The work was supported in part by funding from Alberta Livestock and Meat Agency and Natural Science and Research Council Discovery grant to S. D. Proctor.

ビデオと原稿の両方で説明した実験動物でのすべての手順は、制度的動物実験委員会によって承認され、動物ケアのカナダ人評議会によって設定されたガイドラインに従いました。 1.外科麻酔や新生児ブタの外科的準備<ol><li>アザペロン（0.3ミリグラム/キログラム）、塩酸ケタミン（10mg / kgの）、デクスメデトミジン（15μgの/キログラム）：分離控室では、25キロの含有筋肉内鎮静麻酔薬カクテルと首の付け根の近くに豚をpremedicate。 注：強化された術中疼痛管理のためのプレ麻酔薬カクテルにブプレノルフィン（0.005から0.02ミリグラム/キログラム）を追加します。 </li><li>フェイスマスクを使用して（500ミリリットル、千ミリリットル/分のO 2で四から五パーセントイソフルラン）吸入イソフルラン吸入ガスと豚を麻酔。獣医の喉頭スコープ（17〜25センチメートル長いストレート刃）を使用して、声帯を視覚化し、ボーカルへの局所10％のリドカインの去勢を適用コー​​ド。リドカインスプレーは声帯の痙攣および気道閉塞の可能性を低減するために、挿管に30-60秒前に声帯に連絡することを許可します。 </li><li>声帯の間（5.0〜7.0ミリメートル内径（ID））カフ付き気管内チューブを渡すことで豚を挿管し、イソフルランガスで麻酔を維持（1,000〜2,000 mlの0.5から2.0パーセントイソフルラン/分O 2）が閉じを使用して、手術全体の麻酔システムを再呼吸回路。顎トーンによって麻酔のレベル、およびペダルと眼瞼反射応答の両方を評価します。期限切れの麻酔ガスを掃気し、手術室の外に排出されます。 </li><li> 70％イソプロピルアルコールリンスに続いて2.0％クロルヘキシジン手術用スクラブ溶液で耳の外面を清掃してください。手術中に、20 G静脈カテーテルを用いて、静脈内輸液（5〜10ミリリットル/ kg /時間乳酸リンゲル液）を提供するために、耳静脈をカテーテルを挿入。メッドの舌の粘膜表面にパルスオキシメータを確保iCalのテープは、心拍数および末梢血酸素飽和度（SpO 2）を監視します。 </li><li>背臥位で麻酔をかけた豚を置き、恥骨の腹側面に半ば胸部尾側から腹側腹部を剃ります。 2交互に2.0％クロルヘキシジン手術用スクラブし、滅菌水洗浄でこのエリアを清掃してください。 </li><li>手術室に麻酔豚を移し、70％イソプロピルアルコールリンスの最終的な手術用スクラブを適用し、それを乾燥させ、その後、動物を羽織ります。 </li><li>体温を監視するために、直腸内に、直腸温度プローブ約2〜4センチ挿入します。外科手術中に正常な体温（38-40°C）を維持するために、水の循環加熱パッド上豚を置きます。 </li><li>腹部の周りの上層象限パターンに配置された4タオルのカーテンで豚を羽織ります。約5℃、腹部の外側面に沿って第2のドレープ、剣状突起全体で最初のドレープを配置腹部正中線にメートルの横。骨盤の回腸稜と第四ドレープ全体で三ドレープを配置し、（反対側が）二ドレープのように、腹部の外側面腹部正中線に横約5cmに沿って配置されています。 </li><li>根底にあるタオルドレープの上に、手術部位へのアクセスを可能にするスリット開口と、大きなテーブルのドレープを置き、豚、全体の手術台をカバーしています。最終的なドレープは、大きなテーブルのドレープの上に配置された使い捨てsteriドレープです。 </li></ol>腸リンパ本幹の2腹部手術やカテーテル<ol><li>根本的な腹筋を公開するために手術用メスの刃で20センチの皮膚切開を行います。壁側腹膜を露出させるためにモノポーラ電気メス（20ワットの設定）で腹部の筋肉層を切開。腹部内臓やリンパ管にアクセスするためにMetzenbaumのはさみで頭頂腹膜20cmの直線セグメントを開きます。 </li><li&gt;手術中は開いた腹腔を維持するために外科的切開の頭蓋側面でリトラクタを配置します。 </li><li>全体の外科的処置のための暖かい（37°C）滅菌生理食塩水ですべての組織を湿ら。静かに腹腔から結腸、盲腸、回腸および空腸を含む小腸の大部分を持ち上げ、上腹部、肝臓やリンパ管にアクセスするには、豚の左脇腹にそれを体外に出します。優しく腸をサポートするためにスリングを形成するために追加のタオルドレープとの位置に体外に腸を固定します。 </li><li>リンパ管の位置を確認し、それが約4センチメートル右腎静脈、6センチメートル尾-medial-大静脈職長の腹側および膵臓22,36,37の近くに右肝葉の内臓の側面の下の頭蓋-内側を産みます。門脈36,37の右腹側セグメントに並置された半透明の構造としてリンパ管を識別します。 </li><li>リンパヴェスを切り離しエル優しくQチップアプリケーターで付着した組織を離れてからかうことにより、周囲の筋膜から。容器の外側の側面は、周囲の組織から分離されると、微鈍鉗子をひっくり返して容器の下の「トンネル」の開口部を作成します。 </li><li>細かいピンセットでリンパ管の下に3 2-0絹縫合糸を渡します。 、閉塞拡張とリンパで容器を埋めるために最初に最も尾側の縫合糸を結紮。意図的にリンパ管にカテーテルを固定する（4センチ）比較的長いこの縫合糸の端を残します。他の二つの縫合糸を配置し、互いから1.0センチメートルを分離し、保護された尾縫合糸に頭蓋約1.0〜1.5センチメートルです。血管内にカテーテルを速く確保を可能にするために &#39;一緩い結紮ネクタイ&#39;でこれら2本の縫合糸を残します。 注：（2頭蓋縫合の）ほとんどの尾側結紮縫合糸と中間縫合糸の間に位置するリンパ管のセグメントは、カテーテル挿入のためのサイトです。 suコマンドについて必要に応じて、トゥーレ材料は、2-0ポリグラクチン縫合糸は、2-0絹縫合糸の代わりにすることができます。 </li><li>アイリスハサミで容器に小さな穴をカットし、微鈍鉗子で血管を拡張。容器内に傾斜した端部に特殊なカテーテルチューブ（4.06外径（OD）X 2.31ミリメートルのID）の約1.0〜1.5センチメートルを挿入し、所定の位置にカテーテルを固定するための2つの頭蓋縫合糸を結びます。容器にカテーテルを固定するために尾縫合糸の長い縫合糸の端を使用してください。 </li><li>温かい生理食塩水のおびただしい量の体外に腸を洗浄し、静かに腸の正しい解剖学的な位置決めを確実に腹腔に戻します。 </li><li>左ミッド脇腹（腹側paralumbar窩5〜10センチメートル）でカテーテルを体外に出します。メスで皮膚切開を行い、カテーテルの具象化するための開口部を作成するために、皮膚表面に腹腔からトロカールを通過します。腹部CAVからカテーテルを体外に出すために、大きなケリー鉗子を使用しますトロカールの貫通開口性。 </li><li>ラウンド（テーパー）針で2-0ポリグラクチン縫合糸の単純連続縫合パターンで壁側腹膜を閉じます。ラウンド針に2-0ポリグラクチン縫合糸でシンプルな結節縫合パターンで腹部の筋肉層を閉じます。 </li><li>切断針に2-0ポリグラクチン縫合糸で表皮下のパターンで皮膚を閉じます。巾着縫合パターンおよび切断針上の2-0ナイロン縫合糸で皮膚に体外カテーテルを固定します。 </li><li>まだその配置を容易にし、リカバリ時に豚のストレスを軽減するために麻酔しながら、豚に特化したジャケットを置きます。 </li></ol> 3.手術後の回復とリンパコレクション<ol><li>前吸入麻酔の中止へ約10分で、即時術後の鎮痛を提供するために、筋肉内にbupenorphine（0.1ミリグラム/キログラム）を管理。 bupenorphineを続行（0.1ミリグラム/キログラム）24〜48時間を維持するために12時間ごとに術後の鎮痛。 </li><li>手術後の合併症のために7日間の期間ごとに8-12時間を豚を監視します。 </li><li>エチレンジアミン四酢酸（EDTA）でコーティングし、抗生物質を補充した500 mlのポリプロピレン製の洗瓶でリンパ液を集めます。ペニシリン（6000 IU）、ストレプトマイシン（6 mg）をアムホテリシンB（3 mg）を7日間ごとに12時間。 </li></ol>リポタンパク質ApoB48、トリグリセリド、コレステロールと総タンパクの4定量リンパから収集<ol><li> 4℃で5分間、1800×gでリンパサンプルを遠心します。上清を収集し、トリグリセリド、コレステロールおよび総タンパク質の定量化のためにそれを使用しています。 </li><li>希釈していないサンプル、サンプル希釈1：20蒸留水と1希釈した最終サンプル：蒸留水で100 3サンプルに上清を分割します。 </li><li>市販のキットを用いて、コレステロールレベルを測定するために希釈されていない上澄みを使用してください。 </li><li> 1時20分と午前1時10分を使用します0は、それぞれ、市販のキットおよびビシンコニン酸総タンパク質アッセイを用いてトリグリセリドおよび総タンパク質レベルを測定するため、サンプルを希釈しました。 </li></ol>リンパ38から収集したリポタンパク質ApoB48 5.定量化、 <ol><li>適応免疫ウエスタンブロッティング法38でリポタンパクApoB48の濃度を決定します。 3〜8％トリス - 酢酸塩、ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動（SDS-PAGE）で区切り、総リンパ。 </li><li>ポリフッ化ビニリデン膜（0.45μmの）に分離したタンパク質を移し、アポB（1：4000）にヤギポリクローナル抗体でそれらをインキュベートし、その後、抗ヤギ二次抗体とそれをバインドします。 </li><li>精製されたげっ歯類ApoB48タンパク質標準との線形濃度測定の比較を用い​​て、化学発光とリポ蛋白ApoB48を定量化します。 </li></ol>

The authors have nothing to disclose.

腸リンパを収集する様々な実験動物モデルにおいて、脂質8-12に関与する機構と薬物13-15代謝、癌転移16,17、細胞輸送及び免疫機能18-26を調査するための優れた方法です。実際に、長期間にわたって、末梢（求心性）と中央（遠心性と大きなトランク船）リンパを大量に収穫する能力は、免疫調節剤を18-22と課題以下の細胞集団で発生する時間的な変化を...

リンパ系は、生理の​​understudied領域です。リンパカテーテル法の前臨床モデルは、異なる動物種1-8で発生し、脂質8-12および薬物代謝13-15、実験的治療17と癌転移16に関与するメカニズムを調査するために製薬産業や研究機関によって使用され、免疫機能18 -26。この研究は、リポタンパク質代謝の成分を測定するために、国内のブタモデルにおける腸管リンパトランクカテーテルの使用を探ります。リポタンパク質代謝を産生および分泌カイロミクロンの、ならびに関連する脂質および総蛋白質の変化に関与しています。一般的に使用される齧歯類モデルと人間の間のような脂質代謝の主要な違いがあるように、これらは私を、脂質を研究するためのより多くの比較可能な情報を提供することができる腸のリンパ節を収集するために豚のモデルを採用し、重要な考慮すべき事項であります人々 27-31でtabolism。 いくつかの外科技術は、大規模な動物種における腸管リンパを収集するために使用されている：頭蓋肩のアプローチ（ すなわち、胸管カテーテル法）5、横上部脇腹のアプローチ32-34、および腹側正中線またはparamedianアプローチ22,35。このビデオでは、具体的に腸のリンパトランクのカテーテル挿入のための腹側正中外科的アプローチを使用して、ブタにおいて外科的手順を説明します。慎重な手術法は、長期間にわたって大リンパの量とその成分を収集するためにリンパカテーテル法のこの方法が可能になります。 この技術は、様々な生理学的機能を調べる多くの分野への応用の無数を開きます。アプリケーションが含まれる可能性があり、これらに限定されないが、全身リポタンパク質と脂質代謝、免疫学、腫瘍の起源及び転移、腸機能、およびdevelopm耳鼻咽喉科や腸の炎症性疾患の進行。

<table border="0" cellpadding="0" cellspacing="0" width="567">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Miller laryngoscope blade</td>
			<td style="width:71px;">Welch Allyn</td>
			<td style="width:119px;">68044</td>
			<td style="width:167px;">182 mm length</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Surgivet advisor: Vital signs monitor</td>
			<td style="width:71px;">Surgivet</td>
			<td style="width:119px;">V9203</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Rectal temperature probe</td>
			<td style="width:71px;">Surgivet</td>
			<td style="width:119px;">V3417</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Mono-polar electrosurgery generator</td>
			<td style="width:71px;">Valley Lab</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Metzenbaum scissors</td>
			<td style="width:71px;">Fine Science</td>
			<td style="width:119px;">14518-18</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:211px;">Tuffier retractor</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-11-676</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Mosquito forceps</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-7-10</td>
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			<td height="20" style="height:20px;width:211px;">Kelly forceps-curved (14cm)</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-7-38</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Allis tissue forceps</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-7-38</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Forceps dressing-eye (10.2cm)</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-18-780</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Forceps dressing-Adison (12.1cm)</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-17-2510</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:211px;">Needle Drivers</td>
			<td style="width:71px;">Stevens</td>
			<td style="width:119px;">162-V98-42</td>
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			<td height="40" style="height:40px;width:211px;">Iris scissors</td>
			<td style="width:71px;">Fine science</td>
			<td style="width:119px;">14058-11</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:211px;">Circulating water pump</td>
			<td style="width:71px;">Jorvet</td>
			<td style="width:119px;">J-783X</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Maxitherm-Vinyl blanket</td>
			<td style="width:71px;">Jorvet</td>
			<td style="width:119px;">J-784C</td>
			<td style="width:167px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Q tip applicators</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-037-960</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Catheterization&#160; tubing (4.06 OD X 2.31 ID)</td>
			<td style="width:71px;">Braintree Scientific Inc.</td>
			<td style="width:119px;">MRE-160</td>
			<td rowspan="2" style="width:167px;">Micro-Renethane implantation tubing</td>
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			<td height="20" style="height:20px;width:211px;">2-0 silk suture</td>
			<td style="width:71px;">Ethicon</td>
			<td style="width:119px;">LA556</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:211px;">2-0 polyglactin suture</td>
			<td style="width:71px;">Ethicon</td>
			<td style="width:119px;">J443H</td>
			<td style="width:167px;">2-0 vicryl</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Large animal jacket</td>
			<td style="width:71px;">Lomir Biomedical Inc.</td>
			<td style="width:119px;">SSJ2YC</td>
			<td style="width:167px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Polypropylene wash bottles</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">03-409-22C</td>
			<td style="width:167px;">500 ml</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Penicillin-Streptomycin</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">D4333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">EDTA</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">60-00-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Amphotericin B</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">A2411</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Azaperone</td>
			<td style="width:71px;">Elanco Animal Health</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Stresnil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Dexmedetomidine hydrochloride</td>
			<td style="width:71px;">Zoetis</td>
			<td style="width:119px;">6295</td>
			<td style="width:167px;">Dexdomitor</td>
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		<tr height="60">
			<td height="60" style="height:60px;width:211px;">Isoflurane</td>
			<td style="width:71px;">Abbott Animal&#160; Health</td>
			<td style="width:119px;">05260-5</td>
			<td style="width:167px;">IsoFlo</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:211px;">Ketamine hydrochloride</td>
			<td style="width:71px;">Zoetis</td>
			<td style="width:119px;">2626</td>
			<td style="width:167px;">Ketaset</td>
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		<tr height="100">
			<td height="100" style="height:100px;width:211px;">Bupenorphine hydrochloride</td>
			<td style="width:71px;">Champion Alstoe Animal Health</td>
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			<td style="width:167px;">DIN:02347510</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:211px;">6 mm Endotracheal tube</td>
			<td style="width:71px;">Jorvet</td>
			<td style="width:119px;">J-165d</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">10% Lidocaine spray</td>
			<td style="width:71px;">AstraZeneca</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">DIN:02003767</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">4 % Chlorhexidine surgical scrub</td>
			<td style="width:71px;">Partnar Animal Health</td>
			<td style="width:119px;">PCH-011</td>
			<td style="width:167px;">Diluted: 2.0% solution</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:211px;">3M Surgical steri- drape</td>
			<td style="width:71px;">3M Health Care</td>
			<td style="width:119px;">1040</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">SDS page gel</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">EA0375BOX</td>
			<td style="width:167px;">3-8 % tris acetate</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Polyvinylidene fluoride membrane</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">IPVH00010</td>
			<td style="width:167px;">0.45 &#956;m pore size</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:211px;">ApoB antibody&#160;</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">AB742</td>
			<td style="width:167px;">1:4000 dilution</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Donkey anti-goat IgG-HRP</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">Sc-2304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:211px;">ECL Prime Western Blotting Reagent</td>
			<td style="width:71px;">GE Healthcare LifeSciences</td>
			<td style="width:119px;">RPN2232&#160;&#160;&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Triglyceride Kit</td>
			<td style="width:71px;">Wako Pure Chemicals</td>
			<td style="width:119px;">998-40391/994-40491</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:211px;">Total Cholesterol Kit</td>
			<td style="width:71px;">Wako Pure Chemicals</td>
			<td style="width:119px;">439-17501</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:211px;">Total Protein</td>
			<td style="width:71px;">&#160;Pierce&#160;</td>
			<td style="width:119px;">23225</td>
			<td style="width:167px;">Bicinchoninic Acid Assay</td>
		</tr>
	</tbody>
</table>

long-term catheterization of the intestinal lymph trunk and collection of lymph in neonatal pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

<html>新生児の豚の腸リンパ本幹のリンパカテーテル法は、7日間にわたって中央リンパの約1 L / 24時間の収集を可能にします。この実験で収集さリンパは、脂質代謝、すなわち総リンパタンパク質、ApoB48リポタンパク質、トリグリセリド、総タンパク質、およびコレステロールの成分を含有していました。表1のハイライト3匹のブタのプールされたリンパサンプルから?...

Watch this Scientific Journal Video about Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs at JoVE.com

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

新生豚のリンパ節の腸リンパトランクやコレクションの長期カテーテル法

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

long-term-catheterization-intestinal-lymph-trunk-collection-lymph

Research

JoVE Journal

Medicine

8.6K ビュー.  University of Alberta. We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

ビデオ: Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

婦人科腫瘍におけるセンチネルリンパ節の術中検出のためのマルチスペクトルのリアルタイム蛍光イメージング

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

医学

Basic Surgical Techniques in the G&ouml;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.The size of the G&ouml;ttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6-8. The aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research, by demonstrating how to perform tracheal intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion. 
Endotracheal Intubation should be performed whenever a minipig undergoes general anesthesia, because it maintains a patent airway, permits assisted ventilation and protects the airways from aspirates. Transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures. Furthermore, urinary catheterization can prevent contamination of delicate medico-technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters. Arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters. Catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access, when performing experiments involving frame-based stereotaxic neurosurgery and brain imaging. When performing vessel catheterization in survival studies, strict aseptic technique must be employed to avoid infections6. Transcardial perfusion is the most effective fixation method, and yields preeminent results when preparing minipig organs for histology and histochemistry2,9. For more information about anesthesia, surgery and experimental techniques in swine in general we refer to Swindle 2007. Supplementary information about premedication and induction of anesthesia, assisted ventilation, analgesia, pre- and postoperative care of G&ouml;ttingen minipigs are available via the internet at <a href="http://www.minipigs.com">http://www.minipigs.com</a>10. For extensive information about porcine anatomy we refer to Nickel et al. Vol. 1-511.

Basic Surgical Techniques in the G&#246;ttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion

The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of G&ouml;ttingen minipigs in biomedical research.

ゲッティンゲンミニ豚の基本的な手術テクニック：挿管、膀胱へのカテーテル挿入、大腿血管カテーテル法、およびTranscardial灌流

The emergence of the G&ouml;ttingen minipig in research of topics such as neuroscience, toxicology, diabetes, obesity, and experimental surgery reflects ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

豚における心筋梗塞および機能的結果の評価

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

次世代シーケンシングのための収集と唾液DNAの抽出

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

マウス子宮頸部リンパ節の高周波超音波イメージング

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

豚の長期心血管運動テストのためのカテーテルの外科的配置

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

マカクにおける肝臓及び腸間膜リンパ節のシリアル収集のための腹腔鏡技術

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

変形性関節症モデルにおけるマウス脾臓、骨髄、リンパ節および滑膜組織における免疫細胞サブセットのフローサイトメトリー解析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets.
Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (&gt;1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. 
Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Here we present a step-by-step protocol for a semiautomated approach to analyze murine long-term electrocardiography (ECG) data for basic ECG parameters and common arrhythmias. Data are obtained by implantable telemetry transmitters in living and awake mice and analyzed using Ponemah and its analysis modules.

マウスの不整脈を検出するための長期心電図記録の分析

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ...

Robot-assisted Total Mesorectal Excision and Lateral Pelvic Lymph Node Dissection for Locally Advanced Middle-low Rectal Cancer

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex procedures. The high-quality 3-D visual, multijoint arm and natural tremor filtration allow the surgeon to expose and dissect more accurately with minimal invasion. Total mesorectal excision is the standard surgical technique for the treatment of resectable rectal cancer. To reduce the lateral recurrence rate, lateral pelvic lymph node dissection can be performed, as it is a safe and feasible procedure for locally advanced middle-low rectal cancer with a high possibility of metastasis to the lateral lymph nodes. However, the complexity of the anatomic structures and the high postoperative complication rate limit its application. Recently, several surgeons have increasingly used robotic techniques for total mesorectal excision and lateral pelvic lymph node dissection. Compared with open and laparoscopic surgery, the robotic technique has several advantages, such as less blood loss, fewer blood transfusions, minimal trauma, shorter postoperative hospitalization, and quicker recovery. A robotic approach is generally regarded as a reasonable alternative for complicated procedures such as lateral pelvic lymph node dissection, although there are a limited number of high-quality prospective randomized controlled studies reporting direct evidence. Here, we provide the detailed steps of robot-assisted total mesorectal excision and lateral pelvic lymph node dissection performed at the First Affiliated Hospital of Xi&#39;an Jiaotong University.

The robotic technique described herein aims to detail a stepwise approach to robot-assisted total mesorectal excision and lateral pelvic lymph node dissection for locally advanced (T3/T4) rectal cancer located below the peritoneal reflection.

局所進行中低直腸癌に対するロボット支援全中腸切除および横骨盤リンパ節切断

Since their approval for clinical use, da Vinci surgical robots have shown great advantages in gastrointestinal surgical operations, especially in complex ...