Uptake of ¹¹C-Choline in Mouse Atherosclerotic Plaques

Animals

At the age of 15 mo, male mice deficient for both low-density lipoprotein receptor and apolipoprotein B48 (LDLR^−/−ApoB^100/100) (strain 003000; Jackson Laboratory) were fed for 2 mo with a Western-type diet (TD.88137, Adjusted Calories Diet; Harlan). The normally fed male control mice (C57BL) were 13 ± 2 mo old. The study design was approved by the Laboratory Animal Care and Use Committee of the University of Turku, Finland.

Synthesis of ¹¹C-Choline

¹¹C-Choline was produced through the reaction of N,N-dimethyl-2-aminoethanol and ¹¹C-methyl triflate using high-performance liquid chromatography purification and analysis procedures described by Rosen et al. (10), with high radiochemical yield (>70%) and radiochemical purity (>99.5%).

Biodistribution and Blood Metabolism of ¹¹C-Choline in Atherosclerotic and Control Mice

Six atherosclerotic LDLR^−/−ApoB^100/100 mice (mean weight ± SD, 38 ± 3 g) and 5 C57BL control mice (42 ± 6 g) were intravenously injected with ¹¹C-choline (27 ± 10 MBq) via the tail, and after 10 min, the animals were sacrificed under isoflurane anesthesia. Samples of whole blood and tissues (Table 1) were dissected, weighed, and measured for radioactivity using an automatic γ-counter (1480 Wizard 3″; EG&G Wallac). The data were corrected for background and decay. The radioactivity that had accumulated in the tissues was expressed as a percentage of the injected activity per gram of tissue. The plasma was further analyzed for radiometabolites as described earlier (11).

Autoradiographic Analysis of ¹¹C-Choline in Aortic Cryosections

The distribution of ¹¹C-choline to the aortic tissue was studied with digital autoradiography as described before (12). Briefly, after 1 h of exposure the imaging plates were scanned (Imaging Plate BAS-TR2025 [Fuji]; Analyzer BAS-5000 [Fuji]), and the images of aortic sections were analyzed for count densities (photostimulating luminescence units [PSL]/mm²) with an image-analysis program (Tina 2.1; Raytest Isotopenmessgeräte GmbH). Three types of regions of interest (ROIs) were defined according to the histology: plaque (excluding the medium), adventitia (containing the adjacent adipose tissue), and healthy vessel wall (Figs. 1A and 1B). The background count densities were subtracted from the image data. ROIs for all visible plaques were drawn over the areas where the plaque was easily identifiable.

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FIGURE 1. 

Ex vivo autoradiography analysis. (A) Hematoxylin and eosin–stained 20-μm section showing aortic arch and branches. (B) Autoradiography image of same section as in A, with superimposed contour image and delineated ROIs: R1 and R3 = plaque, R2 = adventitia including adjacent adipose tissue, and R4 = wall. (C) Eight-micrometer section showing higher magnification of R1 plaque in brachiocephalic trunk and immunohistochemical staining of macrophages (Mac-3) (bar = 100 μm). (D) Consecutive 8-μm section of same plaque as in C. Immunohistochemical staining with Ki-67 shows few proliferative cells in plaques (bar = 100 μm). Inset shows 1 positive cell at higher magnification (bar = 5 μm).

Histology and Immunohistochemistry

After autoradiography, the 20-μm sections were stained with hematoxylin and eosin and studied for morphology under a light microscope. Consecutive 8-μm sections were immunostained with Mac-3 (clone M3/84; BD Pharmingen) or Ki-67 (clone Mib-1; Dako) for the detection of macrophages and proliferating cells, respectively.

Randomly selected plaque areas (n = 35) were semiquantitatively assessed for the degree of inflammation by estimating the number of nuclei and Mac-3–positive cells in consecutive sections, without knowledge of the corresponding autoradiography results. The ROIs in these plaques were divided into 2 categories: noninflamed, with none or occasional leukocytes in the region, and inflamed, with a high number of nuclei and corresponding Mac-3 staining in the area.

Statistical Methods

All the results are expressed as the mean ± SD. Student t test for nonpaired data and Dunnett test were used to compare the biodistribution of ¹¹C-choline in organs. Univariate correlations were calculated using the Pearson partial-correlation method. Repeated-measures ANOVA with Tukey correction was applied to the autoradiography data. Normality was tested using the Shapiro–Wilkins method. A P value less than 0.05 was considered as statistically significant.

Characterization of Atherosclerotic Plaques

All of the studied LDLR^−/−ApoB^100/100 mice had developed extensive atherosclerosis throughout the aorta. The observed plaques in the aortas of the LDLR^−/−ApoB^100/100 mice contained cell-rich, inflamed areas and acellular necrotic cores. Occasional calcifications were also found. Mac-3 staining revealed areas in the plaques with a moderate number of macrophages (Fig. 1C). Only a few Ki-67–positive cells were detected in the plaques (Fig. 1D).

Ex Vivo Biodistribution and Blood Metabolism of ¹¹C-Choline

The ¹¹C radioactivity measured at 10 min after the injection of ¹¹C-choline was 1.9-fold higher in the aortas of the LDLR^−/−ApoB^100/100 mice than those of the C57BL control mice (n = 6 and 5, respectively, P = 0.0016) (Table 1). In the other measured tissues, no significant differences between the atherosclerotic and the control mice were observed. The ¹¹C radioactivity was highest in kidneys, lung, heart, and liver (Table 1). The aorta-to-blood and aorta-to-muscle ratios of the LDLR^−/−ApoB^100/100 mice were 5.5 ± 2.2 and 3.0 ± 0.9, respectively. The biodistribution of ¹¹C-choline in the circulating blood was not affected by the animal weight or strain (P = 0.08).

The high-performance liquid chromatography radiodetector analysis of the mouse plasma samples (n = 6) revealed 15% ± 7% of unchanged ¹¹C-choline at 10 min after injection. The percentages of total radioactivity were 78% ± 7% for ¹¹C-betaine and 9% ± 3% for another radiometabolite (unidentified).

Autoradiography of Aortic Cryosections

Hematoxylin and eosin–stained longitudinal cryosections throughout the aorta (n = 6–7 sections of each animal) were imaged under a light microscope, and the images were combined with the autoradiographs to define ROIs. The mean uptake of ¹¹C radioactivity in each region was calculated for each mouse (Table 2).

The autoradiography analysis showed a significant uptake of ¹¹C radioactivity in the plaques in comparison to the healthy vessel wall (plaque-to-wall ratio, 2.3 ± 0.6; P = 0.014, n = 6 LDLR^−/−ApoB^100/100 mice). The adjacent adventitial tissue, containing the adipose tissue surrounding the vessel, also showed a substantial uptake (adventitia-to-wall ratio, 1.9 ± 0.5; P = 0.016), which, however, was significantly lower than that in the plaques (P = 0.021). No significant uptake was found in calcifications.

The mean plaque-to-wall ratios were 2.6 ± 0.8 and 1.4 ± 0.5 in inflamed and noninflamed plaques, respectively (P < 0.001) (Fig. 2). Most of the cells in the inflamed plaques were identified as macrophages.

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FIGURE 2. 

(A) Uptake of ¹¹C-choline in mouse atherosclerotic plaques (n = 20 [noninflamed] and 15 [inflamed], *P < 0.001). Plaque-to-wall ratio was calculated for each plaque region against mean PSL/mm² value of healthy wall uptake of same animal. (B) Immunohistochemical Mac-3 staining of noninflamed plaque area, showing only occasional macrophages. (C) Mac-3 staining of inflamed plaque, showing high number of macrophages. Bar = 50 μm.