Serial Noninvasive Targeted Imaging of Peripheral Angiogenesis: Validation and Application of a Semiautomated Quantitative Approach

Previous studies by our group have demonstrated the feasibility of noninvasive imaging of alpha(v) integrin to assess temporal and spatial changes in peripheral and myocardial angiogenesis. In this study, we validate the reproducibility, accuracy, and applicability of a new semiautomated noninvasive approach for serial quantitative evaluation of targeted micro-SPECT/CT images of peripheral angiogenesis in wild-type and endothelial nitric oxide sythase (eNOS)-deficient (eNOS-/-) mice subjected to hindlimb ischemia. Methods: Mice (n = 15) underwent surgical ligation of the right femoral artery to induce unilateral hindlimb ischemia. One week after ligation, a Tc-99m-labeled cyclic-Arg-Gly-Asp peptide targeted at alpha(v) integrin (NC100692, n 5 = 10) or a Tc-99m-labeled negative control (AH-111744, n = 5) was injected, and 60 min later in vivo micro-SPECT/CT images were acquired. Mice were euthanized, tissue from proximal and distal hindlimb was excised for gamma-well counting (GWC) of radiotracer activity, and ischemic-to-nonischemic (I/NI) ratio was calculated. Micro-SPECT/CT images were analyzed using a new semiautomated approach that applies complex volumes of interest (VOIs) derived from segmentation of the micro-CT images onto micro-SPECT images to calculate I/NI activity ratios for the proximal and distal hindlimb. Studies were reprocessed for determination of intra-and interobserver variability. To compare 3-dimensional (3D) VOI analysis with traditional manual 2-dimensional region-of-interest (ROI) analysis of maximum-intensity-projection images, micro-SPECT images were summed onto a single anterior-posterior projection. Rectangular ROIs were manually drawn and I/NI ratio calculated. Our new 3D analysis approach was applied to additional groups of mice (eNOS-/-, n = 5; wild-type, n = 3) imaged before and 1 and 4 wk after femoral artery resection. Results: Our new semiautomated approach for the evaluation of images of alpha(v) integrin targeted with microSPECT/CT demonstrated both a high intra-and interobserver variability (R-2 = 0.997) and an accuracy (R-2 = 0.780) for estimation of relative radiotracer activity relative to GWC. Analysis of serial micro-SPECT/CT images demonstrated a significant increase in relative NC100692 retention in the ischemic hindlimb of both wild-type and eNOS-/- mice at 1 wk after surgery. There was a significant (similar to 25%) decrease in radiotracer uptake in eNOS-/- mice relative to wild-type animals, which was not observed at baseline or 4 wk after ligation. Conclusion: A new semiautomated analysis of images of alpha(v) integrin targeted with micro-SPECT/CT provides a noninvasive approach for serial quantitative evaluation of peripheral angiogenesis. The reproducibility and accuracy of this approach allows for quantitative analysis of serial targeted molecular images of lower extremities, has applicability to other targeted SPECT or PET radiotracers, and may have implications for clinical imaging in patients with peripheral arterial disease.