この作品は、米国国立衛生研究所、R01CA142928からの助成金によってサポートされていました。

1。マウス外側尾静脈内エバンスブルーの静脈注射<ol><li> PBS中のエバンスブルーの0.5％滅菌溶液を調製します。必要に応じて、溶解していない任意の粒子状物質を除去するためのソリューションをフィルター滅菌してください。 </li><li>シリンジに200μlのエバンスブルー溶液を吸引します。注射器の中に逃れた可能性のあるすべての気泡を避けてください。 </li><li>動物が自由に携帯電話ではありませんが、その尾を扱うことができるように、拘束装置に8から12週齢場所マウス。 </li><li>外側尾静脈が簡単に表示されるように、その側にマウスの拘束装置を置き、上向きに直面しています。 </li><li>親指と人​​差し指の間の非利き手と尻尾にしがみつく。 </li><li> 10から15度の角度、ベベルアップ、ヘッドの方向に向かって横方向の尾静脈に事前に針（小ゲージ、27から30）を挿入します。尾に針と注射器を平行に保ってください。 </li><li> Doは、静脈を崩壊かもしれないように適切な配置を確認するために背圧を適用しない。 </li><li>ゆっくりマウスの尾静脈に200μlのエバンスブルー溶液を注入します。 </li><li>これは静脈に針の正しい配置の証拠であるように、プランジャーの進歩と使いやすさを守ってください。 </li><li>そのケージに戻ってマウスを置き、30分間それを観察します。 </li></ol> 2。臓器から臓器コレクションとエバンスブルーの抽出<ol><li>頚椎脱臼を介してマウスを生け贄に捧げる。それは血管透過性と有意な干渉を制限としてマイルズアッセイの目的のために頸椎脱臼が推奨されます。できるだけ速く、同時にすべてのマウスを生け贄に捧げる。死の血管はより透過性になるので、直後に、6匹のマウス以下のコホートで動作します。 </li><li>背中にマウスを置き、ホワイトボードに自分の足を確保します。 </li><li>トールを公開するための腹部および胸腔を開くACIC腹部臓器。 </li><li>エバンスブルー溢出の違いを示すために代表的な写真を撮る。すべてのマウスの場合、同一の照明条件を持たせるために、同じフィールド内のすべてのマウスが含まれています。 </li><li>興味のある臓器を収集し、1.5 mlチューブに入れ</li><li>空のチューブを計量し、ゼロにバランスの値を持って来る。 </li><li>組織試料を移し、それを重み付けする。すべての組織試料について、この手順を繰り返します。組織は、さまざまな器官の間の水分含量の変動を除去するために乾燥空気とすることができます。 </li><li> 。各組織試料チューブに500μlのホルムアミドを追加します。 </li><li> 55℃の水浴またはヒートブロックにすべてのチューブを転送します。組織からエバンスブルーを抽出するために24〜48時間インキュベートする。 </li></ol> 3。エバンスブルーの定量化は、間質組織に溢出<ol><li>ペレット残っている組織片へホルムアミド/エバンスブルーの混合物を遠心分離します。 </li><li> 610で吸光度を測定nmである。ブランクとして500μlのホルムアミドを使用しています。 </li><li> mgの組織ごとに溢出ngのエバンスブルーを計算します。 </li><li>グラフ内のすべてのデータをプロットします。 </li><li>有意差を決定するために統計分析を実行します。 </li></ol>

<ol>
	<li>Beck, K. F., 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=Inducible+NO+synthase:+role+in+cellular+signaling.">Inducible NO synthase: role in cellular signaling.</a> J. Exp. Biol. 202, 645-653 (1999).</li><li>Bertglia, S., Giusti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+capillary+perfusion+and+oxygen+delivery+regulation+during+systemic+hypoxia.">Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.</a> Am. J. Physiol. Heart Circ. Physiol. 288, H525-H531 (2005).</li><li>Miles, A. A., Miles, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+reactions+to+histamine,+histamine-liberator+and+leutaxine+in+the+skin+of+guinea+pigs.">Vascular reactions to histamine, histamine-liberator and leutaxine in the skin of guinea pigs.</a> J. Physiol. (London). 118, 228-257 (1952).</li><li>Weis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+cardiovascular+disease+and+cancer.">Vascular permeability in cardiovascular disease and cancer.</a> Curr. Opin. Hematol. 15, 243-249 (2008).</li><li>Kumar, P., Shen, Q., Pivetii, C. D., Lee, E. S., We, M. H., Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endothelial+hyperpermeability:+implications+in+inflammation.">Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.</a> Expert Rev. Mol. Med. 30, 11-19 (2009).</li><li>Viazzi, F., 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=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertens. Res. 31, 873-879 (2008).</li><li>Scheppke, ., 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=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> J. Clin. Invest. 118, 2337-2346 (2008).</li><li>Blanchet, M. R., 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=Loss+of+CD34+Leads+To+Exacerbated+Autoimmune+Arthritis+through+Increased+Vascular+Permeability.">Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability.</a> J. Immunol. 184, 1292-1299 (2010).</li><li>Dvorak, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+mediators+of+enhanced+microvascular+permeability,+vascular+permeability+factor/vascular+endothelial+growth+factor,+histamine,+and+serotonin,+cause+leakage+of+macromolecules+through+a+new+endothelial+cell+permeability+organelle,+the+vesiculo-vacuolar+organelle.">Mast cell-derived mediators of enhanced microvascular permeability, vascular permeability factor/vascular endothelial growth factor, histamine, and serotonin, cause leakage of macromolecules through a new endothelial cell permeability organelle, the vesiculo-vacuolar organelle.</a> Chem. Immunol. Allergy. 85, 185-204 (2005).</li><li>Le Guelte, A., Gavard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell-cell+junctions+in+endothelial+permeability.">Role of endothelial cell-cell junctions in endothelial permeability.</a> Methods Mol. Biol. 763, 265-279 (2011).</li><li>Martins-Green, M., Petreaca, M., Yao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+for+in+vitro+detection+of+permeability+in+human+"endothelium".">An assay system for in vitro detection of permeability in human "endothelium".</a> Methods Enzymol. 443, 137-153 (2008).</li></ol>

著者らは、利害の衝突や競合する金融利害関係はありません。

血管透過性は、血管の状態のための重要なマーカーである。増加した血管透過性は、糖尿病、高血圧症、自己免疫疾患6,7,8を含むいくつかの全身性疾患、に存在することが示されている。増加した血管透過性がせん断応力によって媒介されることが示されており、血管内皮細胞増殖因子と線維芽細胞増殖因子、セロトニン、ヒスタミン、ブラジキニン9のような炎症性メディエ...

選択透過性障壁の形成と維持には、適切な臓器の開発と性能1,2のために不可欠です。内皮細胞ライン血管内腔と血液とすべての器官の間質空間との間の選択的輸送に不可欠である半透過性の障壁を形成する。十分な透過障壁は固く成長因子、サイトカインやその他のストレス関連分子3によって制御されているタイトな細胞から細胞への結合を介して維持されています。内皮細胞障壁の破壊は透過性の増大と血管漏出をもたらすことができます。これらの効果は、様々な疾患状態で見られると下線分子シグナルの理解が学際的方法は4,5が必要とされます。この記事では、我々はマウスモデルを用いて血管の透過性を測定するin vivo法について説明します。 またマイルスアッセイとして知られている我々が説明している検定は、よくestablですin vivoで血管透過性をテストするためのメソッドをished。アッセイは、基底の生理的条件下で、アルブミンは、内皮関門を通過しない、という事実に基づいている。エバンスブルー、アルブミンに対して高い親和性を有するアゾ色素は、実験動物の血流中に注入され、生理的条件下では、血管内に制限されることが期待されています。血管透過性の刺激が加えられると、どちら局所的または全身的に血管がアルブミンに結合しているエバンスブルーはまた、このようにタンパク質をリークし始めると。血管透過性を持っている組織の急速な青みがかった色でこの結果。 マウス外側尾静脈に色素の注射は成功した実験の良い結果のために重要である。尾静脈注射技術は広範囲の練習を必要とし、実験を開始する前に習得しなければならない。 血管の透過性は、年齢や体重に大きく依存しているimalので、異なるマウス系統を比較するときには、マウスまたは他の被験者が同じ誕生日と体重の近くに持っていることが不可欠です。内皮関門の透過性に影響を与えるその他の要素には、温度、湿度などの環境条件であり、非常に重要なのは、マウスのハンドリングストレス。それは実験を少なくとも3回実行される統計解析が繰り返されることを常に推奨されている実験の結果に影響を与えることができ、多数の要因に起因する。 このアッセイは、血管遺伝的に改変されたマウスの透過性だけでなく、さまざまな遺伝的背景を持つマウスを比較したり、使用することができます。透磁率が変調されている遺伝子の機能に応じて、刺激の有無で評価することができる。このアッセイは、血管透過性の異なる化合物の効果をテストするために使用したりすることができます。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> 試薬 </td></tr><tr><td>エバンスブルー</td><td> SIGMA </td><td> E2129 </td><td></td></tr><tr><td>ホルムアミド</td><td> INVITROGEN </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> 0.2Mリン酸 1.5MのNaCl pH7.4の</td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>分光光度計</td><td>エッペンドルフ</td><td> 952000006 </td><td></td></tr><tr><td>マウス拘束装置</td><td> HARVARD装置</td><td> 340012 </td><td></td></tr><tr><td>シリンジ</td><td> BD </td><td> 309659 </td><td></td></tr><tr><td>針</td><td> BD </td><td> 305106 </td><td>針のゲージは、動物の大きさに依存します。 </td></tr><tr><td> BALANCE </td><td> DENVER INSTRUMENT </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

このメソッドは、テストモデル動物としてマウスでのエバンスブルーの静脈注射に基づいています。エバンスブルーはアルブミンと結合する色素である。生理的条件下では内皮細胞は血管内に限定アルブミンので、エバンスブルーアルブミン結合した遺骨に対して不透過性である。増加した血管透過性内皮細胞を促進する病態では部分的にそれらの密接な接触を失い、内皮は、アルブミンのような小さなタンパク質に透過性になる。この条件は、組織中のエバンスブルーの漏出を可能にします。健康な内皮細胞は、隣接する血管化組織における色素の漏出を防ぐことができます。透過性の増大を持つ器官が無傷の内皮細胞と器官に比べて大幅に増加した青色呈色​​が表示されます。血管透過性のレベルは、単純な可視化によって、または実験動物/組織vsコントロールの組織のミリグラムあたり組み込ま色素を定量的に測定することによって評価することができる。二つポウこのアッセイのrful側面は、そのシンプルさと定量的な特性です。エバンスブルー色素をホルムアミドの組織の具体的な金額をインキュベートすることにより、組織から抽出することができます。 620 nmの吸光度の最小値である最大のエバンスブルーの吸光度は740 nmである。エバンスブルーの標準曲線を用いることで、光学濃度の測定は、組織のミリグラム当たりミリグラム捕捉色素に変換することができます。統計解析は、血管透過性の有意差を評価するために使用されるべきである。

我々は、8〜12週齢のマウスでは血管漏出をテストするために生体透過性アッセイに使用。このテストでは、異なる遺伝的背景や血管系に影響を与える処理を施したマウスの単一株の動物との間の相対的な血管透過性を比較するのに有用である。我々の結果は、我々は我々の研究室で作成された遺伝子改変マウスの株は野生型マウスと比較して、より透過性の内皮細胞があること?...

Watch this Scientific Journal Video about An in vivo Assay to Test Blood Vessel Permeability at JoVE.com

An in vivo Assay to Test Blood Vessel Permeability

我々は提示している<em生体内で&gt;</em&gt;血管透過性をテストするためのアッセイ。このアッセイは、間質空間への拡散の染料およびその後可視化の静脈注射に基づいています。

アン<em生体内で&gt;</em血管透過性をテストする&gt;アッセイ

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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Research

JoVE Journal

Medicine

An in vivo Assay to Test Blood Vessel Permeability

63.7K ビュー.  Fox Chase Cancer Center. 我々は提示している血管透過性をテストするためのアッセイ。このアッセイは、間質空間への拡散の染料およびその後可視化の静脈注射に基づいています。

ビデオ: An in vivo Assay to Test Blood Vessel Permeability

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化学療法誘発性血管毒性 - リアルタイム<em&gt;インビボ</em船舶減損の&gt;イメージング

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

医学

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

ex vivoで腸嚢は、胃腸疾患のモデルに粘膜透過性を評価するために

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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 ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

ヒト胎盤生理学の統合研究への4容器サンプリングアプローチ<em&gt;インビボ</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

アメロイド収縮を使用して豚の慢性心筋虚血モデル

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

再門脈部分肝移植のラット体内血管指向 70% 肝切除

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

腸血液関門と微生物叢製品の肝代謝を評価するためのIn Vivo法

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

開放血管窓と生体内蛍光顕微鏡を用いたマウス蝸牛の心房血流測定

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

前生体肺の構造と収縮性研究のための効率的なツールとしての精密切断肺スライス

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...