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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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。

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			<td height="31" style="height:31px;width:441px;">CO2</td>
			<td style="width:248px;">Airgas</td>
			<td style="width:167px;">CD USPE</td>
			<td style="width:539px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">RPMI 1640</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td>SH30027FS</td>
			<td>with L-Glutamine</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">RNAlater Stabilization Solution</td>
			<td style="width:248px;">ThermoFisher Scientific</td>
			<td>AM7021</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">Penicillin-Streptomycin</td>
			<td style="width:248px;">Sigma Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">Liberase TM Research Grade</td>
			<td style="width:248px;">Sigma Aldrich</td>
			<td>5401119001</td>
			<td>Commercially available colleganse solution</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">Dnase I from bovine pancrease</td>
			<td style="width:248px;">Sigma Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">70 &#181;m cell strainers</td>
			<td style="width:248px;">Morganville Scientific</td>
			<td>CSS0200</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">5mL Syringe</td>
			<td style="width:248px;">BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:248px;">ThermoFisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">Fetal Bovine Serum</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td>SH3007003</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">5mL Round-Bottom polystyrene tubes</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td>14-959A</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">PBS</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td>SH3025601</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">LIVE/DEAD Aqua Fixable Dead Cell Stain Kit</td>
			<td style="width:248px;">ThermoFisher Scientific</td>
			<td>L34957</td>
			<td>Amine dead cell stain</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">CD45-PerCP (clone D058-1283)</td>
			<td style="width:248px;">BD Biosciences</td>
			<td>558411</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">PE-CF594 Mouse anti-Hu CD3 (clone SP34-2)</td>
			<td style="width:248px;">BD Biosciences</td>
			<td>562406</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BV605 Mouse Anti-Hu CD4 (clone OKT4)</td>
			<td style="width:248px;">Biolegend</td>
			<td>317438</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">APC-H7 Mouse anti-Hu CD8 (clone SK1)</td>
			<td style="width:248px;">BD Biosciences</td>
			<td>560179</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">PE-Cy5 Mouse Anti-Hu CD20 (clone 2H7)</td>
			<td style="width:248px;">BD Biosciences</td>
			<td>555624</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BV786 Mouse Anti-Hu CD14 (clone M5E2)</td>
			<td style="width:248px;">BD Biosciences</td>
			<td>563698</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">16% PARAFORMALDEHYDE AQ SOLUTN</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td>50-980-487</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:441px;">LSR II Flow Cytometer</td>
			<td style="width:248px;">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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermic的技术<em&gt;前体内</em&gt;肝脏灌注的存储，评估和修复边际移植肝

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

科研

医学

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

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 ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

胰腺癌的腹腔镜根左造影切除术:手术策略与技术视频

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

骨关节炎模型中骨髓、骨髓、淋巴结和合成组织内免疫细胞子集的流动细胞测量分析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic resection and reconstruction techniques, it is difficult to perform LPD for patients with locally advanced pancreatic head cancer after neoadjuvant therapy. Our team initiates LPD using the in-situ No-Touch isolation technique. The innovation and optimization of this modified No-Touch isolation technique emphasize exploring the distal section of superior mesenteric vein&#160;(SMV) and the left side of the superior mesenteric artery (SMA) prior to evaluating the resectability by subcolonic mesenteric approach, which is an ideal exploring approach. After that, we use the median-anterior, and left-posterior of SMA approaches to cut off the blood flow of the pancreatic head to make the tumor isolated intact, then move and dissect the tumor. It is a process fitting the surgical principle of tumor-free. This article aims to demonstrate the feasibility and safety of performing LPD using the in-situ No-Touch isolation technique, which might elevate the R0 resection rate. It is an oncological ideal operation process.

Laparoscopic Pancreatoduodenectomy for Pancreatic Cancer Using In-Situ No-Touch Isolation Technique

No-Touch isolation procedures might prevent the dissemination of cancer cells from the primary tumor. However, these techniques are not widely accepted in laparoscopic pancreatoduodenectomy (LPD) by now. We herein present in-situ No-Touch isolation LPD with partial resection and reconstruction of the superior mesenteric vein (SMV) for pancreatic cancer after neoadjuvant therapy.

腹腔镜胰十二指肠切除术使用原 位 非接触式隔离技术治疗胰腺癌

Laparoscopic pancreatoduodenectomy (LPD) is a standard radical operation for pancreatic head malignant tumors by now. Due to the complex laparoscopic ...

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise.

荧光腹腔镜中央肝切除术使用吲哚菁绿阴性染色治疗肝癌

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ...

Application of Laparoscopic Hepatectomy Combined with Intraoperative Microwave Ablation in Colorectal Cancer Liver Metastasis

Laparoscopic hepatectomy is a common treatment for colorectal cancer liver metastasis. Previously, a sufficient number of functional liver masses had to be maintained during laparoscopic hepatectomy, with a residual liver volume of &gt;40% in cirrhotic patients and &gt;30% in non-cirrhotic patients. The high incidence of complications such as bleeding, bile leakage, or liver failure due to the exposure and difficulty of the resection of specific liver segments such as S2 and S7 reduces the success rate of liver resection. At present, microwave ablation is mainly applied in the treatment of liver metastasis using a percutaneous approach, which makes it difficult to identify hidden parts or small lesions. For some liver segments, the percutaneous puncture of liver segment 7 (S7) is likely to pass through the thoracic cavity, and the percutaneous puncture of liver segment 2 (S2) adjacent to the diaphragm is likely to injure the diaphragm and heart; these issues restrict the application of percutaneous ablation in colorectal cancer liver metastasis. Considering multiple lesions, laparoscopic microwave ablation combined with hepatectomy was performed in this study. The location of the lesions was determined by contrast-enhanced ultrasound under laparoscopy, and small lesions that were difficult to detect before the operation were identified. For the scattered lesions, which had diameters less than 3 cm and were difficult to resect, ablation was adopted to substitute hepatectomy. This technique helped to more explicitly locate the tumors, simplified the operation procedures, reduced the risk of complications such as bleeding and bile leakage, shortened the operation time, accelerated the postoperative recovery, significantly improved the success rate of operation, and enhanced the clinical prognosis of colorectal cancer liver metastasis by surgical resection.

This protocol describes the laparoscopic resection of colorectal cancer liver metastases combined with ultrasound-guided microwave ablation. This technique can safely, effectively, and accurately treat refractory liver metastases &lt;3 cm, reduce postoperative complications, and accelerate the postoperative rehabilitation of patients.

腹腔镜肝切除术联合术中微波消融术在结直肠癌肝转移中的应用

Laparoscopic hepatectomy is a common treatment for colorectal cancer liver metastasis. Previously, a sufficient number of functional liver masses had to ...