本研究得到了兰州大学第二医院翠英计划项目（批准号：The Cuiying Plan Project）的支持。PR0121015）和甘肃省泌尿系统疾病研究重点实验室（批准号：0412D2）。

本研究遵循了《实验动物护理和使用指南》第 8 版中概述的指南13。该研究获得了兰州大学第二医院伦理委员会的批准，并按照ARRIVE指南2.014进行了记录。12-16周龄的12只健康Wistar大鼠（6只雄性体重290-330 g，6只雌性体重250-280 g）在兰州大学GLP动物实验室进行3天实际实验。使用的鼠笼为R5型，尺寸为545 mm x 395 mm x 200 mm，并配备了高压灭菌垫料。所有大鼠都可以不受限制地获得食物和水。实验室保持平均湿度为25%，平均温度为24°C，昼夜交替（上午7：00/晚上7：00）。在研究结束时，所有动物都使用过量的异氟烷被人道地安乐死。有关本研究中使用的材料和仪器的全面信息，请参阅 材料表。
1. 样本量计算和动物选择
<ol><li>选择资源方程方法15 ，使用方程（1）估计动物样本量。 E = 动物总数 − 组总数 （1） 其中 E 是方差分析的自由度 （ANOVA），范围为 10 到 20。 注意：在这项研究中，将12只动物分为 A 组和 B 组（每组三只雄性和三只雌性）。</li>
	<li>将本研究的主要结果定义为单个人重复采血的成功率和时间消耗。</li>
	<li>将次要结果指标定义为大鼠体重、食物和水摄入量的变化，以及不良事件的发生率（如锁骨骨折、皮下血肿、气胸和死亡率）。</li>
	<li>将成功的采血定义为满足以下标准：i） 单次采血少于三次;ii） 总时间（从大鼠约束到完成采血）不超过 5 分钟;iii）在获得透明血浆的同时达到目标血容量。将任何偏离这些标准的行为视为抽样失败。</li>
</ol>2. 动物约束和采血
注意： A 组和 B 组大鼠的血液样本由两名经验丰富的研究人员收集，他们都抽取了至少100份血液样本。在4天内从两组大鼠中收集血液样本共96次。这种采血方法不需要对大鼠进行麻醉或额外的约束装置。然而，它需要熟练的处理技术。
<ol><li>在采血前一天（第1天）上午8：00，将每只大鼠分配到其单独的笼子中，同时称量其食物和水。然后，让另一位研究人员对测量结果视而不见，从第 1 天开始每天上午 8：00 记录大鼠的体重、食物消耗量和饮水量。</li>
	<li>要遵循此方案，首先在每天上午 10：00 抽血，然后在晚上 10：00 抽血，从两侧锁骨下静脉交替采集 0.15 mL 血液。 注意：要收集的血液量由体重最低的大鼠在一周内可以忍受的最大体积决定。</li>
	<li>用肝素钠（25 U / mL）冲洗注射器，并用酒精对注射部位进行消毒。</li>
	<li>轻轻抚摸大鼠的背部皮肤并反复捏住它的脖子，以帮助大鼠放松（视频1）。</li>
	<li>使用非惯用手的拇指和食指，牢牢抓住并抬起大鼠的颈部皮肤（图1A 和 视频1）。</li>
	<li>在惯用手的协调下，用剩下的三个手指和非惯用手的手掌固定大鼠的背部皮肤并固定其前肢（图1B，C和视频2）。 注意：如果老鼠反抗或挣扎，可以重复几次此过程，以帮助老鼠习惯处理。以下步骤是成功采血的关键。</li>
	<li>使用非惯用手的食指，轻轻向下推大鼠的头部皮肤，而其他手指和手掌则帮助向外旋转肩关节。在此过程中，用惯用手完全伸展大鼠的肩关节（图1D-F和视频2）。</li>
	<li>用非惯用手牢牢抓住大鼠，使大鼠的头部和身体对齐成一条直线（图1G，H）。然后，用惯用手定位锁骨的位置并确认穿刺部位（图1I，视频2和视频3）。 注意：没有必要给老鼠剃毛。在 图 1 中，剃须只是为了在更大程度上显示锁骨和穿刺位置。当约束大鼠，尤其是大鼠>350克时，让大鼠将脚放在坚实的表面上将有助于支撑它们的体重。此外，约束器在采血时应监测每只大鼠的呼吸频率，以确保约束装置不会太紧，这可能会导致呼吸窘迫。</li>
	<li>用惯用手握住与大鼠身体平行的注射器，针尖朝上，注射器秤朝向实验者，与大鼠身体中线保持约15°角。将针插入锁骨切迹下方0.5厘米处（锁骨近端三分之一和胸骨的交界处），确保针头与大鼠的身体保持平行（图1J 和 视频3）。 注意： 应特别注意针头插入的角度和深度，以免刺穿血管或对相邻血管造成意外损伤。</li>
	<li>轻轻抽出注射器以产生负压，通常伴有进入血管时可触及的突破感（在初始采血期间尤为明显）。保持这个姿势，并根据需要以恒定速度收集0.1-1.0mL血液（遵循IACUC指南，每周约4-5.3mL / kg血液1）（图1K 和 视频3）。</li>
	<li>如果穿刺时没有血，请尝试轻轻调整针头的角度和深度或轻轻旋转注射器（视频 3）。如果在同一侧连续三次尝试均不成功，请停止所有出血，然后切换到另一侧进行穿刺。 注意：建议快速刺穿皮肤，以防止大鼠因不适而挣扎。</li>
	<li>用棉签止血，然后将大鼠放回笼子里（视频4）。</li>
	<li>根据实验要求处理血液样本。</li>
</ol>3. 血样处理
<ol><li>将注射器针头倒入锐器容器中。将收集的血液转移到先前用肝素冲洗的 1.5 mL 微量离心管中。将管放入离心机中，将其设置为4°C和1,200× g，并离心10分钟以分离血浆。使用1.0mL巴斯德移液管将血清转移到干净的微量离心管中，并将其储存在-80°C。 注意： 为防止因压力而溶血，必要时请取下针尖。在血浆抽吸过程中，避免从试管底部抽取血细胞。有时，注射器的表面可能会收集老鼠的皮毛;注意不要让任何毛皮进入管子，因为它会导致凝血。</li>
</ol>4. 统计分析
<ol><li>将所有数据呈现为均值±标准差，并检验它们的方差均匀性。</li>
	<li>使用 Fisher 精确检验来比较组间的成功率。</li>
	<li>使用双样本独立 t 检验来比较两组之间的总体均值。</li>
	<li>使用方差分析 （ANOVA） 进行连续测量，例如血液采样时间、体重、食物摄入量和饮水量。考虑 P < 0.05 具有统计学意义。</li>
</ol>

锁骨下静脉穿刺法对清醒大鼠重复采血

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虽然尾静脉采血是大鼠重复采血的最常见方法，但可能会受到麻醉药物的影响，并且由于尾静脉体积小，单次可采集的血液量有限，导致采血时间较长4,5。尽管高效液相色谱 （HPLC） 串联质谱 （MS/MS） 系统与大鼠尾静脉的毛细管显微采样 （CMS） 相结合可以减少大鼠的血液使用量11，但并非所有机构都配备了这种昂贵的设备。从球后丛...

<a href="https://app.jove.com/t/6595">This corrects</a> the article <a href="https://dx.doi.org/10.3791/66075">10.3791/66075</a>

An erratum was issued for: <a href="https://www.jove.com/t/66075">Subclavian Vein Blood Sampling in Conscious Rats</a>. The Discussion section was&#160;updated.

Erratum: Subclavian Vein Blood Sampling in Conscious Rats

大鼠是最常见的实验动物之一，获取血液样本的方法有很多种。对于在结束阶段涉及单次采血的实验，可以通过心脏穿刺或腹主动脉采血获得足够量的血液1。然而，一些研究需要从大鼠身上重复采血以进行常规血液或生化分析，特别是在药代动力学和毒理学研究中，需要重复采血以确定药物的吸收、分布和代谢2。
目前，虽然尾静脉采血是大鼠采血最常见的方法，尽管不需要麻醉，但这种方法对于重复采集可能具有挑战性，并且采集的血液量相对较小3,4。此外，虽然可以从隐静脉和静脉收集血液，但获得的血液量有限，并且需要麻醉1,5。此外，从下颌下静脉丛以及舌下、颈静脉和锁骨下静脉采集的血液样本可提供更高质量的样本，但通常需要麻醉或多人的协助 1,6,7,8,9。最后，眶后窦/耳道采血不仅需要麻醉，还可能对大鼠造成伤害和压力9.
通常从主要静脉获得的血液样本的质量通常达到最高标准1。目前，一些研究发现，通过颈静脉连续显微取样是一种非常适合大鼠毒理学研究的方法，尽管这种方法通常需要颈静脉导管插入术10,11,12。因此，如何在不进行手术干预的情况下，按照动物研究的3R原则获得高质量的血液样本，是值得探索的。本研究的目的是提出一种有效从大鼠锁骨下静脉中提取血液的方法。该技术无需麻醉即可通过单人手术快速收集令人满意的样本。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.75% normal saline</td>
			<td>Gansu Fuzheng Pharmaceutical Technology Co., Ltd.</td>
			<td>&#8212;&#8212;</td>
			<td>Prepared heparin sodium solution</td>
		</tr>
		<tr>
			<td>1 mL Pasteur pipette&#160;</td>
			<td>Biosharp</td>
			<td>BS-XG-01-NS</td>
			<td>Blood collection</td>
		</tr>
		<tr>
			<td>1 mL syringe (26 G, 0.45 mm x 12 mm)</td>
			<td>Shinva Medical Instrument Co.,Ltd.</td>
			<td>0.45*12RWLB</td>
			<td>Blood collection&#160;</td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tube</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td>Blood storage and collection</td>
		</tr>
		<tr>
			<td>75% medical alcohol</td>
			<td>Shandong Lircon Medical Technology Co., Ltd.</td>
			<td>&#8212;&#8212;</td>
			<td>Disinfection of rat blood collection site</td>
		</tr>
		<tr>
			<td>Centrifuge tube holder</td>
			<td>Biosharp</td>
			<td>BS-05/15-SM60</td>
			<td>&#8212;&#8212;</td>
		</tr>
		<tr>
			<td>Electronic scale</td>
			<td>Shanghai PUCHUN Measure Instrument Co., Ltd.</td>
			<td>JE1002</td>
			<td>Weigh</td>
		</tr>
		<tr>
			<td>Heparin sodium injection</td>
			<td>Hebei Changshan Biochemical Pharmaceutical Co., Ltd.</td>
			<td>&#8212;&#8212;</td>
			<td>Rinse the syringe and EP tube; dilute with normal saline to 25 U/mL</td>
		</tr>
		<tr>
			<td>Low temperature centrifuge</td>
			<td>HuNan Xiang Yi Centrifuge Instrument&#160; Co., Ltd.</td>
			<td>&#160;H1750R</td>
			<td>Separation of serum</td>
		</tr>
	</tbody>
</table>

subclavian vein blood sampling in conscious rats

有几种既定的方法可以从大鼠身上获取重复的血液样本，最常用的方法是无麻醉的侧尾静脉取样和麻醉的颈静脉取样。然而，这些方法大多需要辅助和麻醉设备，有时在采血或血液样本质量差方面造成困难。此外，当需要对大量大鼠进行重复采血时，这些采血方法会消耗大量的时间和人力资源。本研究提出了一种由单个熟练个体对非麻醉大鼠进行重复采血的技术。通过刺穿锁骨下静脉可以获得非常令人满意的血液样本。该方法显示出令人印象深刻的 95% 的总体成功率，从大鼠约束到完成采血的中位时间仅为 2 分钟。此外，在指定范围内进行连续采血不会对大鼠造成任何伤害。这种方法值得推广用于血液采集，特别是在大规模的药代动力学研究中。

Rats are one of the most common experimental animals, and there are many ways to obtain blood samples. For experiments involving a single blood collection at the concluding stage, a sufficient amount of blood can be obtained through cardiac puncture or abdominal aorta blood collection1. However, some studies require repeated blood collection from rats for routine blood or biochemical analysis, especially in pharmacokinetics and toxicology studies, where repeated blood collection is required to determine the absorption, distribution, and metabolism of drugs2.
Currently, although tail vein blood collection is the most common method for blood sampling from rats, despite not requiring anesthesia, this method can be challenging for repeated collections, and the volume of blood collected is relatively small3,4. Additionally, although blood can be collected from the saphenous and penile veins, the amount of blood obtained is limited, and anesthesia is required1,5. Furthermore, blood samples collected from the submandibular venous plexus, as well as sublingual, jugular, and subclavian veins provide higher-quality samples but typically require anesthesia or the assistance of multiple individuals1,6,7,8,9. Finally, retro-orbital sinus/canal blood collection not only necessitates anesthesia but may also potentially cause injury and stress to the rats9.
The quality of blood samples typically obtained from major veins is generally of the highest standard1. At present, some studies have found that continuous microsampling through the jugular vein is a very suitable method for toxicological research in rats although this method usually requires jugular vein catheterization10,11,12. Therefore, it is worth exploring how to obtain high-quality blood samples in accordance with the 3R principle of animal research without surgical intervention. The objective of this study was to present a method for efficiently extracting blood from the subclavian vein in rats. This technique enables the swift collection of satisfactory samples through a single-person procedure without the need for anesthesia.

作者聚焦：在非麻醉大鼠中开发一种高效的血液采集方法 

有意识大鼠的锁骨下静脉血液采样

The scope of our research is centered on devising infection methodologies for collecting blood samples from rats. Our aim is to ensure rapid and set procedures that align with our principle, utilizing minimal manpower and resources. One of the current experimental challenges is to effectively immobilize the rat in a manner that simultaneously maintains the subclavian vein enclosed proximately to the skin surface for optimal blood collection.The advantages of our protocol is that high quality blood samples can be obtained quickly by a person without anesthesizing the rats.

如图2A所示，高质量的等离子体标本呈现淡黄色调、清晰度和透明度，没有任何红色调或凝血。图2B分别显示了由于程序不当导致的溶血（左侧）或凝血（右侧）。在4天内的96次采血过程中，A组和B组的平均单次采血时间分别为119.87±33.62 s和123.28±30.96 s。两组每日采血时间无显著差异（t = 0.66，P = 0.54，表1）?...

Watch this Scientific Journal Video about Developing an Efficient Blood Collection Method in Non-Anesthetized Rats at JoVE.com

在这里，我们介绍了有效的大鼠限制和锁骨下静脉穿刺方法的组合，可以在没有麻醉的情况下在大鼠中快速、安全和重复采血。

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein ...

我们的研究范围集中在设计用于收集大鼠血液样本的感染方法。我们的目标是确保快速和制定符合我们原则的程序，使用最少的人力和资源。目前的实验挑战之一是有效地固定大鼠，同时保持锁骨下静脉靠近皮肤表面以实现最佳采血。我们方案的优点是，一个人可以在不麻醉大鼠的情况下快速获得高质量的血液样本。

subclavian-vein-blood-sampling-in-conscious-rats

Research

JoVE Journal

Medicine

Subclavian Vein Blood Sampling in Conscious Rats

1.2K 次观看.  Lanzhou University Second Hospital. 我们的研究范围集中在设计用于收集大鼠血液样本的感染方法。我们的目标是确保快速和制定符合我们原则的程序，使用最少的人力和资源。目前的实验挑战之一是有效地固定大鼠，同时保持锁骨下静脉靠近皮肤表面以实现最佳采血。我们方案的优点是，一个人可以在不麻醉大鼠的情况下快速获得高质量的血液样本。

视频: 作者聚焦：在非麻醉大鼠中开发一种高效的血液采集方法 

There are several established methods for obtaining repeated blood samples from rats, with the most commonly employed methods being lateral tail vein sampling without anesthesia and jugular vein sampling with anesthesia. However, most of these methods require assistance and anesthetic equipment and sometimes pose difficulties in terms of blood collection or the poor quality of blood samples. In addition, these methods of blood collection consume significant time and human resources when repeated blood sampling is required for a large number of rats. This study presents a technique for repetitive blood sampling in non-anesthetized rats by a single proficient individual. Highly satisfactory blood samples can be obtained by puncturing the subclavian vein. The method demonstrated an impressive overall success rate of 95%, with a median time of merely 2 min from rat restraint to the completion of blood collection. Furthermore, performing consecutive blood collections within the designated range does not inflict any harm on the rats. This method is worth promoting for blood collection, especially in large-scale pharmacokinetic studies.

Here we present a combination of effective rat restriction and subclavian vein puncture methods that enable rapid, safe, and repeated blood collection in rats without anesthesia.

科研

医学

Subclavian Vein Puncture Blood Collection and Observation of Secondary Outcome Measures in Rats

On the day of the blood sampling, place the rat in the cage at 8:00 AM.Weigh the food and water and place them in the cage. Define successful blood sampling based on the following criteria. Have a blinded researcher record the rat's weight, food consumption, and water intake daily at 8:00 AM.Next, gently stroke the rat's back skin and pinch its neck repeatedly to help the rat relax.Using the non-dominant hand's thumb and index finger, grip and lift the rat's neck skin. Using the remaining three fingers and the palm of the non-dominant hand, secure the rat's back skin, and with the help of the dominant hand, immobilize its front limbs. Using the non-dominant hand's index finger, gently push down on the rat's head skin.Rotate the shoulder joint outwardly with the other fingers along the palm while the dominant hand fully extends the shoulder joint. Grip the rat firmly with the non-dominant hand to align its head and body in a straight line. Locate the clavicle with the dominant hand and confirm the injection site.Disinfect the site with alcohol, then flush the syringe with sodium heparin. Hold the syringe parallel to the rat's body and the dominant hand with the needle tip facing upwards and the syringe scale toward the experimenter. Insert the needle 0.5 centimeters below the clavicle notch.Gently withdraw the syringe to create negative pressure, collecting 0.1 to 1 milliliters of blood at a constant speed. Apply a cotton swab for hemostasis and return the rat to its cage. Average blood collection times for groups A and B were similar over 96 hours.Blood collection success rates were 93.8%for group A and 95.8%for group B, with no significant difference. Group A had a shorter average blood collection time on the second day compared to the fourth day. The number of attempts for successful blood collection averaged 1.21 for group A and 1.17 for group B.More attempts and longer puncture times indicated higher failure rates.Both groups showed steady weight gain over four days. Water and food intake remained relatively constant among rats of the same sex.