Investigating the Effects of Infrared Therapy on Bone after High-intensity Treadmill Exercise Training

Summary

Prolonged high-intensity treadmill exercise training inhibits bone formation and promotes bone resorption in mice, resulting in decreased bone mass. Here, we present a protocol to examine the effects of infrared therapy on high-intensity exercise-induced alterations in bone metabolism. Further mechanistic studies are required to elucidate the underlying molecular pathways.

Abstract

Bone tissue is an important load-bearing organ of the human body. Moderate exercise enhances bone mass through mechanical loading, while high-intensity exercise may suppress it. Infrared therapy improves circulation, reduces pain/inflammation, and aids tissue repair. This paper presents a protocol to elucidate the modulatory effects of infrared therapy on high-intensity exercise-induced skeletal metabolic disorders and their molecular mechanisms. In this study, 24 6-week-old mice were randomly divided into control (CTR, N = 8), high-intensity treadmill exercise training (HIT, N = 8), and infrared therapy after high-intensity treadmill exercise (IRT, N = 8) groups. The latter two groups were trained for 8 weeks. In the first week, the mice underwent treadmill acclimation training. In the second week, the mice exercised at the rate of 24 m/min for 50 min, which increased at the rate of 1 m/min and 10 min every week until it reached 28 m/min for 80 min, with a gradient of 5° for 6 days. At the fifth week, HIT group mice were subjected to whole-body infrared therapy intervention, with the temperature controlled between 42 °C and 45 °C. The whole treatment process lasted ~40 min, 3x a week. Results showed that in the HIT group compared with those in the CTR group, the bone volume/total volume and trabecular number were significantly decreased, the trabecular separation was significantly increased, the osteogenic-related genes of Atf4, Runx2, and Ocn were downregulated, and the osteoclast-related genes of Ctsk and Mmp9 were upregulated. The micro-CT results of the IRT group mice showed no significant difference, but the osteogenesis-related genes Alp, Atf4, Runx2, Ocn, and Osx were upregulated in the femur. In addition, the osteoclast-related gene Ctsk was downregulated. Long-term high-intensity treadmill exercise may reduce bone mass. While infrared therapy does not affect bone mineral density, it may improve bone metabolism.

Introduction

Osteoporosis (OP) is one of the most common systemic metabolic bone diseases, particularly prevalent among the elderly, and represents a major global health issue¹. OP is characterized primarily by decreased bone mineral density (BMD), bone quality, and bone strength, which increases the risk of bone fractures². The prevention and treatment of OP are crucial for improving human quality of life, encompassing approaches such as pharmacological therapy, adequate intake of calcium and vitamin D, reduction of smoking and alcohol consumption, and early physical exercise to increase and maintain peak bone mass level^3,4.

Bone, as the structural framework of the body, provides fundamental shape and protects internal organs. Under the regulation of the nervous system, bone facilitates various movements during muscle contractions. Throughout life, bone undergoes continuous formation, resorption, and remodeling. Osteoclasts continually resorb old bone, while osteoblasts form and mineralize new bone. These processes are tightly coupled and mutually regulated, maintaining a dynamic balance in bone turnover⁵. Numerous studies have shown that appropriate physical exercise (such as aerobic exercise and resistance training) can effectively prevent and treat OP⁶. Exercise influences bone metabolism by modulating mechanical stress on the skeleton (such as ground impact, shock, and muscle attachment site tension) and biochemical factors in blood circulation (such as hormones and cytokines), thus promoting bone health⁷. However, prolonged high-intensity exercise might negatively affect bone health, leading to bone loss. For example, after 8 weeks of treadmill running at high, moderate, and low intensities (at a speed of 15 m/min) in a rotator cuff injury mouse model, the bone volume/total volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were lowest in the high-intensity group compared with those in the control and moderate/low-intensity groups, while trabecular separation (Tb.Sp) was highest. This finding suggests that high-intensity training has detrimental effects on rotator cuff recovery and bone mass⁸.

Infrared therapy (IRT) is one of the most widely used principles in rehabilitation physical therapy devices in China. It promotes local blood circulation, accelerates the absorption of inflammatory substances, and enhances the supply of nutrients^9,10. As a non-invasive physical therapy, IRT has gained widespread attention in the fields of sports medicine, rehabilitation science, and bone metabolism research in recent years. IRT is more commonly used in clinical settings than other interventions. Studies have shown that bone tissue healing is a complex process, including the resolution of inflammation and cellular regeneration, which can be divided into three stages: inflammation, proliferation, and remodeling. The inflammatory phase is primarily characterized by angiogenesis; the proliferative phase involves the proliferation of osteoblasts and the formation of new bone; and the remodeling phase is marked by the gradual increase in newly formed trabeculae within the callus, with the bridge callus across the fracture gap completely undergoing ossification¹¹. After a fracture, blood flowing from vessels into tissues needs to be rapidly absorbed, which stimulates osteoblast proliferation and bone formation. These vital processes require a favorable internal environment and sufficient nutrients for support. A certain degree of thermotherapy can effectively facilitate these functions¹². In clinical practice, infrared therapy can be used as an auxiliary means to treat osteoporosis and help alleviate related symptoms, but its research on bone mass is relatively limited. This paper presents a protocol to elucidate the modulatory effects of infrared therapy on high-intensity exercise-induced skeletal metabolic disorders and their molecular mechanisms. Therefore, this work, built upon previous research¹³, constructs a high-intensity treadmill exercise model in mice to observe changes in bone mass and further investigate whether thermotherapy can mitigate bone loss induced by high-intensity treadmill exercise. The aim is to provide a reference for exercise-based prevention and treatment of OP.

Protocol

This study received ethical approval from Shanghai University of Sport's Laboratory Animal Ethics Committee (No. 102772023DW016) and complied with national/institutional animal research regulations. Twenty-four 5-week-old male C57BL/6 mice were housed in an SPF-grade animal facility at the Shanghai University of Sport. Mice were housed at 22 ± 2 °C, 40-70% humidity with 12 h light/dark cycles and provided ad libitum food/water. All biological materials, including bones, blood, and RNA extraction reagent-treated samples, should be collected in Biohazard Level II autoclave bags, sterilized at 121 °C for 60 min prior to disposal, with proper documentation in laboratory waste logs.

1. Animals

1. After 1 week of acclimatization, stratify the mice by body weight and randomly assign them to three groups: the control (CTR group, N = 8), high-intensity treadmill exercise training (HIT, N = 8), and infrared therapy after high-intensity treadmill exercise (IRT, N = 8) groups.

2. High-intensity treadmill exercise training and thermotherapy intervention protocol

1. Feed the CTR group under static conditions without intervention.
2. After1 week of acclimatization, subject the mice in the HIT and IRT groups to 8 weeks of high-intensity treadmill training. The first week is considered the adaptation phase: make the mice in the treadmill group run at speeds of 12, 15, and 18 m/min for 20, 30, and 40 min, respectively, with a 0° incline. In the second week, make the mice exercise at the rate of 24 m/min for 50 min, and then increase their exercise at the rate of 1 m/min and 10 min every week until it reaches 28 m/min for 80 min, with a gradient of 5°. Conduct this exercise regimen 6 days a week, with rest on Sundays (Table 1).
3. For the IRT group, after 4 weeks of treadmill exercise, introduce infrared therapy (IRT) in the fifth week.
   NOTE: During infrared irradiation, strictly maintain mouse body temperature between 42 °C and 45 °C.
   1. Anesthetize the mice by intraperitoneal injection to induce unconsciousness.
   2. Irradiate the whole body of the mice using the infrared therapeutic apparatus, ensuring that the temperature of the mice is kept between 42 °C and 45 °C (detected by a temperature detector). Carry out the whole treatment process for ~40 min, 3x a week (Monday, Wednesday, and Saturday), for 4 weeks until the samples are taken.

3. Tissue collection

NOTE: Dissected mouse bone samples must be immediately preserved in liquid nitrogen or at -80 °C to maintain RNA integrity.

1. Serum sample collection and preservation
   1. Weigh all mice using an electronic balance.
   2. Anesthetize the mice via an intraperitoneal injection of tribromoethanol.
      NOTE: When handling tribromoethanol, always prepare fresh solutions under a chemical fume hood while wearing nitrile gloves and eye protection, store at 4 °C in light-protected amber vials, and dispose unused portions through approved chemical waste channels.
   3. Collect blood samples aseptically using cardiac puncture to obtain 0.5-0.6 mL of blood. First, shave the chest hair of the mouse and disinfect with iodophor. Subsequently, palpate the point of maximal cardiac pulsation, and insert a 4-5 G needle attached to a syringe into the left thoracic cavity between the third and fourth intercostal spaces. Finally, when blood is observed to flow spontaneously into the syringe, apply gentle aspiration to complete the blood collection procedure.
   4. Allow the blood samples to clot for 10 min at room temperature, then centrifuge at 1,000 × g for 15 min. Aliquot and store the supernatant (serum) at -80 °C.
2. Bone sample collection and preservation
   1. Use sterile micro scissors and tweezers to dissect both femurs and tibias. Gently peel off the muscles and tendons from the bone surface along the longitudinal axis, without scraping the periosteum, and pay special attention to protecting the growth plate.
   2. Rinse all samples with 1x PBS (pH 7.4). Fix the left femur in 4% paraformaldehyde (PFA) for 24 h. Use the left femur for bone mineral density measurement before further analysis.
   3. Place the right femur in prelabeled 1.5 mL microcentrifuge tubes and store at -80 °C.
   4. Use the right femur and both tibiae for bone metabolism assays.

4. Measurement of parameters

1. Blood glucose level measurement
   1. Prior to tissue collection, obtain blood samples from the tail vein of the mice. Measure blood glucose levels using a portable glucose analyzer.
2. Blood lactate level measurement
   1. Immediately after the final treadmill exercise session, collect blood samples from the tail vein. Determine the blood lactate levels using a portable lactate analyzer.
3. Rotarod test
   NOTE: After all interventions were completed, we performed rotarod tests on mice from each group. When conducting rotarod testing, we choose different groups of mice to be tested simultaneously to ensure consistent rest time, rather than testing one group of mice first, so we can exclude differences caused by rest time.
   1. Place the mice on a rotarod apparatus with the head facing the opposite direction of rotation, allowing for one adaptation trial.
   2. Set the rotarod speed to 30 rpm; after starting, the apparatus accelerates uniformly to the set speed.
   3. Record the time until the mouse first falls. Use this measure to assess the mice's motor coordination ability.
4. Measurement of femoral trabecular bone mass
   1. Remove muscle and connective tissue attached to the bones using sterile micro-scissors and forceps and wash with PBS.
   2. Perform micro-CT scanning to assess the parameters of the trabecular bone in the left femur using the following scanning parameters: micro-CT scanning was performed at 55 kVp/145 µA (8 W) in continuous rotation mode with 9 µm isotropic voxels, 31.0 mm FOV, 250 ms exposure, native resolution (388 layers/segment), and 1,200 mgHA/cc BH calibration.
   3. Measure the following outcomes of the trabecular and cortical microarchitecture.
      1. Import the reconstructed images into CT-vox for three-dimensional (3D) rendering and visualization.
      2. Using multiplanar views (coronal, sagittal, and transverse planes), separate and extract cortical and trabecular bone into distinct tissue regions to enable independent analysis of their morphometric characteristics.
      3. Quantify key microstructural parameters, including Bone Volume/Total Volume (BV/TV), Trabecular Number (Tb.N), and Trabecular Thickness (Tb.Th) Trabecular Separation (Tb.Sp), to evaluate bone development and mineralization status.
5. Paraffin sectioning and histological staining
   1. Wash three left femora from each group with PBS and decalcify them in 10% EDTA (pH 7.15) until complete decalcification occurs.
   2. Dehydrate the samples through graded ethanol concentrations (70%, 90%, 95%, and 100%), clear with xylene, infiltrate with paraffin, and embed them (see Table 2).
   3. Cut serial sections of 5.0 µm thickness.
   4. Hematoxylin and eosin (HE) staining
      1. Subject the slices first to xylene dewaxing, gradient ethanol rehydration, and then soak them in hematoxylin for 5 min.
      2. Wash to remove excess stain, differentiate in 1% hydrochloric acid alcohol for 3 s, stain with eosin for 3 min, dehydrate through graded ethanol, clear with xylene, and mount with neutral gum (see Table 3).
      3. Capture microscopic images to observe bone tissue structure and cell morphology and distribution in the bone marrow cavity.
   5. Tartrate-resistant acid phosphatase (TRAP) staining
      1. Dewax the slices with xylene and rehydrate with gradient ethanol.
      2. Incubate the slices with TRAP reagent in a 37 °C oven for 1 h, dehydrate, and transparentize them (see Table 4).
      3. Apply a drop of neutral resin onto the glass slide, then carefully lower the coverslip at a 45° angle from one edge to minimize bubble formation. Allow the mounted slide to dry completely in a fume hood before proceeding with microscopic observation and imaging.
6. Gene expression analysis in tibial bone tissue
   1. Bone marrow Isolation
      1. Dissect mouse tibiae and remove excess muscle/fascia using sterile tools.
      2. Place the cleaned tibiae in a 1.5 mL microcentrifuge tube.
      3. Cut open both ends of the bone with sterile scissors.
      4. Flush the bone marrow into a grinding tube containing sterile grinding beads using 1x PBS.
         NOTE: Flush the bone marrow cavity with PBS until the cavity appears pale and all visible marrow tissue is removed.
   2. RNA extraction
      NOTE: Chloroform/isopropanol waste must be segregated in designated halogenated solvent containers and never mixed with bleach due to toxic gas formation.
      1. Add 1 mL of RNA extraction reagent per tube. Homogenize the samples using a benchtop homogenizer (30 s pulses, 4 °C). Incubate the lysates at room temperature for 5 min.
      2. Add 200 µL of chloroform, vortex for 15 s, and incubate at 4 °C for 30 min.
      3. Centrifuge at 12,000 × g for 30 min at 4 °C.
      4. Transfer the upper aqueous phase to a new microcentrifuge tube. Add 500 µL of isopropanol, invert 10x to mix, and incubate at 4 °C for 20 min.
      5. Centrifuge at 12,000 × g for 30 min at 4 °C. Discard the supernatant carefully without disturbing the RNA pellet.
      6. Wash the pellet with 1 mL of 75% ethanol (prepared with DEPC-treated water). Vortex briefly and centrifuge at 7,500 × g for 5 min at 4 °C.
      7. Air-dry the pellet for 5-10 min in a fume hood (avoid overdrying).
      8. Dissolve RNA in 20-50 µL of DEPC-treated water.
      9. Measure concentration and purity (A260/A280 ratio ≥ 1.8) using a spectrophotometer.
   3. cDNA synthesis
      1. Synthesize cDNA using 1 µg of total RNA and a reverse transcription kit.
      2. Store cDNA at −20 °C for qPCR analysis.
   4. qPCR analysis
      1. Analyze gene expression for osteogenic markers: Alp, Atf4, Runx2, Ocn, Osx; osteoclast markers: Nfatc1, Mmp9, Ctsk; cytokines: Tnf-α, Il-1β, Il-10 (see Table 5).
      2. Use primer sequences listed in Table 6 (include annealing temperatures).

5. Statistical analysis

1. Present all data as mean ± standard error of the mean (mean ± SEM).
2. Use Bonferroni and Tamhani for post-hoc tests.
3. Conduct one-way analysis of variance to assess differences among groups.
4. Consider P < 0.05 statistically significant.

Results

Fatigue due to prolonged high-intensity treadmill exercise training
We established a long-term high-intensity platform running exercise model to study the skeletal changes under exercise and the effects of infrared therapy on bones (Figure 1A). At the end of the intervention, mice were observed for changes in body weight. Compared with the body weight of the mice in the CTR group, no significant change was determined in the HIT group, while a significant decrease occurre...

Discussion

Of all metabolic bone diseases, OP is the most common and a major global public health problem¹¹. OP arises when osteoblastic and osteoclastic functions are dysfunctional, and its prevalence is high¹⁸. Exercise has been increasingly emphasized by physicians and researchers as a potential strategy for the prevention and treatment of OP considering its low cost and few adverse effects and its great potential in the future treatment and prevention of OP. The mechanisms by whic...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Far infrared therapy instrument	Cofoe Medical Technology		Infrared radiation was then applied as a heat source to warm the blood in the subcutaneous capillary network. The therapy temperature was controlled between 42 °C and 45 °C, and the entire treatment lasted approximately 40 min, 3 times per week
Micro-CT	SCANCO Medical AG	VIVACT80	Micro-CT scanning was performed to assess parameters of the trabecular bone in the left femur, including BV/TV, Tb.N, Tb.Th, Tb.Sp, and cortical thickness (Ct.Th).
Mouse treadmill	Anhui Zhenghua Biological Apparatus Facilities Co., Ltd		High intensity treadmill running in mice
Paraffin embedding machine	Leica		The paraffin embedding machine can embed tissues
paraffin slicer	Leica		The paraffin microtome can slice tissues
PrimeScript RT Reagent kit
SPSS
Tribromoethanol		Avertin
TRIzol	Accurate Biology	AG21101/AG21102