Name: supramaximal intensity hypoxic exercise and vascular function assessment in mice
Uploaded: 2019-03-15T12:30:00
Description: Watch this Scientific Journal Video about Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice at JoVE.com

The authors would like to thank Danilo Gubian and Stephane Altaus from the Lausanne University Hospital (CHUV) mechanical workshop for helping create the hypoxic setup. The authors would also like to thank Diane Macabrey and Melanie Sipion for their help with training the animals.

The local state&#39;s animal care committee (Service de la Consommation et des Affaires V&#233;t&#233;rinaires [SCAV], Lausanne, Switzerland) approved all experiments (authorization VD3224; 01.06.2017) and all experiments were carried out in accordance with the relevant guidelines and regulations.
1. Animal housing and Preparation
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	<li>House 6 to 8 week-old C57BL/6J male mice in the animal facility for at least 1 week prior to the beginning of the experiments in order for the mice to ge...

<ol>
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The first objective of this study was to assess the feasibility of hypoxic high-intensity training in mice and to determine the adequate characteristics of the protocol that would be well tolerated by mice. Purposely, since there is no data using supramaximal (i.e., more than Vmax) intensity training in mice, we had to perform trials based on previous protocols developed with athletes, which consisted of four to five sets of five all-out sprints (about 200% of Vmax), interspersed with 20 s active re...

In hypoxia, the decreased inspired fraction of oxygen (O2) leads to hypoxemia (lowered arterial pressure in hypoxia) and an altered O2 transport capacity1. Acute hypoxia induces an increased sympathetic vasoconstrictor activity directed toward skeletal muscle2 and an opposed &#39;compensatory&#39; vasodilatation.
At submaximal intensity in hypoxia, this &#39;compensatory&#39; vasodilatation, relative to the same level of exercise under normoxic conditions, is well established3. This vasodilation is essential to ensure an augmented blood flow and maintenance (or limit the alteration) of oxygen delivery to the active muscles. Adenosine was shown to not have an independent role in this response, while nitric oxide (NO) seems the primary endothelial source since significant blunting of the augmented vasodilatation was reported with nitric oxide synthase (NOS) inhibition during hypoxic exercise4. Several other vasoactive substances are likely playing a role in the compensatory vasodilatation during a hypoxic exercise.
This enhanced hypoxic exercise hyperemia is proportional to the hypoxia-induced fall in arterial O2 content and is larger as the exercise intensity increases, for example during intense incremental exercise in hypoxia.
The NO-mediated component of the compensatory vasodilatation is regulated through different pathways with increasing exercise intensity3: if &#946;-adrenergic receptor-stimulated NO component appears paramount during low-intensity hypoxic exercise, the source of NO contributing to compensatory dilatation seems less dependent on &#946;-adrenergic mechanisms as the exercise intensity increases. There are other candidates for stimulating NO release during higher-intensity hypoxic exercise, such as ATP released from erythrocytes and/or endothelial-derived prostaglandins.
Supramaximal exercise in hypoxia (named repeated sprint training in hypoxia [RSH] in the exercise physiology literature) is a recent training method5 providing performance enhancement in team- or racket-sport players. This method differs from interval training in hypoxia performed at or near maximal speed6 (Vmax) since RSH performed at maximal intensity leads to a greater muscle perfusion and oxygenation7 and specific muscle transcriptional responses8. Several mechanisms have been proposed to explain the effectiveness of RSH: during sprints in hypoxia, the compensatory vasodilation and associated higher blood flow would benefit the fast-twitch fibers more than the slow-twitch fibers. Consequently, RSH efficiency is likely to be fiber-type selective and intensity dependent. We speculate that the improved responsiveness of the vascular system is paramount in RSH.
Exercise training has been extensively studied in mice, both in healthy individuals and in pathological mouse models9,10. The most common way to train mice is using a rodent treadmill, and the traditionally used regimen is low-intensity training, at 40%&#8211;60% of Vmax (determined using an incremental treadmill test11), for 30&#8211;60 min12,13,14,15. Maximal intensity interval training and its impact on pathologies have been widely studied in mice16,17; thus, interval training running protocols for mice have been developed. Those protocols usually consist of about 10 bouts of running at 80%&#8211;100% of Vmax on a rodent motorized treadmill, for 1&#8211;4 min, interspersed with active or passive rest16,18.
The interest in mice exercising at supramaximal intensity (i.e., above the Vmax) in hypoxia comes from previous results that the microvascular vasodilatory compensation and the intermittent exercise performance are both more increased at supramaximal than at maximal or moderate intensities. However, to our knowledge, there is no previous report of a supramaximal training protocol in mice, either in normoxia or in hypoxia.
The first aim of the present study was to test the feasibility of supramaximal intensity training in mice and the determination of a tolerable and adequate protocol (intensity, sprint duration, recovery, etc.). The second aim was to assess the effects of different training regimen in normoxia and hypoxia on the vascular function. Therefore, we test the hypotheses that (1) mice tolerate well supramaximal exercise in hypoxia, and (2) that this protocol induces a larger improvement in vascular function than exercise in normoxia but also than exercise in hypoxia at lower intensities.

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			<td height="21" style="height:21px;width:260px;">Cotton swab</td>
			<td style="width:191px;">Q-tip</td>
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			<td height="67" style="height:67px;width:260px;">Gas mixer Sonimix 7100</td>
			<td style="width:191px;">LSI Swissgas, Geneva, Switzerland</td>
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			<td style="width:165px;">Gas-flow: 10 L/min and 1 L/min for O2 and CO2, respectively</td>
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			<td height="21" style="height:21px;width:260px;">Hypoxic Box&#160;</td>
			<td style="width:191px;">Homemade</td>
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			<td>Made in Plexiglas</td>
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			<td height="21" style="height:21px;">Motorized rodents treadmill Panlab LE-8710</td>
			<td style="width:191px;">Bioseb, France</td>
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			<td height="63" style="height:63px;width:260px;">Oximeter Greisinger GOX 100</td>
			<td style="width:191px;">GREISINGER electronic Gmbh, Regenstauf, Germany</td>
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			<td height="21" style="height:21px;width:260px;">Sedacom software</td>
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			<td height="42" style="height:42px;width:260px;">Strain gauge</td>
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supramaximal intensity hypoxic exercise and vascular function assessment in mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

To our knowledge, the present study is the first to describe a program of supramaximal intensity training in normoxia and in hypoxia for mice. In this protocol, mice ran four sets of five 10 s sprints with a 20 s recovery in between each sprint. The sets were interspersed with 5 min of recovery periods. It was unknown whether the mice would be capable of sustaining such a protocol and complete it properly. However, according to Figure 5, the body weight gain ...

Watch this Scientific Journal Video about Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice at JoVE.com

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Our protocol is significant because it describes for the first time how to perform the Supramaximal Intensity Training in mice, especially in hypoxia, and if in induces specific vascular endothelium adaptations.The main advantage of using this technique with mice is that it draws further investigation of vascular mechanism.Exercise training is an important strategy for maintaining health and preventing many chronic disease.Combining exercise with hypoxia leads to specific cellular and molecular adaptations, with potential therapeutic application.For example, in patients with peripheral artery disease, or hypertension.Demonstrating this procedure will be Jessica Lavier, a PhD student from the Laboratory of the Angiology Division, Steeve Men

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Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

Assessment of Sensorimotor Function in Mouse Models of Parkinson&#039;s Disease

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson&#8217;s disease.

Exercise may alleviate behavioral impairments associated with neurodegenerative diseases. Studies of the mechanisms underlying exercise-mediated effects have increased in disease models, but the neurobiological background accompanying these diseases, specifically aging, is likely not considered. Here, we illustrate measures to increase the likelihood of a successful exercise paradigm in aged rats.

There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

Assessment of Sexual Behavior of Male Mice

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral pattern. The purpose of this article is to describe the hormone-induced estrus ovariectomized female model and the experimental procedure for the assessment of sexual behavior of male mice. The most important sexual behavioral elements are demonstrated in the video and illustrations. The critical steps, advantages, and limitations of the sexual behavior test are explained as well. Finally, the behavior parameters are presented, and mounting, intromission, and ejaculation processes in mating are distinguished. Behavioral parameters are assessed in terms of the occurred duration and counts during the test period.

This article describes how to perform sexual behavior tests in male mice.

Sexual behavior is highly species-specific. Although rodents have slightly different sexual behaviors, mice and rats have a similar sexual behavioral ...

Impact of High-intensity Interval Exercise and Moderate-Intensity Continuous Exercise on the Cardiac Troponin T Level at an Early Stage of Training

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has been described. The exercise-induced cTnT response distorts the diagnostic role of the cTnT assay. Although high-intensity interval exercise (HIE) is growing in popularity and concerns remain about its safety, available data related to cTnT release after HIE is limited, which hampers the use of HIE as a health intervention. Here, we present three representative HIE protocols [traditional HIE (repeated 4 min cycling at 90% V&#775;O2max interspersed with 3 min rest, 200 kJ/session); sprint interval exercise (SIE, repeated 1 min cycling at 120% V&#775;O2max interspersed with 1.5 min rest, 200 kJ/session); and repeated sprint exercise (RSE, 40 x 6 s all-out sprints interspersed with 9 s rest)] and one representative MCE protocol (continuous cycling exercise at an intensity of 60% V&#775;O2max, 200 kJ/session). Forty-seven sedentary, overweight young women were randomly assigned to one of four groups (HIE, SIE, RSE, and MCE). Six bouts of respective exercise were performed by every single group, with each being 48 h apart. Meanwhile, for four groups, the duration of the entire testing period was identical, being 10 days. Before and after the first and final exercise bouts, an assessment was conducted of cTnT. The current study provides a frame of reference giving a clear picture of how a specific exercise session affects the circulating cTnT concentration at the early stage of training. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise, especially for HIE.

Here, we present protocols of high-intensity interval and moderate-intensity continuous exercise to observe the response of circulating cardiac troponin T (cTnT) concentration to acute exercise over 10 days. The information may assist with clinical interpretations of post-exercise cTnT elevation and guide the prescription of exercise.

An elevation in cardiac troponin T (cTnT), as a highly specific biomarker of cardiomyocyte damage, after moderate-intensity continuous exercise (MCE) has ...