Hindlimb Suspension-Induced Muscle Atrophy

At the end of the experimental period, rats were anesthetized with pentobarbital sodium, and blood was immediately collected through cardiac puncture. Rats were euthanized, and the soleus muscles were excised, weighed, and used for ex vivo collection of muscle EVs (right) or dissected, weighed, frozen in liquid nitrogen, and stored at − 80 °C for later biochemical analyses (left). For immunohistochemistry (IHC) analyses, the soleus muscles were covered in Tissue-Tek optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA), frozen in liquid nitrogen-cooled isopentane, and stored at − 80 °C. The soleus muscles were used in these experiments because in rats, the soleus, which is almost exclusively type I, is especially susceptible to hindlimb suspension-induced muscle atrophy [25 (link), 26 (link)]. Furthermore, the smaller size of the soleus limits issues of oxygen diffusion during ex vivo muscle assessments [27 (link)]. The gastrocnemius muscles were used for single-cell RNA sequencing (scRNA-seq) as described in [28 (link)]. The serum was isolated by allowing blood to clot at room temperature for 30 min before centrifugation for 10 min at 2000g at 4 °C. The serum supernatant was collected and stored at − 80 °C until analysis. The number of animals used in each experiment is listed in the figure legends.