Ischemia-Reperfusion Injury: Molecular Markers

The effect of IPC on skeletal muscle damage due to ischemia-reperfusion injury was studied using immunohistochemical staining of neovascular factors (VEGF), oxidative DNA damage (8-hydroxyguanosine (8-OHdG)), and inflammatory markers (cyclooxygenase 2 (COX-2)). In all groups, femoral sections were prepared, and sections obtained from the femoral proximal medial diaphysis of each group were deparaffinized with xylene and ethanol. For antigen retrieval, the sections were autoclaved at 121 °C for 15 min. Using 0.3% H₂O₂, endogenous peroxidase was eliminated, blocking was performed using a mouse or goat normal serum, and the primary antibody was made to react. As the primary antibody, anti-Bax rabbit polyclonal antibody (Abcam, Cambridge, UK), anti-VEGF rabbit polyclonal antibody (Proteintech, Rosemont, IL, USA), anti-8-OHdG mouse monoclonal antibody (Abcam), and COX-2 rabbit polyclonal antibody (Proteintech) were used at 1.25, 5.0, 20.0, and 5.0 µg/mL, respectively. The reaction time was overnight at 4 °C in a cool dark room. After reacting with the primary antibody, the secondary antibody (biotin) reacted with an enzymatic agent (streptavidin). After 5 min immersion in DAB to allow for color development, the nuclei were stained. Photographs were taken using a BX53 microscope (Olympus, Tokyo, Japan) and a DP71 camera (Olympus).
VEGF and COX-2 were randomly extracted from their respective muscle tissues with 10 fields of view (400 times) and quantified using ImageJ. In 8-OHdG, cell nuclei expressed by 8-OHdG were extracted from 10 fields of view per sample, and the IPC (+) group was compared with the IPC (−) group.