Annexin V imaging detects diabetes-accelerated apoptosis and monitors the efficacy of benfotiamine treatment in ischemic limbs of mice

The role of apoptosis imaging for monitoring treatment response in ischemic limbs has not been properly explored. In this study, we investigated the ability of annexin V (AnxV) imaging to assess the efficacy of antiapoptotic treatment in ischemic limbs of diabetic mice. Normal C57BL/6 mice and streptozotocin-induced diabetic mice were subject to hindlimb ischemia. AnxV-conjugated fluorescent streptavidin probes were intravenously injected, and optical imaging was performed. Tissue apoptosis was quantifiedbyhistochemistry and Western blotting. The AnxV probes showed specific targeting to apoptotic cells on confocal microscopy and flow cytometry. Intravenous AnxV probes displayed substantially greater accumulation inischemic limbsof diabetic mice. Benfotiamine (BFT) treatment of diabetic mice ledto better perfusion recoveryonlaser Doppler imaging and reduced AnxV binding on optical imaging. TUNEL staining and cleaved caspase-3 Western blots confirmed accelerated apoptosis by diabetes and its suppression by BFT treatment. Furthermore, AnxV-SAv-PEcy5.5 uptake in the ischemic limbs closely correlated to cleaved caspase-3 expression. Thus, AnxV imaging may be useful for monitoring the efficacy of therapeutic agents designed to suppress ischemia-induced apoptosis.