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

注:已经按照我们机构的指导方针和德国巴伐利亚州的当地政府进行下面的步骤。所有尝试重现此协议必须在坚持进行与动物的人道待遇当地的指引，由当地动物护理和使用委员会的决定。例如，许多研究机构需要无菌手套被啮齿类手术期间磨损。此外，病毒工作必须按照当地的规定进行，注意个人和环境的安全。必须小心，以适当处理的被污染的废物和清洁工具和工作区（ 即，通过高压灭菌，并根据制造商的说明用适当的抗病毒消毒剂清洗）。 1.准备开始手术前<ol><li>消毒手术器械（即通过高压灭菌）。 </li><li>稀释病毒的appropr莱特浓度，这将取决于所使用的特定病毒的毒性和大鼠菌株的敏感性而变化。 注意:例如，使用在雄性布法罗大鼠水泡性口炎病毒时，我们通常注入在1毫升体积，这是在该模型中的最大耐受剂量10 7 pfu的。 <ol><li>预填病毒（使用无菌技术）的1ml注射器，并附加地下30针，注意从注射器和针头消除空​​气。 </li></ol></li><li>制备具有所需的所有材料和设备的外科空间，并用乙醇消毒喷雾的区域。填写一个小插座用无菌生理盐水（0.9％氯化钠）为湿润的纱布拭子和棉签涂抹。 </li></ol> 2.鼠的制备<ol><li>施用镇痛（如由当地动物护理和使用委员会指定），在适当的剂量30分钟开始的外科手术之前。例如，管理安乃近高剂量（100-200毫克/千克）。 </li><li>麻醉用吸入（异氟烷）或注射（ 即，美托咪定（0.15毫克/千克的混合物中），咪达唑仑（2毫克/千克），和芬太尼（0.005毫克/千克），也被称为MMF）麻醉大鼠。通过蒸发器在1-3％的管理isolflurane并根据需要调整，以维持正常的呼吸和心脏率。 <ol><li>在开始之前，确保大鼠完全镇静，无法按捏脚垫和观察的反应感到疼痛。 </li><li>应用兽医眼膏，以防止在麻醉过程中干燥。 </li></ol></li><li>从胸骨刮胡子腹部下降到只有用小兽推剪生殖器以上。请注意，以达到死里逃生不损伤皮肤。 </li><li>固定大鼠使用上fore-和后肢胶带手术床上，而且，如果采用吸入麻醉，将鼻子成适宜尺寸的鼻锥体。放置一个加热垫大鼠下方，以保持体温。消毒用外科手术级消毒剂腹部区域，接着是防腐涂料的醇冲洗和应用。 </li></ol> 3.剖腹<ol><li>用小弯手术刀，如＃15，请直接沿中线皮肤层的小纵切口。延长切口到xyphoid过程。接着，用一对斜角肌挤压钳使手术刀切开轻轻抬起切开肌肉层，然后延长切口到手术剪刀xyphoid过程。小心不要刺破腹腔的器官或损坏的xyphoid过程两侧的小血管。 </li><li>将卷收器穿过腹部传播肌肉和皮肤和揭露整个腹部区域。 </li><li>滋润2 7.5×7.5 cm 2的纱布拭子用无菌生理盐水和分布在手术的最上面的一个开口和一个在底部，如在图2A所示。 </li></ol> 4.隔离肝动脉<ol><li>使用用无菌生理盐水浸湿2湿棉签拭子，轻轻提起肠子和盲肠出腹腔，并将它们放置在较低的纱布拭。折叠的纱布上，以保持湿润的器官，并将其持有的出路。 </li><li>使用两个浸湿棉签拭子，轻轻抬起左外侧肝叶，这将是鉴于在这个阶段最主要的耳垂。翻转叶向上，而且，使用一个小的一对弹簧剪刀，通过纤维膜的波瓣的侧切割，允许叶向上被完全翻转并停留在先前放置纱布拭子的顶部。折叠的纱布拭过耳垂举行到位。 </li><li>与下一个叶（前尾状），这是目前在视图中最前叶继续。随着日在解剖显微镜和细镊子电子辅助，以释放它并允许它向上可自由翻转解剖薄膜周围的瓣。与左外叶放置纱布拭下的叶在一起。 </li><li>观察后尾叶，现在将是可见的。重复步骤4.3完全剖析后尾状叶和翻转它与先前放置肝叶向上纱布里面在一起。 </li><li>只是尾状叶的原始位置下方，发现肝总动脉，它现在将可见并通过其强脉动容易识别。继动脉解剖权，观察厚膜材料遮挡从视图中的胃和适当的肝动脉。用细镊子，提起这种材料，轻轻撕开，注意避免这方面的任何小血管。用棉签拭子停止任何可能发生的出血。 <ol><li>找到一个rterial分支，这将是清晰可见用解剖显微镜的帮助下，当膜被正确解剖。 </li></ol></li><li>使用两个细，无损伤钳子，小心解剖肝总动脉，胃十二指肠动脉，以及适当的肝动脉，使得它们从周围的膜（ 图2B）被释放。 </li></ol> 5.制备动脉注射液<ol><li>通过胃十二指肠动脉下方7-0普理灵缝合，并使用微针器在远端绑一个永久的结扎，只是分叉（参见图1为妥善安置）以上。允许残留在两端的缝合材料的大约2英寸，并用针固定器拉教导的动脉，如在图2C夹紧它们。 </li><li>放在肝总动脉暂时阻止血液流动（ 图2C）一个小夹子。 注:这是小鬼ortant到一旦针头从动脉除去防止大量出血。此外，动脉血流的堵塞将允许更慢病毒施用和更好的转导效率。 <ol><li>重要的是:迅速地工作来限制血流停止的时间量 - 5分钟夹紧共同肝动脉内执行动脉的注入和后续的结扎。 </li></ol></li><li>如果可能的话，增加对解剖显微镜的放大倍数，使得胃十二指肠动脉处于锐聚焦，并出现大到足以精确地插入地下30针。放置注射器的针头插入胃十二指肠动脉的内腔和对柱塞慢慢推。 注意:在针的正确位置将导致直接通过适当的肝动脉和朝向肝中流动的注射溶液。 <ol><li>进行注射非常缓慢最大限度地传导效率。 </li></ol></li> &lt;利&gt;一旦整个注射体积已经施用，慢慢缩回从动脉针头。 注意:不应该有从注射部位出血。在该出血确实发生的情况下，它表明该夹具没有正确放置并应快速调整。 </li></ol> 6.闭幕<ol><li>通过第二个7-0普理灵缝合胃十二指肠动脉下方，领带注射部位上方的第二永久结​​扎。 </li><li>除去从肝总动脉的夹子。确认通过对肝脏的肝固有动脉血流已恢复。 <ol><li>切连字有始有终。 </li></ol></li><li>确认有返回肠和肝脏腹腔前无出血。以阑尾在其原来的方向返回的照顾。 </li><li>缝合使用4-0薇乔缝合材料用弯针独立层肌肉和皮肤。地方continuou小号缝合1mm左右分开，紧紧地拉。 <ol><li>在缝合阶段，施用的镇痛如丁丙诺啡（0.05毫克/千克）。 </li></ol></li><li>从麻醉中移除老鼠。如果异氟烷已被使用，大鼠应恢复在几分钟内意识。如果MMF已被用作可注射麻醉，用皮下注射阿替美唑（0.75毫克/千克），氟马西尼（0.2毫克/千克），和纳洛酮（9.12毫克/千克）的拮抗它，这将逆转麻醉的效果并让动物在20分钟内恢复意识。保持温暖的大鼠与红外暖灯在初始阶段觉醒。 <ol><li>无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。 </li><li>不要返回已动过手术，以公司的其他动物，直到完全康复的动物。 </li></ol></li><li>监测大鼠术后遇险的病理体征，和后续与止痛药的要求当地动物护理和使用委员会。例如，丁丙诺啡可以在0.05毫克施用/ kg，每12小时。 </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 a clinical setting.
An obvious disadvantage of hepatic arterial delivery of tumor therapies in preclinical rodent models is the invasiveness of the procedure and the time required, both for the surgical intervention and the post-operative care of the rats. Researchers wishing to establish this technique must invest a substantial amount of time in becoming proficient at the procedure to produce reproducible results and a high survival rate. Furthermore, due to the extremely small diameter of the hepatic artery, to date there have been no reports of hepatic arterial infusions in mice. This is unfortunate, since the genetic models of HCC are primarily generated in mice, and we are therefore limited to testing hepatic arterially applied therapies in implantation or chemically induced HCC models in rats. Chemical induction of HCC is also associated with general liver toxicity, which would potentially preclude the rats from eligibility for receiving this therapeutic intervention due to safety concerns.
As an alternative to the invasive laparotomy approach to hepatic arterial virus delivery, it is theoretically feasible to feed a catheter through the femoral artery and up through the hepatic aorta and into the hepatic artery in rats, which is the standard procedure in patients. Although this approach would provide the huge benefit of avoiding surgical intervention, it is also technically challenging to establish and would require the aid of an experienced interventional radiologist.
Finally, it is noteworthy to mention that the procedure described here, although established for the purpose of efficient delivery of oncolytic viruses to orthotopic HCC, can be applied to any therapeutic agent for locoregional delivery to hepatic lesions, such as chemotherapy, immune-modulatory drugs, or small molecules. This method allows for a sophisticated pre-clinical model for testing novel therapies for clinical translation.

肝细胞癌（HCC）是世界上第五最常见的癌症，和与癌症有关的死亡的第三大原因，使其成为一个显著健康关注1,2。对于谁是病人没有资格肿瘤切除术，或那些等待肝移植，局部治疗包括经动脉栓塞（TAE）或肝动脉化疗栓塞（TACE）应用于标准姑息治疗3,4。这些疗法利用在肝脏肿瘤即是由肝动脉血流几乎完全喂双重血供的独特功能，而周围的肝脏受到多数来自门静脉5,6其供血。 由于肝癌的治疗确立的极为有限的功效，溶瘤病毒已成为有前途的替代疗法。 JX-594，最近改名为Pexa-VEC，是胸苷激酶缺失牛痘病毒，具有粒米武装acrophage细胞集落刺激因子（GM-CSF），它已经完成了肝癌7 II期临床试验。最近，表达人干扰素-β的重组水泡性口炎病毒载体（VSV）已进入I期临床试验为索拉非尼耐火肝癌（NCT01628640）。作为溶瘤病毒靠拢经批准用于患者的临床应用为肝细胞癌，需要一个有效的给药途径对目标多焦点疾病是显而易见的。而全身性递送是很大程度上是无效的，由于低效肿瘤转导，瘤内的应用程序可能会限制疗法所注入的肿瘤的功效，留下uninjectable微观病变易患疾病的进展。 我们已经建立了分离大鼠肝动脉施用溶瘤病毒疗法在一个局部的方式定位到原位HCC的方法。我们已经证明，这种给药途径导致安全和effecti已经多焦点肝癌结节转导，导致免疫活性大鼠8-10显著存活延长。这里，我们描述访问，解剖，并注入大鼠肝动脉的方法。该过程的图解示于图1（先前公布9）

<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.

有经验的，一个近100％的成功率（意味着动脉被成功解剖并注射，和动物存活手术）就可以实现。然而，在手术前（肿瘤负荷程度，潜在肝功能等 ）的大鼠的健康状况将很显然，在外科干预的结果发挥作用。手术后，大鼠可以预期经历短暂的体重减轻和肝酶的轻微，短暂升高，即使单独的缓冲液，无病毒，注射。 我们先前已经证明了溶瘤性水泡性口炎病毒（VSV）或者在有效的肿瘤转导和肿瘤特异性病毒的复制，在原位，多灶性肝癌模型-8,9-新城疫病毒（NDV）的结果的反式肝动脉给药。虽然这两种病毒在其肿瘤内动力学不同病毒复制的，在最大的病毒的复制，病毒繁殖的众多灶，就证明了LacZ报告基因阳性酶染色的各时间点，可专门内肝​​癌结节观察到的，而周围的肝脏没有表现出病毒的证据复制（ 图3和4中 ，此前公布的数据8,9）。此外，随机选择的VSV-处理的肿瘤结节的形态测定分析表明，肿瘤转导效率不与肿瘤的大小（ 图3）相关。 此外，通过肝动脉递送治疗剂的能力，还提供了治疗与栓塞剂作为替代TACE结合的可能性。我们已经证明，通过在一个1混合溶瘤VSV:1的比例用淀粉降解的微球（DSM）栓塞剂和注入该悬浮液THRough肝动脉，我们可以实现大规模的大鼠肿瘤坏死非常显著的生存期延长轴承多发性结节肝癌11。在DSM的反式肝动脉给药导致肿瘤结节的几乎完全栓塞，而对正常肝组织的灌注的影响最小，如通过动态对比增强磁共振成像（DCE-MRI）使用钆造影证实剂（ 图5，此前公布的数据11）。 <img alt="图1" src="/files/ftp_upload/53757/53757fig1.jpg" /> 图1.肝动脉输注过程的示意图。肝血管（肝动脉，肝固有动脉，和胃十二指肠动脉）解剖用解剖显微镜的帮助。胃十二指肠动脉结扎和肝总动脉的时间块之后，1ml的PBS或VSV载体是通过适当的肝动脉经由胃十二指肠动脉给药。注射后，胃十二指肠动脉的近端部位结扎，以防止出血，肝总动脉的块被释放，肝血流量的recommencement被确认。这个数字，已经出版9， <a href="https://www.jove.com/files/ftp_upload/53757/53757fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53757/53757fig2.jpg" /> 图2.肝动脉灌注过程的照片，一个剖腹鼠演示腹壁牵开器和纱布拭子（A）的位置。起重出肝叶，肝总动脉（指定CHA）和胃十二指肠动脉（指定GA）被识别和DIS后剪刀剪下（B）。结扎胃十二指肠动脉的远端后，将肝总动脉被暂时夹持（℃）。胃十二指肠动脉现在可以进行注射。 <a href="https://www.jove.com/files/ftp_upload/53757/53757fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53757/53757fig3.jpg" /> 图3.转导和肝动脉给药后VSV的肿瘤选择性复制大鼠轴承多灶性肝癌。动物套装（N = 3 /时间点）在后第1天，病毒在30分钟和（B）牺牲（A）输液，并冷冻肝切片进行β-gal的表达。代表性的部分被以低（左）和更高的放大倍数（右）示出。箭头指示肿瘤病灶和HEPA之间的边界抽动实质。肿瘤大小与转染效率之间的（C）的关系。 HCC-含有肝切片进行形态测定分析进行分析以定量总肿瘤和X-gal的阳性区域。这个数字，已经出版9， <a href="https://www.jove.com/files/ftp_upload/53757/53757fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53757/53757fig4.jpg" /> 图4. NDV / F3aa（L289A）处理导致肿瘤特异性的病毒复制在肝细胞癌（HCC）-bearing大鼠雄性布法罗大鼠轴承灶性原位HCC结节用rNDV / F3aa（L289A）（10 8 TCID 50）处理肝动脉灌注，并在指定的时间点杀后处理（N = 3）。肝脏切片含肿瘤受到对β-gal染色。代表性的部分是在5X（概述）和20X（肿瘤和肝段的放大倍数）的放大倍率所示。病毒滴度通过肝和肿瘤裂解物的TCID 50分析进行定量，并表示为平均值±SD。 NDV，新城疫病毒; TCID 50，50％组织培养感染剂量。请注意，这个数字已经从原来的形式8修改。这个数字，已经出版8。 <a href="https://www.jove.com/files/ftp_upload/53757/53757fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53757/53757fig5.jpg" /> 栓塞肝癌。MCA-RH7777荷瘤鼠，无论是nonembolized或肝动脉栓塞后，帝斯曼30分钟， 图5.动态对比增强磁共振成像与DCE-MRI进行成像。 （&lt;STRONG&gt; A）轴向T1称重平扫（A，C）及增强（B，D）图像显示缺乏栓塞肿瘤结节（D）的对比。 （B）中的数据进行定量中的肿瘤结节和邻近的正常肝组织的利益大小相等的区域测量的灰阶信号强度进行分析。一位代表数据集显示。这个数字，已经出版11。 <a href="https://www.jove.com/files/ftp_upload/53757/53757fig5large.jpg" target="_blank">请点击此处查看本图的放大版本。</a>

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.

溶瘤病毒的Transarterial管理局大鼠原位肝癌的局部治疗

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.4K 次观看.  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.

视频: 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.

前溶瘤病毒的活组织的体内感染

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

科研

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.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

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.

在癌症的生产和检测活性氧（ROS）的

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.

大鼠原位小肠移植

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.

大鼠原位肝移植

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.

一种新的手术方法气管内注射生物活性剂在胎鼠模型

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.

光动力疗法治疗骨肉瘤细胞的细胞毒功效<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.

处理棉鼠的研究为溶瘤病毒的临床前评估

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.

设计，制造管理的手，和活动感觉测试（急速）

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.

一种使用加速度计量化上肢表现在日常生活的方法

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. ...