Name: laparoscopic technique for serial collection of liver and mesenteric lymph nodes in macaques
Uploaded: 2017-05-02T15:00:00.000+00:00
Duration: 11 min 6 s
Description: Watch this Scientific Journal Video about Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques at JoVE.com

这项工作是由美国国立卫生研究院授权号P51OD010425支持。

<html>动物饲养和协会照顾实验动物护理的评估和认证国际（AAALACi）认可的设施，并根据华盛顿大学的机构动物护理和使用委员会（IACUC）批准方案进行所有动物程序华盛顿国家灵长类动物研究中心。 1.手术准备，麻醉，和镇痛<ol><li>镇静和感应<ol><li>稳重猕猴为10mg / kg氯胺酮和0.015毫克/千克美托咪定（组合在一个注射器）肌肉内注射。之前从笼子去除确保无撤退反射的。 </li><li>插管的动物，并使用异氟烷维持动物在全身麻醉的平面（1.0 - 2.5％）和100％的氧气。参见关于猕猴17插管全身麻醉进一步的参考信息，REF"&gt; 18,19。 </li></ol></li><li>麻醉支持和监控<ol><li>插入外周静脉导管和整个麻醉和手术提供以5毫升/公斤/小时的速率静脉内等渗流体（如乳酸林格溶液）。应用无菌眼科润滑剂，以防止角膜干燥。 </li><li>提供痛觉缺失，如丁丙诺啡的缓释制剂（0.2毫克/千克，皮下）。 注意:一个NSAID（如美洛昔康或酮洛芬）也可以恢复麻醉期间提供，条件是该实验的协议允许NSAID给药和该动物是血压正常和麻醉期间充分水合。 </li><li>监测麻醉深度（ 例如颚音），心脏，呼吸率，EKG，血压，脉搏血氧，呼气末CO 2，和体温整个麻醉和手术。 注意:当腹部被吹气，动物可能减少通气。提供机械通气如果呼吸率降低，或呼气末二氧化碳水平是高于45个毫米汞柱。 </li><li>手术后，管理用于右美托咪逆转阿替美唑（0.15毫克/千克，肌肉注射）。 </li></ol></li><li>手术准备<ol><li>执行多个温水灌肠剂，以减少粪便的容积在结肠中过程20期间，以帮助可视化和肠操作。 </li><li>拥有一批40片用指甲刀从手术野清除头发。从沿海脊耻骨和从左侧到右侧腹刮胡子。 </li><li>在无菌条件下使用合适的防腐剂，例如氯己定交替 - 醇或聚维酮碘的醇制备磨砂手术野。 </li><li>在背侧和右侧卧之间的角度中途在其背部上手术台的动物的位置，。用绳子绑在胳膊和腿伸展位置。提供最终chlorhexi用餐和醇制备一旦动物移动到手术室和定位进行手术。 </li></ol></li></ol> 2.手术注:对于小动物腹腔镜进一步信息，请参见参考文献21。 <ol><li>腹腔镜仪器准备<ol><li>悬垂性用无菌开窗悬垂性动物。 </li><li>随着非无菌助手的帮助下，包裹的数码相机与摄像机无菌悬垂性和电缆连接到塔。附加硬性镜的摄像头。 </li><li>无菌光电缆连接到所述刚性范围和电缆连接到光源。怀特根据制造商的说明平衡相机。 </li><li>装上无菌吹入管的吹入。 </li></ol></li><li>腹腔镜进入<ol><li>使用＃15手术刀，做一个〜5毫米穿刺切口，通过皮肤的腹部肌肉，应用的深度roximately 1 - 2厘米至肚脐的左侧。 </li><li>使用非惯用手，抬起腹壁颅刺切口部位。用优势手，将气腹针的尖端通过切口和角度尖端颅侧。 注:气腹针的穿透角度会根据动物的身体状况。例如，在一个动物脂肪，气腹针就需要几乎垂直于腹壁插入。在薄的动物，气腹针应当颅成角度，以减少内脏接触的风险。 <ol><li>保持气腹针的轴（未在轮毂），牢固地将针穿透腹壁进入腹腔。一个明显的"点击"，并作为尖锐尖端穿透腹膜和钝头气腹针弹簧向前进入腹腔降低电阻将被感觉到。 注意:如果针头适当地传递到日ë腹腔，会有小的阻力，当它是先进的，抽出1 - 2厘米。 </li></ol></li><li>所述CO 2吹入管连接到针和打开活塞。加压腹部不超过10-12毫米汞柱创建气腹。 注意:腹部已取得理想吹气时，它具有对称扩大，有沿海利润率的正常尖锐轮廓的损失。压力应为插管/套管针的插入足够的空间，而不接触内脏的风险。 </li></ol></li><li>套管定位<ol><li>一旦腹部被完全吹入，使〜5 - 用＃15解剖刀刀片7毫米的切口通过左侧颅腹部的皮肤的腹部肌肉〜2 - 4厘米尾到最后肋。 </li><li>放置一个5毫米的套管针套管组件通过皮肤切口和角它〜45 - 60度caudomedially。穿透腹腔至1-2厘米的深度。 SCRE瓦特螺纹套管到腹部通过转动套管（〜0.5 - 再进行1厘米）到1.5的深度 - 3厘米，并取出套管针。 </li><li>拆下气腹针吹入管，关紧旋塞，并取出针。吹入器管路连接到所述套管并且在不超过10打开活塞保持气腹 - 12毫米汞柱。 </li><li>通过套管和进入腹腔插入硬性镜。 </li><li>在摄像头的可视化，继续做一个小切口，通过在用气腹针插入前面的皮肤切口的腹部肌肉和腹膜＃15手术刀刀片。相机用于观察切口的腹部侧，以避免容器和最小化出血。 注:确保穿过肌肉切口和腹膜足够大，以促进所述探针的进入和外化肠系膜（约0.5厘米）的。较大的切口可被要求exteriorize mesentERY动物过度肠系膜脂肪。 </li><li>放置动物在头低脚高位置21与约15升高脚-头30度以上。这是可选的，但允许对结肠的肠系膜淋巴结更容易获得，因为小肠道会移动到一个更颅位置。 </li></ol></li><li>肠系膜淋巴结活检<ol><li>下摄像机可视化，通过在步骤2.3.5所作的腹部切口插入固体探针。 </li><li>放置网膜和肠之间的探针，并使用一个扫描运动轻轻扫网膜脱肠，以允许下面的肠系膜的可视化。继续使用的清扫运动，从尾至颅腹部。网膜可放置在右颅腹所以它是从术野。 </li><li>使用探头向左侧或右侧腹壁移动降结肠。这将支持T的更好的可视化他肠系膜在寻找肠系膜淋巴结，以帮助。 </li><li>使用探头通过肠系膜扫来定位沿着结肠和肠系膜血管肠系膜淋巴结。 注:沿结肠的肠系膜边界运行结肠节点，左结肠节点可以在血管分支点附近发现，和下肠系膜淋巴结可沿肠系膜血管被发现。肠系膜淋巴结是瘦动物容易看到。当埋在肠系膜脂肪他们往往会借给一个略有提高，晶莹的外观上覆肠系膜和可能更多的是更灰棕褐色，然后周围的肠系膜脂肪。此外，肠系膜淋巴结趋向于沿着淋巴管和肠系膜血管所在。 </li><li>一旦一个淋巴结被识别，除去探针，并插入马里兰棘轮镊子进入腹腔。使用镊子把握肠系膜相邻目标节点。朝INCI拉肠系膜锡永，并从套管的范围。 </li><li>关闭吹入，离开套管旋塞阀开来减压腹部，以促进肠系膜的外化。通过切口拉出体外的肠系膜。一旦外置，掌握具有弯曲蚊止血剂或拇指钳子肠系膜，以确保通过切口访问。 </li><li>通过触诊用于更牢固淋巴结和/或灰暗颜色或淋巴结的结节状外观的直接可视化识别形象化肠系膜内的淋巴结（多个）。 <ol><li>一旦被识别，使一个小开口 - 在使用弯曲止血的节点附近的肠系膜（〜3.5毫米）。免费附件使用钝性分离与弯曲止血肠系膜。结扎用4-0必要非吸收性缝合丝血管。用锋利的解剖用手术刀以除去淋巴结。在广场的Roswell Park纪念研究所（RPMI）1640培养基上的节点冰。 </li></ol></li><li>检查出血的形象化肠系膜。如果出血注意，通过，或通过使用烧灼的闭塞与止血剂或结扎线（4-0不可吸收的单纤维缝合线）的船只提供适当的止血。松开肠系膜和吹气腹部回到10 - 12毫米汞柱，使肠系膜返回到腹腔。 </li><li>重复步骤2.4.1至2.4.8以获得附加的淋巴结活检。 </li></ol></li><li>肝活检<ol><li>返回表到水平位置。 </li><li>将一个带螺纹的套管到腹部通过先前用于淋巴结活检集合（切口在步骤2.3.5。制造）的切口。确保切口足够大（约5 - 7毫米），以允许套管放置而不套管针插入。通过这个插管朝向颅腹腔插入刚性范围和可视化的颅腹部和肝脏。 </li><li>下摄像机可视化，插入活检˚F通过套管orceps先前放置在左侧颅腹（切口在步骤2.3.1。制造）。 </li><li>为了获得肝活检，放置朝向肝颚的可动部所选择的肝叶余量下方的活检钳，打开钳子，并允许余量落入打开钳口。确保没有大网膜是肝脏和活检钳之间。调节钳子的位置，并关闭仪器在期望的样品区域。 <ol><li>保持压力为约20 - 30秒，以确保止血。与镊子关闭时，由通过插管轻轻拉动镊子向上取出样品。这会轻轻撕离肝活检。放入RPMI 1640培养基肝活检冰上。 注:肝活检尺寸为约5毫米×2毫米×2用5毫米活检钳毫米。 </li></ol></li><li>检查活检部位使用相机的可视化出血。如果发现出血，放置盐水moisteneð消毒棉签通过插管和施加压力活检部位。另外，包装用无菌凝胶泡沫的活检部位，以帮助止血。 </li><li>重复步骤2.5.3至2.5.5，以获得额外的肝活检。 注意:在此，收集每过程动物3个活组织检查，这足以对所有下游分析。 </li></ol></li><li>手术闭合<ol><li>检查出血的腹腔。 注意:这还没有发生的日期。然而，如果过度出血注意，确定出血的部位，并通过用无菌棉签或使用凝胶泡沫的压力提供适当的止血。 </li><li>关闭注入器并打开插管旋塞。适用于温和的手动压腹部，充分减压腹腔。取出套管。 </li><li>与每个站点1至2简单间断缝合关闭每个腹壁切口。建议4-0可吸收线缝合。 </li><li>通过局部镇痛皮肤闭合之前放置0.1〜0.3立方厘米布比卡因在每个切口进行溅块。 </li><li>关闭皮肤与1 - 每个站点2埋间断缝合（推荐4-0可吸收缝线）。组织胶可以被应用。 </li></ol></li></ol> 3.肝活检处理和淋巴细胞分析<ol><li>存储若干片肝组织（大约0.5mm×0.5毫米×0.5mm的尺寸）在冷冻保存管，在优选的RNA存储溶液，和10％福尔马林未来分子和组织学分析。 <ol><li>放置剩余的肝脏活组织检查中在无菌的250mL塑料杯中用盖子补充有1X青霉素/链霉素，40μg/ mL的可商购的胶原酶溶液中，4微克/毫升DNA酶50毫升的RPMI 1640培养基。剧烈搅拌，使用磁力搅拌棒，在37℃下搅拌板上1个小时。 </li></ol></li><li>均匀地通过倾倒消化的组织分布。 GRIND消化的组织（来自步骤3.1.1）在两个70微米的过滤器装配到2个50毫升锥形管中成使用无菌5毫升的注射器的端部的单细胞悬液。带来两个管的至50mL的体积在补充有10％胎牛血清和1X青霉素/链霉素（R10）1640培养基。 </li><li>离心将细胞在840×g下在4℃下6分钟。倒出上清液。通过从在5mL R10两个管悬浮细胞和组合到一个50毫升锥形管中浓缩细胞成一个单一的50mL锥形管中。带来体积使用R10 50毫升。计数使用血球以确定蜂窝产量细胞。 <ol><li>离心将细胞在840×g下在4℃下6分钟。重悬细胞在1mL R10，均匀地分配到两个5毫升圆底聚苯乙烯试管并且使每个管的体积，以4毫升R10，为&gt; 500,000个细胞每管定位。 </li></ol></li><li>离心将细胞在840×g下在4℃下6分钟。倒出的Supernatant和重悬细胞沉淀于4mL磷酸盐缓冲盐水（PBS）。离心机在840×g下在4℃下6分钟。 </li><li>倒出上清液，并通过温和涡旋重悬在100μLPBS中的细胞。添加一个可以解决的死细胞染色的1.5微升。在室温下孵育5分钟。 </li><li>添加以下未稀释的表面染色的抗体，由生产商推荐的滴度:CD45-PerCP（克隆D058-1283），CD3-PE-CF594（克隆SP34-2），CD20-PE-Cyanin5（克隆2H7），CD4-BV605（克隆OKT4），CD8-BV570（克隆RPA-T8），以及CD14-BV785（克隆M5E2）。孵育在4℃下20分钟。 </li><li>在840×g下添加4毫升PBS中，并离心6分钟，4℃。倒出上清液和重悬细胞于PBS中的1％多聚甲醛的250μL。 注意:多聚甲醛是有毒的。穿戴合适的个人防护装备。 </li><li>在流式细胞仪22参考所描述的分析使用流动细胞群体。 </li></ol>

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G., 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=Plasma+levels+of+soluble+CD14+independently+predict+mortality+in+HIV+infection.">Plasma levels of soluble CD14 independently predict mortality in HIV infection.</a> J Infect Dis. 203 (6), 780-790 (2011).</li><li>Klase, Z., 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=Dysbiotic+bacteria+translocate+in+progressive+SIV+infection.">Dysbiotic bacteria translocate in progressive SIV infection.</a> Mucosal Immunol. 8 (5), 1009-1020 (2015).</li><li>Evans, T. I., 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=SIV-induced+Translocation+of+Bacterial+Products+in+the+Liver+Mobilizes+Myeloid+Dendritic+and+Natural+Killer+Cells+Associated+With+Liver+Damage.">SIV-induced Translocation of Bacterial Products in the Liver Mobilizes Myeloid Dendritic and Natural Killer Cells Associated With Liver Damage.</a> J Infect Dis. 213 (3), 361-369 (2016).</li><li>Fish, R., Danneman, P. J., Brown, M., Karas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anesthesia and Analgesia in Laboratory Animals. 2 edn. , (2011).</li><li>Flecknell, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Animal Anaesthesia. 3 edn. , (2009).</li><li>Tranquilli, W. J., Thurnom, J. C., Grimm, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lumb and Jones' Veterinary Anesthesia and Analgesia. , (2007).</li><li>Ford, R. B., Mazzaferro, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Kirk and Bistner's Handbook of Veterinary Procedures and Emergency Treatment. 9 edn. , (2012).</li><li>Fransson, B. A., Mayhew, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Small Animal Laparoscopy and Thoracoscopy. , (2015).</li><li>Hensley-McBain, T., 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=Effects+of+Fecal+Microbial+Transplantation+on+Microbiome+and+Immunity+in+Simian+Immunodeficiency+Virus-Infected+Macaques.">Effects of Fecal Microbial Transplantation on Microbiome and Immunity in Simian Immunodeficiency Virus-Infected Macaques.</a> J Virol. 90 (10), 4981-4989 (2016).</li><li>Smedley, 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=Laparoscopic+Technique+for+Serial+Collection+of+Para-Colonic,+Left+Colic,+and+Inferior+Mesenteric+Lymph+Nodes+in+Macaques.">Laparoscopic Technique for Serial Collection of Para-Colonic, Left Colic, and Inferior Mesenteric Lymph Nodes in Macaques.</a> PLoS One. 11 (6), 0157535 (2016).</li><li>Park, 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=Short-chain+fatty+acids+induce+both+effector+and+regulatory+T+cells+by+suppression+of+histone+deacetylases+and+regulation+of+the+mTOR-S6K+pathway.">Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mTOR-S6K pathway.</a> Mucosal Immunol. 8 (1), 80-93 (2015).</li><li>Ziegler-Heitbrock, H. W., Ulevitch, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD14:+cell+surface+receptor+and+differentiation+marker.">CD14: cell surface receptor and differentiation marker.</a> Immunol Today. 14 (3), 121-125 (1993).</li><li>Brenchley, J. M., 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=Microbial+translocation+is+a+cause+of+systemic+immune+activation+in+chronic+HIV+infection.">Microbial translocation is a cause of systemic immune activation in chronic HIV infection.</a> Nat Med. 12 (12), 1365-1371 (2006).</li></ol>

作者宣称没有竞争的经济利益。

这里，我们证明了MLN的串行收集和肝活检微创技术，曾在本研究中没有描述这些和其他动物100％的成功率。此外，跨时间对同一动物使用这种技术还没有与任何不良事件。事实上，已经有因使用这一技术在动物体内的其他同伙感染了SIV，猿/人类免疫缺陷病毒（SHIV），或兹卡病毒（未公布数据）而导致的任何并发症。同样，这种手术甚至在曾接受腹部大手术，动物和动物的血小板（未公布数据）证明是成功的。我们证明，这些样品可以通过相同的两个端口通过倒车摄像头的位置，距离MLN切换到肝脏，避免额外的切口需要时收集。影响MLN收集因素已有报道13。 <p类="jove_content"&gt;最多的每3个MLN和3肝活检4个集已经跨越时空，无并发症被执行的动物个体，改变的成功率，或改变数据收集。此外，该腹腔镜MLN /肝收集技术已经成功地与其他程序如外周淋巴结，内窥镜上和下GI活检，骨髓穿刺，脑脊液采集的采集，和静脉穿刺在相同的麻醉事件允许的广泛评估组合病毒学和以串行方式免疫反应（未发表数据）。 整个过程通常需要30 - 45分钟来完成，并通过2〜0.5厘米切口来完成的。在肥胖动物，可能有必要使一个较大的切口（〜1 - 1.5厘米），以检索MLN通过，并且可能需要额外的时间来完成。具有丰富的肠系膜脂肪的动物常常需要到D显著经验从周围的脂肪ifferentiate万。 MLN往往沿肠系膜血管中发现，似乎是在附近的血管分支点更为普遍。这种技术的一个重要方面是，它要求所选择的MLN具有足够的流动性在肠系膜通过腹部切口被外置。其结果是，该技术不能被用来收集MLN靠近肠系膜根部。由于放大，它是有时容易与摄像头，以确定MLN，并在靠近抓的MLN可以定位和识别节点一旦外置帮助。 在头低脚高位的使用可能并不总是需要MLN集合，如果MLN可在不使用的头低脚高位轻松地检索它能使肝活检更容易，因为它会阻止器官从头侧转移。有时，在头低脚高位放置后，肝脏上移向膜和绝<html>轻轻地操纵回活检前收集位置。作为MLN采集端口的位置是在左边，肝脏活组织检查典型地从左侧横向和左内侧肝叶作为它们更接近插管。 收集MLN和肝脏活组织检查提供充足的细胞产量为各种下游分析的，并显示所收集的细胞的高生存力。在这里，细胞进行染色白细胞和淋巴细胞群的流式细胞仪分析，这两个重要的免疫器官之间的主要差异，阐明。例如，MLN高度富集为相比于肝CD4 + T细胞，由于MLN作为用于收集和传播适应性免疫应答，以及对CD4 + T的重要性中心点的重要性用于管理炎性和到环境致耐受性抗原的响应从饮食中摄取和GI-驻留微生物的S = "外部参照"&gt; 24。然而，肝显著富集CD14 +白细胞相比万。 CD14主要表达在单核细胞和巨噬细胞，并且是受体复合物对细菌脂多糖（LPS）25的一个关键组成部分。实际上，增加了可溶性CD14的血浆浓度与微生物移位和在HIV免疫激活和病原感染SIV 26相关联。因此，相比于当MLN在肝脏中CD14 +白细胞较高频率可能在从该器官更大的细菌负荷的结果。 这里，我们证明了MLN的串行收集和使用只有两种腹腔镜条目并没有导致任何不良后果的小切口肝活检快速，微创手术技术。这些样品提供了一个有用的途径，以评估采取解放军关键免疫，病毒学，和微生物方法CE在MLN和肝脏，这是从系统免疫单独且唯一的。类似地，肝脏活组织检查是用于HIV / SIV，药物疗法，和易位微生物的作用，这常常结合诱导显著肝损伤16长期评价是至关重要的。此外，由于MLN和肝脏暴露于GI微生物免疫器官的能力，以评估跨越时间在这些器官的免疫提供了理解的是，微生物在维持机体免疫稳态中扮演的角色一个很好的平台。总之，肝脏的评价和MLN是非常重要的疫苗和治疗疗效，SIV病毒清除水库，和一般的粘膜免疫的情况下。通过微创方法来收集这些样品的能力意味着存在与比目前公布的技术和对动物的生理影响较小存在的程序炎症的风险。在福TURE，我们将评估潜在的通过相同的两个端口位置，以MLN的收集和肝与脾的收集相结合，以便对已被证明在发挥显著作用，对免疫系统的另一个重要的不同部分的采样许多疾病模型。

胃肠（GI）道是人体最大的粘膜表面和暴露于从食品，病原体和内共生细菌群落衍生抗原的无数通常被称为微生物组1,2。肠系膜淋巴结（MLN）排列在胃肠道和是免疫功能的用于促进对这些不同的抗原炎性或致耐受性反应的主要位点。在MLN的物理组织创建了一个隔间系统，可以在本地而不会引起全身反应3抗原反应。同样，血液从胃肠道排至通过门静脉的方式肝脏返回到循环，并因此暴露于从胃肠道易位到固有层和进入血流4的微生物和微生物产品之前。肝还用作二级淋巴器官中一第二有大量免疫细胞，包括专门巨噬细胞，以去除易位微生物5,6。因此，MLN和肝脏是从胃肠道暴露于共生和病原性细菌的主要免疫器官，以及来自其它来源的抗原的环境，使得它们独特和重要的从免疫学的观点。 该MLN是人类免疫缺陷病毒（HIV）或致病性的猴免疫缺陷病毒（SIV）感染的病毒学和免疫效果评价的关键点，并有可能涉及以下直肠挑战SIV的早期传播。肠道淋巴组织被称为是持久的HIV复制的主要场所，尽管现代的抗逆转录病毒治疗（ART）7有效控制病毒血症。在SIV感染模型，MLN已被证明是潜在的病毒的重要水库和可能是主要的贮存器8，9。肝脏也是慢病毒感染重要由SIV特异性CD8 + T细胞在猕猴肝脏的急性SIV感染10在累积所证实。此外，负责从循环11清除病毒的主要器官。 HIV和SIV感染与改变的GI微生物群相关联，打乱GI上皮完整性和增加的微生物和微生物产物的易位从结肠到周边和循环12，13。这些过程与局部和全身免疫激活有关，在HIV感染的个体14增加发病率和死亡率。在SIV感染，易位细菌已经在MLN 15观察到的，而麦克风的积累robial产品在肝脏已显示导致炎症和损害器官16。因此，在HIV和SIV感染的上下文中，MLN和肝可以是用于理解通过GI-常住菌驱动的炎性过程高度信息。 这里，我们提出了用于在非人类灵长类动物（NHP中）的MLN的连续取样和肝脏微创腹腔镜技术。我们证明两个健康雌性恒河猴（RMS）这一技术的成功表现，采样每个动物的两倍，为160天，每个手术间。我们继续使用这些样本来评估并用流式细胞仪检测各器官中比较关键的白细胞和淋巴细胞群体，并展示了两个时间点之间的高度一致的数据。

<table><tbody><tr><td>Artficial Tears Lubricant Ophthalmic Ointment</td><td>Patterson Veterinary</td><td>97-890-8152</td><td></td></tr><tr><td>Priority Care Chlorhexidine 2% Scrub</td><td>Patterson Veterinary</td><td>07-842-6153</td><td></td></tr><tr><td>Vedco Alcohol, Isopropyl 70%</td><td>Patterson Veterinary</td><td>07-869-6434</td><td></td></tr><tr><td>Vedco Vetadine Scrub</td><td>Patterson Veterinary</td><td>07-869-6772</td><td></td></tr><tr><td>Hospira Lactated Ringer&#039;s 250 mL</td><td>Patterson Veterinary</td><td>07-800-9325</td><td></td></tr><tr><td>Adolescent Laparotomy Surgical Drape</td><td>Medline</td><td>DYNJP3004</td><td></td></tr><tr><td>Access 40 L Insufflator</td><td>Dyonics</td><td>7205832</td><td></td></tr><tr><td>300XL Xenon Light Source</td><td>Dyonics</td><td>7206084</td><td></td></tr><tr><td>460P 3 &amp;ndash; CCD Digital Camera with Head</td><td>Dyonics</td><td>72200086</td><td></td></tr><tr><td>Universal Camera Drape, 7&amp;rdquo; x 96&amp;rdquo;</td><td>Endoscopy Support Services</td><td>ESS-6920</td><td></td></tr><tr><td>Universal Light Guide, 7.5 Ft</td><td>Endoscopy Support Services</td><td>US.OU225</td><td></td></tr><tr><td>Insufflator Tubing</td><td>Endoscopy Support Services</td><td>TM-102</td><td></td></tr><tr><td>Multipurpose Rigid Scope, 2.7 mm x 187.5 mm, 0 degree</td><td>Endoscopy Support Services</td><td>B30-0007-00</td><td></td></tr><tr><td>Exam and Protective Sheath for 2.7 mm x 187.5 mm scope with one fixed stopcock</td><td>Endoscopy Support Services</td><td>B10-0025-00</td><td></td></tr><tr><td>Rigid scope, 5 mm x 300 mm</td><td>Endoscopy Support Services</td><td>B30-0071-00</td><td></td></tr><tr><td>Veress Needle, 2 mm x 80 mm&amp;nbsp;</td><td>Endoscopy Support Services</td><td>8302.08</td><td></td></tr><tr><td>Solid Probe with Markings, 5 mm x 320 mm</td><td>Endoscopy Support Services</td><td>8383.661</td><td></td></tr><tr><td>Maryland Grasping and Dissecting Forceps with Double Action Ratchet Handle, 3.5 mm x 240 mm&amp;nbsp;&amp;nbsp;</td><td>Endoscopy Support Services</td><td>8390.2054</td><td></td></tr><tr><td>Biopsy Forceps, 3.5 mm x 310 mm</td><td>R. Wolf</td><td>8391.4063</td><td></td></tr><tr><td>Plastic Threaded Cannula with Insufflator Port, 3.5 mm x 60 mm</td><td>Endoscopy Support Services</td><td>8903.072</td><td></td></tr><tr><td>Plastic Threaded Cannula with Insufflator Port, 5.5 mm x 60 mm</td><td>Endoscopy Support Services</td><td>8919.333</td><td></td></tr><tr><td>Blunt Tip Trocar</td><td>Endoscopy Support Services</td><td>8903.101</td><td></td></tr><tr><td>Pyramidal Tip Trocar</td><td>Endoscopy Support Services</td><td>8903.121</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td>CD USPE</td><td></td></tr><tr><td>RPMI 1640</td><td>Fisher Scientific</td><td>SH30027FS</td><td>with L-Glutamine</td></tr><tr><td>RNAlater Stabilization Solution</td><td>ThermoFisher Scientific</td><td>AM7021</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P4333</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Sigma Aldrich</td><td>5401119001</td><td>Commercially available colleganse solution</td></tr><tr><td>Dnase I from bovine pancrease</td><td>Sigma Aldrich</td><td>D5025</td><td></td></tr><tr><td>70 &amp;micro;m cell strainers</td><td>Morganville Scientific</td><td>CSS0200</td><td></td></tr><tr><td>5 mL Syringe</td><td>BD</td><td>309646</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>ThermoFisher Scientific</td><td>339652</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>SH3007003</td><td></td></tr><tr><td>5 mL Round-Bottom polystyrene tubes</td><td>Fisher Scientific</td><td>14-959A</td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3025601</td><td></td></tr><tr><td>LIVE/DEAD Aqua Fixable Dead Cell Stain Kit</td><td>ThermoFisher Scientific</td><td>L34957</td><td>Amine dead cell stain</td></tr><tr><td>CD45-PerCP (clone D058-1283)</td><td>BD Biosciences</td><td>558411</td><td></td></tr><tr><td>PE-CF594 Mouse anti-Hu CD3 (clone SP34-2)</td><td>BD Biosciences</td><td>562406</td><td></td></tr><tr><td>BV605 Mouse Anti-Hu CD4 (clone OKT4)</td><td>Biolegend</td><td>317438</td><td></td></tr><tr><td>APC-H7 Mouse anti-Hu CD8 (clone SK1)</td><td>BD Biosciences</td><td>560179</td><td></td></tr><tr><td>PE-Cy5 Mouse Anti-Hu CD20 (clone 2H7)</td><td>BD Biosciences</td><td>555624</td><td></td></tr><tr><td>BV786 Mouse Anti-Hu CD14 (clone M5E2)</td><td>BD Biosciences</td><td>563698</td><td></td></tr><tr><td>16% PARAFORMALDEHYDE AQ SOLUTN</td><td>Fisher Scientific</td><td>50-980-487</td><td></td></tr><tr><td>LSR II Flow Cytometer</td><td>BD</td><td>NA</td><td></td></tr></tbody></table>

laparoscopic technique for serial collection of liver and mesenteric lymph nodes in macaques

的肠系膜淋巴结（MLN）和肝脏暴露于从胃肠（GI）道的微生物和微生物产物，使它们免疫唯一的。胃肠道和相关MLN是人类免疫缺陷病毒（HIV）感染早期病毒复制的站点和MLN是怀有即使经过长时间的抗逆转录病毒疗法（ART）潜伏感染的细胞可能重要的水库遗址。肝脏已经显示出在免疫反应中显著作用，慢病毒，并出现在流通病毒的清除发挥显著作用。非人灵长类动物（NHP）模型，艾滋病毒和获得性免疫缺陷综合征（AIDS）紧密地模仿HIV感染和病毒的复制，并在这个模型中与之相关的免疫应答的原发部位的序列纵向抽样的这些方面将有助于阐明感染的关键事件，发病机制，以及对这些事件的各种干预策略的影响。 CURR耳鼻喉科已公开的技术来样肝和MLN一起涉及重大手术和/或剖检，这限制了调查以串行方式这些重要站点在同一动物的能力。之前我们已经描述的腹腔镜技术用于MLN的集合。在这里，我们描述了通过用于MLN的收集所需的相同的两个端口位置为肝脏和MLN的串行纵向采样微创腹腔镜技术。使用相同的两个端口作为不需要额外切口影响最小的动物。这种技术可以增加采样频率可用于比较大的腹部手术，减少了手术并发症的可能性和相关的可能复杂化结果的解释局部和全身炎症反应。这个过程有潜力促进涉及NHP模型，同时提高动物福利的研究。

在这些器官中用于腹腔镜收集MLN的处理的详细方法，以及细胞的产率，和白细胞频率之前已经报道13。 图1示出比较MLN细胞产量和活力数据和肝脏活组织检查在两个时间点，从使用这种腹腔镜技术两个健康女性的RM收集隔开160天。我们发现，从所述蜂窝MLN产量比来自肝脏（P = 0.0021）显著更高，分别获得33.5×10 6和6.74×10 6细胞的平均值。通过流式细胞术死细胞染色和分析表明&gt; 90％的来自两个器官中分离的细胞是存活的。 利用流式细胞仪，我们评价和比较在MLN和肝脏中的白细胞和淋巴细胞的丰度。对肝脏的代表性选通方案中被示出<strong&gt;图2，在那里我们第一选通单细胞排除聚集体，随后只有活细胞，然后选择CD45 +细胞，最后CD3 +细胞的选择。从CD3 +人口，我们评估CD4 +和CD8 + T细胞的丰度，而我们评估从CD3-人群CD14 +和CD20 +细胞的丰度。代表性流式细胞术图，显示在第0天及160天CD45 +和从MLN CD3 +人群和这两种动物的肝脏显示于图3，而图4示出了用于两种动物的两个采样白细胞和淋巴细胞的丰度。的MLN显示CD45 +细胞的显著更高的丰度比没有肝（平均76.9％和活细胞，分别7.66％，P = 0.0002）。毫不奇怪，MLN显示CD3 + T细胞的显著较高比例（P0; 0.0001）和CD4 + T细胞（P = 0.0004）。相反地，将肝脏显著富集了CD14 +白细胞相比，MLN（P = 0.0036）。该MLN表现出较高的平均CD8 + T细胞，但这并不显著。出人意料的是，对于这两种动物都表现出器官CD20 + B细胞的类似丰度。蜂窝产量和从MLN不同的白细胞和淋巴细胞亚群的丰度在这项研究证实了先前的报告值23。 <img alt="图1" src="/files/ftp_upload/55617/55617fig1.jpg" /> 图1.总细胞产率（顶部）和细胞活力（底部）从串行收集MLN和肝活检组织。该MLN表现出显著高于细胞产量比没有肝脏。然而，这两种类型的样品提供了充足的细胞下游分析。细胞活力，通过流式细胞测量cytometrically使用可固定死细胞染色胺，表明，两种类型的样品典型地产生了组织离解后&gt; 90％的细胞生存力。误差棒代表样本之间的标准偏差。 <a href="http://ecsource.jove.com/files/ftp_upload/55617/55617fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55617/55617fig2.jpg" /> 图2.代表性的流式细胞仪门控策略。首先，聚集的细胞用可固定死细胞染色胺染色后排除，随后唯一可行的细胞的选择。接着，选择的CD45 +白细胞，随后CD3 + T细胞的选择。从CD3 +人口，CD4 +和CD8 + T细胞进行了分析。从CD3 -人口，CD14 +和CD20 +细胞进行分析。氏<html>小号门控策略也用于万。 <a href="http://ecsource.jove.com/files/ftp_upload/55617/55617fig2large.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/55617/55617fig3.jpg" /> 图3.从MLN和两只动物肝纵向流式细胞仪情节。 MLN和肝脏标本采集间隔160天两种动物。 （A）。 MLN CD45 +细胞（白细胞）; （B）肝CD45 +细胞（白细胞）; （C）MLN + CD3 +细胞（T细胞）; （D）肝的CD3 +细胞（T细胞）。对于这两种动物的MLN表明CD45 +和CD3 +细胞的比例较高相比肝脏。星移斗转，这些人群的频率分别为这两种动物类似。S / ftp_upload / 55617 / 55617fig3large.jpg"目标=‘_空白’&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55617/55617fig4.jpg" /> 图4. CD45 +细胞（左）的频率和不同的白细胞和淋巴细胞亚群（右）23个对面单独的取样。所述MLNS显示CD45 +，CD3 +，和CD4 +细胞的显著更大的频率，而肝显示CD14 +白细胞显著更大的频率，这表明每个器官的独特功能。误差棒代表样本之间的标准偏差。 <a href="http://ecsource.jove.com/files/ftp_upload/55617/55617fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques at JoVE.com

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

在这里，我们描述了用于在猕猴肝脏和肠系膜淋巴结（MLN）的连续取样，可以增加采样频率，并与执行剖腹时减少了手术并发症的潜力微创腹腔镜技术。

腹腔镜技术在猕猴肝和肠系膜淋巴结的串行采集

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

laparoscopic-technique-for-serial-collection-liver-mesenteric-lymph

Research

JoVE Journal

Medicine

51.1K 次观看.  Washington National Primate Research Center, University of Washington. 在这里，我们描述了用于在猕猴肝脏和肠系膜淋巴结（MLN）的连续取样，可以增加采样频率，并与执行剖腹时减少了手术并发症的潜力微创腹腔镜技术。 

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

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

小鼠浅表淋巴结手术

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

科研

免疫与感染

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Application of the En Bloc Concept Combined with Anatomic Resection in Laparoscopic Hepatectomy

Laparoscopic hepatectomy has been reported in many studies, and it is the mainstream method of liver resection. In some particular cases, such as when there are tumors adjacent to the cystic bed, surgeons cannot palpate the surgical margins through the laparoscopic approach, which leads to uncertainty about R0 resection. Conventionally, the gallbladder is resected first, and the hepatic lobes or segments are resected second. However, tumor tissues can be disseminated in the above cases. To address this issue, based on the recognition of the porta hepatis and intrahepatic anatomy, we propose a unique approach to hepatectomy combined with gallbladder resection by en bloc anatomic resection in situ. Firstly, after dissecting the cystic duct, without cutting the gallbladder primarily, the porta hepatis is pre-occluded by the single lumen ureter; secondly, the left hepatic pedicle is made free by the gap of the Laennec membrane and Hilar plate; thirdly, the assistant is asked to drag the fundus of the gallbladder, and the liver parenchyma tissue is resected using a harmonic scalpel along the ischemia line on the liver surface and intraoperative ultrasound. The whole middle hepatic vein (MHV) and its tributaries appear completely; lastly, the left hepatic vein (LHV) is disconnected, and the specimen is taken out from the abdominal cavity. The tumor, gallbladder, and other surrounding tissues are resected en bloc, which meets the tumor-free criterion, and a wide incisal margin and R0 resection are achieved. Therefore, the laparoscopic hepatectomy with the combination of the en bloc concept and anatomic resection is a safe, effective, and radical method with low postoperative recurrence and metastasis.

Numerous studies have demonstrated the advantages of anatomic resection. Nonetheless, whether anatomic resection can increase R0 resection rates remains controversial. Consequently, the present study describes an innovative procedure involving the en bloc concept combined with anatomic resection in laparoscopic hepatectomy, which can reduce postoperative recurrence and metastasis.

整体概念联合解剖切除在腹腔镜肝切除术中的应用

Laparoscopic hepatectomy has been reported in many studies, and it is the mainstream method of liver resection. In some particular cases, such as when ...

癌症研究

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating肝白细胞的选择性收获

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

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

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

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

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

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

医学

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

<meta charset="utf8"></head><body>在分期肝切除术动物模型中研究 ALLPS 的肝脏再生

A Mouse Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy Procedure Aided by Microscopy

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative mortality, severely impacting patient outcomes. In a robust hepatic environment, the future liver remnant (FLR) must exceed 25%, and in cases of cirrhosis, this requirement increases to over 40%. The inadequacy of FLR is currently a major obstacle in the progression of hepatic surgery.
Traditional methods to enhance FLR hypertrophy mainly focus on portal vein embolization (PVE), but its effectiveness is considerably limited. In recent years, there have been numerous reports on a novel biphasic hepatectomy method involving hepatic partitioning and portal vein ligation, known as associating liver partition and portal vein ligation for staged hepatectomy (ALPPS). ALPPS surpasses PVE in efficiently and considerably inducing FLR hypertrophy. However, the detailed mechanisms driving ALPPS-facilitated hepatic regeneration are not fully understood. Thus, replicating ALPPS in animal models is crucial to thoroughly investigate the molecular mechanisms of hepatic regeneration, offering valuable theoretical and practical insights.

<meta charset="utf8"></head><body>作者聚焦：通过 ALPPS 小鼠模型推进肝脏再生研究

Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) has been shown to result in remarkable liver regeneration after several weeks post first-stage surgery. Animal models that have undergone ALPPS have been developed to explore potential liver regeneration ability and achieve favorable clinical outcomes.

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

Hepatectomy is widely regarded as the primary treatment for hepatic malignancies; yet, postoperative liver failure remains a major cause of perioperative ...

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

The present protocol describes a detailed surgical protocol for isolating flowing intestinal lymph in response to intraduodenal nutrient infusions in mice. This allows for the physiological determination of total intestinal lipid absorption and chylomicron synthesis and secretion in response to various experimental nutrients.

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, ...

腹腔镜技术在猕猴肝和肠系膜淋巴结的串行采集

摘要

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此视频中的章节

小鼠浅表淋巴结手术

在肠系膜淋巴管空心大鼠模型:应用肠淋巴药物运输的评估

整体概念联合解剖切除在腹腔镜肝切除术中的应用

Marginating肝白细胞的选择性收获

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学

腹腔镜技术在猕猴肝和肠系膜淋巴结的串行采集

摘要

探索更多视频

此视频中的章节

小鼠浅表淋巴结手术

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

整体概念联合解剖切除在腹腔镜肝切除术中的应用

Marginating肝白细胞的选择性收获

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

肝分型与门静脉结扎大鼠分期肝切除 (ALPPS) 手术模型的建立

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

显微镜辅助分期肝切除术的关联肝分区和门静脉结扎的小鼠模型

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的 体内 动力学

在肠系膜淋巴管空心大鼠模型:应用肠淋巴药物运输的评估

分离小鼠肠系膜流动淋巴液以量化膳食脂质吸收和乳糜微粒分泌的体内动力学