This work is supported by the SFB 824 subprojects C6 and C7 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany.

注：次の手順は、我々の施設やバイエルン、ドイツの地方政府のガイドラインに沿って行われてきました。地元の動物のケアと使用委員会によって決定されるように、このプロトコルを再現するすべての試みは、動物の人道的な扱いのために地元のガイドラインに遵守で行う必要があります。例えば、多くの機関は、滅菌手袋が齧歯類手術中に着用されている必要があります。また、ウイルスの仕事は、個人と環境の安全のために注意しながら、地域の規則に従って実行されなければなりません。ケアが適切に汚染された廃棄物を処分すると（オートクレーブ処理し、製造元の指示に従って適切な抗ウイルス消毒剤で洗浄することにより、すなわち ）楽器やワークスペースをきれいにするために注意しなければなりません。 手術を開始する前に1.準備<ol><li> （オートクレーブ処理によって、すなわち ）手術器具を滅菌します。 </li><li> approprにウイルスを希釈使用されている特定のウイルスの毒性およびラット株の感受性に応じて変化するであろうiate濃度、。 注：雄バッファローラットにおける水疱性口内炎ウイルスを使用する場合、例えば、我々は、典型的には、このモデルにおける最大許容用量は1 mlの容積中に10 7 PFUを注入します。 <ol><li> 1ミリリットル注射器に（無菌法を使用して）ウイルスを事前入力し、注射器や針から空気を排除するために注意しながら、30 Gの針を取り付けます。 </li></ol></li><li>必要なすべての材料および機器と手術用のスペースを用意し、消毒するためにエタノールでエリアをスプレー。ガーゼ綿棒や綿棒アプリケーターを加湿するための無菌食塩水（0.9％NaCl）で小さな容器を埋めます。 </li></ol>ラットの作製<ol><li> （地元の動物のケアと使用委員会によって指定されるように）適切な用量30分の前に外科手術を開始するには、鎮痛剤を投与します。例えば、メタミゾール（100-200ミリグラム/キログラム）の高用量を投与します。 </li><li>吸入（イソフルラン）または注射（ すなわち、また、MMFとして知られているメデトミジンの混合物（0.15ミリグラム/キログラム）、ミダゾラム（2ミリグラム/キログラム）、およびフェンタニル（0.005ミリグラム/キログラム）、）麻酔を用いてラットを麻酔。百分の1から3で気化器を通してisolfluraneを管理し、正常な呼吸と心拍数を維持するために、必要に応じて調整。 <ol><li>先に進む前に、ラットは完全に鎮静させ、フットパッドをつまんし、反応のために観察することによって痛みを感じることができないされていることを確認してください。 </li><li>麻酔中の乾燥を防ぐために獣医の眼軟膏を適用します。 </li></ol></li><li>ほんの少しの獣医のバリカンを使用して、性器の上にダウンに胸骨から腹部を剃ります。肌を傷つけることなく、深剃りを実現するために注意してください。 </li><li>前足と後肢にテープを使用して手術台にラットを固定し、かつ、吸入麻酔を使用している場合は、適切なサイズノーズコーンに鼻を置きます。加熱パッドを配置ラットの下に体温を維持します。防腐塗料のアルコールリンスやアプリケーションに続いて、外科的グレードの消毒剤を使用して腹部を、消毒。 </li></ol> 3.開腹<ol><li>このような＃15のように、小さな湾曲したメスを用いて、直接正中線に沿って表皮層に小さな縦切開を行います。剣状突起までの切開部を拡張します。次に、静かにメス切開を行い、その後、手術用ハサミで剣状突起までの切開部を拡張するためにAdsonのピンセットで持ち上げて筋層を切開。腹腔の臓器を穿刺するか、剣状突起の両側に小さな血管を傷つけないように注意してください。 </li><li>筋肉と皮膚を広げ、全体の腹部を露出させるために腹部全体に開創器を置きます。 </li><li>滅菌生理食塩水で2 7.5×7.5 cm 2のガーゼ綿棒を湿らせ、手術の上部に1を広げます図2（a）に示すように 、開いて、一番下の1。 </li></ol>肝動脈の4.絶縁<ol><li>滅菌生理食塩水で湿らせた2湿らせた綿棒綿棒を使用して、静かに腹腔から腸と盲腸を持ち上げ、下のガーゼ綿棒上に置きます。湿った臓器を維持するために、道のそれらを保持するために、ガーゼの上に折ります。 </li><li> 2湿らせた綿棒綿棒を使用して、静かにこの段階でのビューの中で最も優勢な葉になります左側肝葉を、持ち上げます。ローブが完全に上向きに反転し、以前に配置されたガーゼ綿棒の上に載ることができるように、葉の下側に繊維質膜を通ってカット春のはさみの小さなペアを使用して、上向きに葉を裏返し、と。所定の位置に保持するために、葉の上にガーゼ綿棒を折ります。 </li><li>今ビューの中で最も前葉である、次のローブ（前方尾状）に進みます。 THと解剖顕微鏡で微細な鉗子の電子援助は、それを解放し、それが自由に上向きに反転されることを可能にするために、葉の周囲の薄い膜を分析します。左側葉と一緒にガーゼ綿棒の下葉を置きます。 </li><li>今表示されます後部尾状葉を、確認します。完全に後方尾状葉を分析し、以前に配置された肝葉と一緒に上向きにし、ガーゼの内側にそれを反転するための手順を繰り返し4.3。 </li><li>ただ、尾状葉の元の位置の下に、今目に見える、簡単にその強い脈動によって認識される、総肝動脈を見つけます。解剖学的右の動脈に続いて、視界から胃十二指腸と固有肝動脈を曖昧に厚い膜状物質を観察します。細かい鉗子を使用して、この材料を持ち上げ、ゆっくりと、この領域内の任意の小血管を避けるように注意しながら、それを引き裂きます。発生する可能性の出血を止めるために綿棒綿棒を使用してください。 <ol><li> Aの位置を確認します膜が適切に解剖されたときに解剖顕微鏡の助けを借りてはっきりと見えるようになりますrterial分岐します。 </li></ol></li><li> 2罰金、非外傷性鉗子を使用して、慎重に総肝動脈、胃十二指腸動脈、彼らは周囲の膜（ 図2B）から解放されるように、適切な肝動脈を解剖。 </li></ol>注射用動脈の5準備<ol><li>胃十二指腸動脈の下に7-0プロレン縫合糸を渡し、そしてちょうど分岐（適切な配置については、 図1を参照）の上に、先端部に永久結紮糸を結ぶために、マイクロニードルホルダーを使用します。両端に残った縫合材料の約2インチを許可し、 図2Cのように、教えられた動脈を引っ張って針ホルダでそれらをクランプします。 </li><li>一時的に血流（ 図2C）をブロックするための一般的な肝動脈に小さなクランプを置きます。 注：これはインプです針が動脈から除去されると大量の出血を防止するためortant。また、動脈血流の遮断が遅いウイルス投与と優れた形質導入効率を可能にします。 <ol><li>重要： - 一般的な肝動脈をクランプの5分以内に、動脈の注射およびその後の連結を行う血流が停止している時間の量を制限するために迅速に作業。 </li></ol></li><li>可能な場合は、胃十二指腸動脈が鮮明であり、正確に30 G針を挿入するのに十分な大き見えるように、解剖顕微鏡の倍率を上げます。胃十二指腸動脈の内腔に注射器の針を置き、ゆっくりプランジャーを押します。 注：針の適切な配置は、適切な肝動脈を通って肝臓に向かって直接流れる注入した溶液になります。 <ol><li>形質導入効率を最大にするために非常にゆっくりと注入を行います。 </li></ol></li> &lt;リー&gt;は全注入量が投与された後、ゆっくりと動脈から針を後退させます。 注：注射部位からの出血があってはなりません。出血が発生した場合には、クランプが適切に配置されていないとすぐに調整されるべきであることを示しています。 </li></ol> 6.閉会<ol><li>胃十二指腸動脈の下に第7-0プロレン縫合糸を渡し、および注射部位の上に第二の永久結紮糸を結びます。 </li><li>総肝動脈からクランプを取り外します。肝臓に向けた適切な肝動脈を通る血流が回復されたことを確認します。 <ol><li>合字の緩い端をカットします。 </li></ol></li><li>腹腔に腸や肝臓を返す前に出血がないことを確認してください。付録には、元の位置に戻されるように注意してください。 </li><li>湾曲した針と4-0ビクリル縫合材料を使用して、別々の層に筋肉と皮膚を縫合。プレイスcontinuousの縫合糸は、約1ミリメートル離れて、しっかりと引き出します。 <ol><li>縫合フェーズでは、そのようなブプレノルフィン（0.05ミリグラム/キログラム）のように鎮痛剤を投与します。 </li></ol></li><li>麻酔からラットを削除します。イソフルランが使用されている場合は、ラットは、数分以内に意識を取り戻す必要があります。 MMFは、注​​射可能な麻酔として使用されている場合には、麻酔の効果を逆転される、アチパメゾール（0.75ミリグラム/ kg）を、フルマゼニル（0.​​2ミリグラム/ kg）で、およびナロキソン（9.12ミリグラム/ kg）を皮下注射とそれに拮抗そして動物は20分以内に意識を取り戻すことができます。初期の覚醒段階の間に赤外線加温ランプで暖かいラットを保管してください。 <ol><li>それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。 </li><li>完全に回復するまで、他の動物の会社に手術を受けた動物を返さないでください。 </li></ol></li><li>苦痛の病理学的徴候のために、手術後のラットを監視し、フォロー地元の動物のケアと使用委員会で必要とされる鎮痛剤とアップ。例えば、ブプレノルフィンは0.05ミリグラムで投与することができる/ 12時間ごとのkgです。 </li></ol>

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	<li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence-based+health+policy+-+lessons+from+the+Global+Burden+of+Disease+Study.">Evidence-based health policy - lessons from the Global Burden of Disease Study.</a> Science. 274, 740-743 (1996).</li><li>Parkin, D. M., Bray, F., Ferlay, J., Pisani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+world+cancer+burden:+Globocan+2000.">Estimating the world cancer burden: Globocan 2000.</a> Int J Cancer. 94, 153-156 (2001).</li><li>Bruix, J., Sala, M., Llovet, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoembolization+for+hepatocellular+carcinoma.">Chemoembolization for hepatocellular carcinoma.</a> Gastroenterology. 127, 179-188 (2004).</li><li>Miraglia, R., Pietrosi, G., Maruzzelli, L., Petridis, I., Caruso, S., Marrone, G., Mamone, G., Vizzini, G., Luca, A., Gridelli, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+transcatheter+embolization/chemoembolization+(TAE/TACE)+for+the+treatment+of+single+hepatocellular+carcinoma.">Efficacy of transcatheter embolization/chemoembolization (TAE/TACE) for the treatment of single hepatocellular carcinoma.</a> World J Gastroenterol. 13, 2952-2955 (2007).</li><li>Breedis, C., Young, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+supply+of+neoplasms+in+the+liver.">The blood supply of neoplasms in the liver.</a> Am J Pathol. 30, 969-977 (1954).</li><li>Nakashima, T., Kojiro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathologic+characteristics+of+hepatocellular+carcinoma.">Pathologic characteristics of hepatocellular carcinoma.</a> Semin Liver Dis. 6, 259-266 (1986).</li><li>Heo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Randomized+dose-finding+clinical+trial+of+oncolytic+immunotherapeutic+vaccinia+JX-594+in+liver+cancer.">Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer.</a> Nat Med. 19, 329-336 (2013).</li><li>Altomonte, J., Marozin, S., Schmid, R. M., Ebert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+newcastle+disease+virus+as+an+improved+oncolytic+agent+against+hepatocellular+carcinoma.">Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma.</a> Mol Ther. 18, 275-284 (2010).</li><li>Shinozaki, K., Ebert, O., Kournioti, C., Tai, Y. S., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolysis+of+multifocal+hepatocellular+carcinoma+in+the+rat+liver+by+hepatic+artery+infusion+of+vesicular+stomatitis+virus.">Oncolysis of multifocal hepatocellular carcinoma in the rat liver by hepatic artery infusion of vesicular stomatitis virus.</a> Mol Ther. 9, 368-376 (2004).</li><li>Shinozaki, K., Ebert, O., Woo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+advanced+hepatocellular+carcinoma+in+rats+via+repeated+hepatic+arterial+infusions+of+recombinant+VSV.">Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV.</a> Hepatology. 41, 196-203 (2005).</li><li>Altomonte, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+antitumor+effects+of+transarterial+viroembolization+for+multifocal+hepatocellular+carcinoma+in+rats.">Synergistic antitumor effects of transarterial viroembolization for multifocal hepatocellular carcinoma in rats.</a> Hepatology. 48, 1864-1873 (2008).</li></ol>

The authors have nothing to disclose.

Although direct intratumoral injection is undoubtedly the simplest method to result in efficient tumor transduction of a single tumor nodule, hepatic arterial infusion represents an ideal administration route to target multifocal, orthotopic HCC. This method has proven to be both safe and effective for treating HCC in immune competent rats with oncolytic viruses. Furthermore, since HCC patients are routinely treated by transarterial application of chemoembolization, the method described here is readily translatable to ...

肝細胞癌（HCC）は重大な健康問題1,2作り、世界中で5番目に普及している癌、および癌関連死因の第3位です。腫瘍切除の対象とならない患者、または肝移植を待っている人のために、動脈塞栓術（TAE）または肝動脈化学塞栓療法（TACE）を含む局所領域療法は、標準的な緩和ケア3,4として適用されます。周囲の肝臓は門脈5,6からの血液供給の大部分を受け取りながら、これらの治療法は、腫瘍は肝動脈血流によってほぼ独占的に供給され、それによって肝臓でのデュアル血液供給のユニークな機能を活用します。 HCCの確立された治療法の非常に限られた有効性のために、腫瘍溶解性ウイルスは、有望な代替治療薬として浮上しています。 JX-594、最近PEXA-VECの名前を変更し、顆粒球メートルで武装し、チミジンキナーゼ欠損ワクシニアベクターでありますHCC 7のための第II相臨床試験を完了したacrophageコロニー刺激因子（GM-CSF）、。さらに最近では、ヒトインターフェロンβを発現する組換え水疱性口内炎ウイルスベクター（VSV）は、ソラフェニブ難治性HCC（NCT01628640）のための第I相臨床試験に入りました。腫瘍溶解性ウイルスは患者におけるHCCの臨床応用のための承認を得るに近づくように、多焦点疾患を標的とするための効果的な投与経路の必要性は明らかです。全身送達が非効率的な腫瘍伝達にほとんど効果があるが、腫瘍内のアプリケーションは、疾患の進行の影響を受けやすいuninjectable顕微鏡的病変を残して、注入された腫瘍に対する治療の有効性を制限することができます。 私たちは、同所性HCCをターゲットとする局所領域の方法で腫瘍溶解性ウイルス療法を管理するために、ラットにおける肝動脈を単離する方法を確立しました。我々は実証されていること、安全かつeffectiでこの投与経路結果免疫適格ラット8-10で有意な生存の延長で、その結果、多焦点HCC結節の伝達をVEの。ここでは、アクセス解剖、およびラットにおける肝動脈内に注入する方法について説明します。手順のスキームを図1に示されている（以前は9日公開）

<table border="0" cellpadding="0" cellspacing="0" width="597">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">Veterinary clippers</td>
			<td style="width:157px;">Aesculap</td>
			<td style="width:88px;">GT415</td>
			<td style="width:208px;">Small, cordless trimmer ideal for removing fur from surgical area</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Stereomicroscope</td>
			<td style="width:157px;">Zeiss</td>
			<td style="width:88px;">Stemi SV6</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">30G Needles</td>
			<td style="width:157px;">Braun</td>
			<td align="right" style="width:88px;">4656300</td>
			<td style="width:208px;">30G x &#189;&#8221;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">1ml syringes</td>
			<td style="width:157px;">Braun</td>
			<td style="width:88px;">9161406V</td>
			<td style="width:208px;">Tuberculin syringe</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Disposable scalpel</td>
			<td style="width:157px;">Feather</td>
			<td style="width:88px;">2975#15</td>
			<td style="width:208px;">#15 blade&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Standard surgical scissors</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">14001-13&#160;</td>
			<td style="width:208px;">Sharp/blunt, for opening skin and muscle</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Adson forcep</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">1101-12</td>
			<td style="width:208px;">With teeth, for grasping skin and muscle</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">Alm retractor</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">17008-07</td>
			<td style="width:208px;">With blunt teeth, for spreading abdominal cavity open during surgery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Gauze swabs</td>
			<td style="width:157px;">Lohmann &#38; Rauscher</td>
			<td align="right" style="width:88px;">18504</td>
			<td style="width:208px;">7.5 x 7.5 cm, should be autoclaved prior to use</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Cotton-tipped applicator swabs</td>
			<td style="width:157px;">Lohmann &#38; Rauscher</td>
			<td align="right" style="width:88px;">11970</td>
			<td style="width:208px;">Sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Fine-tipped foreceps</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">11063-07</td>
			<td style="width:208px;">0.4mm, angled tip, for dissecting hepatic artery</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Vannas spring scissors</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">91500-09</td>
			<td style="width:208px;">For delicate cutting</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Micro-needle holder</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">12076-12</td>
			<td style="width:208px;">For ligating gastroduodenal artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">Needle holder</td>
			<td style="width:157px;">Fine Science Tools</td>
			<td style="width:88px;">12005-15</td>
			<td style="width:208px;">Tungsten carbide jaws</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:144px;">7-0 Prolene sutures</td>
			<td style="width:157px;">Ethicon</td>
			<td style="width:88px;">8648H</td>
			<td style="width:208px;">Polypropylene suture with curved needle, for ligating gastroduodenal artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:144px;">4-0 Vicryl sutures</td>
			<td style="width:157px;">Ethicon</td>
			<td style="width:88px;">V3040H</td>
			<td style="width:208px;">With curved needle attached</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:144px;">Infrared warming lamp</td>
			<td style="width:157px;">Beurer</td>
			<td style="width:88px;">IL11</td>
			<td style="width:208px;">For maintaining body temperature post-operatively</td>
		</tr>
	</tbody>
</table>

transarterial administration of oncolytic viruses for locoregional therapy of orthotopic hcc in rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

経験と、（動脈が正常に解剖し、注入し、動物は手術を生き延びたことを意味する）はほぼ100％の成功率を達成することができます。ただし、（ など腫瘍負荷の程度、基礎となる肝機能、）手術前にラットの健康状態は明らかに外科的介入の結果に役割を果たします。手術後、ラットは緩衝液のみても、一過性の体重減少及び肝酵素のわずかな、一時的な上昇...

Watch this Scientific Journal Video about Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats at JoVE.com

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

ラットにおける同所性肝細胞癌の局所領域治療のための腫瘍溶解性ウイルスのTransarterial管理

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

transarterial-administration-oncolytic-viruses-for-locoregional

Research

JoVE Journal

Medicine

9.4K ビュー.  Klinikum rechts der Isar der TU München. Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

ビデオ: Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

腫瘍溶解性ウイルスと生きている組織のex vivoでの感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

がんの生産と活性酸素種の検出（ROS）

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

ラット同所性小腸移植

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

ラット同所性肝移植

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

骨肉腫細胞における光線力学療法の細胞毒性有効性<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

腫瘍溶解性ウイルスの前臨床評価のための研究のコットンラットの取扱い

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe)

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of outcome measures of upper extremity motor performance has grown. However, clinicians and researchers lack practical, quantitative measures of the hand&#8217;s natural role as a receptor of the environment. The Hand Active Sensation Test (HASTe), developed by Williams and colleagues in 2006, is a valid and reliable measure of haptic performance. Though not available commercially, the HASTe can be fabricated from inexpensive materials, and it has been used successfully to identify impairments in haptic touch in individuals with stroke. (Williams, 2006). This paper presents the methods of design and fabrication of the HASTe testing kit, as well as a visual screen to be used during administration, and instructions for the tests administration and scoring.

Design, Fabrication, and Administration of the Hand Active Sensation Test HASTe

The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe.

ハンドアクティブ感覚テスト（ヘイスト）の設計、製造、および管理

The concept of personalizing neurologic rehabilitation, based on individual impairments, has experienced a recent surge. In parallel, the number of ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

加速度計を使用して日常生活に上肢のパフォーマンスを定量化する方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...