Figure 5.

Evaluation of mitochondrial toxicity following HLV injection. (A) COX staining of skeletal muscle following HLV injection. Frozen cross-sections (10 μm thickness) of HLV injection performed skeletal muscle were prepared, followed by COX staining. The section was then observed by microscopy; saline administered muscle (a) and condensed pDNA administered muscle (b). Scale bars, 100 μm. In this experiment, cells were stained brown when the cells have the cytochrome oxidase activity (COX-positive cells). We also calculated the ratios of COX-positive cells and the values for each image as indicated. Data represent the mean ± S.D. (n = 3). Statistical analysis was performed by a two-tailed unpaired Student’s t-test (p = 0.95). (B,C) Evaluation of mitochondrial membrane potential in skeletal muscles following HLV injection. At 24 h post HLV injections, the soleus (B) or gastrocnemius (C) of crural muscles were harvested, and mitochondria were then stained with TMRM (red color; a,c,e) and MTDR (cyan pseudo color; b,d,f). The staining of mitochondria with TMRM is dependent on the membrane potential, while MTDR can stain mitochondria even when membrane potential is lost. The muscle tissues were observed using CLSM. Scale bars, 10 μm.