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>

<ol><li>Murray, C. J., Lopez, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence-based+health+policy+-+lessons+from+the+Global+Burden+of+Disease+Study%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Estimating+the+world+cancer+burden%3A+Globocan+2000%5BTitle%5D)+AND+%22Int+J+Cancer%22%5BJournal%5D)">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/pubmed?term=((Chemoembolization+for+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">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/pubmed?term=((Efficacy+of+transcatheter+embolization%2Fchemoembolization+%28TAE%2FTACE%29+for+the+treatment+of+single+hepatocellular+carcinoma%5BTitle%5D)+AND+%22World+J+Gastroenterol%22%5BJournal%5D)">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/pubmed?term=((The+blood+supply+of+neoplasms+in+the+liver%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">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/pubmed?term=((Pathologic+characteristics+of+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Semin+Liver+Dis%22%5BJournal%5D)">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/pubmed?term=((Randomized+dose-finding+clinical+trial+of+oncolytic+immunotherapeutic+vaccinia+JX-594+in+liver+cancer%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer.</a> Nat Med. 19, 329-336 (2013).</li>
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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><tbody><tr><td>Veterinary clippers</td><td>Aesculap</td><td>GT415</td><td>Small, cordless trimmer ideal for removing fur from surgical area</td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi SV6</td><td></td></tr><tr><td>30 G Needles</td><td>Braun</td><td>4656300</td><td>30 G x &amp;frac12;&amp;rdquo;</td></tr><tr><td>1 ml syringes</td><td>Braun</td><td>9161406V</td><td>Tuberculin syringe</td></tr><tr><td>Disposable scalpel</td><td>Feather</td><td>2975#15</td><td>#15 blade&amp;nbsp;</td></tr><tr><td>Standard surgical scissors</td><td>Fine Science Tools</td><td>14001-13&amp;nbsp;</td><td>Sharp/blunt, for opening skin and muscle</td></tr><tr><td>Adson forcep</td><td>Fine Science Tools</td><td>1101-12</td><td>With teeth, for grasping skin and muscle</td></tr><tr><td>Alm retractor</td><td>Fine Science Tools</td><td>17008-07</td><td>With blunt teeth, for spreading abdominal cavity open during surgery</td></tr><tr><td>Gauze swabs</td><td>Lohmann &amp;amp; Rauscher</td><td>18504</td><td>7.5 cm x 7.5 cm, should be autoclaved prior to use</td></tr><tr><td>Cotton-tipped applicator swabs</td><td>Lohmann &amp;amp; Rauscher</td><td>11970</td><td>Sterile</td></tr><tr><td>Fine-tipped foreceps</td><td>Fine Science Tools</td><td>11063-07</td><td>0.4 mm, angled tip, for dissecting hepatic artery</td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>For delicate cutting</td></tr><tr><td>Micro-needle holder</td><td>Fine Science Tools</td><td>12076-12</td><td>For ligating gastroduodenal artery</td></tr><tr><td>Needle holder</td><td>Fine Science Tools</td><td>12005-15</td><td>Tungsten carbide jaws</td></tr><tr><td>7-0 Prolene sutures</td><td>Ethicon</td><td>8648H</td><td>Polypropylene suture with curved needle, for ligating gastroduodenal artery</td></tr><tr><td>4-0 Vicryl sutures</td><td>Ethicon</td><td>V3040H</td><td>With curved needle attached</td></tr><tr><td>Infrared warming lamp</td><td>Beurer</td><td>IL11</td><td>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?...

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 ...

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9.5K 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

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors 

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

高效率转导肝癌细胞的重组腺相关病毒血清型3载体

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

科研

医学

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 ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

部分原位肝移植大鼠模型的肝动脉重建的外科手术

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

门静脉注射结直肠癌类器官研究肝转移基质

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

癌症研究

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

在急性肝衰竭异位辅助性大鼠肝移植流量调节门静脉动脉

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

颅内插管植入小鼠连续局部区域嵌合抗原受体（CAR）T细胞输注

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

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 ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

静脉大鼠部分肝移植的体内血管定向70% 肝切除术

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

应用新型磁吻合技术 Suprahepatic 腔静脉重建大鼠原位肝移植模型的研究

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

矫形肝移植大鼠模型中动脉重联后并发症减少

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...