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Medicina

无创微透析 技术从 大鼠心脏动态连续提取血液

Published: September 13, 2022
doi:
10.3791/64531
, , , , ,
1School of Pharmacy,Chengdu University of Traditional Chinese Medicine, 2Research Institute of Integrated TCM & Western Medicine,Chengdu University of Traditional Chinese Medicine, 3School of Ethnic Medicine,Chengdu University of Traditional Chinese Medicine, 4Innovative Institute of Chinese Medicine and Pharmacy,Chengdu University of Traditional Chinese Medicine

Summary

本协议描述了一种使用微透析技术实时和动态收集大鼠心脏血液的简单有效的方法。

Abstract

血液成分的动态分析对于了解心血管疾病及其相关疾病，如心肌梗塞、心律失常、动脉粥样硬化、心源性肺水肿、肺栓塞和脑栓塞等具有重要意义。同时，亟待突破活鼠心脏血液采样技术，评价特色民族药物治疗的有效性。在这项研究中，通过精确且无创的外科手术将血液微透析探针植入大鼠的右颈静脉。然后通过连接到在线微透析样本收集系统，以2.87 nL/min至2.98 mL / min的速率收集心脏血液样本。更重要的是，获得的血液样本可以暂时储存在4°C的微透析容器中。 基于微透析的大鼠心脏在线连续采血方案极大地保证了血液样本的质量，推进和激发了全身性心血管疾病研究的科学合理性，并从血液学的角度评价民族医学治疗。

Introduction

随着生活节奏的加快和心理压力的增加，心血管疾病（CVD）多见于青年、中、老年人1，2。心血管疾病的发病率和死亡率高，具有发病急、进展快、病程长等特点，严重影响患者安全3.CVD的发生可能与某些血液成分的变化密切相关，如胆固醇、血脂、血糖、心肌酶、蛋白激酶K4，5，6。通过分析常规血液检查项目，可以最快速地管理患者的相关情况。因此，血液样本的质量决定了测试结果的准确性。然而，传统的采血方法存在一些不可避免的弊端，严重影响实验结果，如创伤面积大、采血量小、对操作人员要求高、无法实时反映药物变化、血样预处理繁琐、实验动物消耗量大、不符合动物伦理要求等7，8，9.随着医疗技术的不断进步，采血质量也提出了更高的要求。因此，迫切需要开发一种新的血液采样技术来克服上述缺点。

微透析是一种基于透析原理的体内采样技术10。在非平衡条件下，待测化合物沿浓度梯度从组织中扩散并灌注到嵌入组织中的微量透析探针中，透析液与透析液一起连续去除，达到从活组织取样的目的11，12。与传统的采样方法相比，微透析技术在以下几个方面具有显著的优势13、14、15：连续实时跟踪血液中各种化合物的变化;取样不需要繁琐的预处理，可以真实地代表取样现场目标化合物的浓度;探针可以植入身体的不同部位，以研究目标化合物的吸收，分布，代谢，排泄和毒性;采集的样品不含生物大分子（>20 kD）。因此，更高质量的血液样本确保了对心血管疾病和民族医学治疗机制的更好解释。

微量透析取样系统通常由微注射泵、连接管、无动物运动罐、微透析探头和样品收集器16组成。作为微透析系统装置中最关键的部分，常见的微透析探头包括同心探头、柔性探头、线性探头和分流探头17。其中，柔性探针是软性和非金属探针，主要用于从清醒和自由移动或麻醉动物的血管和外周组织（如心脏、肌肉、皮肤和脂肪）中采集样品13。当与血管或组织接触时，探头可以灵活弯曲，从而避免对探头或采样部位造成不可逆转的损坏。随着探针技术的不断发展，微透析技术在各个领域的应用也在不断深化。本文通过无创微透析技术通过专为采血设计的柔性探针动态连续获取大鼠心脏血液。

Protocol

该动物方案已获得成都中医药大学管委会批准（备案号：2021-11）。将指定的无病原体雄性Sprague Dawley（SD）大鼠（8-10周，260-300g）饲养在独立的通风笼中，将实验室环境保持在22°C和65%相对湿度，并用于本研究。这些动物是从商业来源获得的（见 材料表）。在此期间，所有大鼠均习惯于适应性喂养1周，并有自由水和饮食。 1. 实验准备 组装涉?…

Representative Results

本协议允许根据微透析设备中设置的采样参数从有意识的大鼠获得心脏血液。正常的血液样本必须是鲜红色的，而缺氧、潜在血栓或贫血的动物可能呈深紫色或深红色。通过血液微透析技术获得的样品无色、透明、透明，可用于采用高效液相色谱或质谱分析不同疾病的血清标志物以及药物及其代谢产物的血液分布。参数设置和采集的单血量如 表1所示。 <p class="jove_content" fo:keep-toget…

Discussion

心血管疾病是我国临床上常见的慢性病，发病率逐渐增加，发病年龄趋于年轻化，引起大多数患者的关注和恐慌20，21。心血管疾病是世界上导致死亡的主要原因，可诱发脑梗塞等高死亡率疾病，严重威胁患者健康生命22。CVD，包括缺血性心脏病、心肌病、动脉粥样硬化、高血压、中风和心力衰竭，发生在向心脏供血的动脉变窄或变硬?…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金（82104533）、中国博士后科学基金（2020M683273）、四川省科技厅（2021YJ0175）和四川省科技计划重点研发项目（2022YFS0438）的支持。同时，作者要感谢TRI-ANGELS D&H TRADING PTE的高级设备工程师Yuncheng Hong先生。Ltd.（新加坡新加坡市），提供微透析技术服务。

Materials

Animal anesthesia system Rayward Life Technology Co., Ltd R500IE
Animal temperature maintainer Rayward Life Technology Co., Ltd 69020
Blood microdialysis probe  CMA Microdialysis AB T55347
Catheter  CMA Microdialysis AB T55347
Citrate Merck Chemical Technology (Shanghai) Co., Ltd 251275
Electric shaver Rayward Life Technology Co., Ltd CP-5200
Fep tubing  CMA Microdialysis AB 3409501
Free movement tank for animals  CMA Microdialysis AB CMA120
Glucose Merck Chemical Technology (Shanghai) Co., Ltd G8270
Hemostatic forceps Rayward Life Technology Co., Ltd F21020-16
Isofluran Rayward Life Technology Co., Ltd R510-22
Micro scissors Beyotime Biotechnology Co., Ltd FS221
Microdialysis collection tube  CMA Microdialysis AB 7431100
Microdialysis collector  CMA Microdialysis AB CMA4004
Microdialysis in vitro stand  CMA Microdialysis AB CMA130
Microdialysis microinjection pump  CMA Microdialysis AB 788130
Microdialysis syringe (1.0 mL)  CMA Microdialysis AB 8309020
Microdialysis tubing adapter  CMA Microdialysis AB 3409500
Microporous filter membrane Merck Millipore Ltd. R0DB36622
Non-absorbable surgical sutures Shanghai Tianqing Biological Materials Co., Ltd S19004
Operating table Yuyan Scientific Instrument Co., Ltd 30153
Ophthalmic forceps Rayward Life Technology Co., Ltd F12016-15
Sodium citrate Merck Chemical Technology (Shanghai) Co., Ltd 1613859
Sprague Dawley  (SD) rats Chengdu Dossy Experimental Animals Co., Ltd SYXK(Equation 1)2019-049
Surgical scissors Rayward Life Technology Co., Ltd S14014-15
Surgical scissors Shanghai Bingyu Fluid technology Co., Ltd BY-103
Syringe needle  CMA Microdialysis AB T55347
Ultrasonic cleaner Guangdong Goote Ultrasonic Co., Ltd KMH1-240W8101
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Tags

MicrodialysisBlood ExtractionRat HeartNoninvasiveCardiovascular DiseaseBlood BiomarkersLiquid ChromatographyMass SpectrometryMyocardial InfarctionApneaAtherosclerosisCardiogenic Pulmonary EdemaPulmonary EmbolismCerebral EmbolismJugular VeinCatheterProbeSemipermeable MembraneAnticoagulantFree-movingDialysis Membrane
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Hou, Y., Bai, J., Zhang, Y., Meng, X., Zhang, S., Wang, X. Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique. J. Vis. Exp. (187), e64531, doi:10.3791/64531 (2022).
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