在新生儿猪淋巴的肠淋巴躯干和收集的长期导尿
Summary
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.
Abstract
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.
Introduction
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。
Protocol
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?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Miller laryngoscope blade | Welch Allyn | 68044 | 182 mm length |
| Surgivet advisor: Vital signs monitor | Surgivet | V9203 | |
| Rectal temperature probe | Surgivet | V3417 | |
| Mono-polar electrosurgery generator | Valley Lab | ||
| Metzenbaum scissors | Fine Science | 14518-18 | |
| Tuffier retractor | Stevens | 162-11-676 | |
| Mosquito forceps | Stevens | 162-7-10 | |
| Kelly forceps-curved (14cm) | Stevens | 162-7-38 | |
| Allis tissue forceps | Stevens | 162-7-38 | |
| Forceps dressing-eye (10.2cm) | Stevens | 162-18-780 | |
| Forceps dressing-Adison (12.1cm) | Stevens | 162-17-2510 | |
| Needle Drivers | Stevens | 162-V98-42 | |
| Iris scissors | Fine science | 14058-11 | |
| Circulating water pump | Jorvet | J-783X | |
| Maxitherm-Vinyl blanket | Jorvet | J-784C | |
| Q tip applicators | Fisher Scientific | 22-037-960 | |
| Catheterization tubing (4.06 OD X 2.31 ID) | Braintree Scientific Inc. | MRE-160 | Micro-Renethane implantation tubing |
| 2-0 silk suture | Ethicon | LA556 | |
| 2-0 polyglactin suture | Ethicon | J443H | 2-0 vicryl |
| Large animal jacket | Lomir Biomedical Inc. | SSJ2YC | |
| Polypropylene wash bottles | Fisher Scientific | 03-409-22C | 500 ml |
| Penicillin-Streptomycin | Sigma Aldrich | D4333 | |
| EDTA | Sigma Aldrich | 60-00-4 | |
| Amphotericin B | Sigma Aldrich | A2411 | |
| Azaperone | Elanco Animal Health | Stresnil | |
| Dexmedetomidine hydrochloride | Zoetis | 6295 | Dexdomitor |
| Isoflurane | Abbott Animal Health | 05260-5 | IsoFlo |
| Ketamine hydrochloride | Zoetis | 2626 | Ketaset |
| Bupenorphine hydrochloride | Champion Alstoe Animal Health | DIN:02347510 | |
| 6 mm Endotracheal tube | Jorvet | J-165d | |
| 10% Lidocaine spray | AstraZeneca | DIN:02003767 | |
| 4 % Chlorhexidine surgical scrub | Partnar Animal Health | PCH-011 | Diluted: 2.0% solution |
| 3M Surgical steri- drape | 3M Health Care | 1040 | |
| SDS page gel | Invitrogen | EA0375BOX | 3-8 % tris acetate |
| Polyvinylidene fluoride membrane | Millipore | IPVH00010 | 0.45 μm pore size |
| ApoB antibody | EMD Millipore | AB742 | 1:4000 dilution |
| Donkey anti-goat IgG-HRP | Santa Cruz Biotechnology | Sc-2304 | |
| ECL Prime Western Blotting Reagent | GE Healthcare LifeSciences | RPN2232 | |
| Triglyceride Kit | Wako Pure Chemicals | 998-40391/994-40491 | |
| Total Cholesterol Kit | Wako Pure Chemicals | 439-17501 | |
| Total Protein | Pierce | 23225 | Bicinchoninic Acid Assay |
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Cite This Article
Uwiera, R. R., Mangat, R., Kelly, S., Uwiera, T. C., Proctor, S. D. Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs. J. Vis. Exp. (109), e53457, doi:10.3791/53457 (2016).