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用于小鼠纵向采血的改良锁骨下心脏穿刺技术(英文)

马雨婷, 党青亚, 钟思一, 包鑫, 王诗语, 全海莲, 洪兰^*

延边大学医学院生理与病理生理学教研室，延吉 133002

摘要

小鼠采血是药代动力学研究和慢性疾病建模的重要环节。然而，常规方法如尾静脉和眼眶后采血，采血量有限且频率受限，难以满足长期、大量采血的需求。本研究旨在建立一种可重复、大量采血的小鼠锁骨下心脏穿刺技术，并确定其在不影响全身生理的前提下的最大安全采血量与频率。本研究选取36 只健康成年C57BL/6 小鼠(体重20~30 g，雌雄各半)，随机分为6 组：对照组、假手术组，以及4 个实验组，每组6 只。四个实验组每周按总血量(total blood volume, TBV)的7.5%、15%、22.5%或30%进行采血，持续采血4 周。在异氟烷麻醉下，对小鼠行锁骨下心脏穿刺采血。采血时取仰卧位固定小鼠，于左锁骨中点平行胸壁进针1~1.5 cm，见搏动性回血后缓慢抽取血液可降低气胸、脏器损伤风险；采血器具预先用EDTA-2K抗凝剂润管，根据小鼠体重按72 mL/kg估算TBV后精准计算采血量，可避免采血量偏差造成实验误差。监测指标包括体重、收缩压、饮水量、被毛状况及血液学指标[红细胞计数(red blood cell count, RBC)、血红蛋白(hemoglobin, HGB)、红细胞比容(hematocrit, HCT)、平均红细胞体积(mean corpuscular volume, MCV)、平均血红蛋白量(mean corpuscular hemoglobin,MCH)、平均血红蛋白浓度(mean corpuscular hemoglobin concentration, MCHC)]，并进行血涂片形态学观察。结果显示，30% TBV组可耐受采血2 周；22.5% TBV组可耐受采血3 周；7.5%和15% TBV组均可耐受采血4 周且无不适。30% TBV组小鼠体重增长幅度最小，并于实验后期出现轻度红细胞大小不均，此阶段的血压较采血第0 天基线水平下降，RBC、HGB、HCT和MCHC在采血第21 天低于正常参考范围。22.5% TBV组在实验后期亦出现血压下降，且RBC、HGB和HCT在采血第21 天低于正常参考范围。以上结果表明，改良锁骨下心脏穿刺可实现安全、重复的大量采血(安全采血量达15% TBV)，适用于药代动力学及纵向生物标志物研究，可提高数据可靠性，并减少实验动物使用数量与应激。

关键词： 血液标本采集; 心脏穿刺; 血液检测; 小鼠; 生理监测

A refined subclavian cardiac puncture method for longitudinal blood collection in mice

MA Yu-Ting, DANG Qing-Ya, ZHONG Si-Yi, BAO Xin, WANG Shi-Yu, QUAN Hai-Lian, HONG Lan^*

Department of Physiology and Pathophysiology, School of Medicine, Yanbian University, Yanji 133002, China

Abstract

Murine blood sampling is essential for pharmacokinetic research and chronic disease modeling. However, conventional techniques such as tail vein or retro-orbital blood collection have low sample volumes and limited frequency, rendering these approaches inadequate for long-term, volume-intensive studies. This study aimed to establish subclavian cardiac puncture (SCP) as a technique for repeated, high-volume blood collection in mice, and to define its maximum safe volume and frequency without compromising systemic physiology. Thirty-six healthy adult C57BL/6 mice (20-30 g, with equal male and female numbers) were randomized into six groups: control, sham, and four experimental cohorts receiving weekly blood removal at 7.5%, 15%, 22.5%, or 30% of total blood volume (TBV) for four weeks (n = 6 per group). Under isoflurane anesthesia, blood collection was performed on mice via SCP. Mice were secured in supine position before puncture, with needle inserted 1-1.5 cm horizontally at the midpoint of left clavicle; slow blood aspiration upon pulsatile blood backflow effectively prevented pneumothorax and cardiac trauma. All syringes and collection tubes were pre-rinsed with EDTA-2K anticoagulant, and TBV was calculated by the standard 72 mL/kg body weight formula to guarantee accurate blood withdrawal proportion. Monitoring indicators included weight, systolic blood pressure, water intake, fur condition, and hematological indices, such as red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Morphological observations of blood smears were also conducted. The results showed that the 30% TBV group tolerated blood collection for two weeks; the 22.5% TBV group for three weeks; and the 7.5% and 15% TBV groups for all four weeks without distress. The 30% TBV group showed the smallest increase in body weight and developed mild anisocytosis at the later stage of the experiment; in this stage, blood pressure decreased compared to the baseline level on day 0 of blood collection, and RBC, HGB, HCT, and MCHC values were below the normal reference ranges on day 21 of blood collection. The 22.5% TBV group also showed a later-stage decrease in blood pressure, with RBC, HGB, and HCT values below the normal reference ranges on day 21 of blood collection. These results suggest that the refined SCP enables safe, repeated, high-volume sampling in mice (with a safe blood collection volume of 15% TBV), supports pharmacokinetic and longitudinal biomarker studies, enhances data reliability, and reduces the number of animals used and stress.

Key words: blood specimen collection; cardiac puncture; blood testing; mice; physiological monitoring

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通讯作者：洪兰　　E-mail:

DOI: 10.13294/j.aps.2026.0065

引用本文：

马雨婷, 党青亚, 钟思一, 包鑫, 王诗语, 全海莲, 洪兰. 用于小鼠纵向采血的改良锁骨下心脏穿刺技术(英文)[J]. 生理学报 2026; 78 (4): 943-955.

MA Yu-Ting, DANG Qing-Ya, ZHONG Si-Yi, BAO Xin, WANG Shi-Yu, QUAN Hai-Lian, HONG Lan. A refined subclavian cardiac puncture method for longitudinal blood collection in mice. Acta Physiol Sin 2026; 78 (4): 943-955 (in Chinese with English abstract).