Name: frequent tail-tip blood sampling in mice for the assessment of pulsatile luteinizing hormone secretion
Uploaded: 2018-07-04T14:00:00
Description: Watch this Scientific Journal Video about Frequent Tail-tip Blood Sampling in Mice for the Assessment of Pulsatile Luteinizing Hormone Secretion at JoVE.com

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. 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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. 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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>

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.

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		<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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Neuroscience

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

DREADDs Technology and Automated Blood Sampling

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.

Author Spotlight: Automated Infusion and Blood Sampling for Precise Hormonal Analysis in Conscious Mice

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.

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