Summary

肝硬化大鼠门脉高压综合征的侵袭性血流动力学特征

Published: August 01, 2018
doi:

Summary

在这里, 我们描述了一个详细的协议, 以侵入性测量的血流动力学参数, 包括门压力, 内脏血流量, 系统血流动力学, 以表征门脉高压综合征的大鼠。

Abstract

这是一个详细的协议描述侵袭性血流动力学测量肝硬化大鼠门脉高压综合征的特点。肝硬化门静脉高压症 (PHT) 是导致肝病患者最严重并发症的原因。门脉高压综合征的全貌是由于肝内血管阻力增加 (IHVR), 存在高动力循环, 内脏血流量增加, 门静脉压力增加 (PP)。进展性内脏动脉舒张和增加心脏输出与高心率 (HR), 但低动脉压力特点的门脉高压综合征。

目前正在开发新的治疗方法, 目的是降低 PP 的靶向 IHVR 或增加内脏血流-但副作用的系统性血流动力学可能会发生。因此, 需要对门静脉、内脏和全身血流动力学参数进行详细的描述, 包括测量 PP、门静脉血流量 (PVBF)、肠系膜动脉血流量、平均动脉压 (MAP) 和 HR 是临床前PHT 新疗法的疗效评价。我们的视频文章为读者提供了一个结构化的协议来执行肝硬化大鼠的侵袭性血流动力学测量。特别是, 我们描述了通过回结肠静脉插管的股动脉和门静脉, 并通过周围多普勒超声流量探头测量门静脉和内脏血流量。PHT 了不同大鼠模型的代表性结果。

Introduction

PHT 被定义为门静脉系统的病理增加的血压, 可能导致严重并发症的肝硬化患者, 如静脉曲张出血和腹水1。肝前 (门静脉血栓) 和肝后 (布-加综合征) PHT 少见, 肝硬化肝内 PHT 是 PHT2的最常见病因。

在肝硬化中, PP 主要是由于升高的 IHVR3而增加的。在晚期, PHT 的增加 PVBF 由于增加的心脏输出和减少系统性和内脏血管阻力-定义门脉高压综合征4。欧姆定律 (ΔP = Q R) 意味着 IHVR 和血流与 PP5成正比。在患者中, 直接测量 PP 是危险的, 没有例行执行;相反, 肝静脉压力梯度 (HVPG) 被用作间接测量 PP6,7。HVPG 的计算方法是从楔形肝静脉压 (WHVP) 中减去游离肝静脉压 (FHVP), 用放置在肝静脉中的气囊导管测量8。1–5 mmHg 之间的生理 HVPG 范围, 而 HVPG ≥10 mmHg 定义临床重要的门静脉高压 (CSPH), 表明 PHT 相关并发症的风险增加, 如静脉曲张出血, 腹水和肝性脑病9.尽管 PP (HVPG) 是 PHT 严重性的最相关参数, 但有关 PHT 其他成分的信息, 包括存在高动力循环的严重性 (HR、MAP)、内脏/肠系膜动脉血流和 IHVR, 对于全面了解 PHT 的基本机制。

因此, 与间接测量人的 pp 相比, 引入的方法为大鼠提供了直接测量 pp 的优势, 并允许记录额外的血流动力学参数表征门脉高压综合征。此外, 直接测量 PP 是一个很好的综合读数的肝纤维化的数量 (IHVR 的一个主要决定因素), 克服了一定程度的纤维化量化相关的肝组织抽样错误。

肝硬化 PHT 最常用的啮齿动物模型包括手术胆管结扎术 (BDL)、毒素诱发肝损伤 (四氯化碳、硫乙酰胺或硝胺管理) 和饮食诱导的代谢性肝。疾病模型。肝前性 (非肝硬化) PHT 可由部分门静脉结扎术 (PPVL)10引起。

小啮齿类动物非常适合所提出的方法, 包括老鼠, 仓鼠, 大鼠, 或兔子, 并与相对较低的维护成本。尽管所有的血流动力学评估是可行的, 在小鼠, 更好的准确性和重现性的结果被发现与大鼠或较大的啮齿动物由于明显的优势, 动物的大小。此外, 还需要特定的微仪器和器件来获得类似的小鼠血流动力学参数。最后, 大鼠更健壮, 相关的发病率和死亡率较低, 因此, 大鼠的辍学率可能低于小鼠。

所提出的方法非常适合评估肝病的具体治疗 (抗纤维化或消炎药) 或新的药理学方法, 影响血管张力和/或内皮生物学;因此, 可能影响 PHT 的血流动力学参数。

Protocol

这里描述的所有方法都已获得维也纳医科大学道德委员会和奥地利科学、研究和经济部 (BMWFW) 的批准。由于血流动力学测量代表手术干预, 必须在手术室或类似清洁工作区的无菌条件下执行程序。一般情况下, 建议在无菌条件下工作。当使用吸入麻醉时, 考虑适当的通气手术室的工作安全。在本议定书中提出的所有血流动力学读数的情况下, 必须考虑 40–50 min/动物的时间段。 1. ?…

Representative Results

根据动物模型和肝脏疾病的严重性, 门脉高压综合征的 PHT 程度和严重性不同 (图 7)。 BDL 模型导致胆汁性肝硬化胆汁淤积。因此, 随着时间的推移, PP 的增加和存在高动力的循环发展, 从人力资源的增加和地图的减少看到。在肝硬化动物中, SMABF、PVBF 和 IHVR 也增加和谐肝脏和血流动力学的改变 (<strong class="xf…

Discussion

PP 是评价门脉高压综合征的主要结局参数, 反映肝硬化的严重程度。基质沉积 (纤维化) 和正弦血管收缩 (由于血管收缩剂肝表达的增加和对血管扩张剂的反应减少) 导致 IHVR 增加。聚丙烯的重要性和它对慢性肝病的影响已显示在多个临床前11,12,13,14和临床研究15,<sup cla…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢兽医, 护士和动物饲养者在生物医学研究中心的持续支持, 在我们的研究项目。作者承认本议定书所有审评员的重要投入。其中一些研究由奥地利胃肠病和肝病学会的 “青年科学奖” (ÖGGH) 到 PS 和奥地利内科医学协会的 “斯柯达奖” 资助。

Materials

Instruments
LabChart 7 Pro software ADInstruments, Colorado Springs, CO, USA  - Software
ML870 PowerLab 8/30 ADInstruments, Colorado Springs, CO, USA  - Electronic multichannel recorder
MLT0380/D ADInstruments, Colorado Springs, CO, USA  - Pressure transducer (x2: for Portal Pressure and Arterial Pressure)
ML112 Quad Bridge Amplifier ADInstruments, Colorado Springs, CO, USA  - Bridge amplifier
TS420 Transonic Systems Inc., Ithaca, NY, USA  - Flowmeter module
Biological Research Apparatus 7025 UGO BASILE S.R.L., Comerio, Italy  - Ventilator
Vapor 2000 Dräger Medical AG & Co. KG, Lübeck, Germany  - Isofluran Vaporizer
Perivascular probes (rat) for Transonic systems (Superior Mesenteric Artery) Transonic Systems Inc., Ithaca, NY, USA #MA1PRB Ultrasonic flow probe (1mm)
Perivascular probes (rat) for Transonic systems (Portal Vein) Transonic Systems Inc., Ithaca, NY, USA #MA2PSB Ultrasonic flow probe (2mm)
1st for intubation & 2nd for clean skin incisions  -  - Mayo scissor [x2]
Metzenbaum scissor  -  -  -
Cuticle scissor  -  -  -
e.g. Adson Brown tissue forceps  -  - Tissue Forceps
High precision 45° angle broad point forceps [x2]  -  -  -
Hemostat [x4]  -  -  -
e.g. Mikulicz peritoneal clamp  -  - Curved clamp
e.g. Dieffenbach clamp  -  - Micro clamp
e.g. micro spatula with flat ends, width 4 mm,  -  - Micro metal spatula
for transbuccal suture at intubation  -  - Needle holder
Scalpel grip  -  -  -
selfmade  -  - Intubation desk
blut, flexible and with a suitable diameter for arterial cannula and venflow  -  - Blunt steel wire
modified arterial line 20G with Flowstich Becton Dickinson, Farady Road, Swindon, UK #682245 Arterial line
Heating pad  -  -  -
Rectal temerature probe  -  -  -
Saline heater  -  -  -
Laryngoscope (specific for animal size, e.g. rat)  -  -  -
Inductionbox for inhalation anesthesia  -  -  -
Scale (able to measure mg)  -  -  -
Hair clipper  -  -  -
Name Company Catalog Number Comments
Consumables
e.g. modified BD Venflon Pro Safety 14GA Becton Dickinson Infusion Therapy, AB, SE251 06 Helsingborg, Sweden #393230 Peripheral venous catheter (14G)
Fine-Bore Polyethylene Tubing, ID 0.58mm, OD 0.96mm, Portex, Smiths Medical International Ltd., Kent, UK #800/100/200 Catheter tube (PE-50)
e.g. Omnifix-F Solo B. Braun Melsungen AG, Melsungen, Germany #9161406V Syringe 1mL
e.g. Injekt Solo B. Braun Melsungen AG, Melsungen, Germany #4606051V Syringe 5mL
e.g. Injekt Solo B. Braun Melsungen AG, Melsungen, Germany #4606205V Syringe 20mL
e.g. BD Microlance 3, 18G – 1 1/2" Becton Dickinson S.A., Fraga, Spain #304622 Cannula (18G)
e.g. BD Microlance 3, 23G – 1" Becton Dickinson S.A., Fraga, Spain #300800 Cannula (23G)
e.g. BD Microlance 3, 30G – 1/2" Becton Dickinson S.A., Fraga, Spain #304000 Cannula (30G)
e.g. Leukoplast S BSN medical GmbH, Hamburg,  Germany #47619-00 Adhesive tape
e.g. Gazin RK Mullkompressen (18x8cm) Lohmann & Rauscher, Vienna, Austria #10972 Gauze compress (small)
e.g. Gazin RK Mullkompressen (5x5cm) Lohmann & Rauscher, Vienna, Austria #10961 Gauze compress (big)
Silk Braided black, USP 4/0, EP 1.5 SMI AG, St. Vith, Belgium #2021-04 Suture (Silk 4/0, EP 1.5)
e.g. Mersilk, 2-0 (3 Ph. Eur.), PS-1 Prime Johnson & Johnson Medical GmbH – Ethicon Deutschland, Germany #EH7552 Transbuccal suture
e.g. Cottonbuds (2.2mm, 15cm) Paul Hartmann AG, Heidenheim, Germany #967936 Cotton buds
e.g. Vue Ultrasoundgel Optimum Medical Limited, UK #1157 Ultrasound gel
e.g. Glubran 2 Gem srl, Viareggio, Italy #G-NB2-50 Tissue glue
e.g. Surgical scalpell knife Nr. 10 – carbon steel Swann-Morton, England, B.S. #202 Scalpel Knife
Heparin, 5000 i.E./mL (Natriumheparin) Medicamentum Pharma GmbH, Allerheiligen im Mürztal, Austria  - Heparin
Florane Aesica Queenborough Ltd., Queenborough, UK  - Isoflurane
OeloVital (5g) Fresenius Kabi Austira Gmbh, Graz, Austria  - Eye gel
Ketasol aniMedica GmbH, Senden-Bösensell, Germany  - Ketamine
Rompun Bayer Austria Ges.m.b.H., Vienna, Austria  - Xylazine
Xylocain 10% Pumpspray AstraZeneca Österreich GmbH, Vienna, Austria  - Lidocaine pump spray
Dipidolor Jansen-Cilag Pharma GmbH, Vienna, Austria  - Piritramide
NaCl 0.9% Fresenius, 1L Fresenius Kabi Austira GmbH, Graz, Austria #13LIP132 Physiological saline solution

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Cite This Article
Königshofer, P., Brusilovskaya, K., Schwabl, P., Podesser, B. K., Trauner, M., Reiberger, T. Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats. J. Vis. Exp. (138), e57261, doi:10.3791/57261 (2018).

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