Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

Richard B. McCosh; Michael J. Kreisman; Kellie M. Breen

doi:10.3791/57894

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

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05:58 min

July 4, 2018

DOI: 10.3791/57894-v

Richard B. McCosh¹, Michael J. Kreisman¹, Kellie M. Breen¹

¹Department of Reproductive Medicine,University of California, San Diego School of Medicine

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Overview

This study describes a tail-tip blood sampling protocol for the collection of blood samples in unrestrained mice, focusing on the measurement of pulsatile luteinizing hormone (LH) secretion. The method is designed to enable reliable assessments of LH pulsatile secretion, contributing to research in reproductive neuroendocrinology.

Key Study Components

Area of Science

Neuroendocrinology
Reproductive biology
Animal behavior

Background

Luteinizing hormone pulsatile secretion is critical in reproductive physiology.
Reliable blood sampling techniques are essential for accurately measuring hormonal fluctuations.
Existing methods may not adequately account for the effects of animal stress.
This protocol aims to minimize mouse stress during blood collection.

Purpose of Study

To develop a reliable tail-tip blood sampling protocol for unrestrained mice.
To assess LH pulsatile secretion in response to both acute and chronic treatments.
To improve the accuracy of neuroendocrinological research methods.

Methods Used

The protocol involves daily handling and habituation of mice prior to blood collection.
Adult female C57BL/6 mice were used, with blood collected from the tail tip for LH measurement.
Frequent handling sessions helped facilitate a stress-free environment.
Blood samples were collected over a set timeframe to establish hormonal baselines.

Main Results

The technique led to successful collection of LH levels in control animals.
Psychosocial stress from restraint significantly suppressed LH secretion in experimental groups.
Stress minimized reliable measurements of pulsatile LH secretion, highlighting the method's efficacy.
Environmental disturbances were found to affect hormonal measurements.

Conclusions

This study establishes a novel method for obtaining accurate hormonal measurements in mice.
The protocol allows researchers to explore LH regulation effectively in neuroendocrinology studies.
Understanding the impact of stress on hormonal secretion contributes to broader insights into reproductive physiology and animal behavior.

Frequently Asked Questions

What are the advantages of this blood sampling protocol?

This protocol minimizes stress by allowing mice to remain unrestrained, facilitating more reliable hormonal measurements.

How is the tail sample obtained?

Blood is collected by gently clipping the tail tip and stroking it to form a droplet, which is then sampled.

What is the biological model used in this study?

The study uses adult female C57BL/6 mice, which are well-characterized for neuroendocrinology research.

How does this method contribute to neuroendocrinology?

By providing reliable blood sampling under unrestrained conditions, researchers can better understand LH pulsatile secretion and its regulation.

What are key considerations when using this method?

Minimizing environmental disturbances is crucial, as noise and interruptions can impact hormonal measurements.

Can this method be adapted for other experiments?

Yes, the protocol can be adapted to assess other circulating factors, broadening its applicability in research.

在这里, 我们提出了一个尾巴尖端血液取样协议, 以频繁样本收集在无约束的小鼠。该方法对评估脉动性促黄体激素分泌模式有一定的参考价值, 可用于其他循环因素的分析。

频繁尾尖血液采样的总体目标是获得全血样本，用于测量清醒、非麻醉和行为正常的小鼠的脉冲性黄体生成素分泌，称为 LH。该方法通过提供可靠的方案来评估小鼠的黄体生成素搏动分泌，可以帮助回答可重复性神经内分泌学领域的关键问题。该技术的主要优点是频繁的处理和习惯化将确保在急性或慢性实验治疗后稳定可靠的黄体生成素脉搏测量。

在采血前的五周内，周一至周五每天处理小鼠。在最后两周，周六和周日也要处理老鼠。在生物安全柜中执行小鼠处理制度。

将第一只鼠标从固定架中取出，并将其放在机柜内合适的表面上。通过握住鼠标尾巴的底部，轻轻地将鼠标约束在此表面上。然后用另一只手从根部到尖端用力抚摸尾巴的腹面。

总共执行该作六次，然后将鼠标放回其笼子中。这构成了一个处理集。现在等待 6 分钟，然后执行另一组处理。

以集间间隔继续此模式，每个处理会话执行四个处理集。总共为每个会话提供 24 次尾部划动。如果采样期间的实验性治疗将包括任何其他处理方法，如擦伤或腹膜内注射，那么在处理过程中也要让小鼠适应此类处理活动。

在计划的采血日前两周，将小鼠成对饲养，以尽量减少它们在采样期间的干扰。对于血液采集，准备 0.6 毫升微量离心管用于血液储存。标记试管，对于三个微升血液采集，将 57 微升 ACES 缓冲液分装到每个试管中。

准备好后，将试管存放在 4 摄氏度。在生物安全柜中，准备好以下物品：移液器吸头、垃圾桶、计时器、实验日志、装有采血管的冰桶、锋利的无菌剪刀、永久性记号笔和小纱布。在开始采血前 45 分钟，将第一只小鼠从笼子中取出，并将其放在合适的表面上。

然后标记尾巴，从它的笼子伙伴中快速识别这只老鼠。现在用剪刀去除一到两毫米的尾巴尖端。剪断尾巴后，小心地抚摸尾巴，直到尾部形成一滴血。

然后收集 3 微升血液，将血液和吸头都丢弃到垃圾桶中。接下来确保血流已停止。如果需要，用无菌纱布轻轻按压。

然后将鼠标放回笼子里 6 分钟。现在，在 45 分钟内每隔 6 分钟一次，用力抚摸尾巴，从小鼠身上采集额外的血液样本。如果没有形成血滴，请用浸泡在生理盐水中的纱布垫轻拍尾巴，以帮助清除血凝块。

在预采样期间，丢弃所有血液等分试样。45 分钟后，开始采样期。取样时，将 3 μL 血液转移到采集管中，倒置混合管，然后将其放回冰上。

现在，根据需要继续进行采样。一旦结束，要么对小鼠实施安乐死，要么确保它们的尾血流完全停止，然后为小鼠提供一个干净的笼子。接下来，将血液样本转移到储存盒中，并将样本储存在零下 20 摄氏度，直到可以进行分析。

在频繁的血液样本中测量黄体生成素或 LH，以确定对急性心理社会应激挑战的反应。对成年雌性 C57 black 6 只小鼠进行卵巢切除，并采集血样 90 分钟，以建立 LH 搏动分泌的治疗前基线。为了进行应激处理，在 90 分钟的采样期间，将实验动物放入啮齿动物约束装置中并转移到新的笼子中，而对照组则留在它们的家笼中，没有被约束。

分析表明，对照动物在整个采样期间具有清晰明确的 LH 脉冲。相比之下，约束装置的社会心理压力迅速明确地抑制了实验组的搏动性 LH 分泌。在准备使用这项技术时，重要的是要记住尽量减少环境干扰，例如噪音或其他研究人员的干扰，这可能会给小鼠带来压力并中断脉动的 LH 分泌。

自 Stein 博士和 Chen 实验室的其他人开发以来，这项技术已开始为神经内分泌学领域的研究人员铺平道路，以探索复杂分子和遗传工具实用且可用的物种中的 LH 脉冲调节。

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