Effects of acute- and long-term aerobic exercises at different intensities on bone in mice.

INTRODUCTION: Exercise intensity determines the benefits of aerobic exercise. Our objectives were, in aerobic exercise at different intensities, to determine (1) changes in bone metabolism-related genes after acute exercise and (2) changes in bone mass, strength, remodeling, and bone formation-related proteins after long-term exercise. MATERIALS AND METHODS: Total 36 male C57BL/6J mice were divided into a control group and exercise groups at 3 different intensities: low, moderate, or high group. Each exercise group was assigned to acute- or long-term exercise groups. Tibias after acute exercise were evaluated by real-time PCR analysis. Furthermore, hindlimbs of long-term exercise were assessed by micro-CT, biomechanical, histological, and immunohistochemical analyses. RESULTS: Acute moderate-intensity exercise decreased RANKL level as bone resorption marker, whereas low- and high-intensity exercise did not alter it. Additionally, only long-term exercise at moderate intensity increased bone mass and strength. Moderate-intensity exercise promoted osteoblast activity and suppressed osteoclast activity. After low- and high-intensity exercise, osteoblast and osteoclast activity were unchanged. An increase in the number of ß-catenin-positive cells and a decrease in sclerostin-positive cells were observed in the only moderate group. CONCLUSION: These results showed that moderate-intensity exercise can inhibit bone resorption earlier, and long-term exercise can increase bone mass and strength through promoted bone formation via the Wnt/ß-catenin activation. High-intensity exercise, traditionally considered better for bone, may fail to stimulate bone remodeling, leading to no change in bone mass and strength. Our findings suggest that moderate-intensity exercise, neither too low nor high, can maintain bone health.

Effects of different exercise modes and intensities on cognitive performance, adult hippocampal neurogenesis, and synaptic plasticity in mice.

Exercise can induce beneficial improvements in cognition. However, the effects of different modes and intensities of exercise have yet to be explored in detail. This study aimed to identify the effects of different exercise modes (aerobic and resistance) and intensities (low and high) on cognitive performance, adult hippocampal neurogenesis and synaptic plasticity in mice. A total of 40 C57BL/6J mice were randomised into 5 groups (n = 8 mice per group): control, low-intensity aerobic exercise, high-intensity aerobic exercise, low-intensity resistance exercise, and high-intensity resistance exercise. The aerobic exercise groups underwent treadmill training, while the resistance exercise groups underwent ladder climbing training. At the end of the exercise period, cognitive performance was assessed by the Y-maze and Barnes maze. In addition, adult hippocampal neurogenesis was evaluated immunohistochemically by 5-bromo-2'-deoxyuridine (BrdU)/ neuronal nuclei (NeuN) co-labeling. The levels of synaptic plasticity-related proteins in the hippocampus, including synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95), were analyzed by western blotting. Our results showed no significant differences in cognitive performance among the groups. However, high-intensity aerobic exercise significantly increased hippocampal adult neurogenesis relative to the control. A trend towards increased adult neurogenesis was observed in the low-intensity aerobic group compared to the control group. No significant changes in synaptic plasticity were observed among all groups. Our results indicate that high-intensity aerobic exercise may be the most potent stimulator of adult hippocampal neurogenesis.

Effects of uphill and downhill walking on post-traumatic osteoarthritis development in mice.

Using destabilization of the medial meniscus (DMM) to induce models of osteoarthritis (OA), we sought to clarify how flat, uphill and downhill walking affects OA-related inflammation and articular cartilage degeneration. Thirty-two male C57BL/6J mice 7 weeks old underwent DMM surgery in their right knee and sham surgery in their left knee, and were then assigned to either the no walking after DMM group or the flat, uphill or downhill walking after DMM group (n = 8/group). After creating the knee OA model, the mice in the walking groups were subjected to treadmill walking 1 day after surgery, which included walking at 12 m/min for 30 min/day, 7 days/week, at inclines of 0, 20, or -20 degrees. Knee joints were harvested at the end of the intervention period. Non-demineralized frozen sections were prepared and samples were examined histologically. Osteoarthritis Research Society International scores were significantly decreased in both the uphill and flat-walking groups, compared with the no-walking group. Immunohistochemical staining showed increased levels of aggrecan and Sry-related high-mobility group box9; conversely, decreased levels of matrix metalloproteinase-13 and A disintegrin and metalloproteinase with thrombospondin motifs-5 in both the uphill and flat-walking groups. Micro-CT results showed a higher bone-volume fraction in the uphill and flat-walking groups than that in the no-walking group. Our findings indicate that flat and uphill walking may prevent the progression of OA. KEY POINTS: Flat and uphill treadmill walking can prevent the development of post-traumatic osteoarthritis in mice. Flat and uphill walking increases anabolic proteins and decreases catabolic proteins and inflammatory cytokines in articular cartilage, resulting in protection against cartilage degeneration. Downhill walking increases catabolic proteins and inflammatory cytokines in cartilage, which has negative effects on articular cartilage.

Utilization of Mechanical Stress to Treat Osteoporosis: The Effects of Electrical Stimulation, Radial Extracorporeal Shock Wave, and Ultrasound on Experimental Osteoporosis in Ovariectomized Rats.

Current treatment options for osteoporosis primarily involve pharmacotherapies, but they are often accompanied by undesirable side effects. Utilization of mechanical stress which can noninvasively induce bone formation has been suggested as an alternative to conventional treatments. Here, we examined the efficacy of mechanical stress induced by electrical stimulation, radial extracorporeal shock waves, and ultrasound for estrogen-deficient osteoporosis. Female Wistar rats were divided into following five groups: sham-operated group, untreated after ovariectomy, and treated with electrical stimulation, radial extracorporeal shock wave, or ultrasound starting at 8 weeks after ovariectomy for 4 weeks. Trabecular bone architecture of the femur was assessed by micro-CT and its biomechanical properties were obtained by mechanical testing. The femurs were further evaluated by histochemical, immunohistochemical, and real-time PCR analyses. Radial extracorporeal shock wave and ultrasound treatment improved trabecular bone microarchitecture and bone strength in osteoporotic rats, but not electrical stimulation. The shock wave decreased osteoclast activity and RANKL expression. The exposure of ultrasound increased osteoblast activity and ß-catenin-positive cells, and they decreased sclerostin-positive osteocytes. These findings suggest that mechanical stress induced by radial extracorporeal shock wave and ultrasound can improve estrogen-deficient bone loss and bone fragility through promoted bone formation or attenuated bone resorption.

Exercise-induced interactions between skeletal muscle and bone via myokines and osteokine in mice: Role of FNDC5/irisin, IGF-1, and osteocalcin.

Skeletal muscle and bone interact to maintain their structure and function. Physical exercise is the most effective and easily applicable strategy to maintain their functions; however, exercise-induced interactions by soluble factors remained elusive. Our study aimed to identify exercise-induced interactions between muscle and bone by examining (1) the effects of myokine on bone and (2) the effects of osteocalcin (OCN) on skeletal muscle. To understand the effects of exercise-induced myokines on bone, we examined the effects of FNDC5 for aerobic exercise and IGF-1 for resistance exercise using a muscle-specific myokine overexpression model. To examine OCN effects on muscle, mice were intraperitoneally administered OCN-neutralizing antibody during long-term exercise. Our result showed that aerobic exercise tended to increase serum HA-tag protein attached to FNDC5 in muscle-specific overexpression groups. In addition, osteoblastic activation was increased only after aerobic exercise with HA/FNDC5 overexpression. Resistance exercise did not alter circulating HA-tag (muscle-derived IGF-1) and bone metabolism after IGF-1/HA overexpression. In the OCN study, aerobic exercise enhanced endurance capacity by restoring muscle glycogen content; however, OCN neutralization returned these to baseline. After resistance exercise, OCN suppression inhibited muscle hypertrophy and strength gains by preventing protein synthesis. Our results suggest that aerobic exercise following FNDC5 muscle overexpression promotes osteoblast activity, which may be partially caused by muscle-derived FNDC5 secretion. In addition, OCN was necessary for muscle adaptation in both aerobic and resistance exercises.

Effects of aerobic exercise at different intensities on articular cartilage in mice.

Background: Maintaining intrinsic articular cartilage homeostasis is essential for the health of cartilage. However, the impact of aerobic exercise of varying intensities on the articular cartilage homeostasis has never been studied. This study aims to elucidate the influence of different aerobic exercise intensities on the anabolic and catabolic processes within articular cartilage. Methods: Forty-eight male C57BL/6J mice, aged 7 weeks, were divided into 4 aerobic exercise groups and 1 control group. The aerobic exercise groups were subjected to both acute and chronic exercise protocols with varying intensities of 8, 12, 16, 20, and 24 m min-1. Total RNA from the knee joint cartilage was extracted in both phases to quantify mRNA of anabolic (Sox9, Col2a1, and Acan) and catabolic (MMP-13 and ADAMTS5) markers. In the chronic exercise, articular cartilage thickness and chondrocyte density were histologically assessed. Additionally, immunohistochemical staining quantified relevant molecules involved in cartilage metabolism. Results: In the acute exercise, the 8 m min-1 group exhibited reduced ADAMTS5 expression compared to the control, 16 m min-1, and 24 m min-1 groups. Chronic exercise showed enhanced articular cartilage thickness in both the 8 and 12 m min-1 groups relative to the control group. Moreover, the 8 m min-1 group demonstrated elevated aggrecan levels in comparison to both the control and 24 m min-1 groups. Additionally, the 24 m min-1 group exhibited significantly higher ADAMTS5 levels than the control group. Conclusion: Our findings suggest that consistent low-intensity aerobic exercise suppresses catabolic molecule expression in articular cartilage, thereby fostering anabolic activity. Conversely, continuous high-intensity aerobic exercise can potentially disrupt cartilage homeostasis by enhancing catabolic processes. This dichotomy underscores the need for balanced exercise regimens to maintain cartilage health.

Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1.

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing low-intensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higher-intensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.

Effects of cyclic tensile strain and microgravity on the distribution of actin fiber and Fat1 cadherin in murine articular chondrocytes.

Chondrocytes as mechano-sensitive cells can sense and respond to mechanical stress throughout life. In chondrocytes, changes of structure and morphology in the cytoskeleton have been potentially involved in various mechano-transductions such as stretch-activated ion channels, integrins, and intracellular organelles. However, the mechanism of cytoskeleton rearrangement in response to mechanical loading and unloading remains unclear. In this study, we exposed chondrocytes to a physiological range of cyclic tensile strain as mechanical loading or to simulated microgravity by 3D-clinostat that produces an unloading environment. Based on microarray profiling, we focused on Fat1 that implicated in the formation and rearrangement of actin fibers. Next, we examined the relationship between the distribution of Fat1 proteins and actin fibers after cyclic tensile strain and microgravity. As a result, Fat1 proteins did not colocalize with actin stress fibers after cyclic tensile strain, but accumulated near the cell membrane and colocalized with cortical actin fibers after microgravity. Our findings indicate that Fat1 may mediate the rearrangement of cortical actin fibers induced by mechanical unloading.

Effects of ultrasound, radial extracorporeal shock waves, and electrical stimulation on rat bone defect healing.

Fractures associated with osteoporosis are a major public health concern. Current treatments for fractures are limited to surgery or fixation, leading to long-term bedrest, which is linked to increased mortality. Alternatively, utilization of physical agents has been suggested as a promising therapeutic approach for fractures. Here, we examined the effects of ultrasound, radial extracorporeal shock waves, and electrical stimulation on normal or osteoporotic fracture healing. Femoral bone defects were created in normal or ovariectomized rats. Rats were divided into four groups: untreated, and treated with ultrasound, shock waves, or electrical stimulation after surgery. Samples were collected at 2 or 4 weeks after surgery, and the healing process was evaluated with micro-CT, histological, and immunohistochemical analyses. Ultrasound at intensities of 0.5 and 1.0 W/cm2 , but not 0.05 W/cm2 , accelerated new bone formation. Shock wave exposure also increased newly formed bone, but formed abnormal periosteal callus around the defect site. Conversely, electrical stimulation did not affect the healing process. Ultrasound exposure increased osteoblast activity and cell proliferation and decreased sclerostin-positive osteocytes. We demonstrated that higher-intensity ultrasound and radial extracorporeal shock waves accelerate fracture healing, but shock wave treatment may increase the risk of periosteal callus formation.

Transcutaneous application of carbon dioxide improves contractures after immobilization of rat knee joint.

OBJECTIVE: Joint contractures are a major complication following joint immobilization. However, no fully effective treatment has yet been found. Recently, carbon dioxide (CO2) therapy was developed and verified this therapeutic application in various disorders. We aimed to verify the efficacy of transcutaneous CO2 therapy for immobilization-induced joint contracture. METHOD: Twenty-two Wistar rats were randomly assigned to three groups: caged control, those untreated after joint immobilization, and those treated after joint immobilization. The rats were treated with CO2 for 20 min once a daily either during immobilization, (prevention) or during remobilization after immobilization (treatment). Knee extension motion was measured with a goniometer, and the muscular and articular factors responsible for contractures were calculated. We evaluated muscle fibrosis, fibrosis-related genes (collagen Type 1α1 and TGF-ß1) in muscles, synovial intima's length, and fibrosis-related proteins (Type I collagen and TGF-ß1) in the joint capsules. RESULTS: CO2 therapy for prevention and treatment improved the knee extension motion. Muscular and articular factors decreased in rats of the treatment group. The muscular fibrosis of treated rats decreased in the treatment group. Although CO2 therapy did not repress the increased expression of collagen Type 1α1, the therapy decreased the expression of TGF-ß1 in the treatment group. CO2 therapy for treatment improved the shortening of the synovial membrane after immobilization and decreased the immunolabeling of TGF-ß1 in the joint capsules. CONCLUSIONS: CO2 therapy may prevent and treat contractures after joint immobilization, and appears to be more effective as a treatment strategy for the deterioration of contractures during remobilization.