作者感谢 Drs. 珍妮弗. 杨和亚历山大 (萨沙) 考夫曼为技术协助与这项技术并且许多有用和关键的讨论。血清激素测定是由2017在弗吉尼亚大学的生殖配体检测和分析核心研究中心进行的, 由尤尼斯肯尼迪发育研究院/NIH (NCTRI) 赠款 P50-HD28934 支持。
研究支持来源: R01 发育研究院 86100 (九巴), 成果管理制得到 T32 发育研究院007203的支持。

这里描述的方法是与国立卫生动物护理和使用指南的协议, 并已授权的机构动物护理和使用委员会在加州大学圣地亚哥分校。
1. 处理的驯化
<ol><li>处理过程<ol>
<li>把老鼠笼放在安全柜里。将第一只老鼠从笼子里移开, 放在安全柜内合适的表面 (如干净的笼盖上)。</li>
		<li>用一只手的食指和拇指握住尾部的底座, 轻轻地抑制工作表面上的鼠标。然后, 将尾巴的腹面, 从底座到尾端, 用食指和拇指牢牢地描出尾部。重复此抚摸6次 (即1 集), 并将鼠标返回到其笼中。</li>
		<li>等待6分钟并重复步骤1.1.2。 注: 总计, 步骤1.1.2 将重复4次, 每只鼠标 (即4套6笔画), 使每一个鼠标将收到总共24尾中风的每一天的处理。</li>
	</ol>
</li>
	<li>日常处理频率<ol>
<li>从五至计划采血日前两个星期, 处理鼠标, 如步骤 1.1, 每周五天 (小鼠在星期六或星期日没有处理, 直到最后两周的驯化)。</li>
		<li>如果在取样期间进行的实验性治疗将包括任何额外的处理 (如scruffing 或腹腔注射), 在每次处理过程中使老鼠适应这种处理活动。 注: 为适应注射程序, 使用钝针进行假注射。</li>
		<li>在计划采血日的两周内, 每周处理1.1、七天的老鼠。</li>
		<li>至少在计划的采血日前两周, 小鼠在配对时尽量减少对小鼠在取样过程中的干扰。</li>
	</ol>
</li>
</ol>2、采血管的制备
<ol><li>在玻璃瓶中制备超灵敏 LH 检测缓冲器: 0.2% 牛血清白蛋白和 0.05% Tween-20 在 PBS [0.2 克氯化钾, 8 克氯化钠, 1.44 克 Na2HPO4 (无水), 0.24 克的 PO2, 超纯水到4升, pH 1, 过滤器和蒸压釜], 贮藏在4°c。1 注: 超灵敏 LH 检测缓冲器的体积可根据样品数量和样品体积计算, 如下所示 (2.2 & 讨论)</li>
	<li>准备采血管 (0.6mL 离心管)。标签管和整除检测缓冲器 (每管57µL 缓冲器; 注意, 这一体积可能因所收集的血液量或血液与所需的缓冲液的比例而异)。管可以提前几天准备取样, 贮存在4°c。</li>
</ol>3. 收集血液
<ol><li>收集采血材料的准备工作。<ol>
<li>准备使用的生物安全柜, 并安排以下材料在敞篷或在附近的手推车:10 µL 吸管 (设置为3µL 或需要的血液收集量), 吸管提示, 垃圾桶的使用吸管提示, 定时器, 打印与动物数量和样本数 (有关取样或动物问题的说明), 带有采血管、剪刀和小纱布的冰桶 (2 "x 2")。 注意: 在实验室计时器上的 "计数" 功能是有用的, 因为它没有一个可以干扰老鼠的声音警报。</li>
		<li>使用永久性标记标记每个鼠标的尾部 (靠近底座), 以帮助在取样过程中快速识别正确的鼠标。</li>
	</ol>
</li>
	<li>尾部和采血前准备的修剪。<ol>
<li>在血液收集开始前45分钟, 将第一只老鼠从笼子里移开, 在安全柜内的适当表面 (干净的笼盖工作良好)。</li>
		<li>使用锋利, 无菌剪刀删除1-2 毫米的鼠标尾部的尖端, 以下无菌制剂 (即用酒精和 betadine 擦洗)。 注: 这可以在清醒的动物, 温和克制或在异氟醚麻醉下完成。 此外, 还可以根据当地 IACUC 的建议提供镇痛。</li>
		<li>小心地抚摸鼠标的尾部如上所述 (1.1.2; 1-2 冲程通常是足够的), 直到血滴在尾部尖端形成。</li>
		<li>使用一个干净的提示吸管收集3µL 的血液从尾部尖端和丢弃的吸管尖端与血液。</li>
		<li>在把老鼠送回笼子之前, 要确保血流停止。如有必要, 用无菌纱布轻轻按压。</li>
		<li>对所有老鼠重复步骤 3.2.1 3.2.5。随后的血液样本可以通过牢牢地抚摸老鼠的尾巴来收集, 直到尾巴尖端形成一滴血。 注: 如果血滴在结实的笔触后不形成, 尾巴可以用盐水浸泡的纱布垫擦拭。血栓更可能形成在不太频繁的取样 (即 > 15 分钟间隔)。一旦血栓从静脉中清除, 取样就可以继续进行。</li>
		<li>继续收集每只老鼠3µL 的血液, 如上文所述, 每6分钟约45分钟, 在这个前采血准备期间。扔掉血和吸管尖。</li>
	</ol>
</li>
	<li>采血<ol>
<li>从尾端收集3µL 的血, 如上文所述。注意确保将整个3µL 样品带入吸管尖端 (没有气泡或床上用品污染)。将血液样本喷射到标记管中的化验缓冲器中。混合通过反转, 并返回到冰的管。</li>
		<li>在把老鼠送回笼子之前, 要确保血流停止。如有必要, 用无菌纱布轻轻按压。</li>
		<li>对每个鼠标重复步骤 3.3.1 3.3.2, 每隔6分钟为预定的取样持续时间。监测每一个动物的窘迫迹象 (例如, 部分闭合的眼睑, 驼背的位置 [不是由其他治疗引起的) 或缺乏对处理的反应) 和终止抽样, 如果必要的话。 注意: 行为困扰或其他需要终止取样的并发症在我们的实验室极少发生。</li>
		<li>如果动物在血液取样后不被安乐死, 请确保尾部的血流完全停止。将每只动物笼内的溅出的血液擦掉 (或更换笼子), 并将动物笼返回到外壳架上。</li>
	</ol>
</li>
</ol>4. 样品处理和分析
<ol><li>将血液样本储存在-20 摄氏度直到分析。 注: 在检测缓冲液稀释的全血标本中, LH 测定;在贮存或分析之前, 不需要分离血清或血浆。</li>
	<li>用超灵敏 ELISA 法测定血液样本, 如前所述1 , 并报告全血中 ng/毫升的值, 并对测定缓冲区中样品容积的稀释进行校正。</li>
	<li>识别数据集中的 LH 脉冲。根据数据集的不同, 计算和比较平均脉冲振幅和脉冲频率 (或脉间间隔)。 注: 已发布标准13,14和程序,15,16检测 LH 脉冲可用。</li>
</ol>

<ol>
	<li>Steyn, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+methodology+for+and+assessment+of+pulsatile+luteinizing+hormone+secretion+in+juvenile+and+adult+male+mice.">Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice.</a> Endocrinology. 154 (12), 4939-4945 (2013).</li><li>McArdle, C. A., Roberson, M. S., Plant, T. M., Zeleznik, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Knobil and Neill's Physiology of Reproduction. , (2015).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> Journal of Pharmacology and Pharmacotherapy. 1 (2), 87-93 (2010).</li><li>Steiner, R. A., Bremner, W. J., Clifton, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+luteinizing+hormone+pulse+frequency+and+amplitude+by+testosterone+in+the+adult+male+rat.">Regulation of luteinizing hormone pulse frequency and amplitude by testosterone in the adult male rat.</a> Endocrinology. 111 (6), 2055-2061 (1982).</li><li>Moore, A. M., Prescott, M., Marshall, C. J., Yip, S. H., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+a+robust+arcuate+GABAergic+input+to+gonadotropin-releasing+hormone+neurons+in+a+model+of+polycystic+ovarian+syndrome.">Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome.</a> Proceedings of National Academy of Science U S A. 112 (2), 596-601 (2015).</li><li>Czieselsky, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulse+and+Surge+Profiles+of+Luteinizing+Hormone+Secretion+in+the+Mouse.">Pulse and Surge Profiles of Luteinizing Hormone Secretion in the Mouse.</a> Endocrinology. 157 (12), 4794-4802 (2016).</li><li>Campos, P., Herbison, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+activation+of+GnRH+neurons+reveals+minimal+requirements+for+pulsatile+luteinizing+hormone+secretion.">Optogenetic activation of GnRH neurons reveals minimal requirements for pulsatile luteinizing hormone secretion.</a> Proceedings of National Academy of Science U S A. 111 (51), 18387-18392 (2014).</li><li>Yang, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Psychosocial+Stress+Inhibits+LH+Pulsatility+and+Kiss1+Neuronal+Activation+in+Female+Mice.">Acute Psychosocial Stress Inhibits LH Pulsatility and Kiss1 Neuronal Activation in Female Mice.</a> Endocrinology. 158 (11), 3716-3723 (2017).</li><li>Clarkson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition+of+the+hypothalamic+GnRH+pulse+generator+in+mice.">Definition of the hypothalamic GnRH pulse generator in mice.</a> Proceedings of National Academy of Science U S A. 114 (47), E10216-E10223 (2017).</li><li>Lewis, V. J., Thacker, W. L., Mitchell, S. H., Baer, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technic+for+obtaining+blood+from+mice.">A new technic for obtaining blood from mice.</a> Laboratory Animal Science. 26 (2 Pt 1), 211-213 (1976).</li><li>Abatan, O. I., Welch, K. B., Nemzek, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+saphenous+venipuncture+and+modified+tail-clip+blood+collection+in+mice.">Evaluation of saphenous venipuncture and modified tail-clip blood collection in mice.</a> Journal of the American Association of Laboratory Animal Science. 47 (3), 8-15 (2008).</li><li>Durschlag, M., Wurbel, H., Stauffacher, M., Von Holst, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+blood+collection+in+the+laboratory+mouse+by+tail+incision--modification+of+an+old+technique.">Repeated blood collection in the laboratory mouse by tail incision--modification of an old technique.</a> Physiology and Behavior. 60 (6), 1565-1568 (1996).</li><li>Goodman, R. L., Karsch, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsatile+secretion+of+luteinizing+hormone:+differential+suppression+by+ovarian+steroids.">Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids.</a> Endocrinology. 107 (5), 1286-1290 (1980).</li><li>Clarke, I. J., Cummins, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+gonadotropin-releasing+hormone+pulse+frequency+associated+with+estrogen-induced+luteinizing+hormone+surges+in+ovariectomized+ewes.">Increased gonadotropin-releasing hormone pulse frequency associated with estrogen-induced luteinizing hormone surges in ovariectomized ewes.</a> Endocrinology. 116 (6), 2376-2383 (1985).</li><li>Vidal, A., Zhang, Q., Medigue, C., Fabre, S., Clement, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DynPeak:+an+algorithm+for+pulse+detection+and+frequency+analysis+in+hormonal+time+series.">DynPeak: an algorithm for pulse detection and frequency analysis in hormonal time series.</a> PLoS One. 7 (7), e39001 (2012).</li><li>Merriam, G. R., Wachter, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algorithms+for+the+study+of+episodic+hormone+secretion.">Algorithms for the study of episodic hormone secretion.</a> American Journal of Physiology. 243 (4), E310-E318 (1982).</li><li>Xie, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Deletion+of+Ghrelin-O-Acyltransferase+on+the+Pulsatile+Release+of+Growth+Hormone+in+Mice.">Effect of Deletion of Ghrelin-O-Acyltransferase on the Pulsatile Release of Growth Hormone in Mice.</a> Journal of Neuroendocrinology. 27 (12), 872-886 (2015).</li><li>Koch, C. E., Leinweber, B., Drengberg, B. C., Blaum, C., Oster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+circadian+rhythms+and+stress.">Interaction between circadian rhythms and stress.</a> Neurobiology of Stress. 6, 57-67 (2017).</li><li>Tuli, J. S., Smith, J. A., Morton, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corticosterone,+adrenal+and+spleen+weight+in+mice+after+tail+bleeding,+and+its+effect+on+nearby+animals.">Corticosterone, adrenal and spleen weight in mice after tail bleeding, and its effect on nearby animals.</a> Laboratory Animal. 29 (1), 90-95 (1995).</li><li>. Animal Welfare Inspection Guide Available from: <a target="_blank" href="https://www.aphis.usda.gov/animal_welfare/downloads/Animal-Care-Inspection-Guide.pdf">https://www.aphis.usda.gov/animal_welfare/downloads/Animal-Care-Inspection-Guide.pdf</a> (2017)</li><li>Steyn, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+method+for+the+determination+of+pulsatile+growth+hormone+secretion+in+mice.">Development of a method for the determination of pulsatile growth hormone secretion in mice.</a> Endocrinology. 152 (8), 3165-3171 (2011).</li></ol>

作者没有什么可透露的。

在这里, 我们描述了一个经常性的血液收集的全血尾端样本, 以评估脉冲 LH 分泌的小鼠的协议。该协议可以收集样本, 以检测在受心理社会压力照射后搏动性 lh 分泌物的急性变化, 并且很适合在其他急性或慢性操作下对 lh 脉冲进行评估。
该协议的关键组成部分, 以实现可靠和健壮的 LH 的配置文件是处理驯化期。在文献中推测, 处理压力会损害激素分泌的想法在17 , 但没有明确的测试。然而, 对最近的出版物中描述的 LH 脉冲模式的分析清楚地显示了对处理的彻底驯化的好处。在早期的研究中, 利用10天的处理, lh 脉冲发生在不规则的时间间隔, 长时间内没有发现 lh 脉冲。1在那以后那精的出版物有一个趋向在文学为更长的处理期间 (即3 或4星期每日处理)。5,6,8此处所述的协议涉及总共5周的处理, 但在前3个周末忽略了处理, 从而实现了类似数量的处理会话, 并将实验家的周末工作量降至最低。虽然我们不能为成功检测 LH 脉冲所需的处理会话提供一个经过验证的阈值数, 但我们鼓励这一程序的新从业者考虑延长的驯化期。在某些实验范式下 (例如, 研究年轻动物的 LH 脉冲), 几个星期的处理驯化可能是不切实际的。对于这些实验, 有足够的处理驯化是至关重要的;也许每天多处理会话就足够了。
在采血期间尽量减少环境压力对本议定书的成功也是至关重要的。为了追求这个目标, 我们把老鼠放在 vivarium 的一个安静的房间里, 在整个驯化期内。在取样当天, 在动物房门外放置一个 "安静的请" 标志可以帮助消除意外的中断。在采血期间, 可以在生物安全柜中使用布, 以提供一个地方休息的吸管, 吸管的提示和钢笔, 从而减少了操作这些项目造成的噪音。最后, 建议一个人在实验日处理小鼠并收集所有血样。这个人应该勤奋地处理动物的照顾, 并在一天的一致时间 (这应该是类似的取样时间), 因为生理输入是已知的影响压力反应。18如果 LH 脉冲很少或无法检测到, 则应确定并最小化外部压力源。另一个与在采血期间尽量减少压力有关的程序性细节是尾部修剪和采集样品的时间间隔。在采集样品前, 我们先将每只老鼠的尾部45分钟夹紧。这种延迟服务于世俗地将与尾部修剪相关的压力与取样过程中施加的任何附加处理分开, 因为单独的尾部修剪已经证明可以提高循环皮质酮的浓度。在这个45分钟的窗口中, 19个血样被撤回 (但被丢弃), 以进一步适应小鼠的处理程序, 防止在这种延迟过程中形成血栓。
在使用本议定书进行频繁的小鼠采血时, 最后的考虑是这个物种的小血量会限制收集的每个样本的数量和体积。根据美国农业部动物检查指南, 老鼠每公斤体重有72毫升的血。20为生存实验, 7.5% 的血容量可以收集在一个单一的取样期间, 给予1周的恢复期, 10% 的血容量可以收集到2周的恢复期。我们经常收集 < 7.5% 的估计血量的老鼠使用目前频繁的血液收集协议 (包括这里显示的代表性结果), 没有任何不利影响 LH 分泌模式。在我们的 vivarium, 成年 C57/Bl6 去卵巢小鼠体重约22克 (理论血容量1.58 毫升), 允许118.5 µL 的血液收集。除了7个预收集处理样本 (未化验或分析), 30 个样本收集超过180分钟总共111µL 的血液 (3 µL 每个样本), 这是低于7.5% 血容量限制。应该指出的是, 血液样本量可能需要调整, 以检测 lh 在某些情况下, 当 lh 浓度预计低。在 gonadectomized 小鼠中, 3 µL 的全血足以检测 LH。当 lh 浓度较低时, 应考虑收集更大的血容量, 因此预期更接近 lh 测定的灵敏度。因此, 尽管老鼠的血量小, 可以收集足够的血液样本来评估180分钟内搏动性 LH 分泌物的不折不扣剖面。
总之, 这一频繁的血液取样协议突出了一个延长处理适应期, 以确保评估的脉搏 LH 分泌物的小鼠在一段时间内。我们已经证明, 这个协议足以描述在心理压力暴露之前和之后可以监测的 LH 的健壮和常规脉冲。该协议可用于评估 LH 对其他类型的压力或其他可能改变生殖激素分泌的治疗方法的反应。此外, 这种血液收集协议可以调整, 以测量其他循环因素, 随着时间的推移浓度变化, 如已报告的生长激素。21这是一个显着的贡献, 神经内分泌学的领域, lh 的分泌模式现在可以监测在一个物种, 复杂的遗传和体内分子方法研究 LH 脉冲调节是可用的, 并实际。

哺乳动物性腺功能依赖于促性腺激素分泌, 促黄体激素 (LH) 和卵泡刺激激素, 从垂体腺。促性腺激素是以脉动或浪涌模式分泌促性腺激素释放荷尔蒙 (促性腺素) 的下丘脑分泌物。LH 和 FSH 的合成和分泌都是通过内分泌、分泌和分泌作用来调节的, 包括下丘脑促性腺激素、性腺激素和激活-抑制素-卵泡抑素系统, 以及无数包括应力和能量平衡在内的生理条件。2
lh 在血液中的脉动模式产生于一个相当突然的 lh 放电到外周血, 其次是近似指数消除。该模式的重要特征包括每个 lh 放电的频率和 lh 反应的振幅, 这两者都是由释放促性腺激素. 由于收集下丘脑-垂体门血的困难, 测量搏动性促性腺激素, LH 的取样和测量被用来作为促性腺激素调节下丘脑-垂体-性腺轴的代表。因此, 关键信息编码的频率和幅度的 LH 脉冲, 这不能确定从一个单一的样本。
对搏动性 LH 分泌物的分析历史上仅限于大型哺乳动物 (人类、灵长类动物和绵羊), 原因是它们的血容量大, 而且对频繁的血样采集有耐受性。在啮齿动物中, 频繁的血液取样仅限于大鼠, 通过留置心房导管实现。3,4相对较低的遗传成本和可用性 (例如, CRISPR) 和复杂的神经回路 (如光遗传学、chemogenetics) 操作使小鼠成为具有吸引力的模型有机体;然而, 获得频繁的血液样本和随后的 LH 浓度分析, 直到最近, 证明是难以捉摸的。这项艰巨的任务是由 Steyn 和同事开创的。1从那以后, 几个实验室已经开始使用频繁的血液取样和超敏 lh 检测, 以评估脉冲 LH 分泌物在各种实验范式。5,6,7,8,9应指出的是, 寻求一种实用的方法来收集来自小鼠的多份血样已经进行了至少40年的10 , 并进行了多次改良。11,12
对 LH 脉冲模式 (即频率和振幅) 的评估, 是监测这种遗传听话动物模型中的基础促性腺激素分泌的一个重要的改进。传统上, 小鼠的 LH 浓度在单个血液样本中测定。单点样本的一个缺点是高度可变的数据集, 因为 LH 浓度在每个脉冲期间自然波动。另一个缺点是, 孤立的测量本身就忽略了 LH 脉冲分泌模式所传达的关键信息。因此, 在自由行为, 无约束的小鼠收集频繁的血液样本 (除了在取样过程中温和处理) 的方法将提供增强的信息, 并证明有用的许多实验室研究搏动激素调节。
在这里, 我们描述了一个协议, 收集频繁 (每6分钟) 血液样本从清醒, 无拘无束的老鼠。重要的是, 我们包括一个处理习得协议, 允许强健和连续检测脉冲 LH 分泌物在整个血液样本收集的时间内, 对急性挑战的前期和事后评估可以确定,如对固定化和克制的社会心理压力的反应。对全血标本 LH 浓度进行了有效的测定;1本协议的重点是收集血液样本的 LH 脉冲测量方法。

<table><tbody><tr><td>Biosaftey cabinet</td><td>Lab Products Inc</td><td>L/F-B</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A5403</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Na2HPO4 (anhydrous)</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>KH2PO4</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>Ultrapure water</td><td>Millipore</td><td></td><td>Purified and filtered water</td></tr><tr><td>Broome Rodent Restraint Device</td><td>Harvard Apparatus</td><td>52-0460</td><td>Not necessary for blood collection, but were used in the collection of representative data.</td></tr><tr><td>DynPeak</td><td>n/a</td><td>n/a</td><td>http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039001</td></tr></tbody></table>

frequent tail-tip blood sampling in mice for the assessment of pulsatile luteinizing hormone secretion

在许多内分泌系统中, 循环因子或荷尔蒙不会连续释放, 而是作为一种离散的脉搏分泌, 以响应释放因子。单点取样措施不足以充分理解正常生理条件下或失调条件下脉动激素分泌模式的生物学意义。促黄体激素 (LH) 是由垂体前 gonadotrope 细胞合成的, 在脉动模式下分泌, 需要经常采集血液样本进行脉搏评估。这是不可能的, 直到最近, 由于开发了高灵敏度 LH 检测和先进的技术, 频繁的低量样本收集, 最初描述的 Steyn 和同事。1在这里, 我们描述了一个协议, 从小鼠频繁的外周血标本收集, 充分的处理驯化, 以检测 LH 的脉动分泌。目前的协议详述了一个扩大的驯化期, 允许评估的健壮和连续脉冲的 LH 在多个小时。在这个协议中, 尾巴的尖端被修剪, 血液是从尾部收集使用手持吸管。为了评估 gonadectomized 小鼠的搏动性 LH, 每隔5-6 分钟收集一系列样品, 90-180 分钟. 重要的是, 在清醒、自由行为的小鼠体内, 采集血液和测定 lh 的健壮脉冲, 给予适当的处理驯化和努力, 以尽量减少环境压力。在采血前4-5 周内可以达到足够的驯化。本议定书强调了方法的进展, 以确保收集全血样本, 以评估在老鼠的多小时内脉动 LH 分泌模式, 一个强大的动物神经内分泌研究模型。

典型的 LH 脉冲模式从4小鼠显示在图 1 (数据重印从杨等, 2017)。LH 是在频繁的血液样本测量, 以确定对急性社会压力挑战的反应。成年雌性 C57/Bl6 小鼠被卵巢切除, 并在本议定书中详细处理, 以便频繁采血。为所有动物的前90分钟收集血液样本, 以建立 LH 搏动分泌物的预处理基线。在取样的第二个90分钟内, 控制 (非受压) 动物留在笼子里, 限制压力动物被放置在老鼠的克制装置中, 并被隔离到新的笼子里。控制动物在整个取样期间显示清楚, 明确的 LH 脉冲。与此相反, 约束的心理社会压力迅速而明确地抑制了脉动 LH 分泌物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57894/57894fig1.jpg"> 图 1: 卵巢切除小鼠的典型 LH 脉冲模式。(A、C)控制无应力小鼠。(B、D)用0-90 分钟 (灰色阴影区) 和 LH 测定的压力小鼠。开放数据点表示由 DynPeak 标识的脉冲。15这一数字已从杨等人, 内分泌学 158 (11): 3716-3723, 2017 经内分泌学会许可, 版权所有2017。<a href="/files/ftp_upload/57894/57894fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion at JoVE.com

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

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。

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing ...

frequent-tail-tip-blood-sampling-mice-for-assessment-pulsatile

Nanyang Technological University

Research

JoVE Journal

Neuroscience

14.7K 次观看.  University of California, San Diego School of Medicine. 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。

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

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

真空采血系统和眼镜放大镜从非麻醉小鼠尾静脉中修饰采血

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

科研

行为学

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

发育生物学

Handling Techniques to Reduce Stress in Mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal&#39;s capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

处理技巧，以减轻老鼠的压力

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects ...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-&beta;-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Here, we describe how to identify the stage of the murine reproductive (proestrus, estrus, metestrus, or diestrus) by simple, non-invasive collection and cytological assessment of vaginal smear samples. We further describe how vaginal cytology reflects circulating hormonal levels underlying transition through the murine reproductive cycle.

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the ...

生物学

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 ...

医学

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

评估小鼠全身脂质处理能力

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

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, ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

多囊卵巢综合征小鼠模型中肝葡萄糖产生的评价

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

DREADDs 技术和自动血液采样

Remote Neuronal Activation Coupled with Automated Blood Sampling to Induce and Measure Circulating Luteinizing Hormone in Mice

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of numerous inputs and neuronal populations in the modulation of LH release is still unknown. Measuring changes in LH levels in mice is often a challenge since they are easily disrupted by environmental stress. Current techniques to measure LH release and pulsatility require long-term training for mice to adapt to manipulation stress, certain restraint, the presence of the investigator, and working on individual animals, reducing its usefulness for many research questions.
This paper presents a technique to remotely activate specific neuronal populations using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) technology coupled with automated sequential blood sampling in conscious, freely moving, and undisturbed mice. We first describe the stereotaxic surgery protocol to deliver adeno-associated virus (AAV) vectors expressing DREADDs to specific neuronal populations. Next, we describe the protocol for carotid artery and jugular vein cannulation and postsurgical connection to the CULEX automated blood sampling system. Finally, we describe the protocol for clozapine-N-oxide intravenous injection for remote neuronal activation and automated blood collection. This technique allows for programmed automated sampling every 5 min or longer for a given period, coupled with intravenous substance injection at a desired time point or duration. Overall, we found this technique to be a powerful approach for research on neuroendocrine control.

作者聚焦:自动输注和血液采样对清醒小鼠进行精确的激素分析

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

Circulating luteinizing hormone (LH) levels are an essential index of the functioning of the hypothalamic-pituitary control of reproduction. The role of ...

小鼠经常尾端采血对脉动性促黄体激素分泌的评价

摘要

探索更多视频

此视频中的章节

真空采血系统和眼镜放大镜从非麻醉小鼠尾静脉中修饰采血

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

处理技巧，以减轻老鼠的压力

进行阴道灌洗，结晶紫染色法和阴道细胞学评价小鼠发情周期分期鉴定

小鼠锁骨下静脉穿刺采血

评估小鼠全身脂质处理能力

从大鼠的侧尾静脉血液取样

多囊卵巢综合征小鼠模型中肝葡萄糖产生的评价

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素

远程神经元激活与自动血液采样相结合，以诱导和测量小鼠体内循环黄体生成素