In Vivo Near-Infrared Fluorescence Imaging of Atherosclerosis Using Local Delivery of Novel Targeted Molecular Probes

Abstract

This study aimed to evaluate the feasibility and accuracy of a technique for atherosclerosis imaging using local delivery of relatively small quantities (0.04-0.4 mg/kg) of labeled-specific imaging tracers targeting ICAM-1 and unpolymerized type I collagen or negative controls in 13 rabbits with atheroma induced by balloon injury in the abdominal aorta and a 12-week high-cholesterol diet. Immediately after local infusion, in vivo intravascular ultrasonography (IVUS)-NIRF imaging was performed at different time-points over a 40-minute period. The in vivo peak NIRF signal was significantly higher in the molecular tracer-injected rabbits than in the control-injected animals (P < 0.05). Ex vivo peak NIRF signal was significantly higher in the ICAM-1 probe-injected rabbits than in controls (P = 0.04), but not in the collagen probe-injected group (P = 0.29). NIRF signal discrimination following dual-probe delivery was also shown to be feasible in a single animal and thus offers the possibility of combining several distinct biological imaging agents in future studies. This innovative imaging strategy using in vivo local delivery of low concentrations of labeled molecular tracers followed by IVUS-NIRF catheter-based imaging holds potential for detection of vulnerable human coronary artery plaques.

Figure 1

Atherosclerotic rabbit model. At week 0, 15 male NZW rabbits of 12–13 weeks of age underwent balloon denudation of the distal abdominal aorta (40-mm length), upstream of the aorto-iliac bifurcation. At recovery, they were fed with a high-cholesterol diet (HCD) (0.5% cholesterol) over a period of 12 weeks. In vivo imaging experiments were performed at week 12, which consisted in performing local delivery of labeled NIRF molecular probes (0.04–0.46 mg/kg/probe) using a porous-balloon catheter at the site of the injured aorta. Immediately after local probe infusion, in vivo IVUS-NIRF imaging was acquired and repeated every 10-min for 40 minutes (total of 5 pullbacks/rabbit). Following in vivo imaging, ex vivo fluorescence imaging and histopathology were performed.

Figure 2

NIRF molecular imaging of unpolymerized type I collagen in atherosclerotic balloon injured rabbit abdominal aortas. Collagen-binding hairpin peptide was injected locally (0.04 mg/kg) in the balloon-injured distal abdominal aorta of atherosclerotic rabbits after 12 weeks of 0.5% cholesterol-enriched diet. (a) Shows conventional angiography of the rabbit aorta with the red box indicating the balloon-injured area; (b) Represents IVUS imaging cross-sections of the aorta at two distinct pullback distances from the aorto-iliac bifurcation (27.7 mm and 41.6 mm, respectively). Red arrows delineate eccentric atherosclerotic plaques on IVUS imaging, which are colocalized with higher NIRF signal intensity (orange/red, outer color spectrum); (c) Shows in vivo intravascular NIRF imaging pullback of collagen-binding peptide probe localization in the rabbit abdominal aorta performed 40 minutes after local injection of the NIRF labeled agent; (d) Demonstrates ex vivo en face fluorescence of collagen-binding peptide over the entire rabbit aorta performed 60 minutes after local injection of the NIRF labeled agent. There are two distinct zones of higher NIR-fluorescence signal intensity seen on either in vivo and ex vivo NIRF imaging, localized mostly upstream of the injection area (c–e) Shows the corresponding white light image of the rabbit aorta with the red box indicating the balloon injured area. The distinct zones of higher NIRF signal displayed on both in vivo and ex vivo images appear to be localized in a lipid-rich plaque area (bright white areas); (f) Correlation histopathology of unpolymerized collagen in the distal abdominal aorta following staining with Masson’s trichrome (MTRI). In vivo NIRF color scale is in arbitrary units (yellow/white: higher NIRF signal intensity; red: lower NIRF signal intensity). Ex vivo NIRF color scale displays number of counts according to signal intensity (yellow/white: higher NIRF signal intensity; red: lower NIRF signal intensity).

Figure 3

Ex vivo binding of NIRF molecular probes targeting ICAM-1 and unpolymerized type I collagen to atherosclerotic lesions in rabbit aortas. Atherosclerotic rabbit model. (a) Shows histopathology staining of rabbit denudated aorta sections. Left panel represents neointima staining with hematoxylin and eosin (H&E); mid-panel represents neutral lipids staining with Oil red O (ORO); right panel represents collagen staining with Masson’s trichrome (MTRI). All images are at x4 magnification. Cryosections from atherosclerotic rabbit aorta were stained with: (b) anti-ICAM-1 sdAbs; (c) collagen-binding peptide; (d) anti-ICAM-1 sdAbs control antibody and; (e) collagen-binding control peptide. The right panel shows fluorescence signal from either molecular probe targeting ICAM-1 or unpolymerized type I collagen, or the negative control probes. The left panel shows a merged image of both DAPI and the fluorescent molecular probes. The cell nuclei (blue) were stained with DAPI (4′,6-Diamidino-2-Phenylindole,Dilactate).

Figure 4

Blood clearance of molecular probes in atherosclerotic rabbits. Clearance from the blood of: (a) anti-ICAM-1 sdAbs (red squares) and the corresponding negative control (blue circles) from (n = 5/group) and; (b) collagen-binding peptide (red squares) and the corresponding negative control (blue circles) (n = 5 in the collagen group; n = 4 in the control group). Blood clearance kinetics shows a continuous decrease in NIRF signal intensity, which tends to stabilize 40 minutes after in vivo local injection of all NIR-fluorescence molecular agents. In vivo fluorescence signal detected by IVUS-NIRF catheter of both (c) anti-ICAM-1 sdAbs (red squares) and (d) collagen-binding peptide, by comparison to their negative controls (blue circles). Both imaging probes shows an initial strong NIRF signal that decreases rapidly, followed by a re-increase in signal intensity between 20–40 minutes, mostly seen with anti-ICAM-1 sdAbs. By contrast, a weak fluorescence signal is observed for both negative controls following in vivo local injection in the injured zone, which then becomes undetectable after 45 minutes. Data points are mean ± standard error of the mean (SEM) from the rabbits combined.

Figure 5

Comparison of in vivo and ex vivo near-infrared fluorescence (NIRF) imaging of targeted ICAM-1 and collagen in atherosclerotic rabbit aortas. Comparison of in vivo NIRF imaging and ex vivo fluorescence signal following single-probe local injection of either: (a) anti-ICAM-1 sdAbs, (b) collagen-binding peptide, (c) ICAM-1 negative controls; (d) collagen negative control; (e) dual-probe local injection of anti-ICAM-1 sdAbs combined to collagen-binding peptide probe, or (f) the combined negative controls. In vivo and ex vivo signals were colocalized in both single- and dual-probe injections along the longitudinal direction and delineated by white dot lines. In vivo and ex vivo fluorescence signals were properly aligned, taking into account post-mortem average tissue shrinkage estimated between 25–30%. In both groups, NIRF signal was only detected over the balloon-injured zone with the anti-ICAM-1 sdAbs agent (a,e). However, fluorescence signal was observed upstream from the injection zone with the collagen-binding peptide probe, notably in an aneurysm lipid-rich area of the aorta, as shown in (b,e). There were no NIRF signals detected in the control-injected groups (c,d,f), except in the abdominal and thoracic aorta of 2 rabbits following dual-injection of the control collagen-specific agent. In vivo and ex vivo peak NIRF signal in: (g) ICAM-1 probe- and (h) collagen-peptide probe injected abdominal aortas. White dot lines indicate the injury zone of the distal abdominal aorta, site of molecular probes delivery through the porous-balloon catheter. In vivo NIRF color scale is in arbitrary units (yellow/white: higher NIRF signal intensity; red: lower NIRF signal intensity). Ex vivo NIRF color scale displays number of counts according to signal intensity (yellow/white: higher NIRF signal intensity; red: lower NIRF signal intensity). Scale bar: 10 mm. Data is presented as mean ± standard error of the mean (SEM).

Figure 6

Immunostaining validation of anti-ICAM-1 sdAbs in atherosclerotic rabbit aortas. Colocalization of ICAM-1 in atherosclerotic rabbit aorta by immunofluorescence. Anti-ICAM-1 sdAbs NIRF signal colocalize with ICAM-1 on endothelial cells by staining the sections with primary mouse anti-rabbit ICAM-1 monoclonal antibody (red NIRF signal, images 1 and 2), followed by a secondary labeling using goat anti-mouse IgG1 antibody (green NIRF signal, image 3), The merged image shows specific colocalization of ICAM-1 on endothelial cell surface of atherosclerotic aorta segments (yellow NIRF signal, image 4). The endothelial cell nuclei (blue) were stained with DAPI (image 5). No autofluorescence signal was detected under confocal microscopy (image 6).