The authors thank Drs. Jennifer Yang and Alexander (Sasha) Kauffman for technical assistance with this technique as well as numerous helpful and critical discussions. Serum hormone assays were performed by Yang et al., 2017 at The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core, supported by the Eunice Kennedy Shriver NICHD/NIH (NCTRI) Grant P50-HD28934.
Sources of Research Support: R01 NICHD 86100 (KMB), RBM was supported by T32 NICHD 007203.

The method described here is in agreement with the National Institutes of Health Animal Care and Use Guidelines and have been authorized by the Institutional Animal Care and Use Committee at the University of California, San Diego.
1. Acclimatization to Handling
<ol>
	<li>Handling procedure
	<ol>
		<li>Place mouse cages in the biosafety cabinet. Remove the first mouse from the cage and place on a suitable surface (e.g. a clean cage lid) within the biosafety cabinet.</li>
		<li>Gentl...

<ol>
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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. 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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>

The authors have nothing to disclose.

Here we describe a protocol for frequent blood collection of whole blood tail-tip samples for assessment of pulsatile LH secretion in mice. This protocol enables the collection of samples for detection of acute changes in pulsatile LH secretion following exposure to psychosocial stress and is well-suited for assessment of LH pulses under other acute or chronic manipulations.
A critical component of this protocol for achieving reliable and robust profiles of LH pulses is the handling acclimatiz...

Gonad function in mammals is dependent upon gonadotropin secretion, luteinizing hormone (LH) and follicle stimulating hormone, from the pituitary gland. The gonadotropins are secreted in either a pulsatile or surge pattern in response to hypothalamic secretion of gonadotropin-releasing hormone (GnRH). The synthesis and secretion of both LH and FSH are regulated via endocrine, paracrine and autocrine action from a variety of molecules including hypothalamic GnRH, gonadal steroid hormones, and the activin-inhibin-follistatin system, as well as a myriad of physiological conditions including stress and energy balance.2
The pulsatile pattern of LH in blood arises from a rather abrupt discharge of LH into peripheral blood, followed by approximately exponential elimination. Important features of the pattern include the frequency of each LH discharge and the amplitude of the LH response, both of which are dictated, in part, by the release of GnRH. Due to the difficulty in collecting hypothalamic-pituitary portal blood for measurement of pulsatile GnRH, the sampling and measurement of LH is&#160;used as a proxy for GnRH regulation of the hypothalamic-pituitary-gonadal axis. Therefore, critical information is encoded in the frequency and amplitude of LH pulses, which cannot be determined from a single sample.
Analysis of pulsatile LH secretion has historically been limited to large mammals (humans, primates, and sheep) due to their large blood volume and tolerance for frequent blood sample collection. In rodents, frequent blood sampling was limited to the rat and achieved via indwelling atrial catheter.3,4 The relatively low cost and availability of genetic (e.g. cre-lox, CRISPR) and complex neural circuit (e.g. optogenetics, chemogenetics) manipulations make mice an attractive model organism; however, attainment of frequent blood samples and subsequent analysis of LH concentrations has until recently, proven elusive. This monumental task was pioneered by Steyn and co-workers.1 Since then, several labs have begun to utilize frequent blood sampling and ultra-sensitive LH assays to assess pulsatile LH secretion in a variety of experimental paradigms.5,6,7,8,9 It should be noted that the pursuit of a practical method of collecting multiple blood samples from mice has been in progress for at least 40 years10&#160;with multiple refinements made along the way.11,12
Assessment of LH pulse patterns (i.e. frequency and amplitude) represents a major refinement in monitoring basal gonadotropin secretion in this genetically tractable animal model. Traditionally, LH concentrations in mice were determined in a single blood sample. One weakness of single-point samples is a highly variable data set because LH concentrations are naturally fluctuating during each pulse. Another weakness is that isolated measurements inherently miss critical information conveyed by the patterns of LH pulse secretion. Thus, a method for collecting frequent blood samples in freely behaving, unrestrained mice (except for gentle handling during sampling) will provide enhanced information and prove useful to many laboratories investigating pulsatile hormone regulation.
Here, we describe a protocol for the collection of frequent (every 6 min) blood samples from awake, unrestrained mice. Importantly, we include a handling acclimatization protocol that allows for&#160;robust and continuous detection of&#160;pulsatile LH secretion in whole blood samples collected over a length of time in which pre- and post-assessment to an acute challenge can be determined, such as the response to the psychosocial stress of immobilization and restraint. An effective assay for LH concentrations from whole blood samples has been described previously;1 this protocol is focused on a method for collecting the blood samples for LH pulse measurement.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biosaftey cabinet</td>
			<td style="width:121px;">Lab Products Inc</td>
			<td style="width:119px;">L/F-B</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">A5403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P2287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (anhydrous)</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">S7907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure water</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>Purified and filtered water</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:216px;">Broome Rodent Restraint Device</td>
			<td style="width:121px;">Harvard Apparatus</td>
			<td style="width:119px;">52-0460</td>
			<td style="width:183px;">Not necessary for blood collection, but were used in the collection of representative data.</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">DynPeak</td>
			<td style="width:121px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:183px;">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

In many endocrine systems, circulating factors or hormones are not released continuously, but are secreted as a discrete pulse in response to a releasing factor. Single-point sampling measures are inadequate to fully understand the biological significance of the secretory pattern of pulsatile hormones either under normal physiologic conditions or during conditions of dysregulation. Luteinizing hormone (LH) is synthesized by the anterior pituitary gonadotrope&#160;cells and secreted in a pulsatile pattern which requires frequent collection of blood samples for pulse assessment. This has not been possible in mice until recently, due to the development of a high-sensitivity LH assay and advancement in a technique for frequent low-volume sample collection, initially described by Steyn and colleagues.1 Here we describe a protocol for the frequent peripheral blood sample collection from mice with sufficient handling acclimatization to detect pulsatile secretion of LH. The current protocol details an expanded acclimatization period that allows assessment of robust and continuous pulses of LH over multiple hours. In this protocol, the tip of the tail is clipped&#160;and blood is collected from the tail using a hand-held pipette. For assessment of pulsatile LH in gonadectomized mice, serial samples are collected every 5-6 min for 90-180 min. Importantly, the collection of blood and measurement of robust pulses of LH can be accomplished in awake, freely behaving mice, given adequate handling acclimatization and effort to minimize environmental stressors. Sufficient acclimatization can be achieved within 4-5 weeks prior to blood collection. This protocol highlights advances in the methodology to ensure collection of whole blood samples for assessment of pulsatile LH secretion patterns over multiple hours in the mouse, a powerful animal model for neuroendocrine research.

Representative LH pulse patterns from 4 mice are shown in Figure 1 (data reprinted from Yang et al., 2017). LH was measured in frequent blood samples to determine the response to an acute psychosocial stress challenge. Adult female C57/Bl6 mice were ovariectomized and handled as detailed in this protocol for frequent blood collection. Blood samples were collected for the first 90 min in all animals to establish a pre-treatment baseline of LH pulsatil...

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

Here we present a tail-tip blood sampling protocol for frequent sample collection in unrestrained mice. This method is useful for assessing patterns of pulsatile luteinizing hormone secretion and could be adapted for analysis of other circulating factors.

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

The overall goal of frequent tail-tip blood sampling is to obtain whole blood samples for the measurement of pulsatile luteinizing hormone secretion referred to as LH in awake non-anesthestized and normal behaving mice. This method can help answer key questions in the field of reproducive neuroendochrinology by providing a reliable protocol to assess luteinizing hormone pulsatile secretion in mice. The main advantage to this technique is that frequent handling and habituation will ensure robust and reliable luteinizing hormone pulse measurements following acute or chronic experimental treatments.For five weeks prior to the blood collection, handle the mice on a daily basis from Monday to Friday. In the final two weeks, handle the mice on Saturday and Sunday as well. Perform the mouse handling regime in a bio-safety cabinet.Remove the first mouse from its cage and place it on a suitable surface within the cabinet. Gently restrain the mouse on this surface by holding the base of its tail. Then with the other hand firmly stroke the ventral surface of the tail from the base to the tip.Do this a total of six times and then return the mouse to its cage. This constitutes one handling set. Now wait six minutes and perform another handling set.Continuing this pattern with inter set intervals, perform four handling sets per handling session. In total this provides twenty-four tail strokes per session. If experimental treatments during the sampling period will include any additional handling methods like scruffing or an intraperitoneal injection then also acclimatize the mice to such handling activity during the handling sessions.Two weeks before the planned blood collection day, house the mice in pairs to minimize their disruption during the sampling period. For the blood collections prepare 0.6 milliliter micro centrifuge tubes for blood storage. Label the tubes and for three microliter blood collections aliquot fifty-seven microliters of ACES buffer into each tube.Once prepared store the tubes at four degrees Celsius. At the bio-safety cabinet have the following items ready:pipette tips, a waste bin, a timer, an experiemental log, an ice bucket containing the blood collection tubes, sharp, steril scissors, a permanent marker, and small gauze. Forty-five minutes before starting the blood collection, remove the first mouse from its cage and place it on a suitable surface.Then mark the tail to quickly identify this mouse from its cage partner. Now use scissors to remove one to two millimeters of the tip of the tail. After clipping that tail carefully stroke the tail until a droplet of blood forms at the tip.Then collect three microliters of blood and discard both the blood and the tip into the waste bin. Next ensure that the blood flow has stopped. If needed apply gentle pressure with sterile gauze.Then return the mouse to its cage for six minutes. Now on six minute intervals over forty-five minutes, stroke the tail firmly to take additional blood samples from the mouse. If a blood droplet does not form, dab the tail with a gauze pad soaked in saline to help clear the blood clot.During the pre-sampling period, discard all the blood aliquots. After forty-five minutes start the sampling period. When taking samples transfer three microliters of blood into a collection tube mix the tube by inversion and put it back on ice.Now proceed with the sampling for as long as necessary. Once over either euthanize the mice or ensure that their tail blood flow has completely stopped and then provide the mice with a clean cage. Next transfer blood samples to a storage box and store the samples at minus twenty degrees Celsius until they can be analyzed.Luteinizing Hormone or LH was measured in frequent blood samples to determine the response to an acute psychosocial stress challenge. Adult female C57 black six mice were ovariectomized and blood samples were collected for ninety minutes to establish a pretreatment baseline of LH pulsatile secretion. For the stress treatment during the ninety minute sampling experimental animals were put into rodent restraint devices and moved into a new cage while controls remained in their home cages and were not restrained.The analysis showed that control animals had clear unambiguous LH pulses throughout the entire sampling period. In contrast the psychosocial stress of the restraint rapidly and unambiguously suppressed pulsatile LH secretion in the experimental group. When preparing to use this technique, it's important to remember to minimize environmental disturbances such as noise or interruptions by other investigators which could stress the mice and interrupt pulsatile LH secretion.Since its development by Dr.Stein and others in the Chen laboratory this technique has begun to pave the way for researchers in the field of neuroendocrinology to explore LH pulse regulation in a species where complex molecular and genetic tools are practical and available.

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14.5K Views.  University of California, San Diego School of Medicine. The overall goal of frequent tail-tip blood sampling is to obtain whole blood samples for the measurement of pulsatile luteinizing hormone secretion referred to as LH in awake non-anesthestized and normal behaving mice. This method can help answer key questions in the field of reproducive neuroendochrinology by providing a reliable protocol to assess luteinizing hormone pulsatile secretion in mice. The main advantage to this technique is that frequent handling and habituation will ensure robu...