Preventive effects of transcutaneous CO2 application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model

We previously demonstrated that transcutaneous CO₂ application promotes muscle fiber-type switching, fracture healing, and osteogenesis by increasing blood flow and angiogenesis. Here, we aimed to investigate the preventive effects of transcutaneous CO₂ application on disuse osteoporosis and muscle atrophy in a rat hindlimb suspension model. Eleven-week-old male Sprague-Dawley rats were divided into hindlimb suspension (HS), HS with transcutaneous CO₂ application (HSCO₂), and control groups. HSCO₂ rats were administered transcutaneous 100 % CO₂ gas in their bilateral hindlimbs, five times a week for 20 min. After 3 weeks, we harvested the gastrocnemius, femur, and tibia for assessment. Histological analysis revealed a significant decrease in the gastrocnemius myofiber cross-sectional area in HS rats compared to the control rats, whereas HSCO₂ rats exhibited a significant increase compared to HS rats. Micro-computed tomography showed significant bone atrophy in the trabecular and cortical bones of the femur in HS rats compared to those of the control rats, whereas significant improvement was noted in HSCO₂ rats. Histological analysis of the proximal tibia revealed more marrow adipose tissue in the HS rats than in the control rats. However, in the HSCO₂ rats, fewer marrow adipose tissue and osteoclasts were observed. Moreover, HSCO₂ rats had more osteoblasts and higher expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and vascular endothelial growth factor (VEGF) than the HS rats. The gastrocnemius and distal femur of HSCO₂ rats also exhibited elevated PGC-1α and VEGF expression and upregulation of the myogenesis markers and osteogenesis markers compared to those of HS rats. This treatment effectively prevented disuse osteoporosis and muscle atrophy by promoting local angiogenesis and blood flow. PGC-1α is crucial for promoting this angiogenic pathway. Transcutaneous CO₂ application may be a novel preventive procedure for disuse osteoporosis and muscle atrophy, complementing medication and rehabilitation.