この作品は、分子医学におけるHHMI /バンダービルト大学の証明書プログラムを通じてハワードヒューズ医学研究所（HHMI）からTJWにSSHで接続するには、米国立衛生研究所（NIH）からの助成金（R01-DK078640）、（GRDOT56006779）によってサポートされていましたバンダービルト糖尿病研究·研修センター（P30〜DK020593）とコア·サービスを提供するバンダービルト消化器病研究センター（P30〜DK058404）。

1。カニューレを準備<ol><li>指先でPE10チューブの1インチの長いセクションを温めるとチューブが薄くなるように、それを伸ばす。 注意：カニューレを挿入する血管やダクトのサイズが必要なストレッチの度合いを決定します。カニューレを胆管キャストのためによく伸ばさなければなりませんが、適度に大きい門脈内に収まるように延伸することが必要になることがあります。 </li><li>ベベルチップを生成するために対角線で延伸チューブをカットします。 </li><li> 5から6インチのセグメントを作成するためにチューブのもう一方の端をカットします。 </li><li>チューブの非傾斜端に32ゲージ、½インチの皮下注射針を挿入します。 </li></ol> 2。 PBSでシリンジを準備<ol><li> PBSで3ミリリットル注射器を埋める。 </li><li>先に調製したPE10チューブを引っ張っ添付で注射針にシリンジをねじ込みます。 </li><li>フリックシリンジとプッシュシリンジプランジャPBSは針やチューブを埋めていることを保証し、シリンジ内に気泡がありません。 </li><li>粘土から出状シリンジホルダにPBSを用いてプリペアドシリンジを置きます。チューブがダクト内に挿入されながら、このホルダーは、注射器を安定に保つでしょう。 </li></ol> 3。他のセット·アップ<ol><li>小さ ​​な容器、 例えば 、24ウェルプレートのウェル1に触媒0.1gを計量する。 </li><li> 3ミリリットル注射器に1ミリリットル樹脂を引き出します。 注意：触媒と樹脂の相対量は、硬化時間を変更するように調整することができる。触媒05の量を減少させる一方、鋳造収縮を増加させ、あまり望ましくキャストにつながる。 注：樹脂を取り扱う際に、樹脂ヒュームの樹脂または吸入との接触を避けるための予防措置を取る。常に手袋を着用し、そのような化学ヒュームフードなど換気の良い場所で樹脂を含む手順を実行します。 </li><li>字として使用される約5インチの5.0絹縫合糸の部分を切り取ります。 </li></ol> 4。マウスを準備<ol&gt; <li>成体マウスを生け贄に捧げる。その裏にマウスを置き、腹腔を開く。 </li><li>スコーププラットフォームを解剖にマウスを置きます。肝臓への可視性とアクセス可能性を高めるためにマウスの下に垂直に15 mlコニカルチューブを置きます。 </li><li> PBSで綿棒を湿らせ、肝門脈または総胆管と一緒に肝臓の背部ビューを公開することから上方に離れて肝臓を反転するためにそれを使用。 </li><li>門脈キャストでは、室温でPBSで静脈をフラッシュすることによって血液凝固を防ぐことができます。 PBSで満たされた3 mlの注射器に針を取り付け、限りすることができますように、肝臓から肝門静脈に針を挿入します。そっと門脈を介してPBSを押してください。血液を排出するために総主静脈内にニックネームを作成します。血液の排水性を高める、マッサージ肝臓にぬれた綿棒を使用してください。このステップは、胆管にはお勧めしませんし、すべての血管系に必要でないかもしれません。 注：このステップはまたperfoすることができます代わりにPBSの4％パラホルムアルデヒドでrmed。 4％パラホルムアルデヒドの使用は、特に小さな血管構造の、より正確なキャストで血管の整合性と結果を高める可能性があります。 </li></ol> 5。肝外門脈または総胆管カニューレを挿入<ol><li>肝臓から約¼インチ、門脈や胆管の下に外科的縫合結紮を渡すように湾曲したピンセットを使用しています。 </li><li>緩い共通の結び目に糸を結びます。結び目を締めないでください。 </li><li>あなたはオープンエンドのシステムで作業している場合、それはシステムのもう一方の端に合字を結ぶ締めすると便利かもしれません。あなたは門脈から血液を排出するために総主静脈を開いている場合、門脈内の圧力を増加させ、中央の肝静脈への樹脂漏れを防止するために、その周りに合字を結ぶ。 </li><li>縫合糸の結び目の下約¼インチ、門脈や胆管に小さなカットをする春のはさみを使用しています。このCutは中途半端な門脈や胆管の直径を通じ以上のものを貫通するべきではありません。 </li><li>第5ピンセットを用いて、カットのサイトで門脈や胆管を保持し、胆管にチューブの傾斜端を挿入します。 </li><li>プリタイドリガチャー過ぎ、肝臓に向かってチューブの先端を押してください。 </li><li>カニューレを介してPBSを注入し、それは門脈や胆管に入っているかどうかを観察することで、カニュレーションの精度をテストします。 </li><li>代わりにカニューレを保持するために字を締めます。 </li></ol> 6。樹脂は門脈や​​肝内胆管をキャスト<ol><li>予め測定された0.1グラムの触媒に予め測定された1ミリリットル樹脂を加え、よく混ぜる。気泡を発生させることは避けてください。 </li><li> 3 mlの注射器に戻す樹脂触媒混合物を描画します。優しくシリンジをフリックし、気泡を追い出すことにより、任意の気泡を除去する。 注：小さな構造をキャストするために、それは目から非常に小さな気泡を除去するために真空チャンバーを使用すると便利かもしれませんeは樹脂とキャストの質を向上させる。この手順を実行するための時間を考慮するためには、樹脂の硬化を遅くするために、上記示唆から触媒/樹脂比を低下させることが必要である。 </li><li>慎重にカニューレに取り付けられた32ゲージの針を残して、樹脂含有注射器を含むPBS含有注射器を交換してください。 </li><li>抵抗が増加し、肝臓がいっぱいになるまでゆっくりと優しく門脈や胆管に樹脂を押してください。 </li><li>優しく肝臓をマッサージしても塗りつぶしを奨励するためにぬれた綿棒を使用してください。 </li><li>構造からカニューレを取り外し、速やかに樹脂漏れを防ぐために字を締め直してください。 </li><li>マウスは樹脂が硬化しながら20分間室温で平らにすることができ、その後、肝臓を削除します。 </li><li>どちらかの肝臓をクリアしたり、マセレーションを続行します。 </li></ol> 7。 その場で可視化するための明確な肝<ol><li> 4℃、ロッキングで一晩、4％パラホルムアルデヒド中で肝臓を修正しました。 </li><li>肝臓を洗うPBS中で少なくとも4時間、ロッキングのために。必要であればこの時点では、肝葉を分離することができる。 </li><li>室温で揺らし、少なくとも4時間ごとに50％メタノールにした後、100％メタノールで脱水する。 </li><li>ロッキング、少なくとも24時間ベンジルアルコールと安息香酸ベンジル（Babbの）の1:2溶液中で肝臓をクリアします。長い洗浄時間が必要になることがあります。肝臓は2-3日後には完全にクリアされない場合は、24時間、100％メタノールに戻って移動し、Babbのクリアを繰り返す。 </li><li> 上皮内イメージング用ガラスシャーレ内に沈めBabbの肝。 </li></ol> 8。肝臓組織を柔らかく<ol><li>マウスから肝臓を取り出して、水で50 mlコニカルチューブに入れた。完全に樹脂硬化で1時間待ちます。必要に応じて独立した肝葉。 </li><li>水を捨て、ガラスボトルに肝臓に移動します。 15パーセントのKOHに沈め。室温で一晩にしておきます。動かさないでください。 </li><li>慎重にKOHを捨て、蒸留水でキャストを洗う。注：キャストは、verです壊れやすいとyは、細心の注意を払って取り扱ってください。管から流体を追加または削除するときにキャストを中断することは避けてください。 </li><li>レジンキャストがきれいになったら、そっと格納するコンテナに転送して、乾燥させるキムワイプ上に横たわっていた。 </li></ol> 9。代表的な結果門脈や胆管の正常なキャストは、へそから肝臓周囲に伸びる徐々に小さく枝の連続的なネットワークが表示されます。 図1は、門脈がBabbのクリア。 図1Aは、全体の左肝葉のキャストとキャストの形状とサイズを示した後、その場で可視化としてキャスト。 図1Bは同じキャストの拡大図を示し、実証を示してい肝周囲で最小の門脈枝への樹脂の浸透。 図2は、KOHミリアンペアを遂げている門脈キャストを示していますceration。 図2Aは、全体の左肝葉と図2Bは末梢小型の枝のクローズアップであることを示している。 この手法はまた、正常肝内胆管に適用され、Babbの清算とKOHマセラシオン手法1,2,7の両方を使用して公開されています。 一般的な問題がいっぱいに不完全含む、周囲のシステムまたは組織への樹脂と、オーバーフローで泡。 図3に門脈からの不完全な充填（ 図3A）、気泡（ 図3A）、樹脂のオーバーフローを認識する方法を示してい中心静脈（ 図3B）。 すべての画像はライカMZ-16のFAステレオスコープを使用していて、RETIGA 4000RカメラをQImaging取られる。 <img alt="図1" src="/files/ftp_upload/4272/4272fig1.jpg" /> 図1。肝左葉の門脈のレジンキャストをBabbのクリアされた。 A.レジンキャストは門部から肝周囲に伸びる階層的な分岐構造を示しています。レジンキャストは、青みがかった肝葉内に黄色で表示されます。 B.は近くアップ樹脂塗りが肝周囲に小さな枝に及ぶことを示しています。樹脂はクリア肝臓を通して白っぽいです。スケールバーは= 1ミリメートル。 <img alt="図2" src="/files/ftp_upload/4272/4272fig2.jpg" /> 図2肝臓の組織浸軟樹脂キャストは葉の門脈を残しました。全肝葉のA.レジンキャストは、さまざまなサイズの多くの枝を示しています。 Bのクローズアップは、肝臓の周囲に小さな枝を示しています。小さな枝の外観をフェザリングする正弦スペースの充填樹脂を表し、又は樹脂キャストに表示されない可能性はありますが、存在しなければ明らかに枝の密度が増加します。樹脂の色は白です。スケールバーは= 1ミリメートル。 図3樹脂キャストの一般的な問題。 A.左葉の門脈の不完全な充填は、肝臓周囲に拡張するために失敗し、一部またはすべてのキャストに分岐によって明らかである。このキャストはまた、連続鋳造（矢印）でブレークなど目に見える気泡が表示されます。いくつかの場所で門脈と中心静脈枝の近さのために、Bを、門脈樹脂は、多くの場合、中心静脈を入力して記入します。二つの異なる肺門枝（矢印）と異なるパターンと重複している2つの分岐構造がある場合にこれを認識することができます。技術的なエラーと真の形態学的変化とを区別するには2つの方法で行うことができます。まず、鋳造組織は、2次元解析、または他の方法に基づいて予想される構造に比較されるべきである。第二に、それはいくつかが複製を実行することが重要であり、可能な場合は、構造体を定量UREと異なる実験群と対照群間で統計学的に有意と内の形態学的変化がある場合には、再現性を決定するとするために統計分析を実行します。

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Assessment+of+the+Rat+Intrahepatic+Biliary+System+by+Three-Dimensional+Reconstruction.">Quantitative Assessment of the Rat Intrahepatic Biliary System by Three-Dimensional Reconstruction.</a> The American Journal of Pathology. 158, 2079-2088 (2001).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+Artery+and+Portal+Vein+Remodeling+in+Rat+Liver:+Vascular+Response+to+Selective+Cholangiocyte+Proliferation.">Hepatic Artery and Portal Vein Remodeling in Rat Liver: Vascular Response to Selective Cholangiocyte Proliferation.</a> The American Journal of Pathology. 162, 1175-1182 (2003).</li></ol>

特別な利害関係は宣言されません。

我々は、肝臓の門脈および肝内胆管系はキャストすることができますが、この技術は若​​干の適応と他のほとんどすべての乳管や血管系にどのように適用できるかの具体例を記載している。前の仕事は10,11子ガモマウス1,2,7-9を含む複数種、、、ウサギ12,13、犬14、豚15、および鼻腺14を含む多くの器官における本手法の有効性を実証してきま?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> PE10ポリエチレンチューブ</td><td>フィッシャー·サイエンティフィック</td><td> 1417012P </td><td></td></tr><tr><td> 5から0外科黒編組シルク</td><td> Roboz外科</td><td> SUT-15-1 </td><td></td></tr><tr><td> Steriject 32 G×13 mmの針</td><td>エアタイト</td><td> TSK3213 </td><td></td></tr><tr><td>春のはさみ</td><td>ファイン科学ツール</td><td> 15000から08 </td><td></td></tr><tr><td> 5番鉗子</td><td>ファイン科学ツール</td><td> 91150から20 </td><td></td></tr><tr><td> Mercox IIキット</td><td>ラッド研究</td><td> 21247 </td><td></td></tr><tr><td>ベンジルアルコール</td><td>フィッシャー·サイエンティフィック</td><td> 1816年から1804年</td><td> OBabbのクリアリングのために必要nly </td></tr><tr><td>安息香酸ベンジル</td><td> MPバイオメディカル、LLC </td><td> 154839 </td><td>唯一Babbのクリアリングのために必要</td></tr><tr><td>リン酸緩衝食塩水（PBS） </td><td></td><td></td><td></td></tr><tr><td>粘土</td><td></td><td></td><td></td></tr><tr><td>スケール</td><td></td><td></td><td></td></tr><tr><td>研究所はさみ</td><td></td><td></td><td></td></tr><tr><td> 15mlのキャップポリプロピレンチューブ</td><td></td><td></td><td></td></tr><tr><td> 4％paraformaldahyde </td><td></td><td></td><td></td></tr><tr><td> 15％水酸化カリウム（KOH） </td><td></td><td></td><td>唯一のマセラシオンのために必要</td></tr><tr><td>かみそりの刃</td><td></td><td></td><td></td></tr><tr><td> 100％メタノール</td><td></td><td></td><td></td></tr><tr><td> 3ミリリットルルアーロックシリンジ</td><td></td><td></td><td></td></tr></tbody></table>

3-dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system

臓器、血管および腺管構造の正しいアーキテクチャーは、適切な生理的機能のために不可欠であり、これらの構造体の形成と維持には、高度に制御されたプロセスである。これらの複雑な3次元構造の解析が大幅にセクションまたは染料注入試験のいずれかで2次元検査に依存してきた。これらの技術は、しかし、彼らは明らかにすることを意図している乳管や血管構造の完全かつ定量的な表現を提供することができません。あるいは、3次元プラスチック樹脂キャストの性質は、システムの永続的なスナップショットを作成し、3次元構造とネットワークを可視化し、定量化するための新規かつ広く有用な技術である。 樹脂鋳造システムの重要な利点は、血管やダクトの無傷と接続、または通信し、構造を決定する機能です。血管とDUCの構造TALネットワークは臓器の機能に不可欠であり、この手法にはいくつかの方法で、血管や管網の研究を支援する可能性を秘めています。レジンキャストは、管腔構造の正常な形態と機能アーキテクチャを分析発達形態形成の変化を識別し、正常と病気の状態間の組織構造の形態学的差異を明らかにするために使用することができる。前の仕事は、たとえば、勉強する注型用樹脂を利用している、構造1,2の2次元解析に反映されませんでしたマウスの肝内胆管系内のアーキテクチャと機能の欠陥、アルツハイマー病マウスモデル3の脳血管系の変化、門脈圧亢進性と肝硬変マウス4、未熟と大人の肺の間のラットのリンパ成熟の発達のステップ5、薬害への応答におけるラット肝臓、膵臓、腎臓の即時微小変化で門脈の異常6。 ここでは、門脈と肝内胆管に特に焦点を当て、マウスの血管や管網の3次元レジンキャストを生成する方法を提示します。これらのキャストは、組織をクリアしたり、マセレーションにより可視化することができますし、分析することができます。この手法は、実質的に任意の血管や管システムに適用することができ、3次元の乳管や血管構造の開発、機能、保守、または傷害に問い合わせるどんな研究に直接適用できるであろう。

Watch this Scientific Journal Video about 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System at JoVE.com

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

マウスの肝門脈や肝内胆管の3次元構造を可視化し、定量化する方法が記載されている。このレジンキャスト技術は、他の管または血管系に適用し、可能にすることができます<em&gt;その場で</em&gt;システムの完全な通信アーキテクチャの可視化や定量化。

3次元樹脂鋳造およびマウスの門脈や肝内胆管系の結

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

3-dimensional-resin-casting-imaging-mouse-portal-vein-or-intrahepatic

Research

JoVE Journal

Medicine

17.1K ビュー.  Vanderbilt University. マウスの肝門脈や肝内胆管の3次元構造を可視化し、定量化する方法が記載されている。このレジンキャスト技術は、他の管または血管系に適用し、可能にすることができますその場でシステムの完全な通信アーキテクチャの可視化や定量化。

ビデオ: 3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

モデリングとイメージング、3次元の集団細胞浸潤

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

医学

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

マウスにおける胆管結紮：閉塞性胆汁うっ滞による炎症性肝障害と線維症の誘発

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

多フッ素化された胆汁酸を用いて<em&gt;インビボ</em胆汁酸輸送を測定するために&gt;磁気共鳴イメージング

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Ileectomy-induced Bile Overaccumulation in Mouse Intestine

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer that often results in severe short bowel syndrome-like adverse effects including bile acid diarrhea, dehydration, electrolyte disturbances, and nutrient malabsorption. Here we introduce a murine ileal resection model, termed ileectomy, to evaluate tissue communication and the maintenance of systemic homeostasis. After ileal resection, circulating blood is permanently devoid of the ileum-specific endocrine hormone fibroblast growth factor 15 (FGF15), which releases its endocrinal inhibition of bile acid synthesis in the liver. In combination with the increased production and abolished reabsorption of bile acids after removing the ileum, mice that underwent surgery suffer from bile salt overaccumulation in the intestine and associated diarrhea, morbidity, and mortality. Novel usage of the surgery model introduced in this study may provide mechanistic and functional insights into ileal control of systemic metabolic regulation in physiology and disease.

Small intestine-dependent bile acid reabsorption and feedback inhibition of hepatic bile acid synthesis is important for systemic homeostasis and health. In this study, we describe a mouse model for ileal resection to evaluate ileectomy-induced bile malabsorption, overaccumulation, and toxicity in mouse intestine.

マウス腸管で胆汁の回腸誘起蓄積

Intestinal resection is a common therapeutic approach for human diseases such as obesity, inflammatory bowel disease, Crohn's disease, and colon cancer ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

肝のパーティションを関連付けると段階的な肝切除 (ALPPS) プロシージャの門脈結紮ラットモデル

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Laser Doppler Perfusion Imaging in the Mouse Hindlimb

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is a common, noninvasive, repeatable method for assessing blood flow recovery. The technique calculates overall blood flow in the sampled tissue from the Doppler shift in frequency caused when a laser hits moving red blood cells. Measurements are expressed in arbitrary perfusion units, so the contralateral non-intervened upon leg is usually used to help control measurements. Measurement depth is in the range of 0.3-1 mm; for hindlimb ischemia, this means that dermal perfusion is assessed. Dermal perfusion is dependent on several factors&#8212;most importantly skin temperature and anesthetic agent, which must be carefully controlled to result in reliable readings. Furthermore, hair and skin pigmentation can alter the ability of the laser to either reach or penetrate to the dermis. This article demonstrates the technique of LDPI in the mouse hindlimb.

Here, we present a protocol that demonstrates the technique and necessary controls for Laser Doppler perfusion imaging to measure blood flow in the mouse hindlimb.

マウスの後肢におけるレーザードップラー灌流イメージング

Blood flow recovery is a critical outcome measure after experimental hindlimb ischemia or ischemia-reperfusion. Laser Doppler perfusion imaging (LDPI) is ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

胆管TNBS輸液による慢性膵炎のマウスモデル

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

マウスの肝内膵島移植中の注入後門脈静脈出血の最小化

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

ダクト:3D肝臓解析のためのマイクロコンピュータ断層撮影に続く二重樹脂鋳造

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

9日齢マウス新生児における肝外胆管および胆嚢郭清

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...