This work is supported by the National Institutes of Health (NIH), grant R00-DK114498, and the United States Department of Agriculture (USDA), grant CRIS: 3092-51000-062 to Y. Z.

Animals are housed in standardized conditions following animal-care and experimental protocols approved by the Institutional Animal Care and Use Committee of the Baylor College of Medicine (BCM). Animals are fed a standard or special diet, water ad libitum, and kept with a 12-hour day/night cycle.
1. Measuring of fasting serum lipids
<ol>
	<li>Transfer mice to a new cage after 5 PM and fast with free access to water, overnight (with around 16 hours of fasting before the experiment). The overnight fasting ensures complete emptying of the mice&#8217;s gastrointestinal tracts. 
	NOTE: Mice eat their feces during fasting, so food withdrawal cannot ensure they are adequately fasted.</li>
	<li>The next morning, grasp the mouse by the tail and place it on a surface. Pull back gently on the tail of the mouse to place the mouse in the restrainer. Place the nose restraint to retrain the movement of the mouse while still allowing the mouse to breathe. Tighten the knob of the nose restraint. Monitor chest movements to make sure the animal is breathing normally and minimize the time a mouse spends in restrainers.</li>
	<li>Make a superficial incision (nick) in the tail vein of the free-moving mouse, and draw 25 &#181;L of blood from the incision into a glass capillary (filling about 1/3 of the capillary) without restraining the mouse. Quickly blow the blood into a microcentrifuge tube.</li>
	<li>Stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of&#160;stress.</li>
	<li>Complete the blood withdrawal for all the mice.</li>
	<li>Allow the blood to clot by leaving it undisturbed at room temperature for 1 hour. Spin the clotted blood samples at 2,000 x g at 4 &#176;C for 10 minutes in a refrigerated benchtop microcentrifuge, and transfer supernatant (serum) for analysis. 
	NOTE: Serum can be stored at &#8211;20 &#176;C for several weeks until analysis. For long-term storage, keep the serum at &#8211;70&#176;C.</li>
	<li>Analyze each lipid metabolite using the manufacturer&#8217;s provided protocol.</li>
</ol>
2. Oral Intralipid Tolerance Test
<ol>
	<li>After 5 PM, weigh the mice for the calculation of the intralipid volume to be given to them the next day. Then, transfer the mice into a new cage and fast them overnight (16 hours).</li>
	<li>The next morning, mark tails of the mice housed in one cage to help identify them in the subsequent bleeding steps.</li>
	<li>Make a nick in the tail vein and draw 15 &#181;L of blood from the incision into a glass capillary (filling about 1/5 of the capillary), and quickly blow the blood into a microcentrifuge tube for T = 0 serum. 
	NOTE: There is no need to stop the bleeding during the assay unless the mice show excess bleeding.</li>
	<li>Gavage mice 20% intralipid using an 18G gavage needle at a ratio of 15 &#181;l per gram of bodyweight, using the pre-fasting bodyweight. Stack each mouse by 1 minute. 
	NOTE: Weigh the animal and determine the appropriate gavage needle size. Generally, an 18 gauge gavage needle is appropriate for mice &#62;25 g, and a 20-22 gauge gavage needle would be more appropriate for smaller animals. Scruff the mouse, grasping the skin over the shoulders to hold the animal&#8217;s head in place. Place the gavage needle in the mouth and then gently advance along the upper palate, until&#160;the esophagus is reached. The tube should pass without resistance. Once proper depth is reached, the Intralipid can be slowly administered. Gently remove the needle following the same angle during needle insertion after administrating the Intralipid. Return the animal to the cage and monitor for signs of labored breathing or other distress. 
	NOTE: Researchers who are inexperienced with oral gavage or tail bleeding techniques can stack each mouse by 2 minutes or even longer.</li>
	<li>Draw blood at T = 1, 2, 3, 4, 5, and 6 hours: Draw 15 &#181;L of blood (1/5 capillary) per mouse through tail bleeding, and quickly blow the blood into a microcentrifuge tube.</li>
	<li>Spin the blood samples at 2,000 x g at room temperature for 10 minutes in a microcentrifuge. Transfer the supernatant, including the floating fat layer, to a PCR tube for storage. The supernatant can be stored at &#8211;20 &#176;C for several weeks until analysis. 
	NOTE: The supernatant should be plasma. If some samples have already clotted by the time of centrifugation, it does not affect triglyceride measurement.</li>
	<li>After the last blood collection, stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of extreme stress.</li>
	<li>Load 2 &#181;L of triglyceride standard and collected supernatants into a 96-well plate.</li>
	<li>Add 200 &#181;L of triglyceride reagent and let the plate incubate for 5 minutes at 37 &#176;C for color development.</li>
	<li>Measure the absorbance at 500 nm with a reference wavelength of 660 nm in a laboratory plate reader, and calculate the sample&#8217;s concentration.</li>
</ol>
3. &#946;3 Adrenergic Receptor Agonist CL 316,243 Stimulated Lipolysis Assay
<ol>
	<li>Prepare CL 316,243 as a stock solution of 5 mg/mL (50x) in sterile&#160;saline, and store at &#8211;20&#176;C until use.</li>
	<li>In the morning, weigh the mice to calculate the amount of diluted CL 316,243 solution needed for the experiment. The mouse will receive 10 &#181;L per gram of bodyweight of diluted CL 316,243, for a final dose of 1 mg/kg bodyweight.</li>
	<li>Transfer the mice into a new cage with free access to water, and fast them for 4 hours.</li>
	<li>Make enough 1x CL 316,243 solution from 50x stock using saline. The final concentration of 1x CL 316,243 solution is 0.1 mg/mL. Use saline&#160;for the control treatment group.</li>
	<li>Mark the tails of the mice housed in the same cage for easy identification during the bleeding steps.</li>
	<li>Make a nick in the tail vein, and draw 15 &#181;L of blood from the incision into a glass capillary (filling about 1/5 of the capillary), and quickly blow the blood into a microcentrifuge tube for T = 0 sample. 
	NOTE: There is no need to stop the bleeding during the assay unless the mice show excess bleeding.</li>
	<li>Inject diluted CL 316,243 solution (or control if included in the experiment) intraperitoneally at a volume of 10 &#181;L/g bodyweight. Stack each mouse by 1 minute. Use a maximum of 5 mice for each 60-minute experiment, or 10 mice for a two-person team.</li>
	<li>Draw blood at T = 5, 15, 30, 60 minutes: draw 15 &#181;L of blood (1/5 capillary) per mouse through tail bleeding.</li>
	<li>After the last blood collection, stop the bleeding using styptic powders, refill the feed in the cage, and make sure the mice show no signs of extreme stress.</li>
	<li>Spin blood samples at 2,000 x g at 4 &#176;C for 5&#160;minutes in a refrigerated microcentrifuge. Transfer the supernatant to a PCR tube for storage. The supernatant can be stored at &#8211;20&#176;C for several weeks until analysis.</li>
	<li>Load 1 &#181;L of 2x serially diluted glycerol standards (0.156, 0.312, 0.625, 1.25, and 2.5 mg/ml Trioleine-equivalent concentrations) and collected supernatants into a 96-well plate. Add 100 &#181;L of free glycerol reagent, and let the plate incubate for 5 minutes at 37 &#176;C for the color to develop.</li>
	<li>Measure the absorbance at 540 nm using a laboratory plate reader, and calculate the sample&#8217;s concentration.</li>
</ol>

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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=Molecular+processes+that+handle+--+and+mishandle+--+dietary+lipids.">Molecular processes that handle -- and mishandle -- dietary lipids.</a> Journal of Clinical Investigation. 118 (10), 3247-3259 (2008).</li><li>Burla, B., 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=MS-based+lipidomics+of+human+blood+plasma:+a+community-initiated+position+paper+to+develop+accepted+guidelines.">MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines.</a> Journal of Lipid Research. 59 (10), 2001-2017 (2018).</li><li>Umpleby, A. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+cholesterol+ester+transfer+protein+and+relationship+to+coronary+artery+disease+risk.">Functions of cholesterol ester transfer protein and relationship to coronary artery disease risk.</a> Journal of Clinical Lipidology. 4 (5), 389-393 (2010).</li><li>Singh, A. K., Singh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triglyceride+and+cardiovascular+risk:+A+critical+appraisal.">Triglyceride and cardiovascular risk: A critical appraisal.</a> Indian Journal of Endocrinology and Metabolism. 20 (4), 418-428 (2016).</li><li>Miller, M., 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=Triglycerides+and+cardiovascular+disease:+a+scientific+statement+from+the+American+Heart+Association.">Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association.</a> Circulation. 123 (20), 2292-2333 (2011).</li><li>Dron, J. S., Hegele, R. 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The authors have nothing to disclose.

The three assays described function robustly in the lab, with a few critical considerations. Overnight fasting is required for determining fasting serum lipid levels and oral intralipid tolerance test. For oral intralipid tolerance test, it is critical to spin the blood at room temperature to minimize the formation of a fat layer, especially at the 1- and 2-hour time points; it is important not to discard this fat layer if it forms. Make sure to transfer the supernatant with the lipid layer, and pipet gently to mix them ...

Carbohydrates and lipids are two major substrates for energy metabolism. Aberrant lipid metabolism results in many human diseases, including type II diabetes, cardiovascular diseases, fatty liver diseases, and cancers. Dietary lipids, mainly triglycerides, are absorbed through the intestine into the lymphatic system and enter the venous circulation in chylomicrons near the heart1. Lipids are carried by lipoprotein particles in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase at peripheral organs such as muscle and adipose tissue2. The remaining cholesterol-rich remnant particles are cleared by the liver3. Mice have been widely used in laboratories as a research model to study lipid metabolism. With comprehensive genetic toolsets available and a relatively short breeding cycle, they are a powerful model for studying how lipids are absorbed, synthesized, and metabolized.
Due to the complexity of lipid metabolism, sophisticated lipidomics studies or isotopic tracer studies are usually used to quantify collections of lipid species or lipid-related metabolic fluxes and fates4,5. This creates a massive challenge for researchers without specialized equipment or expertise. In this paper, we present three assays that can serve as initial tests before technically challenging techniques are used. They are non-terminal procedures for the mice, and thus very useful for identifying potential differences in lipid-handling capacity and narrowing down the processes affected.
First, measuring fasting serum lipid molecules can help one ascertain a mouse&#8217;s overall lipid profile. Mice should be fasted, because many lipid species rise after meals, and the extent of the increase is strongly affected by the composition of the diet. Many lipid molecules, including total cholesterol, triglyceride, and non-esterified fatty acid (NEFA), can be measured using a commercial kit and a plate reader that can read absorbance.
Second, an oral intralipid tolerance test evaluates lipid-handling capability as a net effect of absorption and metabolism. An orally administered intralipid&#160;causes a spike in circulating triglyceride levels (1&#8211;2 hours), after which the serum triglyceride levels&#160;return to basal levels (4&#8211;6&#160;hours). This assay offers information about how well a mouse can handle the exogenous lipids. Heart, liver, and brown adipose tissue are active consumers of triglycerides, whereas white adipose tissue stores it as an energy reserve. Changes in these functions will lead to differences in the test results.
Lastly, promoting lipolysis to mobilize stored lipids is considered a possible strategy for weight loss. The &#946;3-adrenergic receptor signaling pathway in the adipose tissue plays an important role in adipocyte lipolysis, and human genetics have identified a loss-of-function polymorphism Trp64Arg in &#946;3-adrenergic receptor&#160;correlated with obesity6. CL 316,243, a specific and potent &#946;3-adrenergic receptor agonist, stimulates adipose tissue lipolysis and the release of glycerol. Evaluation of a mouse&#8217;s response to CL 316,243 can provide valuable information on the development, improvement, and understanding of the efficacy of the compound.
Collectively, these tests can be used as an initial screen for changes in the lipid metabolic state of mice. They are chosen for the accessibility of the instruments and reagents. With the results derived from these assays, researchers can form an overall picture of the metabolic fitness of their animals and decide on more sophisticated and targeted approaches.

<table>
	<tbody>
		<tr>
			<td>20% Intralipid</td>
			<td>Sigma Aldrich</td>
			<td>I141</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile Syringes 1ml</td>
			<td>Shaotong</td>
			<td>B07F1KRMYN</td>
			<td></td>
		</tr>
		<tr>
			<td>CL 316,243 Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C5976</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Feeding Needles (18 Gauge)</td>
			<td>Kent Scientific</td>
			<td>FNC-18-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Free Glycerol Reagent</td>
			<td>Sigma Aldrich</td>
			<td>F6428</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Standard Solution</td>
			<td>Sigma</td>
			<td>G7793</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>999-34691</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)COLOR REAGENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>991-34891</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT A</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>995-34791</td>
			<td></td>
		</tr>
		<tr>
			<td>HR SERIES NEFA-HR(2)SOLVENT B</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>993-35191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Vedco</td>
			<td>50989-161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix Plus Chemistry Reference Kit</td>
			<td>Verichem</td>
			<td>9500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Centrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-222-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Microhematrocrit Capillary Tube, Not Heparanized</td>
			<td>Fisher Scientific</td>
			<td>22-362-574</td>
			<td></td>
		</tr>
		<tr>
			<td>NEFA STANDARD SOLUTION</td>
			<td>Fujifilm Wako Diagnostics</td>
			<td>276-76491</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Triglycerides Reagent</td>
			<td>Fisher Scientific</td>
			<td>TR22421</td>
			<td></td>
		</tr>
		<tr>
			<td>Total Cholesterol Reagents</td>
			<td>Thermo Scientifi</td>
			<td>TR13421</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Henry Schein</td>
			<td>11695-4022-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

We show with three excerpts that each assay offers valuable information about the mice&#39;s lipid metabolism. For C57BL/6J male mice, challenged by eight weeks of high-fat diet (HFD) feeding&#160; starting at eight weeks of age, total cholesterol levels were significantly elevated, while serum triglyyceride and NEFA were not (Table 1), suggesting that triglyceride and NEFA in the blood are not predominantly regulated by a dietary fat challenge. In the second cohort of mice, C57BL/6J and C57BL6/N&#160;su...

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Assessing Whole-Body Lipid-Handling Capacity in Mice

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

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3.8K Views.  Baylor College of Medicine. This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

Video: Assessing Whole-Body Lipid-Handling Capacity in Mice

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...