Indocyanine green enables near-infrared fluorescence imaging of lipid-rich, inflamed atherosclerotic plaques

Abstract

New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. Although molecular imaging agents are available for low-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries. Here, we have demonstrated that indocyanine green (ICG), a Food and Drug Administration-approved near-infrared fluorescence (NIRF)-emitting compound, targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aorta, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.

Fig. 1

ICG rapidly targets atheroma in rabbit arteries and provides NIRF signal enhancement. Atheroma-bearing or normal rabbits were injected with either ICG or saline and then sacrificed after 45 minutes. (A–C) Ex vivo fluorescence reflectance images of aortas obtained at 800 nm (left column, fire color lookup table) and their corresponding white light images (right column). Scale bar, 2 cm. (A) A strong focal arterial NIRF signal colocalizes with atherosclerotic plaques seen in the white-light image of atheroma-bearing animals. (B) The saline-injected, atheroma-bearing group shows minimal NIRF arterial signal. (C) Normal, uninjured rabbits injected with ICG generate reduced, diffuse NIRF signal. All fluorescence images processed and windowed identically. (D) Signal-to-noise ratios (SNRs) were calculated for ICG-injected, atheroma-bearing animals (n=17); saline-injected, atheroma-bearing animals (n=2); and ICG-injected normal animals (n=2). The SNR in atheroma-bearing ICG injected animals was 170–630% higher than in control groups (*p<0.05, one-way ANOVA test across all 3 groups). (E) Plaque target-to-background ratios (TBRs) were 110% higher than the saline control group (*p<0.05, unpaired t-test). Data reported as mean +- standard deviation.

Fig. 2

Histological assessment of ICG targeting of rabbit atheroma. (A) Correlative light microscopy and fluorescence microscopy of ICG deposition in atherosclerosis reveals ICG colocalization with lipid-rich and macrophage-abundant areas of atheroma sections. (1) Oil red O (ORO) stain of neutral lipid shows colocalization with ICG signal (arrowheads highlight the lipid-rich zone). (2) RAM-11–positive macrophages (dark brown stain) also colocalize with ICG, particularly in dense lipid-associated areas. (3) Immunoreactive smooth muscle cells (alpha-actin; stained brown) minimally colocalize with the matched ICG fluorescent area. (4) A few endothelial cells (CD31+) are present at the luminal surface (arrowhead). (5) Collagen (stained blue with Masson’s trichrome) and (6) elastin fibers (stained black with von Gieson’s) show very little colocalization with ICG. All histological slices are adjacent (6 µm). Fluorescence microscopic images of ICG distribution (854 nm) in all sections were obtained for each slice before staining. Scale bar, 200 µm. (B) Histopathological and fluorescence microscopic deposition of ICG in atheroma sections from three different animals. (1–3) From left to right: ICG fluorescence (845 nm, false-colored red), ORO staining of neutral lipid, and RAM-11+ macrophages. Within each group, all images are from adjacent sections (6 µm). Asterisk (*) denotes lumen. Scale bar, 100 µm.

Fig. 3

ICG deposition profile and autofluorescence in various-sized rabbit atheroma. From left to right: hematoxylin and eosin (H&E), for general morphology; Oil red O (ORO); 845 nm fluorescence channel for ICG (false-colored red); and 535 nm channel for autofluorescence from the medial elastic laminae (false-colored blue). Within each group, all images are from adjacent sections (6 µm) [OK?]. (A) ICG colocalizes with ORO-positive areas (arrowheads) in a larger atheroma section. (B) In a section from a small atheroma, ICG colocalizes with ORO (open arrowhead) and also generates a signal in the superficial intima-media in ORO-negative areas (solid arrowhead). (C) In saline-injected animals, minimal NIRF signal is evident, but 535 nm channel autofluorescence remains. Asterisk (*) denotes the location of the lumen. Scale bar, 100 µm.

Fig. 4

ICG atheroma-targeting profile in comparison to the Evans blue vascular permeability agent. (A) Fluorescence microscopy of Evans blue (EB) (710 nm) demonstrates homogenous signal enhancement in atheroma areas, indicating an impaired endothelial barrier. (B) ICG NIRF signal (845 nm) from the same section demonstrates focal signal in the deeper intimal plaque area. (C) A fusion image further illustrates the difference between ICG (red) and Evans blue (green) localization in an atheroma. Asterisk (*) denotes the location of the lumen. Dashed line outlines atheroma. Scale bar, 100 µm.

Fig. 5

ICG binding to acetylated LDL (acLDL) and BSA, and uptake by human macrophages. (A, B) Size-exclusion chromatography with optical absorbance measured at 750 nm for serial elutions. Early elutions (*) consisted of higher molecular weight compounds. (A) Elution profiles of ICG, acLDL, and the pre-incubated ICG-acLDL combination. (B) Elution profiles of ICG, BSA, and the pre-incubated ICG-BSA combination. (C) Fluorescence microscopy of intracellular ICG uptake by foam cells (810 nm; fire color lookup table). Overlay of fluorescence with white light (WL) images is shown on right column. Scale bar, 50 µm. (D) ICG uptake by human macrophages and foam cells detected at 810 nm . Scale bar, 100 µm. (E) Fluorescence measurements (790 nm) of ICG (25 and 125 µM) uptake by human monocyte–derived macrophages and foam cells (average of n=2 experiments). Data reported as mean +- standard deviation.

Fig. 6

In vivo intravascular NIRF guidewire sensing of ICG localized in atherosclerotic plaques in rabbits. (A) X-ray angiographic images of aortic and iliac luminal narrowings (yellow arrowheads). Green and red dotted arrows represent the start and end of the NIRF guidewire pullbacks, respectively. The white asterisks demarcate arterial branch points. Image formed by merging two overlapping angiograms. Angiography, in combination with fiducial landmark coordinates from radiopaque tips on both the optical and IVUS catheters (not shown), allowed for co-registration of the NIRF and IVUS datasets. Scale bar, 1 cm. (B) IVUS longitudinal image obtained from an aortic atheroma-bearing animal (left). A small plaque (intimal thickening) is evident at the lower edge of the dotted yellow line, as seen further on the corresponding axial image (right, yellow arrowheads). (C) Fifteen minutes after ICG injection, a NIRF guidewire signal trace was recorded at 780 nm during automated pullback (left). The NIRF signal (right, false-colored yellow) was fused onto the IVUS-demarcated atheroma in the axial slice shown in (B). All longitudinal images were scaled on the same unit length and matched on the same fiducial markers. (D) Ex vivo fluorescence reflectance images (FRI) show enhanced ICG signal in aortic and iliac plaques (fire color lookup table). (E) Overlay of FRI with the white light (WL) image.

Fig. 7

Focal ICG deposition in rabbit atheroma evolves rapidly after injection and provides stable target-to-background ratios (TBRs). After ICG injection, serial NIRF guidewire pullbacks were performed in vivo through blood, with each pullback initiated distal to plaques and with the starting position confirmed by both IVUS and X-ray angiography. (A) The maximum plaque TBR was 10.4±6.2 in the rabbits (n=5) studied. *p<0.05, paired t-test, versus the maximum pre–ICG-injection TBR. (B) Immediately after ICG injection, the NIRF guidewire signal recorded a saturating signal (10 au) owing to high concentration of ICG in the blood, followed by a rapid exponential decay (upper inset). A representative baseline NIRF signal during manual guidewire pullback, prior to ICG injection is shown (below, left). Fifteen minutes (below, middle) and 25 minutes (below, right) after ICG injection, the NIRF signal presented focal peaks at the site of the atheroma, as visualized on corresponding X-ray angiograms and registered in real-time during X-ray fluoroscopic tracking of the radiopaque NIRF guidewire. Each data point depicts one measurement. The red line shows a best fit with an exponential decay of the data points from 10 to 33 minutes. (C) Over the course of the imaging session (approximately 45 minutes), the ICG peak plaque TBR remained stable (n=5). Error bars show the measurement error of each TBR data point.