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Published: 07 February 2020

A comparison of the metabolic effects of treadmill and wheel running exercise in mouse model

Youn Ju Kim 1 , 2 , 3 na1 ,
Hye Jin Kim 2 , 3 na1 ,
Won Jun Lee 4 &
Je Kyung Seong ORCID: orcid.org/0000-0003-1177-6958 1 , 2 , 3 , 5

Laboratory Animal Research volume 36 , Article number: 3 ( 2020 ) Cite this article

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Aerobic exercise is well known to have a positive impact on body composition, muscle strength, and oxidative capacity. In animal model, both treadmill and wheel running exercise modalities have become more popular, in order to study physiological adaptation associated with aerobic exercise. However, few studies have compared physiological adaptations in response to either treadmill exercise (TE), or voluntary wheel running exercise (WE). We therefore compared each exercise intervention on body composition and oxidative markers in male C57BL/6 N mice. The total distance run was remarkably higher in the WE group than in the TE group. Both forms of exercise resulted in the reduction of body weight, fat mass, and adipocyte size. However, the average for grip strength of WE was higher than for control and TE. Interestingly, PGC-1α expression was increased in the gastrocnemius (glycolytic-oxidative) and soleus (oxidative) muscle of TE group, whereas WE showed a significant effect on PGC-1α expression only in the soleus muscle. However, muscle fiber type composition was not shifted remarkably in either type of exercise. These results suggest that TE and WE may exert beneficial effects in suppressing metabolic risks in mouse model through attenuating body weight, fat mass, size, and increase in mitochondria biogenesis marker, PGC-1α.

Introduction

It is well known that regular exercise can have a substantial positive effect on various health conditions [ 1 ]. In particular, aerobic exercise has emerged as an effective prevention and treatment for metabolic problems [ 2 ]. Therefore, many researchers have tried to utilize the treadmill exercise (TE) or wheel running exercise (WE) in mouse and rat model to detect various physiological and metabolic responses [ 3 , 4 , 5 , 6 , 7 , 8 ]. As is commonly known, TE is required exercise at the appointed time and intensity, while WE is voluntary enhanced activity in mice. However, it is not clear which form of exercise training is more appropriate for the challenges in the study of metabolic changes by aerobic exercise. Our study aimed to compare the effect of 8-weeks of TE and WE training on the basic physiological and metabolic parameters, such as body composition, grip strength, skeletal muscle mitochondrial biogenesis marker (PGC-1α), and skeletal muscle fiber type in male C57BL/6 N mice model.

Materials and methods

Animal and experimental design.

The 7 weeks-old Male C57BL/6 N mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). Mice were randomly divided into the following groups: control (CON, n = 5), treadmill exercise (TE, n = 5), and wheel running exercise (WE, n = 5). Mice were maintained at temperature of (22–24) °C, humidity of (50–60) %, with a 12 h light/dark cycle in a specific pathogen-free barrier facility, and had ad libitum access to a regular chow diet (NIH-31, Ziegler Bros, PA), along with tap water. All animal experimental protocol was performed according to the “Guide for Animal Experiments” (Edited by the Korean Academy of Medical Sciences) and approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (Approval Number SNU-160718-3-4).

Treadmill and wheel running exercise protocol

Before the exercise training, 1 week of adaptation was followed for the TE group mice to become familiarized to the treadmill (Columbus Instruments, Ohio). After the adaption period, a 5 days/week progressive exercise training regime was utilized, such that the speed and intensity were incrementally increased from 60 min at 17 m/min in week 1 to 60 min at 24 m/min by week 8 of training, with the incline of the machine being gradually raised from (5 to 15°) during exercise periods. The WE group performed voluntary wheel running exercise for the same periods, for 8 weeks. The distance of voluntary running per day was recorded by activity wheel running machine. (Activity wheel, TECNIPLAST, Italy).

Grip strength

The grip strength of all mice was measured for maximal muscle strength. Mouse grasped a steel greed connected to a force gauge. Then the mice’s tail was pulled against the steel greed, until its forelimb and hind limb released the steel greed. The force (g) was measured three times, and the maximum grip strength value was used for analysis. Grip strength was measured using a Grip Strength Meter (Bioseb, Vitrolles Cedex, France) at the last week (week 8) of the experiment.

Body composition

Fat and lean body masses were assessed by 1 H magnetic resonance spectroscopy after TE and WE. Body Composition was analyzed by Nuclear Magnetic Resonance (NMR) methods (Minispec LF-50, Bruker BioSpin, MA).

Western blotting

Total proteins were extracted using PRO-PREP buffer (iNtRON Biotechnology Inc., Seoul, Korea) containing proteinase inhibitors and phosphatase inhibitors (GenDEPOT, Barker, TX). Homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C, supernatant were collected, and protein concentration was determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL). Equal amounts of protein were resolved on SDS-PAGE gels, and then transferred to PVDF membranes. Primary antibodies against the following proteins were used: PGC1α (Abcam, Cambridge, UK), Troponin I-SS (C-19), Troponin I-FS (G-7) (Santa Cruz Biotechnology, CA, USA), and GAPDH (Cell Signaling Technology, MA, USA). The membranes were then incubated with anti-rabbit or anti-mouse IgG horse-radish peroxidase-linked secondary antibody (AbClon, Korea), and then visualized with Micro-Chemi 4.2 system (DNR Bio Imaging Systems, Israel). The target protein levels were then normalized against the GAPDH protein levels. Band intensities were measured with image J software (NIH, USA).

H&E staining

Tissues were weighed, and fixed with 4% paraformaldehyde (Biosesang, Korea) at room temperature (RT) overnight. Paraffin-embedded sections of fat were sliced at thickness of 3 μm. Paraffin sections of fat tissues were deparaffinized, and stained with Hematoxylin & Eosin (H&E), following standard procedures. Sectioned tissues were analyzed under a scanner (Pannoramic Scan, 3D HISTECH) and Image-Pro program.

Statistical analysis

All values were performed using Prism 7 software. Data were expressed as the mean ± SEM. Statistical analysis was performed using One-way ANOVA between groups. Turkey’s post hoc test was performed to express the mean difference between groups. p < 0.05 was considered statically significant.

Comparison of running characteristics of the treadmill and wheel running exercise

Table 1 shows that animals exercised significantly longer on WE than on TE. The total distance increased gradually in the (2nd – 5th) weeks of training in TE mice, reaching a plateau by weeks 6–8. In WE mice, the running distance increased rapidly in the 2nd week of training, and decreased gradually until weeks 4–8.

The effect of treadmill and wheel running exercise on body weight, body composition, fat weight, and food intake

Significantly decreased body weight ( p < .05) was recorded in both the TE and WE groups after 8 week of treatment, compared to those in the CON group (Fig. 1 a). Interestingly, food Intake per day of the WE group was the highest compared with those of TE and CON groups, although WE mice had the lowest body weight (Figs. 1 b and c). Nuclear Magnetic Resonance (NMR) recorded significantly decreased fat mass in both the TE ( p < 0.01) and WE ( p < 0.001) animals after 8 weeks of training, compared to those in the CON animals (Fig. 1 d). However, lean mass was not changed by TE and WE (Fig. 1 e).

Effect of treadmill running and voluntary wheel running on body weight, food intake, body composition, and fat weight. a Body weight gain, b Body weight / food intake per week, c Food intake per day, d and e Body composition analysis by NMR spectroscopy, and f Fat (eWAT, iWAT, BAT) weight. Data are presented as the mean ± SEM; n = 5 per group. Significance level set as * p < 0.05; ** p < 0.01; *** p < 0.001. CON, Control; TE, Treadmill Exercise; WE, Wheel Running Exercise; *Compared CON vs TE; # Compared to CON vs WE, & Compared to TE vs WE

Consistent with this result, eWAT and iWAT weights were significantly lower in both TE (eWAT; p < 0.01, iWAT; p < 0.05) and WE (eWAT and iWAT; p < 0.05) groups, compared to that in the CON group. However, BAT weight was not significantly lower in the TE and WE groups, compared to that in the CON group (Fig. 1 f).

Effect of treadmill and wheel running exercise on skeletal muscle weight and grip strength

Figure 2 a shows that significantly increased muscle weight / body weight was recorded in the TE (Gastrocnemius and EDL; p < 0.05) and WE (Gastrocnemius and EDL; p < 0.05) groups, compared to those in the CON group. In addition, significantly increased EDL muscle weight / body weight was recorded in the WE group, compared to that in the TE group. Next, we determined whether increase in muscle weight was associated with increased muscle strength. The grip strength analysis revealed that grip strength per body weight was increased significantly in the WE group, compared to in the CON group. However, it was not considerably increased in the TE group, compared with that in the CON group (Fig. 2 b).

Effect of treadmill running and wheel running on skeletal muscle weight and grip strength. a Skeletal muscle (gastrocnemius, soleus, TA, and EDL) weight, and b Grip strength. Data are presented as the mean ± SEM; n = 5 per group. Significance level set as * p < 0.05. *Significantly different from the following lines. CON, Control; TE, Treadmill Exercise; WE, Wheel Running Exercise

Treadmill and wheel running reduces adipocyte size

Histological analyses also revealed that adipocyte (eWAT) size were decreased in both TE and WE groups (Fig. 3 a). In addition, the frequency (%) of adipocyte distribution was lower among TE and WE groups compared to CON group (Fig. 3 b). However, they were reduced remarkably in the WE group, compared to that in the TE group.

Epididymal white adipose tissue (eWAT) section analysis after 8 weeks of treadmill and wheel running. a Representative images of eWAT sections stained with H&E (scale bar size is 50 μm), and b Adipocyte size distribution Frequency (%) counted by Image-Pro. CON, Control; TE, Treadmill Exercise; WE, Wheel Running Exercise

Effect of treadmill and wheel running exercise on mitochondria biogenesis

To further investigate the process involved in fat mass reduction, peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) protein expression in the soleus and gastrocnemius muscle were determined. PGC1α protein expression in the soleus (oxidative) muscle was significantly increased by TE and WE (both; p < 0.001), compared to that in the CON group (Fig. 4 a and b). However, PGC-1α protein expression in the gastrocnemius (glycolytic-oxidative) muscle showed increase only in the TE group, compared to in the CON group ( p < .05) (Fig. 4 c and d).

Expression of mitochondrial biogenesis marker, PGC-1α in skeletal muscle. Expression of PGC-1α in soleus muscles ( a ) and ( b ). Expression of PGC-1α in gastrocnemius muscles ( c ) and ( d ). Data are presented as the mean ± SEM; n = (3–5) per group. Significance level set as * p < 0.05; *** p < 0.001. *Significantly different from the following lines. CON, Control; TE, Treadmill Exercise; WE, Wheel Running Exercise

Effect of treadmill and wheel running exercise on skeletal muscle fiber type shifting

The effect of TE and WE training on fiber type shift was then investigated using antibodies specific to the Troponin I isoforms Troponin I-FS (type2, white muscle), and Troponin I-SS (type1, red muscle), which are common marker proteins of distinct muscle fiber types. Troponin 1-SS is usually marked in slow-twitch oxidative fiber, such as soleus muscle. In contrast, Troponin I-FS is usually marked in fast-twitch glycolytic fiber, such as EDL. In our study, we determined whether increase in Troponin I-SS was associated with increased exercise-induced oxidative capacity. This analysis revealed that the expressions of Troponin I-SS and Troponin I-FS proteins were not significantly changed in both soleus and gastrocnemius muscle (Fig. 5 a-d).

Effect of Treadmill and Wheel Running Exercise on fiber type changes in skeletal muscles. Troponin 1-SS (Slow skeletal muscle twitch fibers, Type1 fiber) and Troponin 1-FS (Fast skeletal muscle twitch fibers, Type2b fiber) expression levels in soleus muscles ( a ), ( b ) and ( c ), gastrocnemius muscles ( d ), ( e ), and ( f ). Data are presented as the mean ± SEM; n = 3 per group. CON, Control; TE, Treadmill Exercise; WE, Wheel Running Exercise

The present study compared the impact of either TE or WE on body composition, muscle strength, muscle size, fat size, and oxidative capacity of skeletal muscle in C57BL/6 N mice. This study yielded several main findings.

First, in terms of reducing body weight and fat size, both TE and WE are effective exercise modalities. This effect was the largest in the WE group, although the food intake of the WE group was the highest among groups. These results might be due to the fact that the exercise volume of the WE group was much higher than that of TE. In terms of distance, WE mice ran roughly (20–40) times longer. Although the TE group ran much less than the WE group, the magnitude of changes in body composition after TE was similar to those observed in WE. It is known that voluntary wheel running, unlike forced treadmill running allows the animal to freely exercise with minimal or no external stress [ 9 ]. Involuntary treadmill exercise is known to stimulate release of cortisol [ 10 ]. Acute elevation of cortisol after physical exercise stimulates metabolism and catabolism. Therefore, increased level of cortisol induced by stressful involuntary treadmill exercise might be the reason for TE group to have a similar extent of decrease in weight and fat mass observed in the WE group.

Many studies have demonstrated that in response to increased energy demand, exercise-trained athletes and animals increase food intake [ 11 , 12 ]. Furthermore, Koteja et al. (1999) found that food consumption per body mass was positively associated with the number of revolutions run per day [ 13 ]. Based on the fact that our results confirmed that the WE mice consumed more food per day than the CON and TE mice, we also investigated whether a chronic aerobic exercise training increase in weight loss would promote loss of skeletal muscle mass, because loss of grip strength is strongly associated with loss of body weight, muscle mass, and strength [ 14 ]. To answer this question, we performed measurement of muscle mass/body weight and grip strength. Interestingly, grip strength was significantly elevated in the WE group. These results might be due to the fact that although the absolute value of grip strength was similar between groups, the relative value of the grip strength of the WE group was significantly higher than that of the other groups, because of the lowest body weight of the WE group.

Second, both TE and WE had no effect on muscle fiber type composition in the soleus and gastrocnemius muscle. Aerobic exercise training adaptation is characterized by changes in skeletal muscle contractile and structure protein expression toward a more oxidative fiber composition that is better suited for metabolic improvement [ 15 , 16 ]. However, in the current study, both types of exercise training could not alter the muscle fiber type composition of the glycolytic-fast and oxidative-slow muscle.

Third, muscle oxidative capacity determined by PGC-1 α was significantly affected by both TE and WE in oxidative muscle. It is well known that PGC-1α is a key regulator of skeletal muscle mitochondrial number and function, as well as an increase in oxidative muscle fiber [ 17 ]. In addition, PGC-1α has been suggested to be an important factor in mediating exercise training-induced adaptations in mitochondrial biogenesis [ 18 ]. Our results suggest that increased fat oxidation through the induction of PGC-1α by both TE and WE might be partially responsible for the significant reduction of fat size and mass in the TE and WE groups. The remarkable phenomenon was the elevation of PGC-1α expression in gastrocnemius (glycolytic-oxidative) muscle of TE mice, but not in WE mice. This result suggests that the intensity of WE was much lower in comparison to that of TE, in order to recruit type II muscle fiber. In fact, although gastrocnemius muscle is classified as type II muscle, it is actually composed of a mixture of oxidative and glycolytic fiber. Therefore, it is possible that the WE performed in our study might not be sufficient to induce mitochondrial biogenesis through the PGC-1a in glycolytic-oxidative muscle fibers.

It has been known that physiological changes induced by voluntary wheel running were often qualitatively similar, but may often be quantitatively less robust than those achieved by forced treadmill exercise, which is typically done at higher speed and inclination. However, the results of the current study show that physiological adaptations from both TE and WE were similar in terms of reducing body composition and fat size, and increasing muscle mitochondrial biogenesis, because mice undergoing voluntary WE ran substantially farther per night than TE groups. Therefore, although the intensity of forced TE was much higher than that of WE, the greater overall volume of exercise by WE appeared to be sufficient to produce similar adaptational responses.

Our results revealed that both TE and WE contribute to the maintenance of metabolic health. However, the total distance of wheel running exercise is relatively high when compared to forced treadmill exercise. Thus, it is important to consider the different factors that can affect the activity and outcomes of both TE and WE.

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author on reasonable request.

Abbreviations

Brown Adipose Tissue

Extensor Digitorum Longus

Epididymal white adipose tissue

Fast skeletal muscle twitch fibers

Inguinal White Adipose Tissue

Peroxisome proliferator-activated receptor γ coactivator-1α

Slow skeletal muscle twitch fibers

Tibialis Anterior

Treadmill Exercise

Voluntary Wheel Running Exercise

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Acknowledgments

This study was partially supported by the Research Institute for Veterinary Science, Seoul National University.

This research was supported by the Korea Mouse Phenotyping Project (NRF-2013M3A9D5072550) of the Ministry of Science, ICT and Future Planning, through the National Research Foundation.

Author information

Youn Ju Kim and Hye Jin Kim contributed equally to this work.

Authors and Affiliations

Laboratory of Developmental Biology and Genomics, BK21 Program for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, South Korea

Youn Ju Kim & Je Kyung Seong

The Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, 08826, Republic of Korea

Youn Ju Kim, Hye Jin Kim & Je Kyung Seong

Korea Mouse Phenotyping Center (KMPC), Seoul National University, 08826, Seoul, Republic of Korea

Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, 03722, South Korea

Won Jun Lee

Interdisciplinary Program for Bioinformatics, Program for Cancer Biology, BIO-MAX/N-Bio Institute, Seoul National University, 08826, Seoul, Republic of Korea

Je Kyung Seong

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YJK performed experiment, organized and analyzed data. HJK wrote and correct the manuscript. WJL and JKS managed general research and drafting. All authors read and approved this final manuscript.

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Correspondence to Won Jun Lee or Je Kyung Seong .

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Kim, Y.J., Kim, H.J., Lee, W.J. et al. A comparison of the metabolic effects of treadmill and wheel running exercise in mouse model. Lab Anim Res 36 , 3 (2020). https://doi.org/10.1186/s42826-019-0035-8

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DOI : https://doi.org/10.1186/s42826-019-0035-8

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Treadmill Training Improves Aerobic Capacity in Aged Male Mice Compared to Voluntary Wheel Running

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Emily Schmitt, Hunter Graves, Danielle Bruns, Treadmill Training Improves Aerobic Capacity in Aged Male Mice Compared to Voluntary Wheel Running, Innovation in Aging , Volume 5, Issue Supplement_1, 2021, Pages 683–684, https://doi.org/10.1093/geroni/igab046.2570

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Preclinical exercise studies typically use two forms of exercise training protocols: 1) voluntary wheel running and 2) forced treadmill running. Previous work from our group clearly demonstrates that older (18-month-old) male mice do not voluntarily engage in wheel running, especially compared to younger males or female mice. Therefore, we implemented a forced exercise treadmill training protocol to determine if treadmill training was superior to wheel running in improving aerobic capacity in older male mice. PURPOSE: To determine if a 3-week treadmill training protocol improved time to exhaustion (TTE) in older male mice. METHODS: 18-month-old male mice (n=5) were provided a running wheel in their individual cage for 2 weeks or underwent daily treadmill training (n=6) for 3 weeks with increasing speed/incline. At the end of the training period we assessed TTE. RESULTS: Older male mice that trained on the treadmill demonstrated higher TTE compared to wheel (1382 □ 32 seconds versus 500 □ 99 seconds, respectively). In addition, older male mice that trained on the treadmill improved on average ~8% in their TTE test. CONCLUSION: A 3-week treadmill training protocol improves aerobic capacity in older male mice to a greater extent than voluntary wheel running. Ongoing experiments will utilize this training protocol to understand age-related declines in cardiorespiratory fitness, circadian rhythm, and to test exercise as an intervention in the aging population.

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LED therapy modulates M1/M2 macrophage phenotypes and mitigates dystrophic features in treadmill-trained mdx mice

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Published: 04 September 2024

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Valéria Andrade Pereira 1 ,
Heloina Nathalliê Mariano da Silva 1 ,
Evelyn Mendes Fernandes 1 &
Elaine Minatel ORCID: orcid.org/0000-0001-9863-0761 1

The mdx mouse phenotype, aggravated by chronic exercise on a treadmill, makes this murine model more reliable for the study of Duchenne muscular dystrophy (DMD) and allows the efficacy of therapeutic interventions to be evaluated. This study aims to investigate the effects of photobiomodulation by light-emitting diode (LED) therapy on functional, biochemical and morphological parameters in treadmill-trained adult mdx animals. M dx mice were trained for 30 min of treadmill running at a speed of 12 m/min, twice a week for 4 weeks. The LED therapy (850 nm) was applied twice a week to the quadriceps muscle throughout the treadmill running period. LED therapy improved behavioral activity (open field) and muscle function (grip strength and four limb hanging test). Functional benefits correlated with reduced muscle damage; a decrease in the inflammatory process; modulation of the regenerative muscular process and calcium signalling pathways; and a decrease in oxidative stress markers. The striking finding of this work is that LED therapy leads to a shift from the M1 to M2 macrophage phenotype in the treadmill-trained mdx mice, enhancing tissue repair and mitigating the dystrophic features. Our data also imply that the beneficial effects of LED therapy in the dystrophic muscle correlate with the interplay between calcium, oxidative stress and inflammation signalling pathways. Together, these results suggest that photobiomodulation could be a potential adjuvant therapy for dystrophinopathies.

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Acknowledgements

This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; #2020/09733-4), Coordenação de Pessoal de Nível Superior-Brasil (CAPES) – Finance Code 001, CNPq and FAEPEX. V.A.P., H.N.M.S. and E.M. (#140845/2020-8; #130448/2022-2; #303471/2022-0; respectively) are the recipients of a CNPq fellowship. We thank Mrs. Deirdre Jane Donovan Giraldo for the English revision of the manuscript.

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Pereira, V.A., da Silva, H.N.M., Fernandes, E.M. et al. LED therapy modulates M1/M2 macrophage phenotypes and mitigates dystrophic features in treadmill-trained mdx mice. Photochem Photobiol Sci (2024). https://doi.org/10.1007/s43630-024-00626-2

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Treadmill Running of Mouse as a Model for Studying Influence of Maternal Exercise on Offspring

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1 Nutrigenomics and Growth Biology Laboratory, Department of Animal Sciences, Washington State University, Pullman, WA 99164, USA.
2 School of Food Science, Washington State University, Pullman, WA 99164, USA.
PMID: 33659487
PMCID: PMC7842301
DOI: 10.21769/BioProtoc.3838

Epidemiological studies robustly show the beneficial effects of maternal exercise in reducing maternal birth complications and improving neonatal outcomes, though underlying mechanisms remain poorly understood. To facilitate mechanistic exploration, a protocol for maternal exercise of mice is established, with the regimen following the exercise guidelines for pregnant women. Compared to volunteer wheel running, treadmill running allows precise control of exercise intensity and duration, dramatically reducing variations among individual mouse within treatments and facilitating translation into maternal exercise in humans. Based on the maximal oxygen consumption rate (VO 2 max) before pregnancy, the treadmill exercise protocol is separated into three stages: early stage (E1.5 to E7.5 at 40% VO 2 max), mid stage (E8.5 to E14.5 at 65% VO 2 max), and late stage of pregnancy (E15.5 to birth at 50% VO 2 max), which demonstrated persistent beneficial effects on maternal health and fetal development. This protocol can be useful for standardizing maternal treadmill exercise using mice as an experimental model.

Keywords: VO2max; Maternal exercise; Mice; Pregnancy; Protocol; Treadmill running.

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Figure 1.. Calorimeter and software for measuring…

Figure 1.. Calorimeter and software for measuring oxygen consumption.

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Video 1.. Mice during treadmill exercise training

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Seeing what’s going on inside a body is never easy. While technologies like CT scans, X-rays, MRIs, and microscopy can provide insights, the images are rarely completely clear and can come with side effects like radiation exposure.

But what if you could apply a substance on the skin, much like a moisturizing cream, and make it transparent, without harming the tissue?

That’s what Stanford scientists have done using an FDA-approved dye that is commonly found in food, among several other light-absorbing molecules that exhibit similar effects. Published in Science on Sept. 5, the research details how rubbing a dye solution on the skin of a mouse in a lab allowed researchers to see, with the naked eye, through the skin to the internal organs, without making an incision. And, just as easily as the transparency happened, it could be reversed.

“As soon as we rinsed and massaged the skin with water, the effect was reversed within minutes,” said Guosong Hong , assistant professor of materials science and engineering and senior author on the paper. “It’s a stunning result.”

Absorption reduces scattering of light

When light waves strike the skin, the tissue scatters them, making it appear opaque and non-transparent to the eye. This scattering effect arises from the difference in the refractive indices of different tissue components, such as water and lipids. Water usually has a much lower refractive index than lipids in the visible spectrum, causing visible light to scatter as it goes through tissue containing both.

To match the refractive indices of different tissue components, the team massaged a solution of red tartrazine – also known as the food dye FD&C Yellow 5 – onto the abdomen, scalp, and hindlimb of a sedated mouse. The skin turned red in color, indicating that much of the blue light had been absorbed due to the presence of this light-absorbing molecule. This increase in absorption altered the refractive index of the water at a different wavelength – in this case, red. As a result of the absorption of the dye, the refractive index of water matches that of lipids in the red spectrum, leading to reduced scattering and making the skin appear more transparent at the red wavelength.

This research is a new application of decades-old equations that can describe the relationship between absorption and refractive index, called the Kramers-Kronig relations. In addition to this food dye, several other light-absorbing molecules have demonstrated similar effects, thereby confirming the generalizability of the underlying physics behind this phenomenon.

Researchers were able to see, without special equipment, the functioning internal organs, including the liver, small intestine, cecum, and bladder. They were also able to visualize blood flow in the brain and the fine structures of muscle fibers in the limb. The mouse’s beating heart and active respiratory system indicated that transparency was successfully achieved in live animals. Furthermore, the dye didn’t permanently alter the subject’s skin, and the transparency disappeared as soon as the dye was rinsed with water.

The researchers believe this is the first non-invasive approach to achieving visibility of a mouse’s living internal organs.

“Stanford is the perfect place for such a multifaceted project that brings together experts in materials science, neuroscience, biology, applied physics, and optics,” said Mark Brongersma , professor of materials science and engineering and co-author on the paper. “Each discipline comes with its own language. Guosong and I enjoyed taking each other’s courses on neuroscience and nanophotonics to better appreciate all the exciting opportunities.”

The potential future of ‘clear’ tissue

Right now, the study has only been conducted on an animal. If the same technique could be translated to humans, it could provide a range of biological, diagnostic, and even cosmetic benefits, Hong said.

For example, instead of through invasive biopsies, melanoma testing could be done by looking directly at a person’s tissue without removing it. This approach could potentially also replace some X-rays and CT scans, and make blood draws less painful by helping phlebotomists easily find veins. It could also improve services like laser tattoo removal by helping to focus laser beams precisely where the pigment is below the skin.

“This could have an impact on health care and prevent people from undergoing invasive kinds of testing,” said Hong. “If we could just look at what’s going on under the skin instead of cutting into it, or using radiation to get a less than clear look, we could change the way we see the human body.”

For more information

Other Stanford co-authors include Betty Cai, member of the Department of Materials Science Engineering ; Zihao Ou, Carl H. C. Keck, Shan Jiang, Kenneth Brinson Jr, Su Zhao, Elizabeth L. Schmidt, Xiang Wu, Fan Yang, Han Cui, and Shifu Wu, who are also with the Department of Materials Science Engineering and Wu Tsai Neurosciences Institute ; Yi-Shiou Duh of the Department of Physics and Geballe Laboratory for Advanced Materials ; Nicholas J. Rommelfanger of the Wu Tsai Neurosciences Institute and Department of Applied Physics ; Wei Qi and Xiaoke Chen of the Department of Biology ; Adarsh Tantry of the Wu Tsai Neurosciences Institute and Neurosciences IDP Graduate program ; Richard Roth of the Department of Neurosurgery ; Jun Ding of the Department of Neurosurgery and Department of Neurology and Neurological Sciences ; and Julia A. Kaltschmidt of the Wu Tsai Neurosciences Institute and Department of Neurosurgery .

This work was supported by the National Institutes of Health, National Science Foundation, Air Force, Beckman Technology, Rita Allen Foundation, Focused Ultrasound Foundation, Spinal Muscular Atrophy Foundation, Pinetops Foundation, Bio-X Initiative of Stanford University, Wu Tsai Neuroscience Institute, Knight-Hennessy, and U.S. Army Long Term Health Education and Training program.

Disclaimer: The technique described above has not been tested on humans. Dyes may be harmful. Always exercise caution when handling dyes – do not consume them, apply them to people or animals, or misuse them in any way.

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Jill Wu, School of Engineering: [email protected]

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Open access
Published: 08 September 2024

Quercetin inhibited LPS-induced cytokine storm by interacting with the AKT1-FoxO1 and Keap1-Nrf2 signaling pathway in macrophages

Jingyi Xu 1 na1 ,
Yue Li 1 na1 ,
Xi Yang 2 na1 ,
Hong Li 1 ,
Xi Xiao 1 ,
Jia You 2 ,
Huawei Li 3 ,
Lingnan Zheng 2 ,
Cheng Yi 2 ,
Zhaojun Li 4 &
Ying Huang 1

Scientific Reports volume 14 , Article number: 20913 ( 2024 ) Cite this article

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Inflammation

Cytokine storm (CS) emerges as an exacerbated inflammatory response triggered by various factors such as pathogens and excessive immunotherapy, posing a significant threat to life if left unchecked. Quercetin, a monomer found in traditional Chinese medicine, exhibits notable anti-inflammatory and antiviral properties. This study endeavors to explore whether quercetin intervention could mitigate CS through a combination of network pharmacology analysis and experimental validation. First, common target genes and potential mechanisms affected by quercetin and CS were identified through network pharmacology, and molecular docking experiments confirmed quercetin and core targets. Subsequently, in vitro experiments of Raw264.7 cells stimulated by lipopolysaccharide (LPS) showed that quercetin could effectively inhibit the overexpression of pro-inflammatory mediators and regulate the AKT1-FoxO1 signaling pathway. At the same time, quercetin can reduce ROS through the Keap1-Nrf2 signaling pathway. In addition, in vivo studies of C57BL/6 mice injected with LPS further confirmed quercetin's inhibitory effect on CS. In conclusion, this investigation elucidated novel target genes and signaling pathways implicated in the therapeutic effects of quercetin on CS. Moreover, it provided compelling evidence supporting the efficacy of quercetin in reversing LPS-induced CS, primarily through the regulation of the AKT1-FoxO1 and Keap1-Nrf2 signaling pathways.

Molecular mechanism of the anti-inflammatory effects of Sophorae Flavescentis Aiton identified by network pharmacology

Network pharmacology integrated with experimental validation revealed the anti-inflammatory effects of Andrographis paniculata

Network pharmacology and experimental validation to identify the potential mechanism of Hedyotis diffusa Willd against rheumatoid arthritis

Introduction.

Cytokine storm (CS) is a systemic inflammatory syndrome with excessive hyperactivation of immune cells characterized by increased cytokine release, including interleukin-6 (IL-6), tumor necrosis factor α (TNF-α) and monocyte chemotactic protein 1 (MCP-1), which causes severe pathologic complications, such as sepsis, tissue damage, multiple organ failure, and ultimately, death 1 . CS might be stimulated by multiple factors such as pathogens, auto-inflammation, monogenic, or therapeutic intervention and the lungs are the main organ to be affected by CS 2 .

Macrophages play a pivotal role in infection and inflammation as the principal innate immune cells, exerting crucial regulatory functions in pathological inflammation. Within the tissue microenvironment, they exhibit polarization into either the classically activated M1 phenotype, characterized by pro-inflammatory properties, or the alternatively activated M2 phenotype, which demonstrates anti-inflammatory characteristics. Dysregulation in macrophage phenotypes can result in unchecked inflammatory responses, thereby precipitating CS and subsequent tissue damage 3 . Considering the pivotal role of macrophages in CS progression, modulating macrophage overactivation emerges as a promising strategy for CS intervention.

Quercetin, a flavonoid compound, possesses a spectrum of biological properties, including antioxidant, anti-inflammatory, antiviral, and neuroprotective effects 4 , 5 , 6 , 7 . Research indicated that quercetin exerted its anti-inflammatory effects by targeting Syk/Src/IRAK-1 to inhibit LPS-induced macrophage activation, while also preventing LPS-induced oxidative stress and inflammation through pathways NOX2/ROS/NF-κB 8 , 9 . However, the specific targets and signaling pathways through which quercetin regulates CS remain elusive. Therefore, we embarked on an exploration of new targets and potential mechanisms of quercetin for CS treatment, employing network pharmacology, molecular docking, and experimental validation techniques.

The Forkhead box O (FoxO) family of transcription factors assumes pivotal roles in diverse cellular processes encompassing cell growth, metabolism, survival, and inflammation 10 , 11 , 12 . Nonetheless, FoxO1’s nuclear export or phosphorylation culminates in its inactivation, abrogating its capacity to engage with target regulatory elements 13 . Notably, studies have underscored that elevated FoxO1 expression post-inflammatory injury prompts macrophages to unleash an array of inflammatory mediators, thereby exacerbating inflammatory damage 14 , 15 . In LPS-treated mice, macrophages exhibited heightened FoxO1 levels; transfection of FoxO1 into Raw264.7 cells markedly upregulated interleukin-1β (IL-1β) and concurrently downregulated interleukin-10 (IL-10) expression 16 . Furthermore, FoxO1 serves as a direct substrate of AKT, and its activity hinges on AKT phosphorylation. Notably, AKT inhibition in macrophages abolishes FoxO1 phosphorylation and nuclear exclusion, signifying AKT phosphorylation as a pivotal regulatory event governing FoxO1 activity 17 . Conversely, the Keap1-Nrf2 pathway constitutes a principal defense mechanism safeguarding cells and tissues against oxidative stress while upholding homeostasis. Kelch-like ECH-associated protein 1 (Keap1) serves as an electrophilic reagent and sensor of redox damage, whereas Nuclear factor erythroid 2-related factor (Nrf2) acts as a transcription factor modulating various cytoprotective genes. Oxidative stress prompts Keap1 modification, resulting in its inactivation and disassociation from Nrf2. Consequently, stabilized Nrf2 translocates to the nucleus, where it acts as a transcription factor, activating oxidative stress-responsive genes, thereby exerting antioxidant effects 18 .

In this study, we found that quercetin could effectively inhibit inflammatory responses and oxidative stress in vitro and exhibited anti-inflammatory activity in mice model. Likewise, the AKT1-FoxO1 and Keap1-Nrf2 signaling pathways may be involved in quercetin-mediated anti-inflammatory and antioxidant activities.

Materials and methods

Screening for target genes of quercetin and cs.

The two-dimensional (2D) molecular structure and SMILES of quercetin were downloaded from PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ), the world's largest database of chemical information. To predict potential quercetin targets, 2D structures or SMILES were imported into the Swiss Target Prediction Database ( http://swisstargetprediction.ch/ ). The target genes associated with CS were acquired from the OMIM ( https://www.omim.org ), GeneCards ( https://www.genecards.org ), and PharmGKB ( https://www.pharmgkb.org ) databases by using the following keywords; “cytokine storm” and “cytokine release syndrome.” Subsequently, to analyze and screen common target genes of CS and quercetin, the Venny.2.1.0 e-mapping tool ( https://bioinfogp.cnb.csic.es/tools/venny/ ) was used, and then a Venn diagram was drawn.

To acquire a protein–protein interaction network (PPI), the overlapping genes were submitted to the STRING database ( https://cn.string-db.org/ ); the species restriction was “Homo sapiens,” and the confidence level was > 4.0 for exploring their relationship.

The acquired data were imported to Cytoscape 3.9.1 software for visualization. The core target genes were screened via CytoNCA plug-in using the closeness centrality, betweenness centrality, degree centrality, eigenvector centrality, network centrality and local average connectivity.

GO function and KEGG pathway enrichment analyses

The CS and quercetin target genes intersection were converted to the corresponding gene IDs for gene ontology (GO) functional and Kyoto Protocol Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses in DAVID database ( https://david.ncifcrf.gov/ ). GO comprises biological processes (BP), molecular function (MF), and cellular components (CC). KEGG enrichment analysis can forecast some potential signaling pathways involved in biological processes 19 , 20 , 21 . Subsequently, for analyzing the GO and KEGG data, the Bioinformatics ( http://www.bioinformatics.com.cn/ ) platform was employed.

Molecular docking

Molecular docking can predict the binding potential between drugs and targets. Quercetin and the eight core targets were subjected to molecular docking via SYBYL-X2.1.1 software. First, the crystal structures of the protease were retrieved in the RCSB Protein Data Bank (PDB, http://www.rcsb.org/ ) database as the receptors; then the ligand was docked with the receptor by first extraction the ligand, removing the water molecules, modifying the terminal residues, and hydrogenating to generate the active pocket. Lastly, the total score was used to record the strength of the interaction between the small molecule and the target.

Cell viability assay

Murine macrophage cell line Raw264.7 (SC-6005, ATCC) was cultured in 96-well plates (1 × 10 4 cells/well) in DMEM medium with 10% fetal bovine serum and 1% antibiotics (100 unit/ml penicillin and 100 μg/ml streptomycin) at 37 °C and 5% CO 2 overnight. The next day, cells were treated with different concentrations (2.5, 5, 10, 20, and 40 μg/ml) of quercetin for 24 h. Subsequently, cell viability was tested by CCK-8 kit assay (MA0218, meilunbio), per the kits’ instructions.

Enzyme-linked immunosorbent assay (ELISA)

Raw264.7 cells were seeded in 48-well plates at a density of 5 × 10 4 cells overnight; then, in the Control and LPS groups, the media was replaced with 500 μl fresh complete medium, whereas in the drug groups, 500 μl medium containing the corresponding drug concentrations was added. The dexamethasone (Dex) group represented a positive control. Furthermore, LPS was added to each group except the Control to achieve a final concentration of 1 μg/ml. After 24 h of co-culture, the cell supernatant was collected, and several inflammatory mediators, including IL-6, TNF-α, IL-1β, and MCP-1, were measured using ELISA (Dakowei Biotechnology Ltd.), per the manufacturers’ instructions.

Flow cytometry analysis

Raw264.7 cells were propagated and treated as mentioned above, collected after 24 h, and co-stained after probing with CD11b (101207, BioLegend), CD40 (124612, BioLegend), CD80 (104733, BioLegend) antibodies for 20 min at room temperature, while blank and positive controls (single stained tubes for each antibody) were prepared. The cells were then washed with PBS, resuspended in 500 μl PBS, gated, and then analyzed using a flow cytometer.

Detection of nitric oxide (NO)

Raw264.7 was propagated overnight at the density 1 × 10 5 in 24-well plates before receiving the corresponding treatment based on the experimental groups. After 24 and 48 h of co-incubation with the drug and LPS, the cell supernatant was obtained, and the expression level of NO was detected by the Griess method using NO kit (S0021S, Beyotime).

Detection of intracellular reactive oxygen species (ROS)

Raw264.7 (1 × 10 6 /well) were inoculated in 6-well plates and co-culture with LPS and drug for 24 and 48 h before collecting their supernatants. ROS kit (CA1420, Solarbio) detected intracellular ROS levels. The probe DCFH-DA was added to the cell precipitates and incubated at 37 °C for 20 min. DCFH-DA is a non-fluorescent substance, and the kit uses the principle that the probe can enter cells, where it will be subsequently hydrolyzed into DCFH by esterase, and the intracellular ROS will then oxidize non-fluorescent DCFH to produce fluorescent DCF. Flow cytometry and fluorescence microscopy measured intracellular ROS's fluorescence intensity.

Immunofluorescence assay

Raw264.7 cells were propagated and treated as mentioned above, Raw264.7 cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min, and after being closed with 5% BSA for 2 h at room temperature, the cells were incubated with anti-FoxO1 antibody (1:100) at 4 °C overnight. After incubation with Alexa Fluor 488-labeled secondary antibody (1:800 dilution,HA1121, HUABIO), the cellular localization of the cells to FoxO1 was assessed using a confocal microscope (Leica, German).

RNA extraction and quantitative Real-time PCR (qRT-PCR)

The total RNA of treated Raw264.7 cells was extracted using a total RNA extraction kit (RE-03111, FOREGENE). The cDNA was synthesized using the RT Easy™II (With gDNase) kit (RT-01032, FOREGENE), which was then amplified via a Real-Time PCR Easy™-SYBR Green kit (QP-01014, FOREGENE). The relative expression levels of mRNA were calculated by the 2 − △ △ ct method.

Western blot

Cellular proteins were extracted using the RIPA lysis buffer (E-BC-R327, Elabscience), a protease inhibitor (GRF101, epizyme), and a phosphatase inhibitor (GRF102, epizyme). The proteins were quantified using the BCA protein assay kit (P0010, Beyotime), then mixed with sample loading buffer (P0295, Beyotime) and boiled for 10 min. Proteins were separated on 10% SDS–polyacrylamide gels (PG112, epizyme), transferred to PVDF membranes (IPVH00010, Millipore), which were then blocked with 5% skimmed milk at room temperature, and incubated overnight with primary antibodies at 4 °C. The primary antibodies were as follows: anti-TLR2 (ab209216, Abcam), anti-TLR4 (14358, CST), anti-MyD88 (4283, CST), anti-AKT1 (ET1609-51, HUABIO), anti-phospho-AKT1 (ET1607-73, HUABIO), anti-FoxO1 (ET1608-25, HUABIO), anti-Keap1 (10503-2-AP, Proteintech), anti-Nrf2 (16396-1-AP, Proteintech). Subsequently, the membranes were washed and then incubated with a secondary antibody (RS0002, Immunoway) for 1 h. The proteins were visualized by a supersensitive ECL kit (PD203, Oriscience) and a chemiluminescent imaging system.

Anti-CS in vivo

All procedures were conducted following ARRIVE guidelines. The Ethics Committee of West China Hospital has approved this study and confirmed the statement that all methods were performed in accordance with relevant guidelines and regulations. Male C57BL/6J mice (8 weeks old, 23 ± 2 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were first acclimatized with the environment for a week and then categorized into six groups: control, LPS, high quercetin dose (100 mg/kg), medium quercetin dose (50 mg/kg), low quercetin dose (25 mg/kg) and Dex (5 mg/kg). The mice were in 12 h fast condition before the experiment; then, four drug groups were treated with quercetin and Dex at corresponding concentrations by gavage and intraperitoneal injection, respectively. The Control and LPS groups received the same volume of normal saline. After 2 h, mice were anesthetized with an intraperitoneal injection of 0.3% pentobarbital (55 mg/kg), then 50 μl of 5 mg/kg LPS was intratracheally administered in each group (except the Control) to establish the CS model. 4 h after LPS treatment, the mice were killed, the skin on the front of the neck was cut open, the trachea was separated and exposed, the indentation needle was inserted into the trachea and fixed, and the irrigation solution was irrigated with normal saline three times, 0.6 ml each time, with a recovery rate of 80–90%. The BALF was collected for the detection of cytokines.

Histological analysis

To observe the lung's histopathological alterations, mice were sacrificed 24 h after LPS stimulation; lung tissues were dissected, fixed with 4% formaldehyde, embedded in paraffin, sectionalized, and finally stained with hematoxylin–eosin (HE) for visual analysis.

Statistical analysis

All the statistical measurements were performed using GraphPad Prism 9.0, and the acquired data are expressed as means ± standard deviation (SD). Differences between the two groups were assessed using One-way ANOVA analysis and were considered significant at P < 0.05.

Acquisition of quercetin targets against CS

The 2D molecular structure (Fig. 1 A) and SMILES [C1=CC(=C(C=C1C2=C(C(=O) C3=C(C=C(C=C3O2) O) O) O) O) O)] of quercetin were downloaded from PubChem. The PubChem CID of quercetin is 5280343, and the molecular formula and weight are C15H10O7 and 302.23. Subsequently, this structure was uploaded to the Swiss Targets Prediction Database, and 100 genes were identified as quercetin targets. Furthermore, 8390 CS target genes were obtained from three disease databases, including GeneCards, OMIM, and PharmGKB, after screening for disease and removing the duplication. Venn’s diagram (Fig. 1 B) indicated the potential 90 CS target genes selected after matching drugs to target genes.

Acquisition of target genes for quercetin action on CS. ( A ) 2D molecule structure of quercetin. ( B ) Venn diagram of quercetin and CS target genes. ( C ) PPI network analysis in the common target of quercetin and CS. ( D ) Screening of quercetin and CS core genes by CytoNCA Plug-in.

The PPI network (Fig. 1 C), constructed using STRING, consisted of 90 nodes and 375 edges, with PPI enrichment p-value < 1.0e−16. The nodes represent target proteins, and the edges represent predicted and confirmed interactions between proteins. This network was visualized with Cytoscape 9.0 to identify core targets, which were then screened using the CytoNCA plug-in. Based on six parameters-betweenness centrality, closeness centrality, degree centrality, rigenvector centrality, network centrality, and local average connectivity, 8 core target genes, including AKT1, EGFR, SRC, MMP9, KDR, PIK3R1, CDK1, and MMP2 were filtered. These were significant genes associated with the quercetin mechanism, which regulates the occurrence and development of CS (Fig. 1 D).

Potential mechanism and signal pathways of quercetin in regulating CS

GO and KEGG enrichment analyses were performed through the DAVID database to further explore the BP and potential mechanisms of 90 target genes involved in CS. The result of the GO enrichment analysis (Fig. 2 A) displayed 262 BP, 54 CC, and 112 MF. The major BP included protein phosphorylation, negative regulation of the apoptotic process, and protein autophosphorylation. The target genes were mainly associated with the following determined CC: cytosol, plasma membrane, cytoplasm, nucleus, etc. Moreover, MF included ATP binding, protein serine/threonine/tyrosine kinase activity, protein kinase activity, protein serine/threonine kinase activity, etc.

GO and KEGG enrichment analysis. ( A , B ) Analysis of GO enrichment and KEGG potential signaling pathway enrichment for targets of action. ( C ) The PI3K-AKT signaling pathway was identified as the key KEGG pathway for quercetin action on CS. The above KEGG data were obtained from the KEGG database.

The KEGG enrichment assay indicated the possible signaling pathways via which quercetin improves CS, revealing the therapeutic mechanisms of CS (Fig. 2 B). It identified the CS-related key signal pathways involved in the PI3K-AKT, FoxO and ErbB. The specific signal pathways of PI3K-AKT are listed in Fig. 2 C.

Molecular docking results

The interaction of eight core targets-AKT1, KDR, CDK1, EFGR, MMP2, MMP9, SRC, and PIK3R1 with quercetin in the generated active pocket regions was assessed. The higher the total score, the more stable the binding activity. The binding activity is extremely high when the total score is > 7. The docking results revealed that among the 8 core targets, the interaction between the quercetin and AKT1 was the strongest, with a total score of 8.35 (Fig. 3 A–E).

Molecular docking of quercetin with target genes. ( A – D ) The molecular docking of quercetin with AK1, KDR, CDK1, EFGR, respectively. ( E ) The total score for molecular docking of quercetin to the four core targets showed the highest total score for binding to AKT1.

Quercetin attenuated LPS-induced expression of proinflammatory factors in Raw264.7 cells

The effects of quercetin and drug solvent dimethyl sulphoxide (DMSO) on cell viability were assessed by CCK8 assay, which indicated that 2.5, 5, and 10 μg/ml concentrations do not affect cell survival (Fig. 4 A), therefore, these concentrations were selected as low, medium and high doses for subsequent experiments. LPS is an essential component of the Gram-negative bacterial cell wall and induces inflammation, allowing its application in various in vivo and in vitro experiments 22 . Under inflammatory conditions, LPS exposure activates macrophages to produce diverse cytokines and also promotes oxidative stress 23 , 24 . Consequently, a CS model was established utilizing 1 μg/ml LPS(Fig. 4 B). As shown in Fig. 4 C, quercetin significantly inhibited LPS-induced proinflammatory cytokines (IL-6, TNF-α, IL-1β) and MCP-1 in a dose-dependent manner.

Quercetin inhibited LPS-induced inflammatory factors in vitro. ( A ) Survival of Raw264.7 cells after 24 h intervention with different concentrations of quercetin. ( B ) A scheme for quercetin intervention in LPS-induced macrophage activation. ( C ) Quercetin reduced the concentration of IL-6, TNF-α, IL-1β and MCP-1 released by LPS-activated macrophages in a concentration-dependent manner. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the LPS group.

Upon exposure to LPS, activated M1 macrophages secrete a plethora of cytokines 25 . In addition, the surface markers CD40 and CD80, indicative of M1 macrophage activation, undergo alterations (Fig. 5 A). To scrutinize these changes, we conducted flow cytometry analysis. As illustrated in Fig. 5 B, C, the proportion of CD40 + CD80 + cells constituted approximately 70% in the LPS-untreated group, which exhibited a reduction following quercetin treatment. This observation finds validation in quantitative polymerase chain reaction (qPCR) results (Fig. 5 D), wherein mRNA levels of CD40 and CD80 were augmented upon LPS stimulation, a response mitigated by quercetin intervention in a dose-dependent manner. These findings suggested that quercetin could hold promise in ameliorating LPS-induced macrophage polarization.

The ability of quercetin to modulate macrophage polarization in vitro. ( A ) Schematic representation of LPS-stimulated macrophage polarization. ( B ) Flow cytometry results indicated that quercetin treatment decreased the expression of CD40, CD80, surface markers of M1 phenotype macrophages. ( C ) Statistical analysis of flow cytometry results in ( B ). ( D ) Quercetin treatment also decreased mRNA expression of CD40 and CD80. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the LPS group.

Quercetin inhibited LPS-induced NO production in Raw264.7 cells

Nitric oxide (NO) has been implicated in various cellular responses to external stimuli such as ischemia and LPS stimulation 26 . Our NO detection assays unveiled an absence of NO in the resting state at 24 and 48 h, but its significant induction following LPS exposure. Notably, quercetin exhibited a dose-dependent reduction in NO release in the cell supernatant, with the most pronounced effect observed at 10 μg/ml (Fig. 6 A). Inducible nitric oxide synthase (iNOS) is exclusively present under inflammatory conditions and is responsible for sustained NO production. Therefore, we delved deeper into the expression of NOS2 mRNA (encoding iNOS protein) and iNOS protein. Both quantitative polymerase chain reaction (qPCR) and western blot analyses demonstrated that quercetin downregulated the expression of both in a dose-dependent manner compared to the LPS group at 24 and 48 h (Fig. 6 B–F, Supplementary Fig. 2A, B ).

Effects of quercetin on LPS-induced NOS2 mRNA, iNOS protein expression and NO release in Raw264.7 cells. ( A ) At both 24 and 48 h, quercetin reduced LPS-induced NO release in a concentration-dependent manner. ( B ) NOS2 mRNA levels also decreased with increasing drug concentrations after 24 and 48 h of quercetin treatment. ( C , D ) After 24 and 48 h of LPS attack, iNOS expression at the protein level was down-regulated in a concentration-dependent manner in response to quercetin intervention. ( E , F ) The iNOS expression levels at 24 and 48 h were normalized to GAPDH. Original blots are presented in Supplementary Fig. 2A, B . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the LPS group.

Quercetin regulated the AKT1-FoxO1 signaling pathway

The molecular docking results revealed a high binding score of quercetin to AKT1, suggesting its potential to regulate AKT1 activity. In comparison to the LPS-treated group, quercetin administration led to an increase in AKT1 phosphorylation without altering its total protein levels (Fig. 7 A, B, Supplementary Fig. 2C ). Following LPS stimulation, the expression of FoxO1, a transcription factor, and its nuclear translocation are augmented. However, FoxO1 is subject to negative regulation by AKT, as AKT phosphorylation prompts its exclusion from the nucleus, thereby mitigating inflammation 16 , 27 . Immunofluorescence experiments depicted an intensified nuclear fluorescence upon LPS treatment, which significantly decreased following quercetin intervention (Fig. 7 C, Supplementary Fig. 1A ). Correspondingly, western blot analysis illustrated that LPS augmented total FoxO1 expression, a trend reversed by quercetin treatment (Fig. 7 D, E, Supplementary Fig. 2D ). Additionally, quercetin exhibited a dose-dependent downregulation of Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), and MyD88 expression following LPS stimulation ( Supplementary Fig. 1B–G , Supplementary Fig. 2G–I ). In summary, our findings suggested that quercetin could induce AKT1 activation, leading to subsequent FoxO1 inactivation.

Effects of quercetin on AKT1 and FoxO1 expression levels in response to LPS stimulation. ( A ) The level of phosphorylated AKT1 gradually up-regulated after quercetin intervention. ( B ) p-AKT1 levels were normalized to total AKT1 levels. ( C ) Immunofluorescence assessment of FoxO1 intranuclear expression levels in Raw264.7 cells after LPS and quercetin treatment. The FoxO1 was stained as green granular dots, while the nucleus was stained with blue. The highest expression of FoxO1 in the nucleus was observed in the LPS group, and the fluorescence of FoxO1 in the nucleus gradually decreased with the increase of quercetin concentration. ( D ) Western blotting analysis similarly confirmed that quercetin down-regulated FoxO1 expression in Raw264.7 cells. ( E ) FoxO1 levels were normalized to GAPDH. Original blots are presented in Supplementary Fig. 2C, D . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the LPS group.

Quercetin activated Keap1-Nrf2 signaling pathway to mediate antioxidant response

Oxidative stress, emblematic of the imbalance between reactive oxygen species (ROS) and antioxidant defenses, can potentiate inflammatory responses and exacerbate tissue damage 28 . To confirm whether the quercetin-induced antioxidative effect involves ROS inhibition, intracellular ROS levels were assessed through flow cytometry analysis. Results revealed a gradual increase in cellular ROS levels over time in the untreated LPS cells compared to the control group, reaching approximately 70% and 90% at 24 and 48 h, respectively. Moreover, quercetin demonstrated an augmented ability to scavenge ROS with increasing time and drug dosage (Fig. 8 A, B). Green fluorescence, indicative of intracellular ROS content, exhibited a progressive decline with escalating drug concentrations (Fig. 8 C, D). Both flow cytometry and fluorescence microscopy analyses indicated that quercetin alleviated the high ROS levels induced by LPS. Subsequently, we investigated the expression of key factors in the Keap1-Nrf2 axis. Our results showed low or negligible expression of Nrf2 in the control group, with Nrf2 accumulation increasing with higher quercetin doses following LPS stimulation and quercetin intervention. Conversely, Keap1 expression exhibited an inverse trend compared to Nrf2 (Fig. 8 E–G, Supplementary Fig. 2E, F ). Hence, our findings suggested that quercetin could mitigate oxidative stress by activating the Keap1-Nrf2 signaling pathway.

Effects of quercetin on intracellular ROS levels under LPS induction at 24 and 48 h. ( A, B ) Flow cytometry results showed that quercetin enhanced the scavenging of intracellular ROS with increasing concentration and time under LPS induction. ( C, D ) The intracellular ROS content was observed by fluorescence microscopy, and green fluorescence represents ROS. Magnification is 20×. ( E ) Western blotting analysis revealed that Keap1 protein expression was down-regulated and Nrf2 protein expression was up-regulated in Raw264.7 cells treated with LPS and quercetin. ( F, G ) The expression levels of Keap1 and Nrf2 were normalized to their respective GAPDH at 24 and 48 h. Original blots are presented in Supplementary Fig. 2E, F . * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the LPS group.

Quercetin prevented LPS-induced CS in mice

To assess the in vivo anti-inflammatory activity of quercetin, four inflammatory factors in BALF, including IL-6, TNF-α, IL-1β, and MCP-1, were measured by ELISA, which indicated their rapid upregulation after LPS stimulation and quercetin reversed this effect in a concentration-dependent manner under preconditioning (Fig. 9 A, B).

Quercetin inhibited CS in vivo studies. ( A ) Scheme for in vivo induction and intervention of CS. ( B ) Quercetin suppressed the LPS-induced elevation of IL-6, TNF-α, IL-1β and MCP-1 in BALF. ( C ) Histological study of the protective effect of quercetin against LPS-induced CS lung injury. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the LPS group.

As H&E-staining indicates (Fig. 9 C), the control group indicated normal lung structure and clear alveolar morphology, while LPS exposure distinctly caused lung tissue congestion, edema, and extensive inflammatory cell infiltration, destroying the lung structure and preventing normal lung function. Pretreated quercetin mice had improved histopathologic changes induced by LPS in a concentration-dependent manner. The examination of pathological changes in lung tissue and BALF inflammatory factors demonstrated that quercetin could effectively protect mice from LPS attacks.

CS is a systemic immune overreaction. Under normal circumstances, pro-inflammatory and anti-inflammatory factors are in a state of mutual balance, which is disrupted when pathogens invade or medical intervention, resulting in the excessive emission of the cytokines and inducing CS. If not treated properly, it can lead to systemic damage, multi-organ failure, or even death 29 . In CS caused by immune-related pneumonitis and viral infection, activated macrophages produce excessive amounts of IL-6, TNF-α, and IL-1β accompanied by elevated chemokines. IL-6 is a crucial target for CS treatment and is a risk factor for assessing the severity of COVID-19 as it is associated with a high mortality rate 30 , 31 , 32 , 33 , 34 . Corticosteroids and cytokine monoclonal antibodies are essential for CS treatments. However, the optimal dose and duration of corticosteroids in immune-related pneumonia remains undetermined and could exacerbate the risk of opportunistic infections 35 , 36 , 37 . In COVID-19, despite the benefits of corticosteroids, there is some variation in different patients 38 . Furthermore, it has been studied that they are associated with high mortality, hyperglycemia, and infection 39 . Monoclonal cytokines antibodies, such as IL-6R monoclonal antibodies, TNF inhibitors, and IL-1 antagonists, are specific for specific cytokines, and unfortunately, CS comprises multiple cytokines. Some clinical trials have shown that monoclonal antibodies are only effective in some people 40 , 41 .

LPS used to model pneumonia is a classical approach that activates macrophages and monocytes to produce high levels of inflammatory cytokines (IL-6, TNF-α, IL-1β, etc.) while eliciting oxidative stress, with literature suggesting that it can also mimic the CS that occurs in the lungs 42 , 43 , 44 , 45 , 46 . Pneumonia frequently ensues as a consequence of CS and often stems from viral infections. In our investigation, we successfully established in vitro and in vivo CS models utilizing LPS, with in vivo modeling achieved through tracheal infusion of LPS. The groups treated solely with LPS exhibited elevated levels of inflammatory factors (IL-6, TNF-α, IL-1β, MCP-1) alongside inflammatory cell infiltration in lung tissue. Our findings highlighted quercetin's capacity to modulate the inflammatory response induced by LPS-activated macrophages, effectively suppressing the release of inflammatory factors and thereby exerting an anti-inflammatory effect.

The findings from network pharmacology unveiled quercetin's capacity to modulate CS primarily by targeting AKT1, EGFR, SRC, MMP9, KDR, PIK3R1, CDK1, and MMP2, with AKT1 being the most significantly regulated. Moreover, KEGG enrichment analysis indicated a potential association between quercetin's mechanism of action against CS and the PI3K-AKT signaling pathway. AKT1, an intracellular kinase, governs various biological processes such as cell growth, survival, and metabolism, serving as a pivotal signaling node in various tissues and cellular inflammatory responses 47 , 56 , 49 . Within macrophages, AKT1 represents the sole subtype. Macrophages lacking AKT1 exhibit heightened responsiveness to LPS and display a robust pro-inflammatory reaction, while AKT1 ablation induces the production of M1-type macrophages 50 . Furthermore, AKT1 phosphorylates and deactivates downstream GSK3β, thereby diminishing NF-κB activation and fostering the expression of the anti-inflammatory cytokine IL-10 51 . These findings suggest that AKT1 may play a pivotal anti-inflammatory role in inflammation. The PI3K-AKT signaling pathway indicates that AKT1 might mediate the inflammatory response through the downstream FoxO1 signaling pathway. Multiple studies have implicated FoxO1 in promoting inflammatory signaling 16 , 17 , 52 . It has been observed that the TLR4/MyD88/MD2-NF-κB signaling pathway is markedly activated following FoxO1 overexpression, whereas silencing of FoxO1 downregulates levels of inflammatory pathway proteins 15 . However, AKT-mediated phosphorylation of FoxO1 leads to its nuclear exclusion and inhibition of its activity. Sun et al. 53 demonstrated that Schisandrin substantially reversed the high expression of total FoxO1 protein in the nucleus and upregulated AKT phosphorylation following LPS stimulation. Consistent with these findings, our study revealed that LPS stimulation increased FoxO1 protein levels in Raw264.7 cells. Quercetin targeted AKT1 and significantly phosphorylated it, thereby inhibiting the entry of FoxO1 into the nucleus and reducing the expression of pro-inflammatory genes. This inhibitory effect of quercetin on FoxO1 was further confirmed using immunofluorescence assays.

CS can induce severe oxidative stress, leading to heightened production of ROS and subsequent damage to crucial organs. The Keap1-Nrf2 pathway serves as a primary defense mechanism within cells, safeguarding against oxidative stress and preserving homeostasis. Upon encountering ROS, Keap1 undergoes modification, dissociating from Nrf2. This allows Nrf2 to translocate to the nucleus, where it accumulates and counteracts oxidative stress, thereby protecting cells 18 , 54 . Curcumin, a bioactive compound found in turmeric, has been shown to scavenge ROS generated in macrophages, shielding them from oxidative stress by activating the Keap1-Nrf2 pathway 55 . Similarly, studies by Liu et al. 56 demonstrated that Mollugin activated the Keap1-Nrf2 pathway, mitigating oxidative stress in Raw264.7 cells. Additionally, astaxanthin has been found to safeguard against LPS-induced cellular inflammation and acute lung injury in mice by suppressing iron-induced cell death through the Keap1-Nrf2 pathway 57 . In our investigation, we observed a quercetin dose-dependent decrease in Keap1 levels and an increase in Nrf2 protein levels, significantly inhibiting LPS-induced ROS production. This finding suggests a potential contribution of quercetin to antioxidant stress effects.

In summary, the present study demonstrated that quercetin could inhibit LPS-induced inflammation and alleviate cytokine storm in vitro and in vivo. Mechanistically, quercetin exerted its protective effects by regulating AKT-FoxO1 and Keap1-Nrf2 pathway.

This study employed network pharmacology and molecular docking technology to identify the effective target genes of quercetin against CS. It also preliminarily revealed that quercetin might act against CS through signaling pathways such as PI3K-AKT and FoxO. In addition, The in vitro and in vivo experiments confirmed that quercetin could play an anti-inflammatory role. Collectively, it could regulate AKT1-FoxO1 and Keap1-Nrf2 signaling pathways.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Abbreviations

Bronchoalveolar lavage fluid

Biological process

Cellular composition

Coronavirus disease 2019

Cytokine storm

Dexamethasone

Dimethyl sulphoxide

Forkhead box proteins O

Gene ontology

Interleukin-6

Inducible nitric oxide synthase

Kelch-like ECH-associated protein 1

Kyoto encyclopedia of genes and genomes

Lipopolysaccharides

Monocyte chemotactic protein 1

Molecular function

Nitric oxide

Nuclear factor erythroid 2-related factor

Protein–protein interaction network

Reactive oxygen species

Traditional Chinese medicine

Tumor necrosis factor α

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Acknowledgements

The authors thank Ping Lin, Jie Zhang and Qin Lin from the lab of experimental oncology for their great help in this study. The authors gratefully appreciate BioRender's modifications to the figures. The authors would like to thank all the reviewers who participated in the review and MJEditor ( www.mjeditor.com ) for their linguistic assistance during the preparation of this manuscript.

This study was supported by the National Natural Science Foundation of China (NO.82260490), Sichuan Provincial Nature Science Foundation (2022NSFSC1379); Sichuan Science and Technology Programme (2022YFSY0054) and Technology Innovation Project of Chengdu Science and Technology (2020-YF05-00059-SN); Natural Science Foundation of Hainan Province (NO.821QN394); Science and technology research project on novel corona-virus pneumonia outbreak, West China Hospital, Sichuan University (HX-2019-nCoV-069).

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These authors contributed equally: Jingyi Xu, Yue Li and Xi Yang.

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West China School of Basic Medical Science and Forensic Medicine, Sichuan University, No.17, Section3, Renmin South Road, Chengdu, 610044, People’s Republic of China

Jingyi Xu, Yue Li, Hong Li, Xi Xiao & Ying Huang

Department of Medical Oncology, West China Hospital, Cancer Center, Sichuan University, No.37 Guoxue Lane, Chengdu, 610041, China

Xi Yang, Jia You, Lingnan Zheng & Cheng Yi

Department of Integrated Traditional Chinese and Western Medicine, School of Medicine, Cancer Hospital, University of Electronic Science and Technology of China, Chengdu, 610041, China

Department of Radiation Oncology, Hainan Affiliated Hospital of Hainan Medical University (Hainan General Hospital), No.31, Longhua Road, Haikou, 570100, China

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Xu, Li, Yang, Li, Xiao, You, Li, Zheng, Li, Yi, and Huang contributed to this study. Xu, Li,ang contributed equally to this study. Yi, Li andHuang directed the design of this study, supervised its implementation and revised draft. Xu, Li, Yang, Li, Xiao participated in the specific experimental process, data analysis and paper writing. You, Li, Zheng were involved in the charting of the paper. All authors have read and approved the final draft.

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Correspondence to Cheng Yi , Zhaojun Li or Ying Huang .

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Xu, J., Li, Y., Yang, X. et al. Quercetin inhibited LPS-induced cytokine storm by interacting with the AKT1-FoxO1 and Keap1-Nrf2 signaling pathway in macrophages. Sci Rep 14 , 20913 (2024). https://doi.org/10.1038/s41598-024-71569-y

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Assessing Functional Performance in the Mdx Mouse Model

Annemieke aartsma-rus.

1 Department of Human Genetics, Leiden University Medical Center

Maaike van Putten

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions ( e.g . forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Introduction

Duchenne muscular dystrophy (DMD) is the most common neuromuscular disorder affecting 1:5,000 newborn boys. This severe and progressive muscle wasting disease is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the synthesis of functional dystrophin protein. Muscle fibers lacking dystrophin are vulnerable to exercise induced damage. Upon exhaustion of the muscle's regenerative capacity, and due to chronic inflammation of damaged muscle, fibers are replaced by connective tissue and fat, subsequently leading to a loss of function. Generally, DMD patients lose ambulation of the lower limbs early in the second decade. Later, also the muscles of the arms and shoulder girdle are affected and patients often develop thoracolumbar scoliosis due to asymmetric weakening of the muscles supporting the spinal cord. Assisted ventilation is generally required in the late teens or early twenties. Respiratory and heart failure lead to death in the third or fourth decade 1 .

Although the causative gene has been discovered over 25 years ago 2 , there is no cure available for DMD. However, improved health care and the use of corticosteroids have increased life expectancy in the Western world 3 . With the use of animal models like the mdx mouse, major steps forward into the discovery of potential therapeutic strategies have been made. The mdx mouse is the most commonly used DMD mouse model. It has a point mutation in exon 23 of the murine Dmd gene and consequently lacks dystrophin 4 . Over the last couple of years, many proposed strategies have progressed into clinical trials 5-9 . In these trials, improvement of muscle function is the primary endpoint, underlying the importance of testing the benefit of compounds on muscle function in mice during the pre clinical stage of testing.

Like DMD patients, also the dystrophin negative muscle fibers of mdx mice are vulnerable to exercise induced damage and their muscle function is impaired compared to C57BL/10ScSnJ wild type mice. This impairment can be assessed with a variety of functional tests. Some of these tests are noninvasive and do not interfere with muscle pathology ( e.g. forelimb grip strength, hanging tests and rotarod running). Therefore they can be used to monitor the natural history of the disease or determine the effects of compounds on disease progression. To get an in depth picture of the influence of compounds on muscle function in mdx mice, a functional test regime that does not interfere with disease progression consisting of all of these tests can be used 10 .

Alternatively, forced treadmill running can be used to intentionally exacerbate disease progression and test the protective capacities of compounds 11 . The treadmill can also be used as outcome measure in which running time till exhaustion is measured 12 , or as a tool to fatigue mdx mice so that they perform less well in a subsequent functional test ensuring larger differences in performance between treatment groups 13 . When choosing functional tests, their effect on disease progression should be kept in mind especially when testing dystrophic mice like the mdx mouse 14 .

We here describe in detail how to perform the most commonly used functional tests in a reliable and reproducible manner based on available standard operating procedures from the TREAT-NMD network. Click here to visit TREAT-NMD .

The experiments described here were approved by the Animal Ethics Committee (DEC) of the Leiden University Medical Center (LUMC). Mice were bred by the animal facility of the LUMC and kept in individually ventilated cages with 12 hr light dark cycles. They had ad libitum access to water and standard chow.

When performing any of the functional tests described below, experimental conditions have to be strictly controlled to reduce variation. Preferably, age and gender matched mice should be used, as performance differs between age and genders. Mice belonging to the same litter should be randomized over the experimental groups. Animals should be tested by the same operator, who is blinded to the experimental groups. Tests should be performed on the same time of day and weekday, same room to equalize odors, noises, etc. 14 Large variation between individual mice and time points can be observed for all functional tests, therefore 6-8 mice/experimental group should be used. Functional test performance can also largely differ between different inbred wild type strains. Therefore, experimental and control wild type mice should always have corresponding backgrounds (in case of mdx mice use the C57BL/10ScSnJ wild type strain). All data described here have been obtained with the C57BL/10ScSnJ wild type strain, which we refer to as wild type from here on. The tests described here can be used longitudinally from at least 1-19 months of age in mdx and wild type mice. Tests should not be repeated more than once weekly to prevent mice from losing interest and willingness to perform the task.

1. Forelimb Grip Strength Test

Use the forelimb grip strength test to measure the strength of the forelimbs. The test is based on the tendency of a mouse to instinctively grasp a grid when suspended by the tail 15 , and adapted from DMD_M2.2.001.pdf .

Apparatus set up: Attach a grid to a force transducer, which measures the maximum force applied by the mouse on the grid during the pull. Make sure the setting is on Peak tension mode (T-PK) for pulling. The units of force can be adjusted in either ounces-of-force, grams-of-force, pounds-of-force, kilograms-of-force, or Newtons. Note: We prefer to work with grams as unit of values. Multiple meters are commercially available, but only axial transducers give reliable outcomes as lever type force transducers are negatively influenced by the physical laws of the lever effect. Either a nonflexible grid or triangle can be used with bars that are 1-2 mm in diameter.
Prior to the test, assess the body weight of the mouse, to allow normalization for body weight.
Use grams as unit of values. Reset the meter at the start of each recording.
Remove the mouse from its cage by grabbing the tail and moving it horizontally towards the grid.
Check that the mouse grasps the grid tightly with both forepaws.
Pull the mouse away from the grid so that its grasp is broken; the highest force applied to the grid will be shown on the transducer's display, which can be either manually or automatically recorded.
Only take pulls into account in which the mouse shows resistance to the experimenter. Reject measures in which only one forepaw, or the hindlimbs were used and in which the mouse turned during the pull.
Let the mouse pull the grid three times in a row and then return it in the cage for a resting period of at least one minute. Note: between series of pulls a resting period is necessary for the mouse to recover and avoid habit formation.
Then let the mouse perform four series of pulls, each followed by a short resting period. In this way the mouse has pulled a total of 15x (3 pulls x 5 times = 15 pulls).
Determine the maximum grip strength and normalize for body weight by taking the average of the three highest values out of the 15 values collected.
Optional: Determine fatigue by calculating the decrement between the average of the first two and the last two series of pulls 1+2+3=A, 4+5+6=B, 10+11+12=C and 13+14+15=D. The formula: (C+D)/(A+B) gives a value of 1 for mice which are not fatigued. This can be expressed in percentages so that a mouse without fatigue has a value of 0% and a mouse which forelimbs are completely fatigued has a value of 100%.

2. Hanging Tests

With hanging tests, balance, coordination and muscle condition can be assessed. These tests are based on the knowledge that mice are eager to remain hanging on a wire or grid till exhaustion 16 . There are two distinctive hanging tests in which at the start of the test either only the two forelimbs or all four limbs are used, using a wire or grid respectively. The hanging test using the wire and the grid are the longest suspension time method adapted from DMD_M.2.1.004.pdf and DMD_M.2.1.005.pdf respectively. A fixed hanging limit is used of 600 sec. The majority of wild type mice can hang for 600 sec, while dystrophic mice cannot. To reduce time spend performing this test, a maximum hanging time was set in place. Mice that fall off the wire or grid before then are given up to two more tries. This is done to reinsure that mice are really unable to hang and do not fall due to clumsiness.

Apparatus set up: Tightly secure a 2 mm thick metal cloth hanger to a shelf with tape and maintain the hanger around 37 cm above a layer of bedding. Note: alternatively, a 55 cm wide 2 mm thick metallic wire which is tightly secured between 2 vertical stands could be used. The distance of 37 cm is sufficient to encourage mice to remain hanging, but also low enough to prevent mice from injuries when falling down. The wire should not vibrate or displace during the test as this could interfere with the performance of the mouse.
Handle the mouse via the tail and bring it near the wire.
Let the mouse grasp the wire with the two forepaws only, and lower the hindlimbs in such a way that the mouse only hangs with the two forepaws on the wire ( Figure 2B ).
Directly start the timer when the mouse is released. After release, strong mice try to catch the wire with all the four limbs and the tail, which is allowed ( Figure 2C ).
When a mouse shows improper behavior (like balancing on or deliberately jumping off the wire as shown in Figures 2D and 2E ), directly address this by replacing the mouse on the wire without stopping the timer.
When a mouse falls off the wire, stop the timer and record the hanging time.
When mice are able to hang for 600 sec, take them off the wire and return them to the cage. Mice that fall before this limit are given a maximum of two more tries.
Record the maximum hanging time ( i.e. the longest of the trials) and use this for further analysis.
Apparatus set up: Use either a hand made square or the lid of a big cage for a rat or rabbit for this test. Position the grid 25 cm above soft bedding to prevent mice from harming themselves upon falling, but also to discourage mice to intentionally jump off the grid. Tightly secure the grid so that the experimenter does not have to manually hold the grid during the experiment as these movements might interfere with the mouse's performance.
Place the mouse on the grid so that it grasps it with its four paws.
Invert the grid so that the mouse is hanging and directly start the timer.
The test session ends for mice that are able to hang for a duration of 600 sec. Give mice that fall off the grid earlier a maximum of two more tries.
Use the maximum hanging time ( i.e . the longest of the trials) for further analysis.

3. Rotarod Running

With the rotarod test muscle strength, coordination, balance, and condition can be determined 17 .

Apparatus set up: For this test, mice have to run on a rotating tube. Ensure that the steady speed is set at 5 rotations per min (rpm), and that the speed increases from 5-45 rpm in the first 15 sec when started. After this it has to maintain its speed.
Place the mice on the tube of the rotarod when it rotates at a slow steady speed of 5 rpm. Five mice can be tested simultaneously.
Start the run once all mice are positioned. Within the first 15 sec the speed of the tube accelerates from 5-45 rpm after which it maintains that speed.
Monitor the run. The running time is continuously recorded by the software. Running time stops automatically when a mouse falls off the tube as this activates the time bar positioned below the tube. Reposition mice that turn around facing the opposite direction on the tube while running without stopping the tube to rotate.
End the test session for mice that are able to run for a duration of 500 sec. Give mice a maximum of two more tries allowing them to improve their running time, when they fall earlier.
Use the maximum running time ( i.e. the longest of the trials) for further analysis.

4. Treadmill Exercise

The treadmill can be used in three ways as a tool in pre clinical research. Firstly, forced treadmill running can be used to exacerbate disease pathology as described in this protocol (see also: DMD_M2.1.001.pdf ). Secondly, the maximal running capability of mice and the effects of treatments on this can be assessed (See for the method to let mice run till exhaustion DMD_M.2.1.003.pdf ). Finally, treadmill running can be used prior to another functional test to exhaust the mouse so that it performs less well in the second test 13 . This is done by exercising mice twice or three times weekly as described below, directly followed by either one of the functional tests described in protocol 1-3.

Apparatus set up: There are several treadmills commercially available on which several mice can run simultaneously and for which elevation, duration and speed can be adjusted. Some treadmills are equipped with a grid to deliver low intensity shocks to encourage mice to run. However, mdx mice are sensitive to stress and can easily be motivated in a friendlier manner by a gentle push with the hand in the running direction. Therefore, it is strongly encouraged to NOT use the shock grid. Generally, stimulation with the hand is only needed during the first running session.
Place the mice on the horizontal treadmill.
Start the treadmill at a running speed of 12 m/min. Lower speeds (8 m/min) have to be used in old mice (>15 months), where higher speeds easily lead to exhaustion.
During the first session, encourage mice to run by gently pushing them when they are near the end of the belt.
When the mice have run for a duration of 30 min, place them back into their cage.
Repeat this twice weekly for e.g. 12 weeks.
Allow resting periods when needed. For example, some mdx mice have to stop running and should be allowed to rest for a few min. If this happens, turn the belt off, give all mice a resting period of two min, turn the belt on for two min at 4 m/min. After this, increase speed to 12 m/min and allow the mice to finish the protocol. It is important that all mice complete the entire running protocol. Note: In case mdx mice need resting periods, consider a warm up before the 30 min exercise protocol. This warm up session consists of: a 2 min acclimatization period at a speed of 4 m/min, immediately followed by an 8 min warmup at 8 m/min.In our hands 4-16 week old female mdx mice are able to complete the 30 min exercise protocol without resting. Others have reported that in age matched male mdx mice 45% of the mice do need resting periods to finish the exercise. The warm up protocol reduces the amount of stops 12 .

Representative Results

The forelimb grip strength of wild type and mdx mice increases between the age of 4-12 weeks and reduces again in older mice. Impairments in force can already be observed in young mdx mice. Representative data of 9 week old female mice are shown in Figures 1A and 1 B . Although fatigue does not differ between the strains yet at this age, mdx mice are weaker than wild type mice. We do not have data yet on fatigability in older mdx and wild type mice.

To obtain reliable and reproducible results, multiple assessments need to be done by the same experimenter. We here describe to pull 15 times/individual, however smaller numbers of pulls (as low as 5 pulls) also provide reliable data. Careful attention should be drawn to the positioning of the paws on the grid as this can largely influence outcomes. During the pull, only both forepaws should be used and they have to be placed nicely next to each other ( Figure 1C ). When the mouse is not showing resistance to the pull, the value should not be taken into account.

For the two limb and four limb hanging tests, especially young (4-16 weeks old) wild type mice can easily reach the maximum hanging time of 600 sec. Contrastingly, performance of young mdx mice is impaired (they hardly ever achieve maximum hanging time) and also deteriorates with age, even though both strains put all effort in performing these hang tests at their best abilities ( Figures 2A and 3A ). Larger differences in hanging times between mdx and wild type mice are obtained with the wire. Therefore, even small effect sizes of compounds on muscle function can be detected using this test. Hanging performance (or any other type of performance) differs within and between individuals over time resulting in high standard deviation bars. Nonetheless, mdx mice consistently perform worse than age matched wild type mice ( Figure 2A ). Performing multiple assessments can provide more detailed insight in functional improvements upon treatment than only endpoint measurements. It should be kept in mind that in the first session animals learn how to perform a functional test. This learning curve, which is present in all tests, is clearly visible between 4-6 weeks of age. However, because mice also grow rapidly in this age period, a distinction between improvement due to learning and/or growth cannot be made. Gender differences in hanging performance for the two limbs hang test have also been found. Performance of female mdx mice exceeds that of males by ~100 sec, and performance of treadmill challenged female mdx mice is almost comparable to that of the unchallenged males (compare Figures 2A with 4A ). This finding underlines the importance of using age and gender matched mice to avoid bias. We have preliminary data suggesting that differences in performance in both hanging tests between mdx and wild type mice increases in very old (18 months) mice.

Some mice display inappropriate behavior to avoid hanging on the wire like; balancing on the wire, jumping off the wire deliberately etc. ( Figures 2D and 2E ), although the majority of mice comply with the test and hang with either two or four limbs ( Figures 2B and 2C ). Occasionally, strong mice jump off the wire intentionally. They hang prior to jumping with only the two hindlimbs and the tail on the wire and look down to estimate the distance to the ground. Inappropriate behavior that is occasionally seen on the grid during the four limb hanging test consists of deliberately jumping off the grid or climbing on the grid. All inappropriate forms of behavior can be easily distinguished and should not be allowed. Mice that avoid hanging in one of these ways should be directly placed back on the wire or grid without stopping the timer.

On the rotarod, mdx mice hardly ever run for the maximum running time of 500 sec, while a larger proportion of wild type mice do ( Figure 3B ). With age, running performance of both strains decreases. Some mice are able to clamp tightly on to the rotating tube and avoid running by 'cartwheeling' around. This cannot be corrected for and is a severe limitation of the test when multiple mice start doing this for prolonged periods, thereby increasing variation within the experimental groups. Especially for some mice which partly run and partly cartwheel, and during the transition from cartwheeling into running fall. Some mice turn around on the rotating tube while running. This behavior should be addressed for by directly repositioning the mice on the tube, without stopping it. Also this kind of behavior limits the usefulness of this test.

Forced treadmill running is an easy and effective exercise to exacerbate disease pathology in nontreated mdx mice, while wild type mice undergoing the same protocol are not affected. Generally, mice become familiar with the treadmill after an initial training session and are willing to run, especially when multiple mice are running simultaneously. Old mdx mice (over 15 months of age) have difficulties in running and cannot cope with the same running speed of 12 m/min for 30 min used for young mice. Therefore, a slower running speed of 8 m/min for 30 min is recommended enabling all mice to finish the entire protocol. Mdx mice are especially vulnerable to eccentric contractions, therefore downhill running can only be used for a short duration.

Alternatively, other functional tests like the two limb hanging wire test can be performed directly after running ( Figure 4A ). Using this study design, differences between strains or treatment arms are likely to increase as treadmill challenged untreated mdx mice are less capable of performing these tests than sedentary mdx mice 13 .

As mentioned earlier, when studying muscle function in mdx mice, the C57BL/10ScSnJ wild type strain needs to be used which is of the corresponding genetic background. We advise this as even between inbred wild type strains treadmill running performance differs 18,19 . But also in noninvasive functional tests, functional performance is influenced by genetic backgrounds. Figure 5 illustrates this in three representative graphs where performance of mdx mice on a BL/10 background and on a mixed background consisting of BL/10, BL/6J, DBA2 and 129OLA are compared. As can be appreciated the mixed background mice perform better in the hanging wire tests and worse on the rotarod.

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Figure 1. Forelimb grip strength, representative results and correct positioning of the paws. A. Forelimb grip strength normalized for body weight of 9 weeks old female mdx (n=5) and wild type (n=4) mice. Grip strength is already impaired in young mdx mice. Asterisks indicate p <0.05 and data are presented as mean±st.dev. B. Fatigue of the same individuals as shown in A, was on average less than 10% and did not vary between strains. C. To obtain reliable data, attention should be paid to the positioning of the paws during forelimb grip strength analysis. Correct positioning of the mouse; two forepaws are next to each other, hindlimbs are not touching the grid and the mouse is pulling in a straight line. D. Incorrect positioning of the forepaws; the mouse is not pulling in a straight line. When this happens, or when only one forepaw or also the hindlimbs are used, the mouse turns around during pulling or lacks to show resistance, data should be discarded. Please click here to view a larger version of this figure.

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Figure 2. Two limb hanging tests, representative results and appropriate and inappropriate hanging behavior. A. A representative example of the two limb hanging test performed once weekly in male mdx (n=18, 4-10 weeks, n=13, 11 and 12 weeks, n=10, 13 weeks) and age and gender matched wild type mice (n=6). A learning curve is visible for both strains in the first few weeks of testing. Performance of mdx mice was worse compared to that of wild type mice. Data presented as mean±st.dev. Maximum hanging time allowed is indicated by the dotted line. B. The correct starting position of this test is with the two forepaws. C . Depending on the functional ability of the mouse it can also use the hindlimbs and tail. D and E. A small subset of mice, especially strong wild type mice, can occasionally avoid hanging by climbing on the side bars or balancing on the wire. Some mice intentionally jump off the wire. Please click here to view a larger version of this figure.

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Figure 3. Four limb hanging and rotarod running test. A. Four limb hanging performance assessed once weekly in male mdx (n=18, 4-10 weeks, n=13, 11 and 12 weeks, n=8, 13 weeks) and wild type (n=6) mice. Over time, mdx mice hang less long than wild type mice. B. Rotarod running times did not differ between young male mdx (n=18, 4-10 weeks, n=13, 11 and 12 weeks, n=10, 13 weeks) and wild type mice (n=6). Data are represented as mean ± st.dev.

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Figure 4. The effect of forced treadmill running exercise protocol on functional performance and skeletal muscle pathology in female mice. Muscle pathology was deliberately exacerbated by letting mice run on a horizontal treadmill three times a week at 12 m/min for 30 min for a duration of 12 weeks. Directly after running, mice had to participate in the two limb hanging test. While all wild type mice (n=5) remain hanging till the maximum allowed, all mdx mice (n=6) fall off the wire earlier ( p <0.001, data presented as mean± st.dev.). B. The presence of membrane damage was determined by assessing plasma Creatine Kinase (CK) levels that leak out of muscle fibers through tears in the membrane. CK levels were elevated in mdx mice compared to wild type mice before exercise. Treadmill exercise immediately increased levels ( p< 0.01 indicated by asterisk, data presented as mean± st.dev.) in mdx mice, while they remained low in wild type mice. C-D. Muscles of mdx mice are very vulnerable to treadmill exercise, worsening disease pathology extensively after a few weeks of running. These Haematoxylin and Eosin stainings of the quadriceps of a 16 week old nonexercised ( C ) and treadmill exercised ( D ) mdx mouse show that extensive fibrosis and necrosis are developed. E. Muscles of wild type mice undergoing the same running protocol are not affected. Please click here to view a larger version of this figure.

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Figure 5. Effect of a mixed background on functional performance in mdx mice. Differences in genetic background influence functional performance. To illustrate this, performance of male mdx (BL/10 background, n=18, 4-10 weeks, n=13, 11 and 12 weeks, n=10, 13 weeks) and mdx (mixed BL/10, BL/6J, DBA2 and 129OLA background, n=5) mice was compared over time. A. Two limb hanging test performance significantly differed between the two strains. B. Four limb hanging test results were slightly higher in the mixed background mdx mice. C. Rotarod running times also slightly differed between the strains. Data presented as mean± st.dev.

The functional tests presented here are reproducible, easy to perform and applicable to wild type and dystrophic mice independent of their age. The tests provide useful tools to pre clinically assess muscle function, strength, condition, and coordination. When testing the effects of a compound on the natural history of the disease, the noninvasive tests described here (forelimb grip strength, both hanging tests and the rotarod test) can be nicely combined in a functional test regime where these tests are performed on consecutive days. These protocols are not detrimental to mdx mice and can be used in a longitudinal manner 10 . It should be kept in mind that outcomes of each of these tests are generated by different or partly overlapping muscle groups instead of an individual muscle. Therefore, using a combination of multiple tests is recommended to obtain a more complete picture and thereby better insight in the functionality of the experimental groups. Alternatively, functional improvements of a sole muscle can be assessed using muscle physiology measurements 20 .

Like behavioral tests, also functional tests can show extensive variation between different mice, or within a mouse between different assessments. To reduce variation, all tests should be performed by the same experimenter who is familiar with the mice. External variables like smells and sounds in the room, time of the day and the day of the week on which the test is performed should be kept as constant as possible. Mice should be gender and age matched. When using treadmill running to exacerbate disease progression, it is essential to use a standardized protocol in which all running parameters (running time, speed and slope) are kept constant over time for all experimental groups, so that all mice are equally treated. Although the majority of mice are keen to participate in the functional tests and most animals show high levels of willingness, some mice (primarily strong wild type mice) occasionally avoid performing the test and show avoidance behavior. When this behavior is not corrected for, false conclusions could be drawn 21 . Fortunately, these types of behavior are only observed occasionally and can be corrected for by placing the mouse back on the wire, grid or rotarod, or pulling another time on the grip strength meter.

Improvements in one functional test ( e.g. hanging test assessing muscle function) does not necessarily have to co occur with improvements in another test (forelimb grip strength assessing sole muscle strength). In mdx mice, improvements in muscle function can be distinguished earlier than in muscle strength. This is also seen in DMD patients participating in clinical trials where clinically meaningful improvements in the 6 min walk test do not cooccur with improvements in muscle strength 6,7 . However, this may in part depend on the working mechanism of the compound tested and it is possible that other compounds improve strength and not function. Therefore, the results of the tests should be interpreted with the mechanism of action of the compound in mind.

Disclosures

The authors have nothing to disclose.

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

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 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.

Introduction

Fatigue affects a wide range of people, can markedly reduce quality of life, and frequently has an unclear or unknown pathogenesis. Cancer-related fatigue (CRF), for example, is experienced by the majority of cancer patients undergoing treatment and can persist long after cancer treatment has been completed and in the absence of detectable cancer 1 . Moreover, fatigue is also a prominent symptom in numerous other diseases and disorders, including chronic fatigue syndrome, depression, diabetes, and fibromyalgia. Fortunately, there are non-pharmacological interventions that are capable of helping some people experiencing fatigue ( e.g. , exercise can reduce CRF for some breast cancer patients 2,3 ), but many individuals still lack effective treatment. Furthermore, existing drug treatments for CRF have not been found to be broadly, if at all, efficacious 4-7 .

Despite the therapeutic need and lack of drug treatment options, preclinical assays of fatigue to aid in the discovery and development of novel fatigue treatments are lacking, especially in animal models. One of the only preclinical assays of fatigue for rodent studies is voluntary wheel running activity (VWRA) 9-15 , in which mice or other rodents are given free access to a running wheel and their daily running activity is recorded. In many studies, VWRA is the only measure of fatigue-like behavior, with fatigue-like behavior defined (in either VWRA or the current protocol) as a decrease in the measured physical activity in the experimental group. Although VWRA can provide a useful longitudinal measure of fatigue-like behavior, it is a relatively low-throughput assay, running varies considerably between inbred mouse strains 16 , and it requires subjects to be individually housed, which may cause changes in behavior and test performance 17-19 . Other assays, such as home cage behavioral monitoring and analysis, can also provide continuous data collection and some systems may allow for subjects to be housed in pairs 20 . These assays have utility, but may be less sensitive as a means of detecting fatigue-like behavior and, like wheel running, are also low-throughput.

In contrast to VWRA, mouse treadmill tests do not rely upon voluntary activity and can be completed in a short time frame, allowing for higher throughput. In comparison to VWRA, these tests employ external motivators. Specifically, there is usually an electrified metal grid located to the rear of the moving belt to provide mice with an electric shock should they cease to run. In addition to this shock grid, mice may be motivated to run on the treadmill via several other methods, including prodding, poking, or touching them with a hand, brush, or other tool and directing short puffs of air at them. Instead of fatigue, mouse treadmill tests are often used to measure aerobic and/or anaerobic exercise capacity 21-25 . Mice are motivated to run until they are incapable of or unwilling to continue running on the treadmill as a means of escaping further electric shocks. Testing then ends when mice meet the criterion for exhaustion. In these protocols, to ensure that mice reach true physiologic exhaustion, the criterion for exhaustion is often defined as spending five continuous seconds laying on top of the shock grid and failing to continue running in the face of repeated aversive stimuli. Thus, fatigue-like behavior may be masked in typical treadmill tests due to the strong aversive nature of the external motivation and criterion for ending the test. Interestingly, and in contrast to many other studies using rodent treadmills, a recent publication describes another version of a treadmill fatigue test, which was used as part of an examination of the effects of social stress in mice 26 . Although the method used by this group markedly differed from the current protocol ( i.e. , they employed a single-lane treadmill and required 10 sec of electric shock as the criterion for ending their test), their study highlights the utility of and interest in developing a quick, simple fatigue test using the mouse treadmill.

Fatigue is likely to be detectable by means other than wheel running and alterations in routine behaviors. CRF makes patients feel exhausted by a lesser amount of muscle fatigue, as determined by electromyographic analysis, than people without CRF 27 . Additionally, reduced motivation has been noted in and is measured by several scales measuring human fatigue 28,29 . Thus, a useful preclinical assay of fatigue-like behavior should distinguish between healthy and fatigued mice on the basis of a measure other than physiological capability and should not obscure decreases in motivation. To achieve that end while avoiding limitations of VWRA and other assays, the current method was developed by adapting the mouse treadmill test. This method uses a shock grid as the sole external motivator to make mice run on the treadmill. Mice quickly learn that the grid provides an aversive stimulus and will promptly move away from it when placed on the treadmill and maintain some distance from it when running.

When mice fatigue, they spend progressively more time toward the back of the treadmill instead of maintaining speed toward the front end. Therefore, the criterion for test completion in this protocol is spending five continuous seconds in the designated fatigue zone ( i.e. , the rear of the treadmill, ranging from approximately one body length from the shock grid to, and including, the shock grid). This takes advantage of the aversive nature of the grid without requiring mice to receive many or any actual shocks after training. By allowing mice to complete testing using the current criterion rather than exhaustion (as defined above), this method provides a means of using the treadmill to measure fatigue-like behavior rather than its maximal (or near-maximal) physiological capability. Thus, this method can provide a simple, high-throughput assay of fatigue-like behavior in mice and can serve either as an independent or complementary measure to other assays of fatigue-like behavior.

This procedure was approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.

1. Preparation

To allow for rapid identification of each mouse prior to testing, tattoo the tails of all mice to be trained and tested with identifying marks. NOTE: This step is optional. Permanent marker or other methods of identification can be used as an alternative to tattooing.
Prior to training and testing mice, ensure that the treadmill is on a flat surface and set to the treadmill to desired angle of inclination (recommended angle of inclination: 10°, to be kept consistent throughout training and testing) and set the electric shock frequency and intensity appropriately (recommended: 2 Hz, 1.22 mA). NOTE: The electric shock used should produce no more than a mild tingling sensation when touched by an ungloved finger and should be delivered in a pulsatile fashion (with each shock lasting 200 msec).
Place a clean sheet of butcher's paper or an absorbent pad under the treadmill to collect fecal boli and urine during training and testing.
Place a sheet of paper or an absorbent pad over the third of the treadmill housing ( i.e. , the clear plastic lid that covers the treadmill lanes) furthest from the shock grid. NOTE: This step is optional, but will create a darker space and may provide additional encouragement to avoid the lower portion of the treadmill.
If planning to use a wire brush to provide additional motivation during training, ensure that one is readily available prior to beginning training sessions.
Ensure that any drug or method for inducing and/or alleviating fatigue is available and can be prepared or performed during Step 2.14.

2. Training Mice to Use the Treadmill

NOTE: Training is necessary to ensure that mice are familiar with the treadmill and task and can perform appropriately when tested. If the majority of mice being trained are receiving frequent shocks or otherwise performing poorly during any training session, additional training sessions should be performed. On the first day, most mice will be shocked several times. By the second day of training, mice should be rarely making contact with the grid. If a mouse displays consistently poor training performance, it should be removed from the study. For female C57BL/6NCr mice, this is a rare occurrence (less than 1% have been removed from studies due to poor training performance), but it should be noted that other strains may perform differently during training.

With the treadmill off (and speed set to 0 m/min), individually lift the mice by the tail and place mice into separate lanes of a mouse treadmill. Promptly turn on the corresponding grid after placing each mouse on the treadmill. Ensure that mice are placed directly on the treadmill belt. NOTE: The amount of time and distance each mouse is held by its tail should be minimized by placing the cage near the treadmill prior to transferring mice to the treadmill and/or allowing mice to stand on a solid platform ( e.g. , a wire cage lid) until they are near the treadmill and the experimenter is ready to place them in the treadmill.
Allow mice to freely explore the treadmill for 1-3 min or until each mouse has explored its lane and/or received at least one shock from the grid.
Turn on the treadmill and slowly increase the speed until it begins moving (approximately 1.5 to 3.0 m/min). Monitor all mice to ensure that they begin walking. If a mouse does not begin walking or walks toward the shock grid, be prepared to intervene by tapping the mouse with a wire brush or tail tickling.
Slowly increase the treadmill speed to 8 m/min. Start a timer and continue monitoring behavior.
Increase treadmill speed to 9 m/min at 5 min, 10 m/min at 7 min, and stop the treadmill at 10 min.
Allow the mice to briefly explore the treadmill, then remove and return each to its cage.
Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill.
To train additional mice, repeat Steps 2.1 through 2.7. NOTE: Allow alcohol to dry prior to placing new mice on the treadmill.
On the second day of training, repeat Step 2.1. Turn on the treadmill and increase the speed to 10 m/min. Start a timer. NOTE: Treadmill speed can be increased more rapidly than on the first day of training.
Increase treadmill speed to 11 m/min at 5 min, 12 m/min at 10 min, and stop the treadmill at 15 min.
Remove mice and return them to their cages.
Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill. To train additional mice, repeat Steps 2.9 through 2.12.
Perform additional days (3 days) of training in the same manner as the second day. NOTE: This step is optional, but is strongly recommended if most or all mice (of the same sex and strain) being trained display difficulty with the task. Mice can generally perform well in Step 3 when they have been trained for 3 days ( i.e. , with one additional day of training), although additional or fewer days of training may be appropriate depending on their performance during the second training day and the duration of Step 2.14.
Allow at least one full day to pass in which the mice have no exposure to the treadmill before proceeding to Step 3. NOTE: Any drug(s) used to induce and/or alleviate fatigue should be administered during this step. NOTE: This time period can be varied in length and used to induce fatigue and/or test interventions to reduce or eliminate fatigue. If testing mice more than 7 days after completing training, a pilot study is recommended to verify that the mice used will perform during testing.

3. Treadmill Fatigue Test

NOTE: In this test, fatigue-like behavior is defined as spending 5 consecutive seconds in the "fatigue zone". The fatigue zone is defined as the region encompassing the portion of the treadmill belt within approximately 1 body length of the shock grid as well as the grid, itself. Prior to testing, ensure that the point delineating this zone is clear to the experimenter, such as by applying a mark to the top or side of the treadmill lanes.

Set the treadmill speed to 12 m/min. Do not start the treadmill. Ensure that shock grids are turned off.
Individually place mice into separate lanes of the treadmill. Turn on the corresponding grid immediately after placing each mouse on the treadmill.
Simultaneously start the treadmill and a stopwatch. NOTE: Do not intervene during testing except to remove mice that meet the criterion for removal (see Step 3.5).
Increase treadmill speed as indicated in Table 1 . Carefully observe all mice throughout the test. NOTE: The treadmill speeds listed in Table 1 were selected based on observations from adult female C57BL/6NCr mice. Higher treadmill speeds may be appropriate for larger ( e.g. , outbred CD-1 mice) or more athletic mice.
If a mouse remains in the fatigue zone for 5 continuous sec, promptly remove the mouse from the treadmill and record the duration and distance it ran.
When no mice remain on the treadmill, stop the treadmill. Clean the treadmill and grid with alcohol and replace the paper or absorbent pad beneath the treadmill.
To test additional mice, repeat Steps 3.1 through 3.6. NOTE: This step is optional.

Representative Results

This protocol allows fatigue-like behavior to be measured in mice using a treadmill. The data presented in this section was obtained by training and testing 3 separate groups of mice using the current protocol (excluding Figure 1A and 1C ). To induce fatigue, 5-fluorouracil (5-FU), a cytotoxic chemotherapy drug known to cause fatigue in humans 30 and fatigue-like behavior in mice 10,13 , was administered. All data presented are from adult female C57BL/6NCr mice. Mice were 9-10 ( Figure 1 and 2 ) or 9-13 ( Figure 3 ) weeks of age at time of testing.

Figure 1 shows data from mice that were trained for 5 days, then treated with 5-FU (60 mg/kg/day for 5 days), as in a previously published model 10 , to induce fatigue. After completing treatment, they were tested using an exercise capacity test ( Figure 1A ), which used treadmill speeds listed in Table 2 and a wire brush, tail tickling, and air puffs to motivate mice to run until incapable of running. The test ended when a mouse spent 5 continuous seconds on the shock grid. On the following day, mice were tested using the treadmill fatigue test ( Figure 1B ). This protocol can detect a significant difference in the distance run during testing between chemotherapy-treated and control mice ( Figure 1B ), whereas a treadmill exercise capacity test did not ( Figure 1A ). To validate that the difference found in the treadmill fatigue test was measuring fatigue-like behavior, mouse VWRA was measured in a separate experiment. Following acclimation and collection of baseline wheel running activity, VWRA was measured during the dark cycle ("night", when wheel running primarily occurs) during the 5 days of 5-FU treatment and for an additional night beyond completion of 5-FU treatment. Mice undergoing 5-FU treatment displayed fatigue-like behavior by the second night of treatment ( Figure 1C ). This effect increased over the course of the experiment and persisted beyond the end of treatment, indicating that fatigue-like behavior should have been detectable in the mice from Figures 1A and 1B . As the treadmill fatigue test was capable of detecting differences in the distance run by control and 5-FU-treated mice, this supports the conclusion that the treadmill fatigue test is capable of measuring fatigue-like behavior.

The treadmill fatigue test can also detect fatigue-like behavior in mice receiving chemotherapy at different doses and treatment schedules. Mice receiving one 80 mg/kg dose of 5-FU per week for two weeks (for a cumulative dose of approximately half of what mice received in Figure 1 ) displayed fatigue-like behavior, as demonstrated by a decrease in distance run ( Figure 2 ).

As the number of training sessions and/or length of time between training and testing may vary depending upon the mice used and the method used to induce fatigue, it is important that changes in these variables do not prevent the detection of fatigue-like behavior. The experiments shown in Figures 1A and 1B (in which mice received 5 days of training) and Figure 2 (in which mice received 3 days of training) illustrate that fatigue-like behavior is detectable when the number of training sessions and time between training and testing are changed.

In Figure 3 , no chemotherapy drugs were administered, but mice were tested using the treadmill fatigue test weekly. Although mice can be tested repeatedly using this protocol, but they may become less willing to run upon repeated testing ( Figure 3 ). The percentage of mice that would not run during weekly tests increased with every test and, after the second test, at least half of the mice tested would not run on the treadmill. This data suggests that testing with this protocol should be limited to one or two tests to avoid a high rate of non-compliant mice.


0	12
0.5	14
1	16
6	18
30	20
45	22
60	24
75	26

Table 1: Treadmill Speed During Fatigue Testing.


0	10
10	15
15	16.8
18	18.6
21	20.4
24	22.2
27	24
30	25.8
33	27.6
36	29.4
39	31.2
42	33
45	34.8
48	36.6

Table 2: Treadmill Speed During Exercise Capacity Testing.

The current protocol describes how to use a mouse treadmill to measure fatigue-like behavior. This method has several advantages over VWRA, a common preclinical assay of fatigue-like behavior. VWRA requires that mice choose to interact with the test apparatus. As a result, some inbred strains of mice rarely interact with the wheel 16 and run so little that it may be difficult or impossible to identify a fatigue-induced decrease in activity. In contrast, the treadmill fatigue test eliminates that choice and therefore provides a viable alternative assay of fatigue-like behavior for mice that do not run on running wheels. This protocol could be used as a replacement or complement to VWRA and other measures of fatigue-like behavior and may be particularly useful in testing potential drug therapies to reduce fatigue in mouse models. After establishing via a pilot study that fatigue-like behavior is observable in a particular mouse model, potential treatments could be administered to alleviate fatigue and reduce fatigue-like behavior. If a drug treatment attenuates fatigue-like behavior when tested using this protocol, it (or a similar drug) may be of therapeutic value for treating some forms of human fatigue. Moreover, although there are still many necessary steps in transitioning from preclinical studies to clinical trials, this protocol permits a greater number of mice to be tested in a much shorter time frame than VWRA so that fatigue-like effects and potential treatments can be studied and understood faster.

There are several important limitations and considerations to be aware of when using this protocol. First, it should be noted that, as this test requires physical activity to measure fatigue-like behavior, it may not be suitable for testing conditions that induce cachexia or muscle atrophy ( e.g. , advanced cancer). We have also observed that, if the same mice are tested repeatedly, there may be a decrease in overall compliance ( Figure 3B ). This effect may not be observed under all testing schedules or in all types of mice, and drug treatment or other interventions might alter this effect, but it is an important consideration when planning studies using this method. Additionally, there is a risk of injury if a mouse falls into the gap between the treadmill belt and the shock grid while the treadmill is running. To minimize this risk, mice should be carefully observed throughout training and testing to ensure their safety and the use of very young or small (<15 g) mice should be avoided. Lastly, although pilot data collected suggests that female CD-1 and male and female transgenic mice on a 129S1/SvImJ background will perform this task (data not shown), to date, this protocol has primarily been used to test female C57BL/6NCr mice. As such, it should be noted that other sexes and mouse strains may differ in training and test performance. Lastly, although pilot data collected suggests that female CD-1 and male and female transgenic mice on a 129S1/SvImJ background will perform this task (data not shown), to date, this protocol has primarily been used to test 9-10 week-old female C57BL/6NCr mice. As such, it should be noted that mice of different ages, sex, or strains may differ in training and test performance.

During testing, it is crucial that mice meeting the fatigue criteria are efficiently and quickly removed, as poor removal technique may provide additional motivation for a mouse to continue running, causing something other than fatigue-like behavior to be measured. Although the particular method of removal will depend upon experimenter comfort, a simple method of removal involves using the index and middle fingers of one hand. Each finger should be held straight and slightly apart from each other prior to entering the treadmill lane and promptly closed around the tail, near the base, or over the scruff of the mouse. Once securely grasped, the mouse can be readily removed.

It is important for mice to be familiar with the shock grid to provide motivation to run during testing, but frequent shocks during training may be detrimental to test performance. After the first day of training, most mice will walk on the treadmill successfully and respond to a shock by running or hopping away on the treadmill, then resume walking to avoid drifting back toward the grid. Some mice, however, may react strongly to shocks and/or find ways of not performing the task without receiving any. Mice that react strongly to the shock grid may receive more frequent shocks, spend less time walking on the treadmill, and may attempt to escape from the treadmill. With these mice, the experimenter can place a gloved hand at the rear of the lane to gently encourage the mouse to continue running. To avoid walking on the treadmill, some mice may exploit a limitation of the shock grid. The grid requires at least two points of direct skin contact ( i.e. , two or more paws must be touching the grid) to shock an animal. Thus, if a mouse sits on it without allowing two feet to touch the grid, it will not be shocked. If this behavior is observed, the experimenter can gently nudge the mouse to cause it to move its feet and receive a shock or lift the mouse to replace it on the treadmill. If these interventions are successful, the mouse should begin walking on the treadmill more consistently within several minutes and in future training sessions. If this intervention is not successful, the mouse should be removed from the study.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This research was supported by the Intramural Research Program of the NIH, The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Grant 1Z01 DK011006. We wish to thank Michele Allen for providing technical assistance, Eleni Solomos for editorial assistance, and the NIH veterinary and animal care staff for providing care for the mice used in developing this method.

Exer 3/6 Animal Treadmill	Columbus Instruments	1050-RM Exer-3/6
Stopwatch	Daigger	EF24490M
Wire brush	Fisher Scientific	03-572-5
Compressed air	Dust-Off	FALDSXLPW
Absorbent pads	Daigger	EF2175CX
Butcher paper	Newell Paper Company	4620510
Alcohol (70%)	Fisher Scientific	BP82011

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Run the experiment until the mice are exhausted or the maximal speed is achieved. When a mouse becomes exhausted, write down the time, speed, ... To test additional mice clean the treadmill and grid with 70% alcohol and repeat the described procedure. (Click 'Load' in the profile mode screen and select the saved file generated previously in ...
Treadmill Running of Mouse as a Model for Studying Influence of
Before treadmill running, mice are put on the treadmill with indirect open circuit calorimetry system for 5 min to calm down. Treadmill running ... All animal experiments were approved by the Institute of Animal Care and Use Committees (IACUC) at the Washington State University (Protocol number: 6704; valid between Sept. 25, 2020-Sept. 24, 2023
PDF Small Animal Treadmill
The treadmill, model Eco 3/6 from Columbus Instruments, is located in room 7614 of Medical Science Building II. It has the capability of exercising up to three rats or six mice simultaneously in individual lanes. The user can adjust the. running speed from ∼7 to 70 m/min with a resolution of 0.1 m/min, and the running surface can be inclined ...
The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue
To validate that the difference found in the treadmill fatigue test was measuring fatigue-like behavior, mouse VWRA was measured in a separate experiment. Following acclimation and collection of baseline wheel running activity, VWRA was measured during the dark cycle ("night", when wheel running primarily occurs) during the 5 days of 5-FU ...
Evaluation of Muscle Performance in Mice by Treadmill ...
Evaluation of Muscle Performance in Mice by Treadmill Exhaustion Test and Whole-limb Grip Strength Assay Bio Protoc. 2017 Apr 20;7(8):e2237. doi: 10.21769/BioProtoc.2237. Authors Beatriz Castro 1 , Shihuan Kuang 1 2 Affiliations 1 Department of ...
Mouse Fitness as Determined Through Treadmill Running and Walking
Treadmill running is a noninvasive method to evaluate fitness capacity in a longitudinal or cross-sectional manner. High-intensity exercise tests can be used to determine peak physical capacity in mice. However, because aging is associated with a progressive loss of physical capacity the running protocols can be adapted and optimized for aged ...
Exercise Performance Tests in Mice
Maximal exercise performance is a multifactorial process in which the cardiovascular component, the innervation of the musculature, and the contractile and metabolic properties of skeletal muscle all play key roles. Here, protocols are provided for assessment of maximal running capacity of mice on a treadmill, with a combination of short high ...
Mouse treadmill running enhances tendons by expanding the pool of
Mouse Treadmill Running Experiments. For the treadmill running experiments, 10 C57BL/6J female mice (2.5-month old) were divided into treadmill running and control groups, with 5 mice each group. The treadmill running group ran according to the following protocol, which was approved by the University of Pittsburgh Institutional Animal Care and ...
Evaluation of Muscle Performance in Mice by Treadmill Exhaustion Test
Load the mice into separate lanes on the treadmill belt and then click 'OK'. Run the experiment until the mice are exhausted or the maximal speed is achieved. When a mouse becomes exhausted, write down the time, speed, and distance that are displayed at that moment on the screen (see Figure 4B) and turn off the shock grid for its lane ...
Evaluation of Muscle Performance in Mice by Treadmill ...
After three training sessions, mice were placed on the treadmill at a fixed 5% incline with a speed starting at 5 m/min increasing incrementally by 1 m every 3 min up to 11 m/min and then and by 1 ...
Mouse Fitness as Determined Through Treadmill Running and Walking
Treadmill running in mice provides a functional measure of exercise tolerance. While voluntary wheel running is a useful indicator of overall activity level, forced treadmill running utilizes different physiological realms, and has been extensively tested in both mice and humans [1, 2].Mice are placed on a motorized treadmill and encouraged to run to reach the dark covered end of the treadmill ...
Atlas of exercise-induced brain activation in mice
Prior to the terminal treadmill exercise experiment, treadmill runners and treadmill control mice were all acclimatized to a treadmill running system (TSE Systems, GmbH, Germany). The adaptation protocol comprised 10 min at 10.2 m min −1 each day for three consecutive days (with active shocker grid), whereafter mice rested for two days prior ...
Age-dependent impact of two exercise training regimens on ...
Age-dependent impact of two exercise training regimens ...
A comparison of the metabolic effects of treadmill and wheel running
Animal and experimental design. The 7 weeks-old Male C57BL/6 N mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). Mice were randomly divided into the following groups: control (CON, n = 5), treadmill exercise (TE, n = 5), and wheel running exercise (WE, n = 5).Mice were maintained at temperature of (22-24) °C, humidity of (50-60) %, with a 12 h light/dark cycle in a ...
Treadmill Training Improves Aerobic Capacity in Aged Male Mice Compared
CONCLUSION: A 3-week treadmill training protocol improves aerobic capacity in older male mice to a greater extent than voluntary wheel running. Ongoing experiments will utilize this training protocol to understand age-related declines in cardiorespiratory fitness, circadian rhythm, and to test exercise as an intervention in the aging population.
LED therapy modulates M1/M2 macrophage phenotypes and ...
The mdx mouse phenotype, aggravated by chronic exercise on a treadmill, makes this murine model more reliable for the study of Duchenne muscular dystrophy (DMD) and allows the efficacy of therapeutic interventions to be evaluated. This study aims to investigate the effects of photobiomodulation by light-emitting diode (LED) therapy on functional, biochemical and morphological parameters in ...
Gut microbiota‐directed dietary factors ...
This study investigated the effect of dietary interventions targeting gut microbiota on exercise performance in mice. Analysis of the gut microbiota of individuals with varying levels of physical activity revealed enrichment of Eubacterium rectale and Faecalibacterium prausnitzii in the active population. Through in vitro fecal fermentation experiments, dietary factor combinations that promote ...
Treadmill Running of Mouse as a Model for Studying Influence of
Compared to volunteer wheel running, treadmill running allows precise control of exercise intensity and duration, dramatically reducing variations among individual mouse within treatments and facilitating translation into maternal exercise in humans. Based on the maximal oxygen consumption rate (VO 2 max) before pregnancy, the treadmill ...
Age, experience and genetic background influence treadmill walking in mice
Previous studies and our own findings 50 emphasize that for mice treadmill locomotion is a novel behavior that may be particularly sensitive to the testing protocol and ... (2 males excluded, final n=18). In the final experiment mice were tested six times with inter-trial interval of 3-minutes between the first five and a 1 week inter-trial ...
Researchers make mouse skin transparent using a common food dye
Published in Science on Sept. 5, the research details how rubbing a dye solution on the skin of a mouse in a lab allowed researchers to see, with the naked eye, through the skin to the internal ...
Quercetin inhibited LPS-induced cytokine storm by interacting with the
The mice were in 12 h fast condition before the experiment; then, four drug groups were treated with quercetin and Dex at corresponding concentrations by gavage and intraperitoneal injection ...
Dye found in Doritos turns mouse skin transparent. How the ...
Tartrazine, a bright yellow-orange food dye used in Doritos and other foods and cosmetics, can turn a mouse's skin transparent, according to a new study. ... As part of the experiment, the research team rubbed the dye, mixed with water solution, on the head and abdomen of the mouse. After it got absorbed, within minutes they were able to see ...
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Assessing Functional Performance in the Mdx Mouse Model
Generally, mice become familiar with the treadmill after an initial training session and are willing to run, especially when multiple mice are running simultaneously. Old mdx mice (over 15 months of age) have difficulties in running and cannot cope with the same running speed of 12 m/min for 30 min used for young mice. Therefore, a slower ...
Solar Panels for Experiment
Solar Panels for Experiment. $20 $50. Listed 6 weeks ago. 6 weeks ago. in St Petersburg, FL. Message. Message. Save. Save. Share. Details. Condition. Used - Fair. ... Proform Treadmill crosswalk 400e. Independence, KS. $8 {NEW} Oklahoma Sooners 24 Can Cooler or Lunch Bag, Product Details Dimensions: 9.5" x 12.5" x 10.5" ...
The Treadmill Fatigue Test: A Simple, High-throughput Assay of ...
To validate that the difference found in the treadmill fatigue test was measuring fatigue-like behavior, mouse VWRA was measured in a separate experiment. Following acclimation and collection of baseline wheel running activity, VWRA was measured during the dark cycle ("night", when wheel running primarily occurs) during the 5 days of 5-FU ...
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Treadmill Repair Near Me in St. Petersburg, FL| Fitness Repair
Meet Dudley Jacobs. Keeping your fitness equipment in its prime is what we do best! Our Saint Petersburg team specializes in fitness equipment repair and preventive maintenance on a wide range of exercise equipment like treadmills, exercise bikes, weight machines, ellipticals, and more. We proudly provide trained repair services to residential ...

A comparison of the metabolic effects of treadmill and wheel running exercise in mouse model

Introduction

Materials and methods

Treadmill and wheel running exercise protocol

Grip strength

Body composition

Western blotting

H&E staining

Statistical analysis

Comparison of running characteristics of the treadmill and wheel running exercise

The effect of treadmill and wheel running exercise on body weight, body composition, fat weight, and food intake

Effect of treadmill and wheel running exercise on skeletal muscle weight and grip strength

Treadmill and wheel running reduces adipocyte size

Effect of treadmill and wheel running exercise on mitochondria biogenesis

Effect of treadmill and wheel running exercise on skeletal muscle fiber type shifting

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Treadmill Training Improves Aerobic Capacity in Aged Male Mice Compared to Voluntary Wheel Running

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LED therapy modulates M1/M2 macrophage phenotypes and mitigates dystrophic features in treadmill-trained mdx mice

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Photobiomodulation therapy protects skeletal muscle and improves muscular function of mdx mice in a dose-dependent manner through modulation of dystrophin

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Treadmill Running of Mouse as a Model for Studying Influence of Maternal Exercise on Offspring

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