Figure 1

Oxidative damage determined as carbonyl groups (Panels A and B) and HNE adducts (Panels C and D), is increased in gastrocnemius myofibers of patients with peripheral arterial disease (Panels A and C) compared to the control patients (Panels B and D). The control muscle has polygonal myofibers of similar shape and size. The PAD muscle exhibits a wide range of myofiber sizes with a smaller average myofiber size. Additionally, the PAD muscle has fatty infiltration, endomysial fibrosis (increased extracellular matrix between myofibers) and target lesions with evidence of increased oxidative damage (arrows). Levels of oxidative damage varied widely among PAD myofibers but similar patterns of injury were seen with carbonyl and HNE labeling. Oxidative damage in PAD muscle was not limited to the myofiber compartment but was consistently elevated throughout the extracellular matrix where it was present exclusively as carbonyl groups. HNE adducts were confined to the interior of the myofibers and were not detected in the extracellular matrix. Specimens obtained by needle biopsy of the gastrocnemius were fixed in cold methacarn, embedded in paraffin, sectioned at 4 μ and mounted to glass slides. Carbonyl groups in slide-mounted needle biopsy specimens were labeled with biocytin hydrazide plus streptavidin-Alexa Fluor® 488 ( Panels A and B) and HNE adducts were labeled with monoclonal anti-HNE antibody plus goat anti-mouse IgG-Alexa Fluor® 568 ( Panels C and D). Images of each microscopic field were captured with a 10X objective. The white bar represents a length of 50 microns.