Name: long-term catheterization of the intestinal lymph trunk and collection of lymph in neonatal pigs
Uploaded: 2016-03-05T14:00:00.000+00:00
Duration: 6 min 25 s
Description: Watch this Scientific Journal Video about Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs at JoVE.com

The work was supported in part by funding from Alberta Livestock and Meat Agency and Natural Science and Research Council Discovery grant to S. D. Proctor.

在视频稿件和描述都实验动物的所有程序被机构动物护理和使用委员会批准，其次是动物保健的加拿大理事会确定的指导方针。 1.手术麻醉及新生儿猪手术准备<ol><li>在一个分离的前厅，premedicate颈部与含有肌内镇静麻醉药鸡尾酒底部附近25千克猪:二甲苯胺噻嗪（0.3毫克/千克），盐酸氯胺酮（10毫克/千克），右美托咪（15微克/千克）。 注:在麻醉前药物鸡尾酒增强术中疼痛控制添加丁丙诺啡（0.005-0.02毫克/千克）。 </li><li>麻醉与吸入异氟烷吸入气体的猪（4-5％异氟醚在500毫升千毫升/分钟O 2）使用面罩。使用兽医喉范围（17-25厘米长的直叶片）可视化的声带，并应用外用10％的利多卡因绝育的声音线。允许利多卡因喷30-60秒前联系声带气管插管，以减少声带痉挛及气道阻塞的可能性。 </li><li>由声带之间传递铐气管插管（5.0-7.0毫米内径（ID））插管猪并保持与异氟醚气体麻醉（0.5-2.0％异氟醚在1000-2000毫升/分钟O 2）使用关闭电路在整个手术麻醉重复吸入系统。评估麻醉由颚音调水平，并且两个踏板和眼睑反射响应。过期麻醉气体被清理和手术套件之外排出。 </li><li>清洁用2.0％氯己定外科擦洗液耳后跟一个70％异丙醇冲洗的外表面。用20G的静脉导管，导尿耳静脉，以提供静脉注射液（乳酸林格氏液; 5-10毫升/千克/小时）的手术过程中。安全脉搏血氧仪与MED舌粘膜表面的iCal胶带监测心脏速率和外周血氧合的饱和度（ 血氧饱和度）。 </li><li>放置在一个背卧位麻醉猪和剃从中间胸部尾端腹侧腹部到耻骨的腹侧。有两个交替的2.0％洗必泰外科擦洗和消毒水洗清洁这个区域。 </li><li>转移猪麻醉到手术套件和应用70％异丙醇冲洗的最终手术擦洗，晾干，然后再披动物。 </li><li>插入直肠温度探头大约2-4厘米到直肠监测体温。放置在一个循环水加热垫猪保持在手术过程正常体温（38-40℃）。 </li><li>悬垂性与放置在腹部周围的上覆象限模式4毛巾窗帘猪。放置第一个悬垂横跨剑突，第二悬垂沿腹部约5的c外侧面米外侧腹部中线。放置第三悬垂横跨骨盆的回肠波峰和第四悬垂性，像第二悬垂（尽管在相反侧），沿着腹部约5cm外侧到腹部中线的外侧面放置。 </li><li>放置一个大表的悬垂性，具有狭缝开口允许进入手术部位，在底层的毛巾窗帘并覆盖猪和整个手术表。最终的褶皱是一次性免缝悬垂放置在大桌子悬垂性。 </li></ol> 2.腹部手术和导管肠淋巴干的<ol><li>使20cm的皮肤切口，用手术刀刀片以暴露下面的腹部肌肉。切割与单极电灼（20瓦设置）腹部肌肉层以暴露壁层腹膜。打开壁层腹膜的20cm的线段与梅岑鲍姆剪刀进入腹腔脏器和淋巴管。 </li><li&gt;将牵开器在手术切口的颅方面保持腹腔开放手术的持续时间。 </li><li>滋润所有组织用温水（37℃）的无菌盐水整个手术过程。轻轻抬起一大段小肠包括从腹腔结肠，盲肠，回肠和空肠和它exteriorize到猪的左翼访问上腹部，肝和淋巴管。在固定位置上的外置肠道额外的毛巾窗帘形成吊带轻轻支持肠。 </li><li>找到淋巴管，它奠定了约4厘米右肾静脉，腔静脉工头6厘米尾，中间-腹和胰腺22,36,37附近肝右叶内脏方面下的颅内侧。识别淋巴管作为并列门静脉36,37的右侧腹段的半透明结构。 </li><li>分离淋巴VESSEL从周围筋膜轻轻挑逗废除棉签涂抹附加的组织。一旦船只的横向方面从周围组织分离，创建容器下方的"隧道"口用细钝尖镊子。 </li><li>通过三项2-0丝线缝合用细镊子淋巴管下方。首先结扎最尾缝合闭塞，扩张和淋巴填充容器。目的地离开这个缝合相对长（4厘米）的端部到导管固定到淋巴管。放置另外两个缝线彼此隔开1.0厘米和大约1.0-1.5厘米颅到锁定尾缝合。离开这两个缝线具有"单一松散结扎领带"，以允许在导管的更快固定到容器。 注意:位于最尾端结扎缝线和中间缝合（两个颅缝的）之间的淋巴管的段为导尿站点。关于苏TURE材料，如果需要2-0聚乳糖缝线可替代2-0丝线缝合。 </li><li>切一小口与虹膜剪船和扩张细钝钳的容器。用斜切端插入该容器插入约1.0-1.5厘米专门的导管（4.06外径（OD）×2.31毫米ID），和扎两个颅缝以固定导管就位。使用尾缝线的长缝合线端部的导管固定到容器中。 </li><li>用大量温生理盐水冲洗外置肠，轻轻将其返回到腹腔，确保肠道正确的解剖定位。 </li><li> （从paralumbar窝腹侧5-10厘米）Exteriorize导管在左侧中间侧翼。使用手术刀切开皮肤，并从腹腔到皮肤表面传递套针来创建导管的外化的开口。使用大凯利钳从腹部CAV exteriorize导管穿过套管针开放性。 </li><li>用2-0聚乳糖缝合圆（锥形）针一个简单的连续缝合模式关闭壁层腹膜。用一个简单的间断缝合模式以2-0聚乳糖缝合关闭上圆针腹部肌肉层。 </li><li>用2-0缝合聚乳糖合皮肤的表皮下图案切割针。固定形象化导管与荷包缝合模式皮肤上切割针2-0尼龙缝合。 </li><li>将一个专门的夹克猪，同时仍麻醉以减轻其位置，并减少恢复期间猪的压力。 </li></ol> 3.手术后的恢复和淋巴收集<ol><li>吸入麻醉停产前约10分钟，辖bupenorphine（0.1毫克/千克）肌注提供即时的术后镇痛。继续bupenorphine（0.1毫克/千克），每12小时24〜48小时，以保持手术后镇痛。 </li><li>监控猪的手术后并发症7天期每8-12小时。 </li><li>收集在500毫升聚丙烯洗瓶淋巴液，涂有乙二胺四乙酸（EDTA）和补充有抗生素;青霉素（6000 IU），链霉素（6毫克）和两性霉素B（3毫克）为7天期间，每12小时。 </li></ol> 4.脂蛋白ApoB48，甘油三酯，胆固醇和总蛋白定量从淋巴收集<ol><li>离心淋巴样品在1800 XG在4℃下5分钟。收集上清液，并将其用于甘油三酯，胆固醇和总蛋白的定量。 </li><li>划分上清液分成三个样品:未稀释的样品，将样品以1:20稀释蒸馏水稀释1的最终样品:100用蒸馏水。 </li><li>使用未稀释的上清液来测量胆固醇水平，与市售的试剂盒。 </li><li>使用1:20和1:100稀释的样品以测量甘油三酯和总蛋白水平与市售的试剂盒和二辛可宁酸总蛋白分别测定。 </li></ol> 5.定量脂蛋白ApoB48，从淋巴收集38 <ol><li>确定脂蛋白ApoB48的浓度与适于免疫Western印迹方法38。用3-8％三 - 酯 - 十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离总淋巴。 </li><li>转移分离的蛋白质，以聚偏二氟乙烯膜（0.45微米），用山羊多克隆抗体的ApoB（1:4000）孵育它们，然后将其绑定与抗山羊次级抗体。 </li><li>采用量化与啮齿类动物纯化蛋白质ApoB48标准的线性光密度比较脂蛋白ApoB48化学发光。 </li></ol>

The authors have nothing to disclose.

收集肠淋巴是调查参与脂质8-12和13-15的药物代谢，癌症转移16,17，贩卖细胞和免疫功能18-26机制，在各种实验动物模型的好方法。事实上，收获大量任一外围设备（传入）和中央（传出和大型行李箱血管）淋巴的较长时间内的能力一直是特别重要的理解发生在以下与免疫调节剂18-22挑战细胞群随时间的变化，25,26。同样，中央淋巴结收集已划定参与脂蛋白代谢过程8-12有用。淋巴集合的效用取决于导管手术的成功和导管的淋巴管内的通畅。 涉及的主要淋巴管导尿在大型动物的肠道淋巴引流一般采用的三种手术方法之一。第一种技术采用了颅肩方法进入胸导管覆盖普通颈静脉（牛胸导管）5。第二个使用过右肾32,33的中间-侧部纵横的上部侧面的方法。在这个视频中描述的第三种技术采用两种下腹正中或正中22,35剖腹手术暴露胰腺附近的肠淋巴干和并列门静脉22,36,37的右腹方面。虽然上侧面和腹侧中线手术技术使肠淋巴干的导管，我们相信腹侧中线方法允许用于与上腹部内有足够的空间来处理和操纵容器更好曝光容器。 重要的是，如在任何实验手术有步骤是杂色cularly对实验过程的成功结果的关键。有需要使用腹正中入路进入肠道淋巴管小心注意四个关键领域。首先，优良的血液供应形象化肠是必需的，因为延长损害循环于肠诱导缺血性肠粘膜坏死和手术失败。在此过程中一个"毛巾吊带"通过支持肠子确保了出色的组织灌注的重量减少了肠系膜血管的张力。其次，该容器包括仔细操作:在容器的周围的筋膜和脂肪组织，插入并牢固固定在容器用于长期淋巴集合中的导管的释放是成功导尿至关重要。事实上，有人建议，在新生仔猪的血管的结构完整性是不够健壮在成熟动物，因此可以很容易地撕开39,40 </SUP&gt;。使用棉条涂药慢慢地，轻轻地分开的筋膜层，细钝端工具通过缝合下方的容器大大降低了淋巴管的意外刺伤或撕裂。容器粗暴地对待往往造成弱结构制作导尿非常具有挑战性的一个易碎的容器。第三，导尿故障的常见原因是从淋巴管无意"导管的拉出"。直接固定在导管的容器减少导管损失的发生率。这是通过直接将导管与用于扩张血管尾缝合容器来完成的。最后，重要的是要确保良好的淋巴液的流动是存在以下导管及前腹部闭合。事实上，如果淋巴循环是过程中不畅，随着时间的推移这种诱导导管内血栓形成，并明显降低淋巴收集期间。在研究者的EXPERience，与非肠淋巴绵羊41的长期淋巴集合，它是非常困难的从导管移除凝块作为凝块在导管内开发和是根深蒂固的淋巴管内。因此，这将大大降低（约40％）的动物成功地插入导管的长期试验（未公布的数据）的数字。因此，如果淋巴循环减慢，或在操作过程中已经停产了，淋巴导管，应重新插入颅从最初的导尿网站0.5-1厘米。 用于研究的科学问题所有型号都有inherant局限性和肠淋巴干插管也不例外。对于该过程的最显着的限制是不能有效地隔离，导尿的淋巴管，并在容器内稳定导管。因此，这个过程需要与出色的技术和手术技巧的人秒。另一个显著限制是，大型腹部手术期间延长手术时间增加了风险和手术后的发病率和死亡率。值得注意的是，然而，在我们的实验猪迅速从手术恢复，并没有并发症如感染，肠道肠梗阻，过度的组织损伤，或食欲不振以下的步骤。最肯定的，观察猪是光明的，反应灵敏，走动和进食正常后24小时手术，这持续了7天淋巴收集期。值得注意的是，尽管血清化学或全血计数在以下7天的实验，这些猪没有评估，由绵羊41在长期淋巴收集调查先前的工作表明，插管羊从未白细胞减少，淋巴细胞减少，低蛋白血症与电解质失衡或临床上塑造健康状况不佳（未公布数据）。重要的是与如上所述，这一观察关联到catheteriZED猪也显示出良好的健康的临床特点。 有趣的是，研究人员注意到，淋巴流动的量的变化。流动是白天最大时猪饲养活性与此时发生的日常收集淋巴的约70％。淋巴流动的剩余30％的休息和睡眠过程中发生的晚上。 在图1，图2和设置淋巴样品进一步表征表1中 。 图1显示污染的少量的衍生载脂蛋白B100等离子体的约15％。肠淋巴结内血浆脂蛋白的存在是经常出现在猪42的观察。在乳猪，大部分脂蛋白是由肝脏产生的，并在等离子体释放。从等离子体，载脂蛋白B100可能渗入收集乳糜42，然后通过淋巴血管网排入肠道淋巴干。层析的各种方法来评估采集淋巴液样品42-45中胆固醇，甘油三酯和其它磷脂含量。 图2中的单峰清楚地表明了胆固醇和甘油三酯存在于乳糜微粒分数。这种方法是分析淋巴脂蛋白的方便和准确的技术和从肠淋巴45评估乳糜微粒样品的纯度时，应考虑。最后，表1提供了从仔猪7天手术后收集的肠淋巴的生化成分。 总之，肠淋巴躯干的长期导尿是成功的幼猪。利用这种技术，淋巴导管可用于收集和调查脂质代谢的组分。淋巴，产生淋巴的量的纯度和脂质代谢的成分的量类似于目前在猪淋巴结中央在其他实验9,25,26,42金额。结果表明，采用通过精心组织解剖及血管处理，以导尿肠淋巴干仔猪下腹正中手术方法是收集大量的中央淋巴结的好方法。

淋巴系统是生理学的充分研究领域。淋巴导管的临床前模型发生在不同的动物物种1-8和由制药工业和研究机构用于研究参与脂质8-12和药物代谢13-15，与实验治疗17癌转移16的机制，及免疫功能18 -26。本研究旨在探讨在国内猪模型采用肠淋巴导管干线来衡量脂蛋白代谢的成分。脂蛋白代谢参与乳糜微粒的产生和分泌，以及在相关的脂质和总蛋白的变化。这些都是重要的考虑因素，因为有常用的啮齿动物模型与人类之间，因此脂质代谢的主要区别，采用猪模型，收集肠淋巴能为研究脂类我提供更多的可比信息代谢障碍，在人27-31。 若干外科技术用于收集在大动物种属的肠道淋巴:颅骨肩方法（ 即，胸导管导尿）5，横向上侧翼的方法32-34，和腹中线或正中的方法22,35。详细这段视频描述了使用的肠淋巴树干导管下腹正中手术方法在猪的外科手术。细心的手术技术许可淋巴导管的这种方法来收集大量淋巴结及其成员在一段时间过长。 该技术将打开的应用程序，许多学科研究的各种生理功能万千。应用可包括，但不限于，全身脂蛋白和脂质代谢，免疫监视，肿瘤发生和转移，肠道功能，并且developm耳鼻喉科和肠道炎症疾病的进展。

<table><tbody><tr><td>Miller laryngoscope blade</td><td>Welch Allyn</td><td>68044</td><td>182 mm length</td></tr><tr><td>Surgivet advisor: Vital signs monitor</td><td>Surgivet</td><td>V9203</td><td></td></tr><tr><td>Rectal temperature probe</td><td>Surgivet</td><td>V3417</td><td></td></tr><tr><td>Mono-polar electrosurgery generator</td><td>Valley Lab</td><td></td><td></td></tr><tr><td>Metzenbaum scissors</td><td>Fine Science</td><td>14518-18</td><td></td></tr><tr><td>Tuffier retractor</td><td>Stevens</td><td>162-11-676</td><td></td></tr><tr><td>Mosquito forceps</td><td>Stevens</td><td>162-7-10</td><td></td></tr><tr><td>Kelly forceps-curved (14cm)</td><td>Stevens</td><td>162-7-38</td><td></td></tr><tr><td>Allis tissue forceps</td><td>Stevens</td><td>162-7-38</td><td></td></tr><tr><td>Forceps dressing-eye (10.2cm)</td><td>Stevens</td><td>162-18-780</td><td></td></tr><tr><td>Forceps dressing-Adison (12.1cm)</td><td>Stevens</td><td>162-17-2510</td><td></td></tr><tr><td>Needle Drivers</td><td>Stevens</td><td>162-V98-42</td><td></td></tr><tr><td>Iris scissors</td><td>Fine science</td><td>14058-11</td><td></td></tr><tr><td>Circulating water pump</td><td>Jorvet</td><td>J-783X</td><td></td></tr><tr><td>Maxitherm-Vinyl blanket</td><td>Jorvet</td><td>J-784C</td><td></td></tr><tr><td>Q tip applicators</td><td>Fisher Scientific</td><td>22-037-960</td><td></td></tr><tr><td>Catheterization&amp;nbsp; tubing (4.06 OD X 2.31 ID)</td><td>Braintree Scientific Inc.</td><td>MRE-160</td><td>Micro-Renethane implantation tubing</td></tr><tr><td>2-0 silk suture</td><td>Ethicon</td><td>LA556</td><td></td></tr><tr><td>2-0 polyglactin suture</td><td>Ethicon</td><td>J443H</td><td>2-0 vicryl</td></tr><tr><td>Large animal jacket</td><td>Lomir Biomedical Inc.</td><td>SSJ2YC</td><td></td></tr><tr><td>Polypropylene wash bottles</td><td>Fisher Scientific</td><td>03-409-22C</td><td>500 ml</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>D4333</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>60-00-4</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma Aldrich</td><td>A2411</td><td></td></tr><tr><td>Azaperone</td><td>Elanco Animal Health</td><td></td><td>Stresnil</td></tr><tr><td>Dexmedetomidine hydrochloride</td><td>Zoetis</td><td>6295</td><td>Dexdomitor</td></tr><tr><td>Isoflurane</td><td>Abbott Animal&amp;nbsp; Health</td><td>05260-5</td><td>IsoFlo</td></tr><tr><td>Ketamine hydrochloride</td><td>Zoetis</td><td>2626</td><td>Ketaset</td></tr><tr><td>Bupenorphine hydrochloride</td><td>Champion Alstoe Animal Health</td><td></td><td>DIN:02347510</td></tr><tr><td>6 mm Endotracheal tube</td><td>Jorvet</td><td>J-165d</td><td></td></tr><tr><td>10% Lidocaine spray</td><td>AstraZeneca</td><td></td><td>DIN:02003767</td></tr><tr><td>4 % Chlorhexidine surgical scrub</td><td>Partnar Animal Health</td><td>PCH-011</td><td>Diluted: 2.0% solution</td></tr><tr><td>3M Surgical steri- drape</td><td>3M Health Care</td><td>1040</td><td></td></tr><tr><td>SDS page gel</td><td>Invitrogen</td><td>EA0375BOX</td><td>3-8 % tris acetate</td></tr><tr><td>Polyvinylidene fluoride membrane</td><td>Millipore</td><td>IPVH00010</td><td>0.45 &amp;mu;m pore size</td></tr><tr><td>ApoB antibody&amp;nbsp;</td><td>EMD Millipore</td><td>AB742</td><td>1:4000 dilution</td></tr><tr><td>Donkey anti-goat IgG-HRP</td><td>Santa Cruz Biotechnology</td><td>Sc-2304</td><td></td></tr><tr><td>ECL Prime Western Blotting Reagent</td><td>GE Healthcare LifeSciences</td><td>RPN2232&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Triglyceride Kit</td><td>Wako Pure Chemicals</td><td>998-40391/994-40491</td><td></td></tr><tr><td>Total Cholesterol Kit</td><td>Wako Pure Chemicals</td><td>439-17501</td><td></td></tr><tr><td>Total Protein</td><td>&amp;nbsp;Pierce&amp;nbsp;</td><td>23225</td><td>Bicinchoninic Acid Assay</td></tr></tbody></table>

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.

<html>仔猪肠淋巴干的淋巴导管允许大约1升的中央淋巴/ 24小时收集了7天的时间。在这个实验中收集的淋巴含有脂质代谢，即总淋巴蛋白，ApoB48脂蛋白，甘油三酯，总蛋白，和胆固醇的组分。表1列出代表数量从三头猪汇集淋巴结样品这些脂质成分的。值得注意的是，淋巴流和脂质成分是在符合由下列肠淋巴管的导管等研究者报告中央淋巴的值（淋巴流570±158 979±284毫升/ 24小时25 360±120 MLL / 24小时至1080±720毫升/ 24小时26甘油三酯687±110毫克/分升9胆固醇63.1±5.6毫克/分升9）;指示所述淋巴管内的导管的适当的位置。 内容】"FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/53457/53457fig2.jpg" /> 图2.快速蛋白液相色谱（FPLC）的资料表明在新生仔猪肠淋巴乳糜微粒制剂的纯度 。从三头猪使用1.006克/ ml的密度梯度离心协议是分开收集淋巴液。该曲线显示了（A）胆固醇和淋巴样品（B）中甘油三酯的单峰。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53457/53457fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图1" src="/files/ftp_upload/53457/53457fig1.jpg" /> 图1.代表Western blot分析表明ApoB48和载脂蛋白B100脂蛋白的新生仔猪肠淋巴的贡献。淋巴从三个收集猪是使用1.006克/ ml的密度梯度超速离心协议分离。该图显示的衍生载脂蛋白B100等离子的约15％，少量的污染。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53457/53457fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td></td><td>淋巴特点</td></tr><tr><td> ApoB48（毫克/毫升） </td><td> 3022.4±440.4 </td></tr><tr><td>甘油三酯（毫克/分升） </td><td> 607±203.6 </td></tr><tr><td>胆固醇（毫克/ dL）的</td><td> 58.5±9.6 </td></tr><tr><td colspan="2">结果表示为平均值±标准差（n = 3） </td></tr></tbody></table> 从仔猪7天手术后，结果重复MEA 收集肠淋巴表 1. 组件淋巴结祖雷斯贝尔，并表示为平均标准差（n = 3）

Watch this Scientific Journal Video about Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs at JoVE.com

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

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 ...

long-term-catheterization-intestinal-lymph-trunk-collection-lymph

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.7K 次观看.  University of Alberta. 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.

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

科研

医学

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

新生儿窒息了猪模型

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome (ARDS)

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future therapies. Severe lung injury with a reproducible deterioration of pulmonary gas exchange and hemodynamics can be induced in anesthetized pigs using repeated lung lavages with warmed 0.9% saline (50 ml/kg body weight). Including standard respiratory and hemodynamic monitoring with clinically applied devices in this model allows the evaluation of novel therapeutic strategies (drugs, modern ventilators, extracorporeal membrane oxygenators, ECMO), and bridges the gap between bench and bedside. Furthermore, induction of lung injury with lung lavages does not require the injection of pathogens/endotoxins that impact on measurements of pro- and anti-inflammatory cytokines. A disadvantage of the model is the high recruitability of atelectatic lung tissue. Standardization of the model helps to avoid pitfalls, to ensure comparability between experiments, and to reduce the number of animals needed.

Lavage-induced Surfactant Depletion in Pigs As a Model of the Acute Respiratory Distress Syndrome ARDS

Repeated pulmonary lavages in anesthetized pigs induce lung injury resembling major aspects of human acute respiratory distress syndrome (ARDS). For this purpose the lungs are repeatedly lavaged with 0.9% saline at 37 &#176;C. The goal of the protocol is a reproducible mitigation of gas exchange and hemodynamics for research in ARDS.

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

Various animal models of lung injury exist to study the complex pathomechanisms of human acute respiratory distress syndrome (ARDS) and evaluate future ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

导管手术放置了长期的心血管运动试验猪

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

直接插管植入猪的蓄水池

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

神经科学

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

鼠佩耶斑块中淋巴细胞动力学的微观观察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

Intubation, Central Venous Catheter, and Arterial Line Placement in Swine for Translational Research in Abdominal Transplantation Surgery

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries does not solely rely on the research team's surgical skills; perioperative care and management procedures, like intubation, central venous line, and arterial line placement, are necessary and of the utmost importance for favorable experiment results. As it is uncommon for research teams to have anesthesiologists or any other staff other than the surgical team, the surgical team involved in translational research must acquire and/or develop the skills to perform the perioperative care. The purpose of this paper is to show the techniques of intubation, central venous catheter, and arterial line placement used and perfected at the Toronto Organ Preservation Laboratory over the last 10 years, to be used as a reference for future researchers joining either this team or any other lab performing translational research protocols in swine and/or abdominal transplantation.

To obtain the best possible results, surgeons performing translational research experiments should be proficient at intubation and vascular line placement. This paper describes the techniques used by the Toronto Organ Preservation Laboratory to perform these procedures.

Translational surgical research models in swine are crucial for developing safe preclinical protocols. However, the success of the experimental surgeries ...

用于猪长期血管通路的 Hickman 导管

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood monitoring and reliable intravenous fluid and drug administration. Specifically, the tunneled multi-lumen Hickman catheter (HC) is commonly used in swine models due to its lower extrication and complication rates. Despite fewer complications relative to other CVCs, HC-related morbidity presents a significant challenge, as it can significantly delay or otherwise negatively impact ongoing studies. The proper insertion and maintenance of HCs is paramount in preventing these complications, but there is no consensus on best practices. The purpose of this protocol is to comprehensively describe an approach for the insertion and maintenance of a tunneled HC in swine that mitigates HC-related complications and morbidity. The use of these techniques in &gt;100 swine has resulted in complication-free patent lines up to 8 months and no catheter-related mortality or infection of the ventral surgical site. This protocol offers a method to optimize the lifespan of the HC and guidance for approaching issues during use.

作者聚焦：Hickman 导管在临床前猪模型中用于长期血管通路  

A reliable and reproducible approach for the insertion and maintenance of a tunneled Hickman catheter for long-term vascular access in swine is described. Placement of a central venous catheter allows for convenient daily sampling of whole blood from awake animals and intravenous administration of medication and fluids.

Hickman 导管在临床前猪模型中用于长期血管通路

Central venous catheters (CVCs) are invaluable devices in large animal research as they facilitate a wide range of medical applications, including blood ...

在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿

摘要

探索更多视频

此视频中的章节

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

新生儿窒息了猪模型

灌洗诱发的表面活性剂耗尽猪一样的急性呼吸窘迫综合征模型（ARDS）

导管手术放置了长期的心血管运动试验猪

直接插管植入猪的蓄水池

鼠佩耶斑块中淋巴细胞动力学的微观观察

Intubation, Central Venous Catheter, and Arterial Line Placement in Swine for Translational Research in Abdominal Transplantation Surgery

Hickman 导管在临床前猪模型中用于长期血管通路

Hickman 导管在临床前猪模型中用于长期血管通路

Hickman 导管在临床前猪模型中用于长期血管通路