著者の謝辞があります。

原則研究所動物ケアの (健康の国民の協会文書編 8、2011) と同様、動物研究のドイツの立法によって支配される規則に従っていた。政府の動物の世話のオフィス (Landesamt für Natur 環 und Verbraucherschutz ノルトライン ・ ヴェストファーレン州、レックリングハウゼン、ドイツ) から公式許可します。
1. 動物
<ol><li>オープンケージで収容 (25-30 kg) 女性ドイツ在来種豚を使用します。</li>
	<li>5 動物群 (アルゴン ・ ヘリウム) を使用します。</li>
	<li>実験する前に、少なくとも 1 週間、環境に順応する動物を許可します。水への無料アクセスと手術前に 24 h の高速動物。</li>
</ol>2. 麻酔
<ol><li>Premedicate (15 mg/kg 体重 [BW]) ケタミン ・ キシラジン (10 mg/kg BW)、アトロピン (0.1 mg/kg BW) 筋肉内注射と動物麻酔導入前に 10 分。</li>
	<li>22 ゲージ カニューレの耳静脈に配置によって末梢静脈アクセスを設立します。</li>
	<li>プロポ フォール 2 mg/kg の静脈注射による麻酔を誘導します。</li>
	<li>仰臥位で動物を置き、2 cm。 皮下組織の鈍的準備を通じ検索静脈の長さの頸静脈の溝に縦切開を実行します。セルジンガー ワイヤー、カニューレを挿入します。</li>
	<li>カニューレを撤回し、ガイドワイヤー 14 神父カテーテルを挿入します。ガイドワイヤーを撤回します。拡張機能にカテーテルを接続し、ストラップや縫合糸でカテーテルを固定します。</li>
	<li>舌を抜くし、まっすぐ喉頭鏡を挿入します。喉頭鏡のヒントを使用して、喉頭蓋をプルダウンします。声帯からチューブを挿入します。声門下カフを配置し、膨らませます。</li>
	<li>36 と 42 の mm Hg の間呼気終末部分炭素二酸化炭素の張力を維持する 40% 酸素 20 26 呼吸/分で 10 ml/kg の一回換気量と換気します。</li>
	<li>1 1.5% の濃度でイソフルランと 3-4 μ g/kg/h の濃度でフェンタニル麻酔を維持します。</li>
	<li>4 mL/kg の初期速度で乳酸リンゲル液を供給/h、8 mL/kg の持続注入率を開腹後増加/h。</li>
</ol>3. 手術とプラズマ凝固
<ol><li>標準的な手術台に仰臥位に動物を配置します。</li>
	<li>標準的な手術の消毒薬を適用することによって皮膚を消毒 (2-プロパノール 45 g/100 g、1-プロパノール 10 g/100 g、ビフェニル-2-オール 0.2 g/100 g) を 3 回手術綿棒。</li>
	<li>剣状突起からメスと恥骨へ広い正中開腹を実行し、手術リトラクターをインストールします。</li>
	<li>プラズマ凝固装置のスイッチは、使用キャリアガスによって、アルゴンまたはヘリウムのガスのボトルを開きます。3 L/分に応じて選択凝固デバイス出力へのガス流を調整します。 注: プラズマ凝固の両方の気高いガス、アルゴンまたはヘリウムを使用できます。凝固効果はほぼ同等です。詳細については文献7を参照してください。</li>
	<li>上記7として左肝葉でプラズマ凝固を実行します。チタン金型 (正方形開口 1 × 1 cm2) を使用して、凝固ゾーンを標準化します。5 凝固プローブ距離 1 cm で s。それぞれ異なる電源設定と凝集は、短い間隔 5 mm (図 1) の凝集と並行実行できます。</li>
	<li>肝臓の収穫のため肝臓にすべての靭帯接続を分割します。分離し、門脈と胆管の長い部分を残して優れた十二指腸曲げ上肝椎弓根を分割します。大静脈上と下の肝臓を分割し、器官を取得します。</li>
	<li>肝臓を採取後、豚は 0.16 g/kg BW ペントバルビ タールの投与によって安楽死されました。</li>
	<li>バースト圧力実験のための鋭いはさみで左の内側の肝葉の半分を切除します。プラズマ凝固させ、カット面 (100 w 出力) またはフィブリン (図 2) でカット表面をシールします。</li>
</ol>4. 微小循環測定
注: レーザー ドップラー分光は、赤血球の移動によるドップラー シフトの測定を通じて組織の血流を確認できます。レーザー信号移動赤血球の数と相関します。レーザー ドップラー分光法は臨床使用 (例えば移植医学) とされている複数回15を検証します。
<ol><li>レーザー レーザードップラー血流計や分光光度計に切り替えます。フラットのプローブを使用します。</li>
	<li>流れと速度の基準測定値を取る。保存または値に注意してください。</li>
	<li>前述の下 3.5 凝固を実行します。</li>
	<li>速度とメジャー流凝固サイトにフラットのプローブを配置します。もう一度、保存または値に注意してください。</li>
	<li>凝固装置装置のすべての電源設定について繰り返します。</li>
</ol>5. 温度測定
<ol><li>(サーモグラフィ カメラ、ノートブック、および赤外線温度計) にシステムを切り替え、測定を実行する前に少なくとも 1 時間を実行します。</li>
	<li>フォーカスを調整し、凝固敷地サーモグラフィ カメラのフレームを表示します。20 よりも大きい温度解像度 1024 x 768 ピクセルの空間分解能で検出できる赤外線シーケンス mK。考慮する凝固や周囲の組織の地域-熱伝達によって影響を受ける-ビューの途中であります。 注: それは可能な限り最適な空間解像度のフレームのピクセル数を含める必要があります。</li>
	<li>2 分にわたって肝表面にプラズマ凝固装置、サーモグラフィ カメラで凝固プロセスを記録します。</li>
	<li>サーモグラフィー解析ソフトウェアで画像を分析: 興味の領域を定義します。 注: ソフトウェアは、時間をかけて対応する平均温度のコースを計算します。</li>
</ol>6. 凝固深度測定
<ol><li>シャープはさみ左内側肝葉を収穫します。</li>
	<li>1 cm の厚さで凝固サイトを切除します。さらなる処理のため 3 mm 厚の経度方向のセグメントにカットします。</li>
	<li>一晩で中立的な 10% の 4 ° C で修正組織サンプルは、ホルマリンをバッファリングします。パラフィン 2 ° C、融点以上に加熱し、スライスを埋め込みます。一晩プロセス。</li>
	<li>ヘマトキシリン ・ エオシン染色を実行します。<ol>
<li>Deparaffinize し、その後を 2 に浸すことによって組織をメタンハイド レート x キシレン 100% エタノール (エタノール)、95% エタノール、70 %etoh, 脱イオン H2O 2 分。</li>
		<li>3 分のマイヤーのヘマトキシリン液と組織標本を染色します。</li>
		<li>今、5 分間水道水ですすいでください。</li>
		<li>エオシン溶液 3 分間で組織を染色します。</li>
		<li>3 分の 2 の x EtOH 95%、キシレンですすいでください。メディアを標準マウントをマウントします。</li>
	</ol>
</li>
	<li>(顕微鏡接続カメラ、イメージング ソフトウェア) のシステムに切り替えます。40 倍の倍率ですべてのセクションを表示します。</li>
	<li>40 倍の倍率でオブジェクト マイクロメータのイメージを取る。再目的ウィンドウでボタンを押します。手動校正を選択します。入力 0, 1 mm] ダイアログ ボックスでは、100 μ m のマイクロメータ イメージ上に線を描画し、OK を押します。</li>
	<li>ビューで長さを選択 > 解析コントロール > アノテーションや測定ウィンドウ。マウスを使って凝固余白に肝臓表面から測定します。エクスポートまたは結果に注意してください。同じスライド上の別の場所で測定を繰り返します。 注: 凝固深度は、通常肝細胞コードと縮められた細胞質、核濃縮核出血ゾーンと壊死のゾーン間のシャープなマージンによって通常の肝組織から簡単に区別できます。</li>
	<li>2 つの測定の平均値を計算します。</li>
</ol>7. バースト圧力測定
<ol><li>スイッチ (自動ポンプ、圧力計) のシステム。ステップ 3.7 によると肝臓のサンプルを準備します。 注: 3 方法活栓を介して接続された 2 つの並列ポンプを使用します。シングル ポンプで 1,500 の mm Hg の最大圧力が得られません。</li>
	<li>触覚の茎にはさみで門脈、肝動脈、胆管を分離します。門脈を喀鉗子でクランプし、4-0 monofil 縫合糸で結紮します。総肝動脈 overholt 鉗子をクランプし、4-0 monofil 縫合糸で結紮します。</li>
	<li>Ch 16 カテーテルを胆管に挿入し、2-0 絹縫合糸で結紮します。圧力計 (図 3) と三方活栓をインストール、自動ポンプにカテーテルを接続します。</li>
	<li>生理食塩水で灌流注射器を満たしなさい。</li>
	<li>99 mL/h の配信率の自動ポンプを開始します。</li>
	<li>漏れとレコードの破裂圧力の肝カット面と圧力計を監視します。 注: を漏れの簡単に認識、特許の青は生理食塩水 (2 mL 特許ブルー + 18 mL 生理食塩水) を追加できます。圧力計の圧力の損失の時に気付いて、破裂圧力を確認しやすいです。</li>
</ol>

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長い時間16肝臓手術の齧歯動物モデルが確立されます。大動物モデルがある特定の利点を提供するにもかかわらず、: 人間のための標準的な手術機器を適用できる、手術手技、臨床使用に匹敵する、標準的な臨床的評価方法は、顕微鏡の装置は必要ありません実験に移ります。特別な実験室試験方法 (図 10) を必要とせず標準臨床血液検査が実施される?...

アルゴン プラズマ凝固法 (APC) は、三十年1,2以上の腹部手術で広く使われている楽器です。肝臓のシールによる主要な肝切除後の出血3を防ぐために表面をカット後、二次止血の達成のための標準的な技法です。プラズマ凝固法は、高周波電気焼灼器、イオン化されたガスのアークを介して電気エネルギーを提供の特殊な形式です。モノポーラ電熱止血を提供し、この非接触技術組織4に固執する電極を防止する利点があります。イオン化されたガスのビームは自動的に最低の電気抵抗の領域に送られます、抵抗上昇乾燥のため他の地域がまだ乾燥して離れになっています。これは凝固5,6の制服の限られた深さを生成します。凝固効果に影響する要因は活性化凝固装置とプローブから組織までの距離の電源設定の時間します。ヘリウムは別のキャリア ガスをプラズマ凝固7に使用することができます。最近の臨床研究に集中して成績ではなく、機能的な組織学的所見3,8,9,生体外で調査10 に焦点を当てた実験中または分離臓器11実験。
基になるプロトコルにより、豚の人間の標準的な装置を使用して臨床応用に近い大規模な動物モデルのプラズマ凝固法の効果に関する研究: 微小循環がレーザーのドップラー流量計による非侵襲的評価と分光光度計、この表示12,13の標準的な臨床ツールです。凝固過程での温度変化は、赤外線放射温度計とサーモグラフィ カメラで監視されます。凝固の深さは、組織学的ヘマトキシリン ・ エオシン組織サンプルの採取後のセクションを染色で測定されます。二次止血のための他の手段との比較、バースト圧力実験を行った。血圧を除外する explanted 器官で実施されますが前述のテクニック14とは対照的に関連した効果。プラズマ凝固のローカル影響の調査の説明に加えてブタモデルで標準的な血液検査を実施することもできます。

<table border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Xylazine 20 mg/mL</td>
			<td style="width:184px;">Vetoquinol GmbH</td>
			<td style="width:192px;">Xylapan</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Ketamine 100 mg/mL</td>
			<td style="width:184px;">Ceva GmbH</td>
			<td style="width:192px;">Ceva Ketamine Injection</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Atropine 100 mg / 10 mL</td>
			<td style="width:184px;">Dr. Franz K&#246;hler Chemie GmbH</td>
			<td style="width:192px;">Atropinsulfat K&#246;hler 100mg Amp.</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Propofol</td>
			<td style="width:184px;">Fresenius Kabi GmbH</td>
			<td style="width:192px;">Propofol 1% MCT Fresenius</td>
			<td style="width:232px;"></td>
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		<tr height="47">
			<td height="47" style="height:47px;">Fentanyl</td>
			<td style="width:184px;">KG Rotexmedica GmbH</td>
			<td style="width:192px;">Fentanyl 0,5mg Rotexmedica</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Isoflurane</td>
			<td style="width:184px;">Abbot GmbH</td>
			<td style="width:192px;">Forene 100% (V/V) 250 mL</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Ringer&#39;s lactate solution</td>
			<td style="width:184px;">Baxter Deutschland GmbH</td>
			<td style="width:192px;"></td>
			<td style="width:232px;">sodium 131mmol/l, potassium 5 mmol/l, calcium 2 mmol/l, cloride 111 mmol/l, lactate 29 mmol/l</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Surgical disinfactant</td>
			<td style="width:184px;">Sch&#252;lke &#38; Mayr GmbH</td>
			<td style="width:192px;">Kodan Tinktur forte gef&#228;rbt 1l 104804</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Motorized microscope</td>
			<td style="width:184px;">Nikon Instruments Europe</td>
			<td style="width:192px;">Eclipse TE2000-E</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Microscope camera</td>
			<td style="width:184px;">Nikon Instruments Europe</td>
			<td style="width:192px;">Digitalsight DS-Qi1Mc</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Imaging software</td>
			<td style="width:184px;">Nikon Instruments Europe</td>
			<td style="width:192px;">NIS elements Vers. 4.40</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Plasma coagulator</td>
			<td style="width:184px;">S&#246;ring GmbH</td>
			<td style="width:192px;">CPC-1000</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Argon gas</td>
			<td style="width:184px;">Linde AG</td>
			<td style="width:192px;">Argon 4.8&#160;</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Helium gas</td>
			<td style="width:184px;">Linde AG</td>
			<td style="width:192px;">Helium 4.8</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">O2C</td>
			<td style="width:184px;">LEA Medizintechnik GmbH</td>
			<td style="width:192px;">O2C Version 1212</td>
			<td style="width:232px;">with LF-2 or LF-3 probe</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Infrared thermometer</td>
			<td style="width:184px;">Voltcraft</td>
			<td>VOLTCRAFT IR 260-8S</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Thermographic camera</td>
			<td>InfraTec GmbH</td>
			<td>VarioCAM HD head 820</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Thermographic analysis sofrtware</td>
			<td>InfraTec GmbH</td>
			<td style="width:192px;">IRBIS 3</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Mayer&#39;s Hematoxylin solution</td>
			<td></td>
			<td style="width:192px;">Merck 1.09249</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Eosin solution</td>
			<td style="width:184px;">VWR International GmbH</td>
			<td style="width:192px;">Merck 1.09844</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Rollerpump Masterflex L/S easy Load</td>
			<td style="width:184px;">Cole-Parmer Instrument Company</td>
			<td style="width:192px;">model 7518-10</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Perfusorpump</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">Perfusor secura FT</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Digital pressure meter</td>
			<td style="width:184px;">Greisinger electronic</td>
			<td style="width:192px;">GMH 3161</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Perfusorsyringe, 50 mL</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">REF 8728810 F</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Perfusor line, Type IV Standard, PVC Luer lock</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">REF 8722960</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">3-Way stopcock, Dicofix C35C</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">REF 16494 C</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Silk 2-0. 3 metric</td>
			<td style="width:184px;">Resorba</td>
			<td style="width:192px;">REF H5F</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Vicryl 4-0 Sutupak</td>
			<td style="width:184px;">Ethicon</td>
			<td>V1224H</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">NaCl 0.9 %</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;"></td>
			<td style="width:232px;"></td>
		</tr>
	</tbody>
</table>

assessment of plasma coagulation on liver tissue in a large animal model in vivo

プラズマ凝固焼灼の一形態としては、後の段階で出血を防ぐために主要な肝切除後大きい肝カット表面をシールする何十年も肝臓手術で使用されます。プラズマ凝固肝組織の厳密な効果は単に不十分検討しました。ブタのモデルでは、臨床応用に近い凝固効果を調べることができます。組み合わせたレーザー ドップラー流量計、分光光度計ドキュメント微小循環は非侵襲的、組織深さ 8 mm で凝固時変更臨床印象を超えて止血について定量的な情報を提供します。凝固過程で評価サーモグラフィ カメラと赤外線温度計前およびポスト凝固凝固サイトで温度、ガス ビーム温度の測定デバイスの上限のしきい値のために可能ではないです。凝固の深さは、ヘマトキシリン ・ エオシン オブジェクト マイクロメータの校正後染色標本の顕微鏡は測定し、電源設定凝固深度の関係について正確な情報を与えます。シール効果を胆管に調査する大きな血管をシールするプラズマ凝固装置のため不可能です。バースト圧力実験を血圧を除外する explanted 臓器に関連した効果。

微小循環:次のプラズマ凝固止血診断装置を利用したは、微小循環の変化によって発揮できます。142.7 ± 76.08 のベースライン値から毛細血管血流 (任意の単位 (AU) として表示) が減少 57.78 ± 49.57 AU 25 W デバイス、出力 48.5 ± 7.26 で AU AU 5.04 ± 1.31 と 50 W で 100 W (図 4) で AU。
温度:...

Watch this Scientific Journal Video about Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo at JoVE.com

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

実験的肝組織体内のプラズマ凝固法を評価するためにプロトコルをご紹介します。ブタのモデル レーザードップラー微小循環を検討、組織凝固深度を測定、凝固サイト赤外線放射温度計とサーモグラフィ カメラとダクトのシール効果で温度は圧力によって文書化されます。実験。

プラズマ凝固体内大動物モデルの肝臓組織の評価

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

assessment-plasma-coagulation-on-liver-tissue-large-animal-model

Research

JoVE Journal

Medicine

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

8.8K ビュー.  University of Bonn. 実験的肝組織体内のプラズマ凝固法を評価するためにプロトコルをご紹介します。ブタのモデル レーザードップラー微小循環を検討、組織凝固深度を測定、凝固サイト赤外線放射温度計とサーモグラフィ カメラとダクトのシール効果で温度は圧力によって文書化されます。実験。

ビデオ: Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

大型動物モデルの絶縁型ワーキングハートシステム

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

医学

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

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

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

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

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

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

大規模な疫学研究における児童身体計測の評価

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

豚脛骨移植外科的血管新生: 新しい大型動物の骨血管複合移植モデル

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

ARDS の大動物モデルで大動脈と肺動脈の血行動態の侵襲血行動態モニタリング

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Ex Vivo常温肝灌流の小さな動物モデル

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

塩化第二鉄によるイヌ総頚動脈血栓症: 血管損傷の大動物モデル

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

冠動脈血管新生のインビトロモデル

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

移植質評価のための化学バイオプシーの使用

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...