Name: subclavian vein blood sampling in conscious rats
Uploaded: 2023-11-03T13:50:00.000+00:00
Duration: 3 min 52 s
Description: Watch this Scientific Journal Video about Developing an Efficient Blood Collection Method in Non-Anesthetized Rats at JoVE.com

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/鼻窦取血经常引起大鼠的焦虑和疼痛，操作不当甚至会损害大鼠的视力和健康。因此，不推荐将该方法用于大鼠的血液采样9。
锁骨下静脉位于大鼠的胸大肌和三角肌之间，并在锁骨内三分之一处流入颈静脉（图4）。在Yang等人的研究中，熟练的操作者在麻醉下从大鼠锁骨下静脉采集血液的成功率约为90%，从穿刺开始到结束的最短时间为65 s7。在Wang等人的研究中，使用垂直方法从大鼠锁骨下静脉收集血液样本。虽然他们的方法不涉及麻醉，但它确实需要两个人的合作来安全地限制大鼠6。该研究方案显示了采血的良好优势。该协议不需要任何特殊的约束装置或麻醉设施。如果处理得当，大鼠通常表现出最小的抵抗力。从大鼠约束到完成采血的中位时间仅为2分钟，总体成功率高达95%。这种方法大大节省了人力资源，减少了采血所需的时间。此外，获得的血浆样品是透明透明的，溶血和凝血事件的发生率极低，从而最大限度地减少了实验重复。熟练掌握该技术对于管理需要重复采血的大规模大鼠药代动力学和毒理学实验特别有价值。
在我们的研究中，抽血失败的主要发生发生在第 4 天，这可能与反复穿刺造成的静脉损伤有关。反复穿刺可导致血管壁损伤并引起炎症反应，导致血管壁增厚硬化，甚至诱发血管狭窄。如果穿刺后止血不充分，外渗的血液会进一步引起组织水肿和炎症，从而导致瘢痕组织的形成。这些疤痕组织很难穿透，也会拉扯并导致血管位置移动，所有这些都使血管更难定位和刺穿。在我们的研究中，使用26G注射器（0.45mm）进行采血，相对于人体静脉来说，这很好，但仍然对大鼠静脉造成相当大的损害。在第一次抽血期间，当针头穿过血管时，有明显的穿透感，随着抽血次数的增加，这种穿透感会减少，采血时间更长，失败率更高。因此，我们建议使用较细的胰岛素针头进行采血，采血后应施加足够的压力以防止血肿形成，并应进行交替抽血以充分修复静脉。根据我们的经验，训练有素的抽血师可以在24小时内使用26G针头从同一只大鼠的双侧锁骨下静脉交替抽血8-10次，每次抽血之间平均间隔2-3小时。然而，大鼠可以忍受的最大抽血次数、恢复期和抽血周期可能会受到所用针规、不同实验所需的抽血间隔以及抽血师的熟练程度的影响。这些因素需要在未来的研究中进一步探讨。对于药代动力学实验所需的密集血液采样，最好交替从左右锁骨下静脉采集血液。在确实不可用的情况下，可以补充其他采血方法。
体重、饮水量和食物摄入量是评估大鼠健康状况的最基本和最直接的指标16.一项早期研究表明，每天通过颈静脉采集的血液少于0.9毫升不会影响大鼠的血流动力学，也不会导致任何显着的体重减轻。然而，当血液收集量超过 1.5 毫升时，它会导致体重减轻17。在Yokoya等人的研究中，从颈静脉重复显微取样（每次50μL，24小时内6-7次）不影响大鼠体重或食物摄入量10。此外，采血方式可能会影响大鼠的体重和食物摄入量。在之前使用舌下静脉采血的研究中，第一天 24 小时采集 0.5-1.0 mL 血液导致大鼠体重减轻并减少食物摄入量，尽管体重减轻并不显着18.本研究显示，采血期间大鼠体重仍稳步增加，大鼠食水摄入量、采血相关并发症、死亡均无明显变化，表明该方法安全可靠。
需要强调的是，在进行采血之前让大鼠适应约束过程可能会减少大鼠的压力并提高采血的成功率。固定不足和静脉暴露不足会导致采血失败，甚至由于大鼠在疼痛中挣扎而导致局部静脉破裂。在较轻的情况下，这可能会导致明显的皮下血肿，而在严重的情况下，可能会导致大鼠死亡。此外，固定不良可能导致老鼠逃跑并对个体造成伤害。因此，我们强烈建议在进行采血程序之前彻底掌握处理技术。此外，重要的是要谨慎地向外旋转肩关节施加的力，因为压力过大可能导致大鼠锁骨骨折。
本研究的一个局限性是，我们没有通过测量皮质酮水平的变化或笼边监测来系统地评估这种采血方法在大鼠中引起的应激变化，这需要在未来的研究中进行探索。本文的另一个局限性是缺乏替代的采血方法作为对照。在未来的研究中，将与其他采血方法的优缺点进行比较。总而言之，这项研究引入了一种无需麻醉即可从大鼠身上采集单人血液的方法。这种方法提供了一种直接、快速和安全的方法来获取大鼠的血液样本。

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

An erratum was issued for: <a href="https://www-jove-com.bdigitaluss.remotexs.co/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><tbody><tr><td>0.75% normal saline</td><td>Gansu Fuzheng Pharmaceutical Technology Co., Ltd.</td><td>&amp;mdash;&amp;mdash;</td><td>Prepared heparin sodium solution</td></tr><tr><td>1 mL Pasteur pipette&amp;nbsp;</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&amp;nbsp;</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>&amp;mdash;&amp;mdash;</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>&amp;mdash;&amp;mdash;</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>&amp;mdash;&amp;mdash;</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&amp;nbsp; Co., Ltd.</td><td>&amp;nbsp;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。

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）。 最短的个体采血时间分别为78 s和89 s。
A组和B组成功单次采血所需的平均尝试次数分别为1.21次和1.17次。两组之间的尝试次数没有显著差异（t = 0.58，P = 0.60 ，表2）。A组和B组的总体成功率分别为93.8%（45/48）和95.8%（46/48），两组之间的总体成功率无显著差异（P > 0.05，表1）。B组各时间点采血时间无显著差异。A组第2天采血时间小于第4天（105.75 ± 14.22s vs 144.5 ± 25.45 s，t = 12.39，P < 0.01; 表1）。 此外，更多的尝试和更长的穿刺时间通常表明更高的失败率（图3A-C）。第三天，B组遭遇了一次因溶血而失败的失败。第四天，A组遭遇了三次失败:一次是溶血，两次是无法获取血样。B组还因无法获得血液样本而经历了一次失败。
在连续4天的观察过程中，两组大鼠都表现出稳定的体重增加。同性大鼠的水和食物摄入量保持相对稳定。在整个采血过程中，没有发现大鼠死亡的情况，也没有观察到任何明显的并发症，如锁骨骨折、气胸或穿刺伤部位血肿（图3D-F和表3）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66075/66075fig01.jpg"> 图1:大鼠锁骨下静脉的固定和采血方法。 （A-H） 固定的操作;（一）锁骨和采血部位的位置;（J-L）采血和止血的过程。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/66075fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66075/66075fig02.jpg"> 图2:成功和不成功采集的血液样本。 （A） 典型血样和分离血浆;（B）溶血（左）和凝血（右）血样 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/66075fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66075/66075fig03.jpg"> 图3:采血有效性和安全性的评估。 （A） 每天平均采血时间;（二）日均穿刺次数;（三）两组采血成功率和失败率;（D-F） 两组大鼠采血过程中体重、食物摄入量和水摄入量的变化。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/66075fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66075/66075fig04.jpg"> 图4:大鼠颈部血管的解剖结构。 （A） 浅表解剖结构;（二）深层解剖结构。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/66075fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">群</td>
			<td rowspan="2">时间</td>
			<td colspan="4">平均采血时间</td>
		</tr><tr><td>第1天</td>
			<td>第2天*</td>
			<td>第3天</td>
			<td>第4天</td>
		</tr><tr><td rowspan="6">一个</td>
			<td>上午10:00</td>
			<td>92.83 ± 7.38</td>
			<td>100.5 ± 17.36</td>
			<td>117.83 ± 12.02</td>
			<td>146.6 ± 24.76</td>
		</tr><tr><td>下午10:00</td>
			<td>108.67 ± 10.86</td>
			<td>111.00 ± 6.95</td>
			<td>158.33 ± 60.47</td>
			<td>142.40 ± 25.96</td>
		</tr><tr><td>平均时间</td>
			<td>100.75 ± 12.20</td>
			<td>105.75 ± 14.22</td>
			<td>138.08 ± 48.07</td>
			<td>144.5 ± 25.45</td>
		</tr><tr><td>成功率</td>
			<td>100% (12/12)</td>
			<td>100% (12/12)</td>
			<td>100% (12/12)</td>
			<td>75% (9/12)</td>
		</tr><tr><td>总体成功率</td>
			<td colspan="4">93.8% (45/48)</td>
		</tr><tr><td>总体平均时间</td>
			<td colspan="4">119.87 ± 33.62</td>
		</tr><tr><td rowspan="6">B</td>
			<td>上午10:00</td>
			<td>98.17 ± 7.24</td>
			<td>110,17 ± 14,33</td>
			<td>123.67 ± 30.99</td>
			<td>147.2 ± 17.47</td>
		</tr><tr><td>下午10:00</td>
			<td>106.00 ± 14.35</td>
			<td>126.67 ± 17.12</td>
			<td>123.17 ± 17.50</td>
			<td>165.67 ± 49.70</td>
		</tr><tr><td>平均时间</td>
			<td>102.08 ± 12.02</td>
			<td>118.42 ± 17.82</td>
			<td>123.92 ± 25.16</td>
			<td>157.27 ± 39.63</td>
		</tr><tr><td>成功率</td>
			<td>100% (12/12)</td>
			<td>100% (12/12)</td>
			<td>91.7% (11/12)</td>
			<td>91.7% (11/12)</td>
		</tr><tr><td>总体成功率</td>
			<td colspan="4">95.8% (46/48)</td>
		</tr><tr><td>总体平均时间</td>
			<td colspan="4">123.28 ± 30.96</td>
		</tr></tbody></table>表1:两组大鼠的采血次数和成功率。 * A 组第2天采血时间小于第4天（t = 12.39 P < 0.01）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2">群</td>
			<td rowspan="2">时间</td>
			<td colspan="4">平均穿刺次数</td>
		</tr><tr><td>第1天</td>
			<td>第2天</td>
			<td>第3天</td>
			<td>第4天</td>
		</tr><tr><td rowspan="4">一个</td>
			<td>上午10:00</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1.67</td>
		</tr><tr><td>下午10:00</td>
			<td>1</td>
			<td>1</td>
			<td>1.33</td>
			<td>1.67</td>
		</tr><tr><td>平均</td>
			<td>1</td>
			<td>1</td>
			<td>1.17</td>
			<td>1.67</td>
		</tr><tr><td>总体平均</td>
			<td colspan="4">1.21</td>
		</tr><tr><td rowspan="4">B</td>
			<td>上午10:00</td>
			<td>1</td>
			<td>1</td>
			<td>1.17</td>
			<td>1.5</td>
		</tr><tr><td>下午10:00</td>
			<td>1.17</td>
			<td>1</td>
			<td>1.17</td>
			<td>1.33</td>
		</tr><tr><td>平均</td>
			<td>1.08</td>
			<td>1</td>
			<td>1.17</td>
			<td>1.42</td>
		</tr><tr><td>总体平均</td>
			<td colspan="4">1.17</td>
		</tr></tbody></table>表2:大鼠采血的平均穿刺次数。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2" style="min-width: 60px;">性</td>
			<td rowspan="2" style="min-width: 60px;">群</td>
			<td colspan="4" style="min-width: 60px;">重量 （g）</td>
			<td colspan="4" style="min-width: 60px;">食物摄入量 （g）</td>
			<td colspan="4" style="min-width: 60px;">饮水量 （g）</td>
		</tr><tr><td style="min-width: 60px;">第1天</td>
			<td style="min-width: 60px;">第2天</td>
			<td style="min-width: 60px;">第3天</td>
			<td style="min-width: 60px;">第4天</td>
			<td style="min-width: 60px;">第1天</td>
			<td style="min-width: 60px;">第2天</td>
			<td style="min-width: 60px;">第3天</td>
			<td style="min-width: 60px;">第4天</td>
			<td style="min-width: 60px;">第1天</td>
			<td style="min-width: 60px;">第2天</td>
			<td style="min-width: 60px;">第3天</td>
			<td style="min-width: 60px;">第4天</td>
		</tr><tr><td rowspan="4" style="min-width: 60px;">♀</td>
			<td style="min-width: 60px;">一个</td>
			<td style="min-width: 60px;">260 ± 7.5</td>
			<td style="min-width: 60px;">267.7 ± 6.3</td>
			<td style="min-width: 60px;">271 ± 5.4</td>
			<td style="min-width: 60px;">278 ± 6.5</td>
			<td style="min-width: 60px;">13.3 ± 0.79</td>
			<td style="min-width: 60px;">13.5 ± 0.93</td>
			<td style="min-width: 60px;">14.0 ± 0.29</td>
			<td style="min-width: 60px;">14.0 ± 0.77</td>
			<td style="min-width: 60px;">23.9 ± 0.36</td>
			<td style="min-width: 60px;">23.1 ± 0.77</td>
			<td style="min-width: 60px;">24.4 ± 0.70</td>
			<td style="min-width: 60px;">24.6 ± 0.12</td>
		</tr><tr><td style="min-width: 60px;">B</td>
			<td style="min-width: 60px;">262 ± 12.8</td>
			<td style="min-width: 60px;">268.3 ± 14.0</td>
			<td style="min-width: 60px;">272.7 ± 9.4</td>
			<td style="min-width: 60px;">279 ± 7.0</td>
			<td style="min-width: 60px;">14.4 ± 0.45</td>
			<td style="min-width: 60px;">13.9 ± 0.52</td>
			<td style="min-width: 60px;">14.7 ± 0.26</td>
			<td style="min-width: 60px;">14.3 ± 0.56</td>
			<td style="min-width: 60px;">23.6 ± 0.73</td>
			<td style="min-width: 60px;">23.7 ± 0.65</td>
			<td style="min-width: 60px;">24.4 ± 0.91</td>
			<td style="min-width: 60px;">24.1 ± 1.79</td>
		</tr><tr><td style="min-width: 60px;">电汇</td>
			<td style="min-width: 60px;">0.35</td>
			<td style="min-width: 60px;">0.09</td>
			<td style="min-width: 60px;">0.38</td>
			<td style="min-width: 60px;">0.23</td>
			<td style="min-width: 60px;">2.44</td>
			<td style="min-width: 60px;">1.22</td>
			<td style="min-width: 60px;">2.34</td>
			<td style="min-width: 60px;">1.12</td>
			<td style="min-width: 60px;">0.43</td>
			<td style="min-width: 60px;">0.76</td>
			<td style="min-width: 60px;">0.00</td>
			<td style="min-width: 60px;">0.71</td>
		</tr><tr><td style="min-width: 60px;">调整后的 P 值</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">0.68</td>
			<td style="min-width: 60px;">0.98</td>
			<td style="min-width: 60px;">0.71</td>
			<td style="min-width: 60px;">0.99</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">>0.99</td>
		</tr><tr><td rowspan="4" style="min-width: 60px;">♂</td>
			<td style="min-width: 60px;">一个</td>
			<td style="min-width: 60px;">313.7 ± 12.0</td>
			<td style="min-width: 60px;">325.7 ± 9.1</td>
			<td style="min-width: 60px;">329 ± 14.2</td>
			<td style="min-width: 60px;">340 ± 15.6</td>
			<td style="min-width: 60px;">15.9 ± 0.64</td>
			<td style="min-width: 60px;">16.2 ± 0.08</td>
			<td style="min-width: 60px;">15.7 ± 0.70</td>
			<td style="min-width: 60px;">15.9 ± 0.73</td>
			<td style="min-width: 60px;">26.2 ± 0.62</td>
			<td style="min-width: 60px;">27.2 ± 0.9</td>
			<td style="min-width: 60px;">26.9 ± 1.0</td>
			<td style="min-width: 60px;">25.3 ± 1.1</td>
		</tr><tr><td style="min-width: 60px;">B</td>
			<td style="min-width: 60px;">311 ± 16.4</td>
			<td style="min-width: 60px;">322.3 ± 18.0</td>
			<td style="min-width: 60px;">330.7 ± 17.6</td>
			<td style="min-width: 60px;">342 ± 16.9</td>
			<td style="min-width: 60px;">15.3 ± 0.74</td>
			<td style="min-width: 60px;">15.7 ± 0.85</td>
			<td style="min-width: 60px;">15.1 ± 0.33</td>
			<td style="min-width: 60px;">15.3 ± 0.86</td>
			<td style="min-width: 60px;">27.1 ± 0.37</td>
			<td style="min-width: 60px;">25.6 ± 1.27</td>
			<td style="min-width: 60px;">27.5 ± 0.76</td>
			<td style="min-width: 60px;">26.2 ± 0.99</td>
		</tr><tr><td style="min-width: 60px;">q</td>
			<td style="min-width: 60px;">1.50</td>
			<td style="min-width: 60px;">1.88</td>
			<td style="min-width: 60px;">0.94</td>
			<td style="min-width: 60px;">1.13</td>
			<td style="min-width: 60px;">2.34</td>
			<td style="min-width: 60px;">1.85</td>
			<td style="min-width: 60px;">2.10</td>
			<td style="min-width: 60px;">1.97</td>
			<td style="min-width: 60px;">1.90</td>
			<td style="min-width: 60px;">3.57</td>
			<td style="min-width: 60px;">1.24</td>
			<td style="min-width: 60px;">1.82</td>
		</tr><tr><td style="min-width: 60px;">调整后的 P 值</td>
			<td style="min-width: 60px;">0.94</td>
			<td style="min-width: 60px;">0.86</td>
			<td style="min-width: 60px;">>0.99</td>
			<td style="min-width: 60px;">0.99</td>
			<td style="min-width: 60px;">0.71</td>
			<td style="min-width: 60px;">0.87</td>
			<td style="min-width: 60px;">0.79</td>
			<td style="min-width: 60px;">0.83</td>
			<td style="min-width: 60px;">0.86</td>
			<td style="min-width: 60px;">0.33</td>
			<td style="min-width: 60px;">0.98</td>
			<td style="min-width: 60px;">0.88</td>
		</tr></tbody></table>表3:大鼠日体重、食物摄入量和水分摄入量的变化。
视频1:大鼠的镇静和处理。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/Video%201.zip" target="_blank">请按此下载此影片。</a>
视频2:大鼠的约束程序。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/Video%202.zip" target="_blank">请按此下载此影片。</a>
视频3:大鼠的采血程序。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/Video%203.zip" target="_blank">请按此下载此影片。</a>
视频 4:穿刺部位的止血压迫。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/66075/Video%204.zip" target="_blank">请按此下载此影片。</a>

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

作者聚焦:在非麻醉大鼠中开发一种高效的血液采集方法 

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。

有意识大鼠的锁骨下静脉血液采样

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

Subclavian Vein Blood Sampling in Conscious Rats

1.8K 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 

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

股动脉和静脉导管采血，药品监督管理局和意识的血压和心率测量

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

科研

医学

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

培养一个精密的显微外科技术在大鼠颈总动脉，颈外静脉移植：介入

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Repeated Blood Collection from Tail Vein of Non-Anesthetized Rats with a Vacuum Blood Collection System

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the tail vein is thin so that it is sometimes hard to puncture. In addition, the tail vein has low blood flow and requires a long sampling time to get sufficient blood. The present report describes a simple blood sampling method, the vacuum blood collection method, which is usually used to obtain blood samples from patients, here used for non-anesthetized rats. The 22 G butterfly needle tip was inserted into one of the lateral tail veins approximately 2-3 cm from the tip of the tail at an angle of approximately 20&#176;, and blood was collected in the vacuum collection tube by inserting the rubber end of the butterfly needle into the vacuum blood collection tube. The present experimental results show that the success rate was 95% in the experimental group and 90% in the beginner group. The success rate and puncture times were similar between two groups. The sampling duration was significantly shorter in the experimental group compared to beginner group. In conclusion, this vacuum blood collection method for sequential blood sampling from the tail vein of non- anesthetized rats is feasible and easy-to-learn, which might serve as a reliable alternative to other conventionally used blood sampling techniques for rats.

Here, we describe a simple tail vein blood sampling method in non-anesthetized rats using a vacuum extraction tube system. This method reduces the risk of direct exposure to blood and simplifies taking multiple samples from a single venipuncture.

无麻醉大鼠尾静脉血采集系统的研究

Blood can be collected from rats in a number of sampling locations. For instance, the tail vein is a superior location for blood sampling. However, the ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

锁骨下静脉穿刺作为大鼠采血的一种替代方法

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

小鼠锁骨下静脉穿刺采血

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

生物学

Microsurgical Skills of Establishing Permanent Jugular Vein Cannulation in Rats for Serial Blood Sampling of Orally Administered Drug

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, which could potentially affect results. The jugular vein cannulation (JVC) in rats is a widely used model for repeated blood collection but requires adequate training of surgery skills and animal care. This article details the microsurgical procedures for establishing and maintaining a permanent JVC rat model with specific focus on the placement and sealing of the jugular cannula. The importance of monitoring physiological (e.g., body weight, food, and water intake) and hematological parameters, was highlighted with results presented for 6 days post-surgery during the rat&#39;s recovery. The drug-plasma concentration-time profile of orally administered natural phenol ellagic acid was determined in the JVC rat model.

Detailed microsurgical techniques are demonstrated to establish a longer-term jugular vein cannulation rat model for sequential blood collection in the same animal. Physiological and hematological parameters have been monitored during the rat's recovery phase. This model has been applied to study pharmacokinetics of orally administered polyphenol without inducing animal stress.

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

Blood sampling in small laboratory animals is necessary for pharmaceutical lead optimization but can cause great harm and stress to experimental animals, ...

同时记录脑电图的大鼠脑脊液和血液

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

作者聚焦:获取高质量的 CSF 和血液样本以发现癫痫生物标志物 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

有意识大鼠的锁骨下静脉血液采样

副本

摘要

探索更多视频

此视频中的章节

股动脉和静脉导管采血，药品监督管理局和意识的血压和心率测量

培养一个精密的显微外科技术在大鼠颈总动脉，颈外静脉移植：介入

无麻醉大鼠尾静脉血采集系统的研究

锁骨下静脉穿刺作为大鼠采血的一种替代方法

小鼠锁骨下静脉穿刺采血

从大鼠的侧尾静脉血液取样

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

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液

在脑电图记录期间从大鼠的侧尾静脉采集脑脊液和血液