建立大鼠永久性颈静脉插管的显微外科技能，用于口服药物的连续血液采样
Summary
详细的显微外科技术被证明可以建立一个长期的颈静脉插管大鼠模型，用于同一动物的序贯血液采集。在大鼠的恢复阶段监测生理和血液学参数。该模型已应用于研究口服多酚的药代动力学，而不会诱导动物应激。
Abstract
小型实验动物的血液采样对于药物先导物优化是必要的，但会对实验动物造成巨大的伤害和压力，这可能会影响结果。大鼠的颈静脉插管（JVC）是一种广泛使用的重复采血模型，但需要对手术技能和动物护理进行充分的培训。本文详细介绍了用于建立和维护永久性JVC大鼠模型的显微外科手术程序，特别关注颈静脉套管的放置和密封。监测生理（例如，体重，食物和水摄入量）和血液学参数的重要性，在大鼠恢复期间在手术后6天内提供结果得到强调。在JVC大鼠模型中测定口服天然酚鞣花酸的药物 – 血浆浓度 – 时间曲线。
Introduction
从小型实验动物（如啮齿动物、豚鼠和兔子）中反复采集血液样本是药物先导物优化和减少研究中使用的动物数量的重要方面1，2。开发新的诊断工具和药物制剂（例如疫苗）的管道需要获得不同体积的血液，以评估其 在体内的稳健性和性能，例如药代动力学（PK），毒性和敏感性3，4，5。
实验室采集血液样本的方法大致分为两种类型，外科和非手术6。对于研究人员来说，非手术方法相对容易掌握，其中包括常见的技术，例如心脏穿刺，眼眶窦穿刺以及隐静脉和尾静脉出血。通过一些非手术方法可以进行多次血液采样，但样本量很小，会对动物造成身体伤害和心理压力1。另一方面，手术方法是重复静脉穿刺的最爱替代方案，它涉及在动物血管中放置临时或永久性套管7，8，9。在有意识的大鼠中，可以通过套管反复抽取大血容量，同时避免由于处理技术，抑制和麻醉引起的压力和疼痛7，8，10，11。然而，套管植入需要经验丰富的研究人员进行足够的培训，才能成功收集血液。
通过大鼠颈静脉插管（JVC）收集血液是研究药物PK6，10，12，13的最广泛使用的方法。然而，JVC大鼠模型的建立需要仔细练习显微外科技能和术后护理和维护知识。特别是，手术后，大鼠需要给予镇痛药和足够的恢复时间才能达到稳定的生理状态，用于进一步的实验13，14，15。虽然体重增加（即>10g）是大鼠恢复的有效且常用的指标，但大鼠由于脱水，感染和炎症而在术后意外死亡并不罕见，这在早期发病时可能很微妙地注意到14，15。此外，JVC模型中的导管阻塞仍然是采血过程中的一个问题。
本方案已详细演示了麻醉大鼠JVC的显微外科手术，特别关注颈静脉的识别，分离和插管。强调了在恢复阶段对大鼠进行生理和血液学监测的重要性。最后，通过静脉导管收集连续血液样本，研究口服生物利用度差（即全身浓度低）的天然酚鞣花酸的PK，以验证JVC大鼠模型。
Protocol
以下描述的程序是作为西北工业大学机构动物护理和使用委员会（第202101117号）批准的协议的一部分执行的。 1.术前准备（手术前一天） 注意：所需溶液：生理盐水（0.9%w/v氯化钠），肝素化盐水（1%w/v肝素钠），导管锁溶液，非甾体抗炎药（NSAID），如美洛昔康溶液（2mg / mL）。 溶液制备 等分试样200μL预包装导管锁定溶液在1.5mL…
Representative Results
该协议已彻底展示了如何使用显微外科技能建立长期JVC模型以进行连续采血。 图1A 显示了用于进行手术的基本手术器械和材料。还说明了具有三个蓝色标记的PU导管的规格，这有助于指导研究人员在步骤3.3中放置静脉套管，如何使用PU导管上的标记来引导插管（图1B）。同样重要的是要了解建立JVC大鼠模型所需的时间表（图1C</stron…
Discussion
掌握血管插管技术需要大量的练习，并从每次操作中吸取教训。Christakis等人使用累积总和（CUSUM）分析发现，研究人员需要在一年内练习200只大鼠，然后才能准备对候选药物进行PK评估20只。然而，静脉插管所需的手术时间可以通过执行13，20个大鼠的数量显着减少。使用我们的方案，有效套管颈静脉和收集血液样本的成功率从约50%增加…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由国家自然科学基金（82003692号）资助给张磊。西北工业大学最高学术奖学金授予R. Miao。
Materials
| 0.5 mL test tube containing EDTA anticoagulant | Xinkang | N/A | collecting blood samples for hematology test |
| 0.5*20 mm 1.0-mL syringe | KLMEDICAL | N/A | washing or replacing the fluid with saline |
| 0.6*28.5 mm 5.0-mL syringe | HD | N/A | Subcutaneous injection |
| 1.0-mL Blunt tipped syringe (22G) | skillsmodel | S4-PKT22G | Inject the saline and collect blood samples through catheter |
| 1.5 mL sterile microcentrifuge tube | Axygen | MCT-150-C-S | Store sterile catheter lock solution heparinized saline and meloxicam solution |
| 1.5 mL microcentrifuge tubes | Biosharp | BS-15-M | blood collection |
| 1/2 circle cutting 5*12 mm suture needle | skillsmodel | S4-FHZ | Thread the muscle layer to fix the catheter |
| 3/8 circle cutting 7*17 mm suture needle | skillsmodel | S5-FHZ | Suture the incision of rat cortex |
| 6-0 sterile non-absorbable silk suture thread | JUNSHENG | N/A | ligature |
| 75% medical alcohol | HONGSONG | N/A | Disinfection |
| Adhensive tape | LIUTAI | N/A | positioning the rat |
| Autoclave sterilization tape | Biosharp | BS-QT-028 | Mark sterilized items |
| Automated blood cell counter | Sysmex | XN-550 | Hematology test |
| Castroviejo micro scissors | skillsmodel | WA1010 | Cut the opening in the blood vessel |
| Centrifuge | Thermo Fisher Scientific | 75002402 | Plasma preparation |
| Clean cushion | Qingjie | N/A | Prepare the operation area |
| Cotton balls | HC | N/A | Wound disinfection and sterilization |
| Cotton swabs | BEITAGOGO | N/A | Disinfection |
| Curved hemostat | skillsmodel | N/A | ligature |
| DN50 Stainless-steel rat restrainer | skillsmodel | S4-RGDQ1 | Restrict the movement of rats for easy operation |
| Ellagic acid | Aladdin | E102710-25g | natural phenol for oral administration |
| Half-curved forceps | skillsmodel | 53072 | Lift the muscle layer and tissue, isolate the jugular vein and tie the suture |
| Heating pad | Warm mate | N/A | preventing heat loss of animal |
| Heparin sodium | Solarbio | H8060 | anticoagulant |
| Iodophor | Xidebao | N/A | Clean the wound |
| Iris scissors | skillsmodel | 54002 | Bluent separation the muscle layer |
| Isoflurane | RWD | R510-22-16 | anaesthesia |
| LED lamp | EMPERORFEEL | N/A | sugery |
| Liquid chromatography-mass spectroscopy | Thermo Fisher Scientific | VQF01-20001/ TSQ02-10002 | detection of drug concentration in plasma |
| Meloxicam | Hongqiang | N/A | Analgesic |
| Normal saline | KL | N/A | Prepara the solution and protect blood vessels from drying out |
| Pet razor | Codos | 3180 | Shaving the fur |
| Phosphate-buffered saline | ZHHC | PW012 | Preparation of Ellagic acid solution |
| PU catheter | skillsmodel | RJVC-PU | Jugular vein cannulation |
| Small animal operation anesthesia console | RWD | 68620 | Operation workstation |
| Spray bottle | Other | N/A | aseptic workstation |
| Stainless steel plug (22G) | skillsmodel | S4-PKD22G | Plug the catheter to ensure its sealing |
| Stainless steel trochar | skillsmodel | S$-PKDGZ | Guide the catheter exteriorization |
| Sterile lock solution | skillsmodel | SK-FB | lock the catheter to ensure its sterility |
| Straight feeding needle | skillsmodel | N/A | Oral gavage |
| Surgical pouch | BKMAM | N/A | container for sterilization of surgical instruments |
| Surgical scissors | skillsmodel | J21070 | Cut incision on rat skin |
| Vessel dilator balanced forceps | skillsmodel | WA3020 | Expand the blood vessel and guide the cannula to slide in |
| ZS-MV Small animal anesthesia machine | ZSLab | 1057003 | inducing and maintaining anaesthesia |
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Cite This Article
Lu, W., Miao, R., Hu, S., Liu, J., Jin, F., Zhang, R. X. Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug. J. Vis. Exp. (178), e63167, doi:10.3791/63167 (2021).