虚血再灌流障害のためのin vitroモデルとしてOGD-Rはよくニューロン10,11を研究するために設立されました。透過性及びTJの整合9における脳内皮細胞および変更のOGDの影響を示す研究もあります。しかし、我々の研究は、虚血性脳卒中後の発生のインビボ条件における虚血再灌流障害の近い表現である、OGDの影響だけでなく、再酸素化を示しています。 <p class="jov...

虚血再灌流（IR）傷害は、脳卒中、心筋梗塞、外傷、末梢血管疾患および外傷性脳損傷1,2に関連する様々な衰弱性の合併症や死亡の頻繁な原因であることが見出されています。脳血管のIR傷害は、炎症および浮腫3につながる初期の二次的損傷です。酸化および炎症次代謝ストレスの結果として生じる重篤な合併症の一つは、恒常的なフリーラジカル形成をもたらすバランス、血液脳関門の変化（BBB）タイトジャンクション（のTJ）と微小血管透過性4,5の損失です。 現在、BBBにおけるIR傷害の影響を研究するために使用される生体内モデルにおいて 、中大脳動脈閉塞（MCAO）、微小塞栓、およびトランスジェニックまたはノックアウト動物を含みます。 Hossmann 6で説明したようにしかし、それぞれがその欠点と制限があります。 MCAOモデルはeffecを研究するために使用されますレドックスストレスのTS、BBBの接合部コミュニケーションの変化と脳と免疫細胞間の相互作用。しかし、彼らはその中に正確な顕微手順や困難の必要性など、様々な技術的な課題を提示します。脳虚血を研究するためのトランスジェニックまたはノックアウト動物の使用は、梗塞形成上の遺伝子に依存する分子の影響、血管構造の変化と変化させ、体重6のような課題を持っているかもしれないが微小塞栓は、瞬時にBBBを分解します。したがって、虚血のin vitroモデルは、薬物のための機構研究を行う際にその適用に起因する主に最近の関心を増大させる発見しました。しかし、in vitro試験の結果が完全にin vivo試験を表していない場合があり、注意6と解釈されなければなりません。 内皮細胞単層および微小血管透過性の低酸素濃度の反作用効果がありました小川7によって研究。ラット脳微小血管内皮細胞（RBMECs）はインビトロ BBBを開発するために使用されました。このプロトコルで提示酸素-グルコース欠乏と再酸素化（OGD-R）技術はスルエタらと朱ら 8,9によって研究から適応されています。我々は、0％O 2、5％CO 2及び95％N 2を含む、低酸素/無酸素チャンバー中に置くことにより、OGD-Rの脳内皮細胞を曝露しました。細胞は、後にそれぞれの免疫蛍光局在化およびローダミンファロイジンラベルを使用して、TJの整合性とストレスファイバー形成の変化について評価しました。閉鎖帯-1（ZO-1）についての免疫蛍光染色は、ZO-1と、TJの完全性を決定するために実行されることTJタンパク質に結合した重要な足場膜です。ローダミンファロイジン標識が細胞骨格に繊維状アクチン（Fアクチン ）を決定し、内皮細胞におけるアクチンストレスファイバー形成の明確な指標です。 &lt;Pクラス= &quot;jove_contentは&quot;&gt;この方法の目的は、BBB内皮細胞TJの整合性とF-アクチンストレスファイバー形成を研究するためのin vitroでの IRモデルとしてOGD-Rの開発に洞察力を提供することにあります。結果は、TJタンパク質の運命についての情報を提供し、ZO-1およびOGD-R次のストレスファイバー形成します。これらの関係を理解することはOGD-R以下のトリガされる根底にある分子メカニズムを決定し、OGD-R処理後のBBB破壊を増強する潜在的な治療戦略を開発する機会を提供します。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Proox model 110</td>
			<td style="width:167px;">Biospherix</td>
			<td style="width:129px;">Model 110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DMEM, no glucose</td>
			<td style="width:167px;">Gibco, Life technologies</td>
			<td style="width:129px;">11966-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Rhodamine Phalloidin</td>
			<td style="width:167px;">Life technologies</td>
			<td style="width:129px;">R415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">ZO-1 Rabbit Polyclonal Antibody</td>
			<td style="width:167px;">Life technologies</td>
			<td align="right" style="width:129px;">617300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Lab Tek II-CC 8&#160;well sterile, glass&#160;slides&#160;</td>
			<td style="width:167px;">Thermo scientific</td>
			<td align="right" style="width:129px;">177402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-tagged anti-rabbit secondary antibody&#160;</td>
			<td style="width:167px;">Santa cruz</td>
			<td style="width:129px;">sc-2090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">DPBS 1X</td>
			<td style="width:167px;">Thermo scientific</td>
			<td style="width:129px;">SH 30028.03</td>
			<td>Any other PBS available can be used</td>
		</tr>
	</tbody>
</table>

oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction

虚血再灌流（IR）傷害は、虚血性脳卒中に関連する罹患率および死亡率に大きく寄与することが知られています。虚血性脳血管障害は、すべてのストロークの80％を占めています。 IR傷害の一般的な原因は、血液の急性/慢性閉塞次流体、栄養素、フリーラジカルの形成を誘発する組織への酸素の迅速な流入です。 虚血性脳卒中は、血液脳関門（BBB）機能不全および血管原性脳浮腫が続きます。構造的には、内皮細胞間のタイトジャンクショ​​ン（のTJ）は、血液脳関門（BBB）の完全性を維持する上で重要な役割を果たしています。 IR傷害は、非特異的、炎症反応を引き起こす早期二次損傷です。炎症次酸化および代謝ストレスがBBBの透過性とタイトジャンクショ​​ン（TJ）の完全性の破壊などの二次的脳損傷を誘発します。 我々のプロトコルは、in vitroでの提示します&lt;/ em&gt;のラット脳内皮細胞TJの整合性とストレスファイバー形成の酸素 - グルコース欠乏と再酸素化（OGD-R）の例。現在、in vivoモデルでいくつかの実験は、IR傷害の影響を研究するために使用されます。しかしそれらは、手術、遺伝子依存性分子の影響とメカニズムの関係を研究することの困難を行う際に技術的な問題など、いくつかの制限があります。しかし、in vitroモデルは、これらの制限の多くを克服するのを助けることができます。提示されたプロトコルは、潜在的な治療戦略を提供するために、様々な分子機構と機械的な関係を研究するために使用することができます。しかし、in vitro試験の結果は、in vivo試験で 、標準と異なる場合があり、注意して解釈されるべきです。

フィブロネクチンプレコートヌンクIIチャンバースライド上で培養した細胞はBiospherix ProOxモデル110チャンバー内に配置することにより、OGD-Rに供しました。ローダミンファロイジン染色ラベルを使用して、Fアクチンストレスファイバーの形成を示す図2及び細胞骨格の組み立てに示すように、図3に示すように、OGD-Rの細胞を施した後、それらは、免疫蛍光技術を使用?...

Watch this Scientific Journal Video about Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction at JoVE.com

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 ...

oxygen-glucose-deprivation-reoxygenation-as-an-vitro-ischemia

Research

JoVE Journal

Medicine

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

17.8K ビュー.  Texas A&M University Health Science Center College of Medicine. 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. 

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

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

正常体温心停止と心肺蘇生法：虚血再灌流障害のマウスモデル

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

医学

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

マウスにおける急性腎障害とポスト傷害線維の虚血再灌流モデル

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

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 ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

腸管虚血 - 再灌流傷害のマウスモデル

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

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) ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

ラットにおける心筋虚血再灌流後の冠動脈流量の評価

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

再建微小外科における虚血再灌流損傷の研究のための前臨床ラットモデル

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

マウスモデルにおける虚血再灌流損傷後の幹細胞の筋内内出産の遅延

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...