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 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.
Physical exercise is a determinant lifestyle factor for promoting health and decreasing the incidence of the most prevalent chronic diseases as well as some types of cancer in humans1.
Resistance exercise has raised interest because of its overwhelming relevance for health throughout life2, especially due to its benefits in counteracting age-related diseases that affect the locomotor system, such as sarcopenia, osteoporosis, etc3. Moreover, resistance exercise also affects tissues and organs not directly involved in the execution of movement, such as the brain4. This relevance in recent years has encouraged the development of resistance exercise models in animals to study the underlying tissular and molecular mechanisms, when it is not possible in humans or when the animals provide better insight and are a more controlled model.
Unlike resistance exercise in humans, for animal models researchers usually rely on forced procedures. However, aversive stimuli must be avoided in this context, mainly to preserve animal welfare, reduce stress, and decrease the severity of the experimental procedures5. It should be noted that animals enjoy exercise even in the wild6. For these reasons, it is necessary to improve adaptation to the experiment through prolonged stepwise acclimatization.
The devices, materials, and protocols used for resistance training and assessment in experimental animals must allow the precise control and modulation of numerous variables: load, volume, speed, and frequency7. They should also allow different types of muscle contractions to be performed: concentric, eccentric, or isometric. Considering the above, the protocols used should be able to specifically evaluate or train for different applications of strength: maximal strength, hypertrophy, speed, and endurance.
There are several methods of strength training, such as jumping in water8,9, weighted swimming in water10, or muscle electrostimulation11. However, static and dynamic ladders are versatile devices that are widely used12,13,14.
Resistance assessment in experimental animal models provides valuable information for many research settings, such as describing the phenotypic characteristics of genetically modified animals, evaluating the effect of different intervention protocols (dietary components supplementation, drug treatments, microbiota transplantation, etc.), or assessing the effect of training protocols. Training models provide insight into the physiology of adaptation to strength exercise, which helps to better understand the effect of exercise on health status and pathophysiology.
Consequently, there is no universal protocol for resistance training or the functional assessment of strength in animal models, so versatile protocols are needed.
The aim of this study is to identify the most relevant factors to be considered when designing and applying a protocol for resistance training and evaluation using static and dynamic ladders in animal models, as well as provide specific examples.
The methods presented in this protocol have been evaluated and approved by the animal research technical committee (reference PROAE 04/2018, Principado de Asturias, Spain).
1. Planning
2. Devices and materials for resistance exercise
Figure 1: Resistancetraining devices: static and dynamic ladders. (A) Mouse training with external weight on a static ladder. (B) Two mice training with weight on a dynamic ladder. (C) Schematic representation of ladder angles for training and evaluation. Please click here to view a larger version of this figure.
3. Acclimatization
NOTE: Proper acclimatization is essential to avoid exercise rejection and to minimize stress. Acclimatization is a crucial stage before resistance evaluation tests or training protocols are performed. Adequate time should be spent to achieve behavioral signs of comfort in the animals. Details of daily acclimatization with the static and dynamic ladders are shown in Table 1 and Table 2, respectively.
4. Resistance evaluation
5. Resistance training with static ladder
NOTE: Before starting the training period, acclimatization (Table 1) and training planning are necessary. To reduce anxiety, adapt and train the mice in groups of four animals sharing the same cage.
6. Resistance training with dynamic ladder
NOTE: After acclimatization, the training on the dynamic ladder is quite like the static one (Table 2). Training is performed on 2-4 mice at a time.
7. Evaluation of the crossover effect of resistance training on endurance performance
NOTE: For this, an incremental treadmill test is performed4, after 24 h of rest.
8. Animal behavior during procedures
NOTE: Continuous monitoring of the adaptation of mice to training should be performed to detect extreme fatigue, overtraining, or injury.
9. Safety procedures
Results with static ladder
The progressive resistance training protocol used and described by Codina-Martinez et al.4 (Table 4) was tested in a preliminary study consisting of 7 weeks of training on a static ladder with 6-months-old wild-type C57BL6J mice (n = 4). In this preliminary study, incremental tests to assess maximal strength were performed before and after the training period. We observed a 46.4% increase in maximal strength, meaning that at the end of the training period they were able to climb with 1.9 times their body weight (unpublished data).
In the study of Codina-Martínez et al.4, male mice (C57BL6N/129Sv) deficient in Atg4b16 and their corresponding wild-type controls (8 weeks old, n = 36 per genotype) were trained for 14 weeks (Table 4). Incremental tests to assess maximal resistance, before and after the training period, showed a percentage change of 44% in trained wild-type animals and 15.3% in atg4b-/- mice.
In another study, 8-week-old C57BL6N mice were trained for 4 weeks, 5 days/week (n = 8) (unpublished data). All sessions were designed to achieve the same exercise volume through a combination of the number of steps climbed (or distance against gravity) and weight load17 and were based on the results obtained in a maximal strength test prior to the training period. The number of steps per training session varied between 400-2,000 depending on the maximal weight load, which ranged between 25-65% of the maximal weight load at the pre-training test. We selected these maximum weight ranges because it has been described that below 75% of maximal weight there is no velocity loss to climb 1 RM, which is important for standardizing the intensity of submaximal efforts18. Again, before and after the training period, incremental tests to assess maximal strength were performed. The average percentage of variation in this parameter was 40%. Peak strength was reached by a 27 g mouse, which was able to climb 10 RM with 120 g after the training period.
Results with dynamic ladder
To evaluate the dynamic ladder as a tool for resistance training, we conducted an experiment with the aim of assessing the effect of two types of strength training: endurance-resistance training and strength training. The design and results of this study are shown here for the first time. 8-week-old C57BL6N mice were divided into three groups: Non-trained control (C, n = 5), Endurance-Resistance (E-R, n = 8), and Strength (S, n = 7). After a 3-week (12 sessions) acclimatization period (Table 2), mice were trained for 6 weeks, 5 days/week (Monday to Friday), starting at 9:00 am, for a total of 22 sessions. To reduce anxiety, mice were trained in groups of four animals sharing the same cage. Aversive stimuli were avoided, to minimize stress. The E-R group performed three times more repetitions with 1/3 of the weight load compared to the S group, so, they all performed the same accumulated work, with different combinations of load and repetitions. The speed was constant for all groups, set at 5.4 cm/s. The slope was set at 85°.
The normality of the variables was tested using the Shapiro-Wilk test. Results are shown as mean ± standard deviation (SD). t-test and ANOVA (Bonferroni post-hoc) were used for statistical differences. Significant changes were set at p < 0.05. The statistical software R (www.r-project.org) was used for all statistical analyses.
All animals included in the trained and control group completed the study. The mean daily food intake per mouse was 2.8 ± 0.11 g for C, 3.2 ± 0.24 g for E-R, and 3.3 ± 0.13 g for S. Exercised mice had a higher food intake than control mice (p < 0.05). However, there was no difference in body weight after the intervention (C: 28.0 ± 3.18 g, E-R: 28.5 ± 1.93, and S: 28.1 ± 2.52 g).
The significant increase in maximal strength after the training period was observed in S (29.5 ±1 0.9%) and E-R groups (41.5 ± 2.5% increase), while a non-significant increase was observed for C (20.0 ± 4.0%) (Figure 2). Endurance-resistance measured at the end of the training period (Figure 3) was significantly higher in the E-R group as compared to S (122.5 vs 26.9 rungs, p = 0.005) and C groups (122.5 vs 18.8 rungs, p = 0.013).
The cross-training effect of these models and the effect of strength training on endurance was also studied. For that purpose, all animals performed an incremental maximal endurance tests on a treadmill before and after the training period, according to the protocols previously described19. A significant loss in endurance was observed in C (Pre: 1219 ± 133 s vs. Post: 982 ± 149 s, p = 0.004), while no significant changes were observed for S (Pre: 1364 ± 285 s vs. Post: 1225 ± 94 s, p = 0.253), and E-R (Pre: 1139 ± 96 s vs. Post: 1185 ± 84 s, p = 0.164).
Figure 2: Maximal strength measured using an incremental test, before and after a 6-week resistance training period on a dynamic ladder following two training models: Strength and Endurance-Resistance. Legend: * p < 0.05; ** p < 0.01. Please click here to view a larger version of this figure.
Figure 3: Maximal endurance-resistance measured using a maximal endurance-resistance test, before and after a 6-week resistance training period on a dynamic ladder, following two training models: Strength and Endurance-Resistance. Legend: C: Control; S: Strength and E-R: Endurance-Resistance. * p < 0.05. Please click here to view a larger version of this figure.
Table 1: Example of a 10-day acclimatization protocol with a static ladder and wild-type mice. Please click here to download this Table.
Table 2: Example of a 14-day acclimatization protocol with a dynamic ladder and wild-type mice. Please click here to download this Table.
Table 3: Example of a training week with a static ladder. Legend: Rep: repetitions, Steps: number of rungs climbed, Slope: angle with the horizontal plane, and load: weight (g) attached to the tail. Please click here to download this Table.
Table 4: Example of three weeks of training with a static ladder as part of a 14-week training period. Labeled as low (sessions 1-4), medium (10-14), and high load (30-34). Legend: Rep: repetitions, Steps: number of rungs climbed, Slope: angle with the horizontal plane, and load: weight (g) attached to the tail. This table is adapted from Codina-Martinez et al. 20204. Please click here to download this Table.
Table 5: Example of training with a dynamic ladder. Program of two groups of endurance-resistance and strength training. Legend: The warm-up is common to both groups. The slope is set at 85°. Please click here to download this Table.
Training is an intervention with multiple applications in research, apart from the study of exercise itself. Thus, the analysis of its effect on ageing20 or certain pathological conditions and physical therapy21 has received much attention in recent years. In addition, numerous authors have analyzed the effect of pharmacological22 or dietary21 interventions on physical fitness. In this context, interest has arisen in analyzing different exercise modalities separately, with an emerging interest in resistance exercise. Resistance exercise elicits a different molecular response to endurance in numerous tissues23,24 and has also been shown to have a specific effect on a number of pathological conditions21.
The use of animal models for the study of resistance exercise is a tool with multiple applications. It allows the characterization of a specific phenotype in models of pathologies or genetically modified animals, although this description is not usually included. In addition, the implementation of exercise protocols and the evaluation of their impact on these models provides insight into the physiology or pathophysiology of these conditions25.
Some authors have previously conducted resistance training with rats12,13 and mice4,14, using different training models. Some authors have applied isometric muscle contraction protocols to train and assess strength26. Overload jumping in the water and weighted swimming were also applied9,10. Nerve stimulation performed under anesthesia11, and combining resistance training with surgical procedures to cause biomechanical muscle overload and muscle hypertrophy27 have also been done.
However, some of the interventions to improve resistance have some weaknesses. Forced exercise with electric shocks has been shown to interfere with experimental results28. Some of the procedures are stressful because they rely on forced swimming to prevent the animal from drowning9,10. Nerve stimulation is not a volitional muscle contraction and is performed under anesthesia11. The simplest approach to resistance training and assessment is that of non-invasive procedures using concentric/eccentric muscle contractions.
Although the most common devices to apply these protocols are static ladders on which the animals climb with external weights, resistance exercise could also be carried out using dynamic devices. In this regard, Konhilas et al.29 used weighted wheels. However, this approach is more like a high-intensity endurance exercise, so specificity would be lost. In this article, we show, for the first time, protocols for resistance training and resistance evaluation using a dynamic ladder, which allows for very versatile approaches. Results upon their implementation are also included. In addition, the use of a dynamic ladder means less manipulation of the animals, as they can climb with weight continuously, without the need of climbing a series of steps as with a static ladder.
The force assessment of peak forces can be performed using grip strength30 and torque generated by direct nerve stimulation31. The assessment of strength using the ladders is useful for subsequent training planning. The dynamic ladder also allows time-limit tests to be carried out, evaluating the number of steps as a function of the load. This procedure is equivalent to the maximum number of weight repetitions tests performed in humans7.
Furthermore, in relation to training and assessment methods, in this article we emphasize acclimatization as a key factor in avoiding training refusal on both static and dynamic ladders. This acclimatization is not achieved by food reward, as described in Yarsheski et al.13, but by teaching the mice to reach the resting areas at the top of the ladders, so that they are motivated to climb, without the need for food restrictions. Our goal has been to achieve humanized animal exercise, as suggested by Seo et al.32. In this regard, it is also worth noting that, following this protocol, the mice are trained in groups while maintaining social interaction. In the protocols shown in this paper, the animals' refusal of training was non-existent in both the static and dynamic ladders. This could be due to the adaptation protocol.
Our results show that different protocols with different animal models were effective in improving maximal strength. They were also sensitive enough to detect differences between genetically modified animals with alterations in muscle function and wild-type animals, both in maximal resistance and in response to training4. Furthermore, a comparison of the training programs with the dynamic ladder (strength and endurance-resistance) showed that all groups of mice increased their maximal strength, including C. For C, this could be because the mice were young at the beginning of the training period and still growing. Even so, the improvement in the S and E-R groups was much greater, which is evidence of the effect of training. Furthermore, in the post-training endurance test, which consisted of climbing as many steps as possible with the maximum weight obtained in the incremental test before training, the E-R group was clearly superior to the S and C groups. Furthermore, the incremental treadmill test showed that there was no decrease in endurance in any of the trained groups while a decrease was observed in the C group. This is consistent with the cross-training effect of resistance training on endurance previously described33. These results suggest, on one hand, the specificity of the resistance training protocols presented in this study for increasing resistance and endurance capacities. At the same time, both training modalities show a diverse effect on physical fitness34, probably due to a diverse set of molecular mechanisms triggered by each training model, overlapping to some extent23.
Although these training models affected the overall resistance of the groups of animals involved, we have also observed a great heterogeneity both in the starting resistance of the individuals and in the response to training (Figure 2 and Figure 3). This observation is in line with what has been described by other authors35. This should be considered when interpreting the results of the intervention in the different parameters to be evaluated in the samples obtained from these animals.
Finally, the static ladder is also suitable for eccentric training. It can be performed by descending with a near-maximal or supramaximal load. The load applied for this procedure must be high (e.g., 90%-100% or above of the maximum incremental concentric test load). When mice carry a near- maximal load, they naturally try to descend. In the case of eccentric training, it is necessary to allow the animals to descend rather than ascend during the acclimatization period. For this reason, it is not easy to combine both concentric and eccentric training in mice, and only one training model is feasible at a given time.
The main limitation of the protocols presented here is that evaluation of some type of strength, such as maximal isometric strength is not possible so other devices and protocols, such as grip strength, must be used.
Conclusively, resistance training and assessment using static and dynamic ladders, is a feasible method in animal research, with a wide range of protocols depending on the objective of the study.
The authors have nothing to disclose.
This work was supported in part by the Ministerio de Economía y Competitividad, Spain (DEP2012-39262 to EI-G and DEP2015-69980-P to BF-G). Thanks to Frank Mcleod Henderson Higgins from McLeod´s English Centre in Asturias, Spain, for language assistance.
Dynamic ladder | in-house production | ||
Elastic adhesive bandage 6 cm x 2.5 m | BSN medical | 4005556 | |
Gator Clip Steel NON-INSUL 10A | Digikey electronics | BC60ANP | |
Static ladder | in-house production | ||
Weights | in-house production | ||
Wire for holding weigths | in-house production |