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Previous studies have suggested that matrix metalloproteinase (MMP) inhibitor uptake may offer a precise estimation of MMP activity in atherosclerotic lesions. In this study, we explored the feasibility of noninvasive detection of MMP-9 activity using technetium-99m-labeled matrix metalloproteinase-9 antibody (99mTc-McAb) in vivo.ApoE-deficient (ApoE−/−) atherosclerosis mice models (n=10) were induced through a high-cholesterol diet following ligation of their left common carotid artery. After 4 weeks, the models were verified through proton density-weighted and T2-weighted images obtained by MRI. C57BL/6 sham mice (n=8) were used as controls. In addition, normal mice (n=20) were used to characterize blood clearance. After radiolabeled McAb administration, single-photon emission computed tomography (SPECT) was performed. Subsequently, left common carotid arteries were harvested for ex-vivo autoradiograph imaging. Then, morphology and activity assays of MMP-9 were histologically and immunohistochemically examined.MRI showed higher signal intensities in the left common carotid arteries with irregular stenoses in the lumen of blood vessels in atherosclerosis mice models in vivo. Atherosclerotic lesions on left common carotid artery specimens were also clearly visualized using SPECT 2 h after 99mTc-McAb administration in vivo. Note that the radiochemistry purity of the 99mTc-McAb used was 85–95%. Biodistribution studies have shown that the clearance of 99mTc-McAb from blood was rapid. In addition, atherosclerotic lesions were clearly visualized on radioautography film shadows ex vivo.MMP-9 activities within the atherosclerotic lesions were noninvasively detected using 99mTc-labeled SPECT in vivo.