Funding for this work was provided&#160;in part&#160;by a research grant awarded to M. F. Salvatore from the National Institute on Aging (AG040261). The authors would also like to acknowledge Victoria Fields for her assistance with the behavioral experiments associated with the work presented&#160;and Mackenzie Orchard for his assistance with construction of the backstop for the treadmill.

All of the following procedures are conducted in accordance with the Institutional Animal Care and Use Committee at LSU Health Sciences Center-Shreveport, and comply with the National Institute of Health guidelines.
1. Pre-exercise Procedures
Note: A timeline of all of the relevant procedures in this forced exercise regimen is diagrammed in Figure 1.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51827/51827fig1highres.jpg" width="500" /> 
Figure 1.&#160;Forced exercise regimen timeline. This timeline details the important events of each phase of this exercise regimen during each week of the study. The pre-acclimation phase (weeks 1-2) involves experimenter handling and baseline locomotor activity. During the acclimation phase (weeks 3-4) all rats undergo 3 days of acclimation training where they sit on the stationary treadmill and then 10 days of acclimation training to the treadmill. A minimum of two rounds of exercise (1 round = 12 consecutive days) followed by a 2-week rest period is required during the exercise training phase (weeks 5-10). Behavioral and/or neurochemical measurements can be made during these rest periods. Furthermore, this regimen can be modified to include multiple rounds of exercise in this phase. Body weight is measured throughout all phases of the study on a weekly basis, and before (first session) and after (last session) each phase of this exercise regimen. Treadmill exercise training scores are assigned after all acclimation and exercise training sessions, and range from 1-4, with 4 being the highest possible score. Briefly, a training score of 4 is assigned when rats exercise the entire training session without assistance. Similarly, assign a training score of 3 when rats require minimal assistance (defined as: assistance for less than 25% of the time of the training session) from the experimenter. Assign a training score of 2 when rats require much assistance (defined as: assistance for greater than 25% of the time of the training session) from the experimenter during the exercise session. Finally, allocate a training score of 1 to rats that are noncompliant and fail to complete an exercise session.
<ol>
	<li>Upon arrival to the animal colony, separate and singly house all subjects into standard shoebox cages. Provide food and water ad libitum to subjects throughout the entire study.</li>
	<li>Maintain a reverse 12-hr light-dark cycle (lights on from 1800 to 0600 hr) in the animal colony, and allow subjects to acclimate to this cycle for a minimum of one&#160;week. Perform all behavioral experiments during the subject&#8217;s active (dark) cycle, and 1-2 hr prior to the light-dark cycle change.</li>
	<li>Beginning two&#160;weeks prior to any behavioral experiments, handle all test subjects for several minutes daily to familiarize the subject with the experimenter, and to decrease novelty-induced stress to unfamiliar environments, such as the locomotor chamber and the treadmill.</li>
	<li>Measure each subject&#8217;s body weight once a week, and immediately before (first session) and after (last session) each major interval of the exercise paradigm.</li>
</ol>
2. Baseline Locomotor Activity Assessments: Determining Forced Exercise Group Assignments
<ol>
	<li>Assess each subject&#8217;s spontaneous locomotor activity for 60 min/day for 5 consecutive days to establish baseline locomotor capabilities. Conduct locomotor activity sessions in automated open-field locomotor activity chambers consisting of a plexiglass box (45 x 45 x 20 cm3) with a grid of infrared beams mounted horizontally and vertically as suggested in the product manual.</li>
	<li>At the beginning of each day of the locomotor activity assessments, load, and set up the computer and software program connected to the open-field locomotor activity chambers in order to record the number of beam breaks (locomotor activity) during each session.</li>
	<li>Add approximately 2 cups of pine-chip bedding to the floor of the locomotor chamber prior to each session.</li>
	<li>Transport the subjects from the animal colony to the locomotor activity chambers in their home cages immediately prior to their locomotor activity session. Load subjects into their assigned locomotor chamber under a red light, and start the 60 min session on the computer once they have been placed into the chamber.</li>
	<li>Upon completion of the locomotor activity session, remove subjects from the locomotor chamber and return them to their home cage under red light. Immediately return the subjects back to the animal colony.</li>
	<li>Remove bedding from the chamber and clean the chamber walls and floor with 50% ethanol after each session.</li>
	<li>Repeat steps 2.3-2.6 for additional locomotor sessions each day as needed until all subjects have been tested. Conduct all locomotor activity sessions in the dark, during the animal&#8217;s active cycle, and 1-2 hr prior to the light-dark cycle change.</li>
	<li>To account for the possibility of habituation to the activity chambers, rotate each subject among the available chambers such that locomotor activity is measured in a different chamber from the previous day.
	<ol>
		<li>If multiple sessions need to be conducted in one day, change the order of testing such that subjects are tested in a different order than the previous session.</li>
	</ol>
	</li>
	<li>From the data generated by the software used to measure locomotor activity, determine each animal&#8217;s baseline locomotor activity with the following 5 locomotor parameters: total distance traveled (cm), horizontal activity (number of beam breaks), movement number (number of initiated movements), time spent moving (sec), and movement speed (cm/sec).
	<ol>
		<li>In order to assign exercise and nonexercise groups, calculate the mean value across all 5 sessions for each of the 5 locomotor parameters listed in step 2.9 above.</li>
	</ol>
	</li>
	<li>From these 5 parameters, determine exercise and nonexercise (control) groups such that an equal range of locomotor activity is represented in both groups. In this way, pair rats together based on similar movement capabilities, and assign one rat to the exercise group and one rat to the nonexercise group. Thus, for example, pair the two rats that exhibit the highest average of all 5 locomotor parameters together, and assign one rat to the exercise group and the other to the nonexercise group (group assignments are chosen at random). Repeat this process for the remaining subjects.
	<ol>
		<li>Maintain pairing throughout the entire forced exercise regimen and for all data analyses.</li>
	</ol>
	</li>
	<li>If the experimenter is interested in the effects of exercise on locomotor activity, measure post-exercise locomotor activity using the same procedures described in steps 2.1-2.8.1 beginning the day after the last treadmill exercise session. NOTE: If the investigator is interested and/or the automated locomotor activity software permits, examine anxiety-related measures (i.e., time spent in periphery vs. the center of activity chamber) to assess the relative anxiety of subjects during locomotor activity assessments.</li>
</ol>
3. Acclimation to the Treadmill
<ol>
	<li>Conduct all treadmill exercise-related procedures on a motorized rodent treadmill with lanes that are separated by clear plexiglass walls&#160;during the animal&#8217;s active cycle, and 1-2 hr prior to the light-dark cycle change.</li>
	<li>In place of the electrical shock coils located at the back of each lane, use a plexiglass backstop designed specifically to fit the treadmill so that subjects can continuously exercise without coming into contact with the electrical shock coils. To prevent the backstop from sliding during the training sessions, hold the backstop in place with a 1.5 in&#160;c-clamp.</li>
	<li>Due to the experimenter handling required for treadmill exercise training, conduct all treadmill exercise sessions with the lights on.</li>
	<li>Transport subjects in their home cage to the treadmill immediately prior to their treadmill exercise training session. When the treadmill is stationary, load rats onto the treadmill in their assigned lane by sliding up the backstop just enough that subjects can maneuver under the backstop. Slide the backstop back down to the resting position once the subject is situated on the treadmill belt. Do not start the session until all subjects have been placed in their assigned lane for that particular session.
	<ol>
		<li>During the first phase of acclimation, place each rat on one of the four lanes of the stationary treadmill (as described in step 3.4) for 5-10 min/day for 3 consecutive days. Increase the time of each session with each successive day (e.g., 5 min on day 1, 7 min on day 2, and 10 min on day 3) as the rats become familiar with the treadmill apparatus and to reduce the potential for novelty stress to the treadmill apparatus. Important: Do not place rats anywhere on the treadmill (e.g.,&#160;deactivated electrical shock coils) except the stationary belt at this stage.</li>
	</ol>
	</li>
	<li>During the second phase of acclimation training, place each rat on one lane of the stationary treadmill in front of the backstop. When all subjects are in place, turn the treadmill on to a slow walking speed (e.g., 6 m/min). Regardless of experimental grouping, train all rats to walk on the treadmill during this phase to ensure that any potential effects observed from the forced exercise regimen result from the exercise training and not simply from the ability to exercise.</li>
	<li>As the belt of the treadmill begins to roll, ensure that all rats are walking in the forward direction. Assist rats as necessary by orienting or gently prodding them until they are moving forward. During the acclimation training period, train rats to walk on the treadmill such that they continuously walk on the treadmill without assistance from the experimenter.
	<ol>
		<li>Acclimate all rats to the treadmill by gradually increasing the speed over a period of 5-10 min/day for 10 consecutive days. To further facilitate compliance to treadmill exercise, train the rats at low speeds (6-8 m/min) for 5-10 min during the first 5 acclimation sessions, and increase to more moderate speeds (9-10 m/min) for 5-10 min during the last 5 acclimation sessions.</li>
		<li>Ensure that all rats can maintain a speed of 9-10 m/min for 10 min by the last session of the acclimation training period.</li>
	</ol>
	</li>
	<li>Upon completion of the treadmill acclimation session, remove subjects from the treadmill in the same manner used to load subjects as described in step 3.4. Immediately return subjects to their home cage and transport them back to the animal colony.</li>
	<li>Wipe down each individual lane including the walls and treadmill belt with 50% ethanol after each individual treadmill exercise training session.</li>
	<li>Ascertain the exercise capabilities of each rat across the exercise sessions by assigning a treadmill exercise score at the conclusion of each daily session. Assign a training score of 4 when rats exercise the entire training session without assistance. Similarly, assign a training score of 3 when rats require minimal assistance (defined as: assistance for less than 25% of the time of the training session) from the experimenter. Assign a training score of 2 when rats require much assistance (defined as: assistance for greater than 25% of the time of the training session) from the experimenter during the exercise session. Finally, allocate a training score of 1 to rats that are noncompliant and fail to complete an exercise session. 
	NOTE: The exercise scoring system is adapted, in part, from other studies19.
	<ol>
		<li>Exclude rats that cannot complete the acclimation phase due to noncompliance and/or an injury that prevents subjects from exercising (e.g., this includes rats that earn a treadmill exercise training score of 1 for more than one&#160;consecutive day of acclimation training).</li>
	</ol>
	</li>
	<li>Average the treadmill scores across each training period (e.g.,&#160;acclimation training and exercise training) to determine the exercise capabilities of each subject. Ensure that all rats are able earn a treadmill exercise score of 4 during a training session by the end of the acclimation period.
	<ol>
		<li>If some rats are not compliant to the top exercise speeds maintained by others throughout a training session, adjust the speed for these rats so that they are able to continue exercising for the same amount of time but at a slower speed throughout the training sessions. In this instance, train slower rats in separate sessions and increase the speed as the subjects adjust to the slower speeds.</li>
	</ol>
	</li>
	<li>To avoid the potential for lane bias on the treadmill during treadmill exercise training, rotate each rat among the available lanes of the treadmill so that they are training in a different lane with each subsequent exercise session.</li>
</ol>
4. Treadmill Exercise Training
<ol>
	<li>Begin the exercise training sessions the day following the last acclimation training session, and continue exercising rats for 12 consecutive days. Conduct these sessions during the animal&#8217;s active cycle, and 1-2 hr prior to the light-dark cycle change.</li>
	<li>Follow the same set-up procedures for each treadmill exercise training session used during acclimation training, as described in steps 3.2 and 3.4.</li>
	<li>At the start of each session, turn on the treadmill to a lower, warm-up speed (8 m/min) than the training speed (9-11 m/min) that will be maintained throughout the remainder of the exercise training session. Continue with this warm-up speed for 5-10 min at the beginning of the session. Adjust the speed to 9-11 m/min for the remainder of the training session. Train subjects an average of 30 min each session for the entire 12 days of exercise training.</li>
	<li>After each exercise training session, assign a treadmill exercise score to all rats in the exercise group using the same exercise scoring system described in step 3.9. Do not assign a training score during the treadmill exercise training period to nonexercise rats.</li>
	<li>Conduct the exercise and nonexercise sessions separately. Place the nonexercise rats on the stationary treadmill for the same duration that their exercise cohort trained during each session.</li>
	<li>As described in step 3.11, rotate each rat among the available lanes of the treadmill so that they are training in a different lane with each subsequent exercise session.</li>
	<li>Upon completion of one round of exercise, rest subjects for 14 days. NOTE: This rest period was chosen based on the results from the pilot study described in Figures 2A and 2B.</li>
	<li>If desired, conduct various behavioral and/or neurochemical assessments at the end of the 12-day training period. Conversely, continue with subsequent rounds of exercise training following the 14 day rest period.</li>
</ol>

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The authors declare no conflicts of interest.

There is evidence to suggest that lifestyle strategies, such as exercise, may reduce the risk for chronic age-related diseases and their cardinal symptoms, including those of the neurodegenerative diseases like Alzheimer&#8217;s disease and PD20. The benefit of forced exercise on a treadmill, as opposed to voluntary exercise on a running wheel, is that the investigator can determine the frequency, the rate, and/or intensity of exercise, and duration of the exercise session. Thus, specific post-exercise effects, such as increased locomotor or cognitive performance, may be predicted or expected following the employment of a given regimen. Furthermore, the translation of results from forced treadmill exercise to the human condition is very probable. In fact, recent studies in PD patients demonstrate that when the patient complies with an exercise regimen set forth by a trainer on a tandem bike, these patients exhibit improved locomotor scores in comparison to PD patients who only exercise at their own pace4.
The incidence of neurodegenerative diseases significantly increases with aging. Thus, age is an important consideration when designing studies that aim to translate any potential results to the human condition. For example, studies that utilize adolescent or adult animal models to study neurodegenerative disease do not take into consideration that aging is the neurobiological background of such diseases. This potentially confounds the translation of any observable effects clinically because the physiological factor of age has not been fully considered in such studies. Therefore, studies examining the effects of exercise on either locomotor or cognitive performance, should consider using aged animal models since cognitive and locomotor impairments are also associated with age-related deficits in neurochemical indices within the central nervous system10,12,16. This would allow for a more germane understanding of the neurobiological effects of exercise on locomotor and cognitive function with aging in either the presence or absence of disease pathology.
Finally, in order to ascertain the impact of exercise alone on post-exercise measures in cognitive or locomotor performance, three practices are essential to obtain nearly 100% compliance to an exercise regimen. First, it is important to maintain a reverse light-dark cycle throughout all phases of the forced exercise regimen. This ensures that test subjects are training during their active (wake) cycle. Second, it is vital that the test subjects are slowly acclimated to both the treadmill environment and the equipment used to determine their locomotor or cognitive performance. Here, the investigator should follow the procedures outlined in Figure 1 in the timeline recommended. Finally, the electric footshock apparatus should never be used during any phase of this forced exercise regimen. Instead, as highlighted in the protocol, a plexiglass backstop can be utilized to encourage subjects to continue exercising during the treadmill exercise sessions. At this juncture, the exercise regimen described should be considered a starting point for any laboratory, with the expectation that the frequency and duration of the exercise regimen can be varied according to the investigator&#8217;s paradigm. The limits to the longevity of this forced exercise regimen certainly have yet to be realized, but one may assume that barring injury, compliant rats should be able to maintain adherence to the exercise regimen for a number of months. However, it should be noted that the speed at which test subjects may execute treadmill exercise within a given time frame (30 min in this regimen) may require adjustment and maximum speeds have not exceeded 15 m/min the aforementioned studies. The best compliance is likely to be realized at speeds that do not exceed 11 m/min. Indeed, if we are to translate the exercise findings to the middle-aged and aging population, we might expect the most representative animal protocol is one that focuses on longevity and compliance, rather than speed.
To summarize, this forced exercise protocol is one that can be employed in virtually any laboratory, particularly for those with a focus on neurodegenerative disease and aging. The test subjects used in this report were all 18 months of age at the start of the study. Furthermore, the essential steps needed to obtain virtually 100% compliance in the test subjects have also been discussed. Obviously acquiring 100% compliance is a significant benefit to the investigator, primarily because one will only have to use the number of subjects required to test a given hypothesis at the desired power. Moreover, achieving 100% compliance in this regimen does not require the employment of electric footshock. Indeed, elimination of electric footshock is imperative in order to interpret the impact of exercise on behavioral measures that depend upon catecholamines, especially dopamine, given the long-known impact of stressors like footshook upon catecholamine biosynthesis17-18.

Noninvasive lifestyle strategies that can either prevent or mitigate impairments to cognition and locomotion, both often associated with aging, are gaining traction as viable practices for maintaining health and well-being1,2. For example, moderate to rigorous exercise on a weekly and consistent basis in middle-aged men can significantly increase locomotor capabilities compared to similarly-aged peers with advancing age3. Furthermore, increasing evidence suggests that these lifestyle strategies, such as exercise, can mitigate or even reverse impairments associated with neurodegenerative diseases such as Parkinson&#8217;s disease (PD)4.
Efforts to understand the molecular mechanisms of aging-related impairments have also been directed toward identifying how noninvasive strategies, like exercise, reverse or attenuate the mechanisms that contribute to such impairments. Treadmill exercise is one such strategy being increasingly employed in models of PD5-7 and cognitive impairment1, wherein the mechanisms behind improvements in locomotor or cognitive function are still being determined. However, it is important to point out that aging is the neurobiological background of PD. Thus, for any potential translation of any exercise benefit in an animal model to be realized in the human condition it must take into consideration the neurobiological background of aging. For example, compensatory mechanisms that stave off locomotor impairment during PD progression could be impaired by the process of aging8. Thus, it stands to reason that exercise paradigms must be developed that not only consider the impact of aging in either the presence or absence of disease pathology, but that also could be initiated and maintained in aged rodents.
Therefore, in consideration of aging on the neurobiological background, the selection of the rat strain should be thoughtfully considered by the investigator. Several rat strains are available for aging studies, notably the Fischer 344 and the Brown-Norway/Fischer 344 F1 (BNF) hybrid. The commonly used Sprague Dawley rat (an outbred strain) is also amenable for such use, as it is commonly used in neurodegenerative disease models, such as the 6-hydroxydopamine PD model. Our laboratory uses both the Sprague Dawley and the BNF strains in aging and neurodegenerative disease work. In this report, we will present results highlighting our exercise protocols using both strains. For those investigators strictly focusing on age-related studies, the BNF strain offers some important advantages. First, it is comparatively less vulnerable to aging-related disorders (such as tumors) and has exceptional longevity (typical life span exceeds 30 months) in comparison to other strains. They also have less variability in a variety of physiological and behavioral outcomes9, and are also suitable to investigate approaches that intervene with the process of aging. Furthermore, experiments such as forced exercise demand considerable handling by the investigator, and the gentle disposition of the BNF strain is advantageous. Aging-related changes in striatal and midbrain dopamine tissue content, as well as locomotor activity changes are similar in BNF rats and primates10-11. Our laboratory also has extensive experience with characterizing and manipulating striatal and midbrain dopamine signaling as well as locomotor activity in the BNF strain12-16. Therefore, because the risk of other neurobiologically-based diseases increases with aging, animal models of neurodegenerative disease should consider the use of rodent strains with an extensive track record of use in aging studies.
Our protocol herein also addresses some critical issues that the investigator must consider in the interpretation of their results obtained from an exercise protocol. The rodent treadmill apparatus (the use of which we will highlight in this report), is typically equipped with electric shock coils at the back of each lane of the treadmill that can be deactivated. However when these electric shock coils are activated, a small footshock is delivered to the subject as it comes in contact with the coils. This strategy is often employed in exercise studies to facilitate compliance to treadmill exercise. This is a critical point for consideration, particularly for those investigators involved in behavioral studies that are influenced by dopamine- or norepinephrine-signaling. Electrical footshock is a physiological stressor, and its impact on both neurotransmitter systems is well-documented, with increased activation of tyrosine hydroxylase17-18. Thus, increased biosynthesis of either neurotransmitter could confound the interpretation of any exercise effect, making the investigator liable for interpreting whether any observed change in behavior after exercise is strictly due to the exercise regimen or the footshock stress. Importantly, the proposed forced exercise regimen does not employ the use of footshock at any point in the treadmill acclimation or exercise training periods.
Successful exercise regimens also require maximum compliance to exercise from test subjects. The employment of footshock in order to achieve compliance could confound interpretation of experimental outcomes when the dependent measure is related to neurotransmitter signaling that is influenced by footshock (as previously discussed). Thus, the challenge is to get rodents, and in particular aged rodents, to comply with an exercise regimen. A desirable goal of any exercise regimen is to achieve nearly 100% compliance, as this will reduce the number of animals necessary to complete the exercise regimen. Specifically, maximum compliance to exercise and interpretation of exercise regimen outcomes can be obtained in our exercise regimen as a result of several pre-exercise procedures including, 1) a reverse light-dark cycle that enables animals to exercise during their active (wake) cycle, 2) ensuring locomotor performance baselines are equal prior to segregation of control and exercise groups, and 3) introducing the test subjects to an acclimation period that gradually introduces them to the requirements of the exercise regimen. Here, the aforementioned experimental considerations and steps necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent (&#62;18 months of age) will be evaluated and presented. Finally, the environment associated with exercise may be stressful, and as such, present potential confounds to determine physiological effects specific to exercise. Our protocol also controls for the exposure to the potential stress-inducing environment of the treadmill by including the nonexercise group in every aspect of the acclimation phase (including modest treadmill running) and placing them on the stationary treadmill during the treadmill exercise training. Thus, this protocol aims to describe the measures necessary to determine the physiological impact of exercise alone.

<table><tbody><tr><td>Exer-4 Treadmill for Rats</td><td>Columbus Instruments&amp;nbsp;</td><td>96157-2</td><td>Columbus has a variety of treadmills for animals with varying numbers of lanes and other options such as metabolic measurements</td></tr><tr><td>Animal Activity Meter: Opto-Varimex 4-Auto Track&amp;nbsp;</td><td>Columbus Instruments&amp;nbsp;</td><td>0170-R4</td><td>Columbus has several options for activity chambers with varying arena sizes, beam spacing, and sensors</td></tr></tbody></table>

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.

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

getting-to-compliance-forced-exercise-rodents-critical-standard-to

Nanyang Technological University

Research

JoVE Journal

Behavior

14.2K Views.  Louisiana State University Health Sciences Center. 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.

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

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 ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

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, ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Developmental Biology

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 ...

Medicine

Tracking Individual Running Metrics in Mice Using a Voluntary Wheel Running Protocol that Minimizes Social Isolation

Voluntary wheel running (VWR) in rodents is used to evaluate performance and endurance and to simulate exercise. In contrast to other exercise paradigms that require aversive stimulus to induce active movement, wheel running is voluntary. VWR generally consists of placing a running wheel inside the mouse home cage. However, analyzing exercise parameters can typically only be conducted when mice are individually housed, while overall cage effects can be reported when mice are group-housed. Therefore, the current models force experimenters to choose between measuring individual exercise parameters in single-housed mice or measuring group effects in group-housed mice; depending on the research question and model, this could be limiting. 
Therefore, the goal of this method is to demonstrate a protocol that assesses VWR in a mouse model by quantifying running parameters in individual mice, with minimal isolation. We report on device design, preoperative preparations, and experiment setup. Results of a 6-week training protocol using this VWR model suggest it provides sufficient stimulus to increase spatial object recognition memory, comparable to the clinical effect of exercise. This simple and inexpensive model facilitates investigation into the effect of VWR on multiple measurements of wheel activity, minimizing factors that could limit or affect experimental outcomes.

This protocol describes an effective voluntary wheel-running model that can be implemented in various research contexts. Unlike other voluntary running models, this method avoids isolation housing while tracking running metrics in individual mice.

Voluntary wheel running (VWR) in rodents is used to evaluate performance and endurance and to simulate exercise. In contrast to other exercise paradigms ...

Dosage-Adjusted Resistance Training in Mice with a Reduced Risk of Muscle Damage

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle mass and strength in healthy individuals and in patient populations. There is a need for precision rehabilitation tools to test the safety and effectiveness of PRT to maintain and/or restore muscle mass and strength in preclinical studies on small and large animal models. The PRT methodology and device described in this article can be used to perform dosage-adjusted resistance training (DART). The DART device can be used as a standalone dynamometer to objectively assess the concentric contractile torque generated by the ankle dorsiflexors in mice or can be added to a pre-existing isokinetic dynamometry system. The DART device can be fabricated with a standard 3D printer based on the instructions and open-source 3D print files provided in this work. The article also describes the workflow for a study to compare contraction-induced muscle damage caused by a single bout of DART to muscle damage caused by a comparable bout of isometric contractions (ISOM) in a mouse model of limb-girdle muscular dystrophy type 2B/R2 (BLAJ mice). The data from eight BLAJ mice (four animals for each condition) suggest that less than 10% of the tibialis anterior (TA) muscle was damaged from a single bout of DART or ISOM, with DART being less damaging than ISOM.

The present protocol describes a unique technique called dosage-adjusted resistance training (DART), which can be incorporated into precision rehabilitation studies performed in small animals, such as mice.

Progressive resistance training (PRT), which involves performing muscle contractions against progressively greater external loads, can increase muscle ...

Neuroscience

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Biology

Versatility of Protocols for Resistance Training and Assessment Using Static and Dynamic Ladders in Animal Models

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way to gain insight into the underlying molecular mechanisms that orchestrate these adaptations. The aim of this article is to describe exercise models and training protocols designed for strength training and evaluation of resistance in animal models and provide examples. In this article, strength training and resistance evaluation are based on ladder climbing activity, using static and dynamic ladders. These devices allow a variety of training models as well as provide precise control of the main variables which determine resistance exercise: volume, load, velocity, and frequency. Furthermore, unlike resistance exercise in humans, this is a forced exercise. Thus, aversive stimuli must be avoided in this intervention to preserve animal welfare. Prior to implementation, a detailed design is necessary, along with an acclimatization and learning period. Acclimatization to training devices, such as ladders, weights, and clinical tape, as well as to the manipulations required, is necessary to avoid exercise rejection and to minimize stress. At the same time, the animals are taught to climb up the ladder, not down, to the resting area on the top of the ladder. Resistance evaluation can characterize physical strength and permit adjusting and quantifying the training load and the response to training. Furthermore, different types of strength can be evaluated. Regarding training programs, with appropriate design and device use, they can be sufficiently versatile to modulate different types of strength. Furthermore, they should be flexible enough to be modified depending on the adaptive and behavioral response of the animals or the presence of injuries. In conclusion, resistance training and assessment using ladders and weights are versatile methods in animal research.

The present protocol describes resistance training and testing using static and dynamic ladders in animal models.

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

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

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