Name: ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease
Uploaded: 2016-02-09T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

This work was funded by National Health and Medical Research Project Grant APP1021582 and a Hunter Medical Research Institute grant sponsored by Sparke Helmore/NBN Triathlon and the Estate of the late Leslie Kenneth McFarlane.

このプロトコルのすべての動物の作業はニューカッスル大学の動物倫理委員会に厳密に準拠して行われている手順を承認しました。 楽器、文化メディアと皿の作製<ol><li>プリ暖かいメディア199（TC199）またはダルベッコ改変イーグル培地（DMEM）メディア37へ °C。 95％O 2/5％CO 2でバブリングすることにより事前含酸素媒体。媒体は7.3の最終pHを持っていることを確認してください。 </li><li>各嚢のための2つの5cmのセクションを切断して縫合糸を準備します。ループ閉じられていない結び目に縫合糸。 </li></ol> 2.解剖および消化管の調製<ol><li>安楽死の前に固形食12時間を撤回。必要であれば、この時間の間に栄養ゲルサプリメントに動物を配置します。 </li><li>施設内倫理プロトに従って頸椎脱臼に続いてペントバルビタールナトリウムの過剰摂取（[200ミリグラム/キログラム]、腹腔内注射）によりマウスを安楽死させますCOLSは、腹部と胸部の上に70％エタノールをスプレーし。 </li><li>はさみを使用して、腹部の真ん中に横切開を行い、腹膜を露出させます。 </li><li>分離し、幽門括約筋で胃から小腸上部を切断し、肛門縁で大腸を切断することにより、消化管を除去するために進んでください。優しく腸間膜を削除するために鉗子を使用します。予め温め、酸素媒体中で腸管を置きます。 </li><li> （ 図1）の透過性について評価する腸の部分を特定し、腸管の残りの部分から自由にこのセクションをカット。 <ol><li>動物間の一貫性を維持する胃十二指腸および空腸の相対的なセクションを測定し、盲腸を結腸及び回腸の相対的なセクションを測定するために。 </li><li>組織セグメントを選択する場合、そのようなパイエル板などの粘膜関連リンパ組織の存在に注意してください。これらは小さなうなずきとして同定することができますルーメンの漿膜側のジュール。 </li><li> 1ミリリットルの注射器を使用して、静かに予熱したPBS（37°C）でペトリ皿に腸セグメントの管腔内容をフラッシュします。これらの糞便内容は-80で廃棄または保存することができます 所望のように将来の分析のためにCを°。 </li></ol></li></ol>腸嚢の調製<ol><li>試験化合物または分子の300μlの容量で1ミリリットル注射器を準備します。粘膜の完全性、FITCデキストランM.Wt.の1mg / ml溶液のための4,400を使用してもよいです。 4,400-70,000ダの大きさの範囲のプローブは、増加した感受性のために使用することができます。安全注射器の上に小さな動物の血管カテーテルにフィット。 </li><li>メジャー5腸セグメントの開口部からセンチ、セグメントをネクタイはしっかりとこの時点で、縫合糸ループで閉じます。静かに腸の開口部の周囲に予め結んだ縫合糸ループを配置し、鈍化カテーテルを挿入します。腸のセグメントを確保するために、閉じた縄を引いてそして、すべての溶液が注入されることを確認して、腸にシリンジから300μlのボリュームをリリース。 </li><li>同時に腸嚢の閉鎖を確保​​するために、縫合糸縄を引きながらゆっくりカテーテルを削除します。腸からの緩い腸嚢をカットし、37に予熱酸素培地20mlを充填した50mlコニカルチューブの中に置きます °C。 </li></ol>透水4.測定<ol><li> 37に設定された加熱水浴中で腸の嚢を含む場所コニカルチューブ °C。 0、30、60、90および120分の時点で、各インスタンスで新鮮な培地100μlでボリュームを交換し、円錐管および転送から96ウェルプレートに100μlのサンプルを取ります。 </li><li>最終的なサンプルが採取された後、粘膜表面を露出させ、縫合糸の時点で、セグメントの長さに沿って開いた嚢をカット。 </li><li>各腸のセグメントの長さと幅を測定します。指名打者の場合赤、スナップ-80セグメントと店を凍結 タンパク質または生化学的解析、あるいは、分子アッセイのためのRNA安定化溶液中の店のためのC°。 </li><li> 1から1×10 -6の範囲内のFITCタグ化分子に対する対数希釈の標準曲線を構築します。 </li><li>蛍光プレートリーダー上のFITC用の測定試料および標準、FITCの励起/発光：/ 519 nmの495 nmの。 </li></ol>各個別の腸サックのための見かけ透水5.計算<ol><li>時間単位は秒に変換します。 </li><li>各時点について、累積濃度、Qを計算します Q tの=（C トンの * Vrを）+（Q tの和 * V s）は 、ここで： 時刻tでのQ トン =累積濃度 CのT =時間tにおける濃度 受信機側でのV R =巻 以前のすべてのQ tのQ tの和 =合計 V S =ボリュームをサンプリング </li><li>プロット対時間Q（T）と傾きを計算：δQ/ΔT </li><li>見かけの透過性を計算する（P アプリ ） P アプリ =（δQ/ΔT）/（*株）、場所： 組織のA =エリア C 0 =初期濃度</li></ol>

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	<li>Goggins, B. J., Chaney, C., Radford-Smith, G. L., Horvat, J. C., Keely, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+Integrin-Mediated+Epithelial+Restitution+during+Mucosal+Inflammation.">Hypoxia and Integrin-Mediated Epithelial Restitution during Mucosal Inflammation.</a> Frontiers in immunology. 4, 272 (2013).</li><li>Otte, J. M., Kiehne, K., Herzig, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+in+innate+immunity+of+the+human+intestine.">Antimicrobial peptides in innate immunity of the human intestine.</a> Journal of gastroenterology. 38, 717-726 (2003).</li><li>Robinson, A., 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=Mucosal+protection+by+hypoxia-inducible+factor+prolyl+hydroxylase+inhibition.">Mucosal protection by hypoxia-inducible factor prolyl hydroxylase inhibition.</a> Gastroenterology. 134, 145-155 (2008).</li><li>Feighery, L., 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=Increased+intestinal+permeability+in+rats+subjected+to+traumatic+frontal+lobe+percussion+brain+injury.">Increased intestinal permeability in rats subjected to traumatic frontal lobe percussion brain injury.</a> The Journal of trauma. 64, 131-137 (2008).</li><li>Keely, S., 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=Chloride-led+disruption+of+the+intestinal+mucous+layer+impedes+Salmonella+invasion:+evidence+for+an+'enteric+tear'+mechanism.">Chloride-led disruption of the intestinal mucous layer impedes Salmonella invasion: evidence for an 'enteric tear' mechanism.</a> Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 28, 743-752 (2011).</li><li>Keely, S., 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=Contribution+of+epithelial+innate+immunity+to+systemic+protection+afforded+by+prolyl+hydroxylase+inhibition+in+murine+colitis.">Contribution of epithelial innate immunity to systemic protection afforded by prolyl hydroxylase inhibition in murine colitis.</a> Mucosal immunology. 7, 114-123 (2014).</li><li>Sourisseau, T., 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=Regulation+of+PCNA+and+cyclin+D1+expression+and+epithelial+morphogenesis+by+the+ZO-1-regulated+transcription+factor+ZONAB/DbpA.">Regulation of PCNA and cyclin D1 expression and epithelial morphogenesis by the ZO-1-regulated transcription factor ZONAB/DbpA.</a> Mol Cell Biol. 26, 2387-2398 (2006).</li><li>Ruehl-Fehlert, C., 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=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55, 91-106 (2003).</li><li>Barthe, L., Woodley, J. F., Kenworthy, S., Houin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+everted+gut+sac+as+a+simple+and+accurate+technique+to+measure+paracellular+transport+across+the+small+intestine.">An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine.</a> European journal of drug metabolism and pharmacokinetics. 23, 313-323 (1998).</li><li>Marks, E., 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=Oral+Delivery+of+Prolyl+Hydroxylase+Inhibitor:+AKB-4924+Promotes+Localized+Mucosal+Healing+in+a+Mouse+Model+of+Colitis.">Oral Delivery of Prolyl Hydroxylase Inhibitor: AKB-4924 Promotes Localized Mucosal Healing in a Mouse Model of Colitis.</a> Inflammatory bowel diseases. 21, 267-275 (2015).</li><li>Keely, S., 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=Hypoxia-inducible+factor-dependent+regulation+of+platelet-activating+factor+receptor+as+a+route+for+gram-positive+bacterial+translocation+across+epithelia.">Hypoxia-inducible factor-dependent regulation of platelet-activating factor receptor as a route for gram-positive bacterial translocation across epithelia.</a> Mol Biol Cell. 21, 538-546 (2010).</li><li>Brayden, D. J., Bzik, V. A., Lewis, A. L., Illum, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CriticalSorb+promotes+permeation+of+flux+markers+across+isolated+rat+intestinal+mucosae+and+Caco-2+monolayers.">CriticalSorb promotes permeation of flux markers across isolated rat intestinal mucosae and Caco-2 monolayers.</a> Pharmaceutical research. 29, 2543-2554 (2012).</li><li>Hubbard, D., Ghandehari, H., Brayden, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+transport+of+PAMAM+dendrimers+across+isolated+rat+jejunal+mucosae+in+ussing+chambers.">Transepithelial transport of PAMAM dendrimers across isolated rat jejunal mucosae in ussing chambers.</a> Biomacromolecules. 15, 2889-2895 (2014).</li><li>Keely, S., 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=In+vitro+and+ex+vivo+intestinal+tissue+models+to+measure+mucoadhesion+of+poly+(methacrylate)+and+N-trimethylated+chitosan+polymers.">In vitro and ex vivo intestinal tissue models to measure mucoadhesion of poly (methacrylate) and N-trimethylated chitosan polymers.</a> Pharmaceutical research. 22, 38-49 (2005).</li><li>Maher, S., 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=Evaluation+of+intestinal+absorption+enhancement+and+local+mucosal+toxicity+of+two+promoters.+I.+Studies+in+isolated+rat+and+human+colonic+mucosae.">Evaluation of intestinal absorption enhancement and local mucosal toxicity of two promoters. I. Studies in isolated rat and human colonic mucosae.</a> European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 38, 291-300 (2009).</li><li>Balda, M. S., 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=Functional+dissociation+of+paracellular+permeability+and+transepithelial+electrical+resistance+and+disruption+of+the+apical-basolateral+intramembrane+diffusion+barrier+by+expression+of+a+mutant+tight+junction+membrane+protein.">Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.</a> The Journal of cell biology. 134, 1031-1049 (1996).</li><li>Behrens, I., Stenberg, P., Artursson, P., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+lipophilic+drug+molecules+in+a+new+mucus-secreting+cell+culture+model+based+on+HT29-MTX+cells.">Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells.</a> Pharmaceutical research. 18, 1138-1145 (2001).</li><li>Stefka, A. T., 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=Commensal+bacteria+protect+against+food+allergen+sensitization.">Commensal bacteria protect against food allergen sensitization.</a> Proc Natl Acad Sci U S A. 111, 13145-13150 (2014).</li><li>Keely, S., 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=Activated+fluid+transport+regulates+bacterial-epithelial+interactions+and+significantly+shifts+the+murine+colonic+microbiome.">Activated fluid transport regulates bacterial-epithelial interactions and significantly shifts the murine colonic microbiome.</a> Gut microbes. 3, 250-260 (2012).</li><li>Barrett, K. E., Keely, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+secretion+by+the+intestinal+epithelium:+molecular+basis+and+regulatory+aspects.">Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects.</a> Annual review of physiology. 62, 535-572 (2000).</li><li>Soni, J., 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=Rat,+ovine+and+bovine+Peyer's+patches+mounted+in+horizontal+diffusion+chambers+display+sampling+function.">Rat, ovine and bovine Peyer's patches mounted in horizontal diffusion chambers display sampling function.</a> Journal of controlled release : official journal of the Controlled Release Society. 115, 68-77 (2006).</li><li>Justino, P. 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=Regulatory+role+of+Lactobacillus+acidophilus+on+inflammation+and+gastric+dysmotility+in+intestinal+mucositis+induced+by+5-fluorouracil+in+mice.">Regulatory role of Lactobacillus acidophilus on inflammation and gastric dysmotility in intestinal mucositis induced by 5-fluorouracil in mice.</a> Cancer chemotherapy and pharmacology. , (2015).</li><li>Tran, C. D., Sundar, S., Howarth, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+zinc+supplementation+and+methotrexate-induced+small+intestinal+mucositis+in+metallothionein-knockout+and+wild-type+mice.">Dietary zinc supplementation and methotrexate-induced small intestinal mucositis in metallothionein-knockout and wild-type mice.</a> Cancer biology &amp; therapy. 8, 1662-1667 (2009).</li><li>Musch, M. W., Wang, Y., Claud, E. C., Chang, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lubiprostone+decreases+mouse+colonic+inner+mucus+layer+thickness+and+alters+intestinal+microbiota.">Lubiprostone decreases mouse colonic inner mucus layer thickness and alters intestinal microbiota.</a> Digestive diseases and sciences. 58, 668-677 (2013).</li></ol>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

ここでは、粘膜バリア機能のex vivoで評価するために分離および腸嚢の調製を詳述しています。腸の嚢の調製物は、主に腸を横断候補薬物の吸収を調べ、薬学研究に利用されています。しかし、このアッセイは、腸疾患の研究のためにも同様に適しています。領域と透過性の部位特異的な評価によって大きく異なる場合があります腸の透過性は、消化器疾患における粘膜の整合性の地域...

消化管の腸上皮障壁はヒト成人で400メートル2で推定粘膜表面積です。したがって、常に微生物、摂取薬、栄養素や細菌毒素から挑戦にさらされています。ホストが許容共生細菌と潜在的な病原体を区別しなければならないだけでなく、同​​時に、栄養素の吸収を可能にしながら、上皮関門を通過するから、これらの種とそれらの分泌分子を防止しなければなりません。このように、腸上皮の役割は、管腔の内容物1に対する選択的障壁として作用することです。これは部分的に、構成的および誘導機構2からなる反応性生物系を介して作用する粘膜における先天上皮防御システムによって、達成されます。 上皮バリア機能の喪失は、胃腸疾患の多数の特徴的な病理である。 インビボでの上皮のバリア機能の検査は、トレーサー分子およびその後の血清の分析3の強制経口投与を介して評価することができます。しかし、この技術は、バリア機能障害のサイトへなどの兆候を提供していません。 vitroおよびトランスウェルシステム3を使用し、それぞれのチャンバ4,5をアッシング経上皮抵抗のex vivoでの評価では、一般的に上皮のバリア機能の代理マーカーとして使用するが、動物モデル6の貢献疾患の生理機能を欠いています。このプロトコルでは、レベルの数で粘膜バリア機能を評価するために使用され得る腸の完全性との直接及び局所的な評価を可能にするエクスビボ組織標本のモデルが記載されています。重要なことに、この手法は、疾患の動物モデルに適用することができる、または薬理学的に粘膜障壁機能障害の深さ質問に可能にするように操作することができます。

<table><tbody><tr><td>Dekantel&amp;nbsp; Non-absorbable Silk suture</td><td>Braintree Scientific</td><td>SUT-S 116</td><td></td></tr><tr><td>Media 199 (TC199)&amp;nbsp;</td><td>Life Technologies</td><td>11043-023</td><td>No phenol red as this interferes with fluorescence</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>21063-045</td><td>No phenol red as this interferes with fluorescence</td></tr><tr><td>N-acetylcysteine</td><td>Sigma Aldrich</td><td></td><td>Use at 10 mM in media</td></tr><tr><td>Small animal vascular cathether: Physiocath</td><td>Data Sciences International</td><td>277-1-002</td><td></td></tr><tr><td>FITC-Dextran 4,400 MW</td><td>Sigma Aldrich</td><td>FD-4</td><td></td></tr><tr><td>FITC-Dextran 20,000 MW</td><td>Sigma Aldrich</td><td>FD-20</td><td></td></tr><tr><td>FITC-Dextran 70,000 MW</td><td>Sigma Aldrich</td><td>FD-70</td><td></td></tr></tbody></table>

ex vivo intestinal sacs to assess mucosal permeability in models of gastrointestinal disease

上皮バリアは、胃腸管の最初の先天的防御であり、選択的基盤となる組織区画に内腔からの輸送を調節し、上皮を通過する小さな分子の輸送を制限し、ほぼ完全に上皮高分子輸送を禁止します。この選択性は、親油性分子との両方の頂端受容体および上皮のタイトジャンクションタンパク質複合体の輸送を制限する粘液ゲル層によって決定される。上皮のin vitro細胞培養モデルは便利であるが、モデルとして、それらが欠け微生物、粘液ゲル、上皮および免疫系の間の相互作用の複雑さ。一方、腸管吸収または浸透性のインビボ評価を行うことができるが、これらのアッセイは、部位特異性の兆候と、全体的な消化管吸収を測定する。「腸の嚢」を用いてエクスビボ透過性アッセイ;腸の部位特異性の付加的な利点と、特定の分子の全体的な腸の整合性や比較のトランスポートのいずれかを測定するの迅速かつ高感度な方法です。ここでは、透過性の研究と見かけの透過性（P アプリ ）の計算のための腸嚢の調製を記載 腸管バリアを通過する分子の。この技術は、薬物吸収を評価する方法として使用することができる、または胃腸疾患の動物モデルにおける地域上皮バリア機能障害を検討します。

このプロトコルは、胃腸疾患の動物モデルにおいて腸のバリア機能の地域の変化を調べるために使用することができます。胃腸管7の変化領域に粘膜表面を横切って傍細胞プローブの流量を測定することにより、上皮のタイトジャンクションの完全性を評価することができます。また、大きさ（ 図2）または疎水性（ 図3）、上皮摂動?...

Watch this Scientific Journal Video about Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease at JoVE.com

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 ...

ex-vivo-intestinal-sacs-to-assess-mucosal-permeability-models

Research

JoVE Journal

Medicine

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

21.0K ビュー.  University of Newcastle. 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.

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

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

人間の腸 Organoids 上皮関門の透過性のリアルタイム計測

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

発生生物学

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

実験的大腸炎の間、腸上皮バリア機能に栄養補助食品の有益な効果を分析

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

医学

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

腸透過性と上皮細胞流れ性能を評価する生体内顕微鏡ベースのアプローチ

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

免疫・感染学

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

標準化腸ループモデルを用いたマウスにおける腸透過性と好中球経上皮移動の機能評価

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

根尖腸管モデルにおけるルシファーイエローを用いた腸透過性の決定

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

生きているオルガノイドにおける腸のバリア破壊の調査

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

生物学

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

<meta charset="utf8"></head><body>免疫蛍光染色を用いた結腸粘膜の微小上皮ギャップの評価と定量化

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

免疫蛍光染色を用いた結腸粘膜の微小上皮ギャップの評価と定量化

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

フルオレセインイソチオシアネート標識デキストランを用いたマウスの腸バリア完全性の評価

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

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

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人間の腸 Organoids 上皮関門の透過性のリアルタイム計測

実験的大腸炎の間、腸上皮バリア機能に栄養補助食品の有益な効果を分析

腸透過性と上皮細胞流れ性能を評価する生体内顕微鏡ベースのアプローチ

標準化腸ループモデルを用いたマウスにおける腸透過性と好中球経上皮移動の機能評価

根尖腸管モデルにおけるルシファーイエローを用いた腸透過性の決定

生きているオルガノイドにおける腸のバリア破壊の調査

腸管上皮細胞の透磁率を決定する体外と体内アプローチ

免疫蛍光染色を用いた結腸粘膜の微小上皮ギャップの評価と定量化

免疫蛍光染色を用いた結腸粘膜の微小上皮ギャップの評価と定量化

フルオレセインイソチオシアネート標識デキストランを用いたマウスの腸バリア完全性の評価