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

Summary

Short session (≤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.

Abstract

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.

Introduction

Regular exercise is effective at preventing or delaying many age-associated diseases such as sarcopenia and frailty^1,2,3,4. However, less than 15% of those 65 and older meet recommendations of 150 min a week of moderate intensity exercise plus strength
training^5,6. As the lack of time and lengthy sessions are common barriers to exercise, high intensity interval training (HIIT) is emerging as an alternative to traditional regimens. HIIT features multiple short bursts of intense activity that are interspersed with brief periods of active recovery, and there has been recent interest in identifying the shortest regimens that still yield beneficial outcomes. Such studies include 3 day a week regimens featuring total session times of 4 min⁷, 2-3 min⁸, 1.5 min⁹, a single min¹⁰, and even 40 s¹¹.

Likewise, there has been substantial interest in HIIT animal models. A majority of studies used mice^{12,13,14,15,16,17,18,19,20,21} or rats^22,23,24,25 and were performed using a treadmill, although a few others used swimming protocols^26,27,28. A majority of these studies use VO₂max to set the initial intensity of the exercise^{13,14,19,21,24}. Additionally, although an often described benefit of HIIT is having shorter regimens, almost all of these identified studies feature regimens that last 30 min or longer^{11,12,13,14,15,18,19,21}, with the exception of one with a slightly longer than 10 min regimen²⁰, and another with 19 min across three different intensities¹⁶. To our knowledge, there are no reported animal studies that examine a 10 min or less HIIT regimen, or tailor the regimen to individual animals, with the exception of our published study¹⁷ that serves as the basis for this protocol.

Here, we describe a protocol for HIIT in aged mice designed to model individualized, short session (≤10 min) variants used recently in human studies^7,8,9,10,11. The method includes a 10 min regimen on an inclined (25°) treadmill with a 3 min warm up, and four 1 min intervals at high intensity, interspersed with three 1 min active recovery segments. Advantages of the protocol include greater clinical relevance as it features strategies for tailoring intensity to individual animals, setting intensities that are not based on VO₂max, thereby avoiding the need for metabolic treadmills, and modular design whereby the number of intervals and timings are easily adjustable. Additionally, within this protocol we provide instructions for two strategies for treadmill assessment, which include flat continuous and uphill interval, to examine endurance. Using these methods, we extend our previous findings that short session HIIT increased functional capacity in aged male mice¹⁷, and now demonstrate HIIT increases treadmill performance in aged female mice.

Protocol

All studies and experimental protocols were approved by and in compliance with guidelines of the University at Buffalo and VA Western New York Animal Care and Use Committees.

1. Experiment Setup and General Advice

NOTE: A total of twenty-four female mice on a C57BL/6J background were used in this protocol starting at 23 months of age. The mice carried a conditional SIRT^{loxp-exon4-loxp} mutation²⁹, however, this was not induced in this experiment.

1. Ensure that mice receive permanent identifiers such as ear tags, RFID chips, or tail tattoos.
   NOTE: It is recommended to also use temporary markings (e.g., permanent marker to mark tails) for quick identification during assessment periods.
2. Clean the treadmill with 0.25-0.5% bleach (v/v) or 70% (v/v) ethanol at the end of the day or to remove feces or urine between experimental trials. Dry solutions completely before initiating a new trial.
   NOTE: Ethanol may increase wear to the treadmill belt. It is recommended that the treadmill be cleaned between each run if working with group-housed male mice in order to minimize in cage fighting. It is recommended to perform treadmill assessments at the same time of day at each time point in longitudinal studies^30,31,32, and if other assessments are being performed, the order should remain the same. The investigator running the experiment should also be blind to the group designations of the mice.

2. Acclimatizing Mice to Treadmill Apparatus

NOTE: Initiate acclimatization of mice 1 month prior to baseline experiments.

1. Set up the initial training program using the treadmill software (v3.4.7) in Manual mode (Figure 1 and Table 1).
   1. Open the treadmill software. Then, click on the file and open the experiment.
   2. Input the values indicated in Table 1 on the Manual tab (Figure 1) under Acclimation.
   3. Input the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 10, and the number of shocks as 20.
      NOTE: Shock intensity for this initial run is set at level 1 (0.46 mA).
2. Set the inclination of the treadmill to 0° (flat).
3. Hold mice by the tail when placing in the treadmill and place mice directly on the belt to avoid starting mice on the shock grid.
4. Use a brush or tongue depressor to keep mice away from the shock grid when training or assessment begins. Nudge mice to begin running to avoid unintended shocks.
5. After each mouse is given the initial training program, repeat the program as above with the shock intensity increased to level 2 (0.59 mA) with at least 15 min between trainings.
6. One to two days after the initial training program administer the uphill treadmill acclimatization.
   1. Open the treadmill software. Then click on the file and then open the experiment.
   2. Input the values on the Basic tab as indicated in Table 2 under Acclimation.
   3. Under Shock Detection, input the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 10, and the number of shocks as 20.
   4. Click on the Profile Mode tab (Figure 2) and for Step 1, input a start speed of 0 m/min and an end speed of 5 m/min with a period of 5 s.
      1. Add a warm up step 2 with a start speed of 5 m/min and end speed of 5 m/min for 30 seconds. Add a transition step that starts at 5 m/min and an end speed of 6 m/min for 5 s.
      2. Add a test speed step that starts at 6 m/min and ends at 6 m/min for 20 s. Add a transition step that starts at 6 m/min and ends at 5 m/min.
      3. Add a recovery interval that starts at 5 m/min and ends at 5 m/min for 20 s (Figure 2).
   5. Repeat step 2.6.4 to add test speed steps as indicated in Table 2 under acclimation.
7. Two weeks prior to baseline assessments, continue acclimatization by providing two treadmill flat aerobic capacity assessments as described in section 3, given on consecutive days.
8. One to two days after the second endurance training, provide two treadmill uphill anaerobic capacity assessments as described in section 4, given on the same day with a minimum of 30 min between trainings.

3. Flat Continuous Treadmill Assessment

1. Create a treadmill program as described in step 2.1, using values indicated in Table 1 under Assessment. Under Shock Detection, set the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 10, and the number of shocks as 20.
2. Monitor the mice throughout the trial and remove mice from the instrument that reach the endpoint criteria.
   NOTE: This step is to avoid mice inadvertently touching the shock grid of neighboring mice and affecting data. Parameters include the total time on belt, the distance travelled, and the shocks to visits ratio. It is recommended to perform two assessments at each time point, separated by at least one day between assessments.

4. Uphill Interval Treadmill Assessment

1. Create a treadmill program as described in step 2.6, using values indicated in Table 2 under Assessment. Under Shock Detection, set the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 5, and the number of shocks as 10.
2. Set the interval field in the treadmill program to 0.5.
   NOTE: This allows for data points to be collected every second instead of every minute, which aids in identifying the speed at endpoint for each mouse. The program will automatically stop after the 50 m/min stage due to software limitations. Parameters include the time on belt, the distance travelled, and the test speed of the last successfully completed stage before endpoint. The latter is used for determining baseline intensity for the HIIT regimen described in step 5. It is recommended to perform two assessments per time point, separated by at least 30 min.

5. Short session High Intensity Interval Training

1. Set the treadmill uphill (25°) and remove the plastic cover.
2. Determine the intensity for each mouse. Use the speed of the last successfully completed stage (Step 4.5) and find the intensity group and corresponding Base, Sprint, and Dash speeds using Table 3.
3. Open the treadmill software and click on file to create a new program.
   1. Click on the Profile Mode tab and for Step 1, input a start speed of 0 m/min and an end speed as indicated under Base speed as indicated in Table 3 with a period of 5 s.
   2. Add a warm up step 2 with a start speed at Base speed and end speed at Base speed for 180 s.
   3. Input 3 intervals each with 1) a transition step that goes from Base to Sprint for 5 s, 2) an step at Sprint speed for 60s, 3) a transition step that goes from “sprint” to Base over 5 s, and 4) a step for a recovery interval at Base speed for 60 s.
   4. Add a transition step that goes from Base to Sprint speed over 5 s and a final step that goes from Sprint to Dash speed over 60 s.
      NOTE: Programs can be saved, modified, and reloaded.
4. Perform exercise three days a week with at least one day of rest between sessions (i.e., Monday, Wednesday, Friday).
5. Rewrite exercise programs to increase Base, Sprint, and Dash speeds 1 m/min every two weeks.
   NOTE: No electric shocks are given during the exercise.
6. Motivate mice to run using a makeup brush (recommended) or tongue depressor to lightly motivate mice that fall near or on the shock grid. If a mouse does not respond retry in 5 s, then 10 s, and then retry every 30 s or during a recovery interval until the end of the session.
   NOTE: consider removing mice from the study that do not run on the treadmill at any intensity or are unable to complete the first three intervals at the intensity defined for the lowest intensity group (Table 3)
7. Move mice that cannot be motivated to complete the first three intervals in two consecutive exercise sessions to lower intensity groups (Table 3).
8. Move mice that do not require motivation in two consecutive exercise sessions to higher intensity groups (Table 3).
9. Take sedentary control mice in cages place beside the treadmill as it runs. Alternatively, sedentary controls can be placed in the lanes of a non-moving treadmill for 10 min.

Results

A total of twenty-five female mice were bred and aged in house. The C57BL/6J background mice carried a SIRT1^{loxp-exon4-loxp} mutation²⁹; however, this conditional knockout was not induced and therefore all mice exhibited full length Sirtuin1 (data not shown). At 24 months of age, mice were assessed for treadmill endurance and uphill sprint capacity prior to and after the administration of two months of HIIT exercise (n = 14), or remaini...

Discussion

Benefits from short sessions are a key aspect of high intensity interval training that captures scientific and public interest. However, animal studies rarely investigate HIIT regimens that are 10 min or less. Here, we describe a protocol for a 10 min short session HIIT uphill treadmill exercise regimen that enhanced treadmill performance in aged female mice and which we previously have shown to increase physical performance in aged male mice¹⁷. A strength of our protocol is that in addition to th...

Materials

Name	Company	Catalog Number	Comments
Exer-3/6 Open Treadmill w/ Shock, Detection, auto-calibration and PC Interface/Software	Columbus Instruments	1055-SDRM	The Columbus Instruments 3/6 treadmill allow up to 6 mice or 3 rats simultaneously. The device comes with controllers to allow manual control of treadmill belt speed and shock intensity, or connections to a computer and software to run and control these elements.
Bleach	Varies	Varies	0.25-0.5% Bleach solution (V/V) is used to clean the treadmill belt between sessions
Ethanol	Varies	Varies	70% ethanol solution (V/V) can alternatively be used to clean treadmill belt between runs and sesions.
Make-up Brush (large)	Varies	Varies	A make-up brush provides a soft surface and ample length to motivate mice to continue exercise.