Evaluation of 18F-Annexin V as a PET Imaging Agent in an Animal Model of Apoptosis

Evaluation of ¹⁸F-Annexin V as a PET Imaging Agent in an Animal Model of Apoptosis

Kevin J. Yagle, PhD¹, Janet F. Eary, MD¹, Jonathan F. Tait, MD, PhD², John R. Grierson, PhD¹, Jeanne M. Link, PhD¹, Barbara Lewellen, CNMT¹, Don F. Gibson, BS² and Kenneth A. Krohn, PhD¹

¹ Department of Radiology, University of Washington, Seattle, Washington
² Department of Laboratory Medicine, University of Washington, Seattle, Washington

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FIGURE 1. PET images of normal and cycloheximide-treated rats. Four hours after injection of 5 mg of cycloheximide per kilogram (intraperitoneally) into treated rat, both animals were injected with approximately 51.8 MBq of ¹⁸F-annexin V (851 kBq/µg protein) and imaged for 2 h starting immediately after injection of radiotracer. In each panel, untreated animal is on left and treated animal is on right. All 3 false-color images were windowed using same logarithmic scale from 0 to 481 kBq/cm³. (A) Transverse image from 10-min time frame beginning at 0 min shows higher uptake in treated liver (R). (B) Coronal image from 10-min time frame beginning at 40 min; urinary bladder shows high uptake in lower abdomen. (C) Transverse image from 10-min time frame beginning at 1 h 30 min shows higher uptake in treated liver (R).

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FIGURE 2. Time–activity curves for rat liver in 2 different imaging studies. (A) Time–activity curve is shown for liver of each animal imaged in Figure 1. Ratio of ¹⁸F-annexin V uptake in liver (treated over control) increased from 1.9 to 8.6 over course of imaging session. (B) Time–activity curves are shown for rat livers in second study, imaged after injection of approximately 44.4 MBq in each animal. Animals were untreated (no Rx), or pretreated for 50 and 150 min with cycloheximide before injection of radiotracer. Ratio of ¹⁸F-annexin V uptake in liver increased from 1.25 to 3.0 (50-min treatment) and from 2.2 to 6.5 (150-min treatment); only ratio line for 150-min treatment is shown.

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FIGURE 3. Correlation of apoptotic morphology and cycloheximide treatment conditions. (A) H&E-stained slides were made from paraffin blocks of liver samples from rats treated with cycloheximide (5 mg/kg) for times indicated. Slides were scored for percentage of apoptotic cells, scoring minimum of 15 fields per slide. Each data point represents single animal; error bars represent SD of mean value of all fields read from same slice. (B) H&E slides were similarly prepared and scored from rats treated for 90 min with the indicated concentrations of cycloheximide, before injection of ¹⁸F-annexin V. Because these rats were also imaged for 2 h after injection of radiotracer, total duration of exposure to cycloheximide was 210 min. Numbers of animals per data point were 4 for no Rx, 2 for 2 mg/kg, and 4 for 5 mg/kg; error bars refer to SD of mean values.

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FIGURE 4. TUNEL assay of liver slices from 3 different imaging studies. Liver slices were subjected to TUNEL assay, and number of positive nuclei was counted (15–25 fields per slide). Untreated liver slices typically showed 1%–2% positive nuclei per field, whereas cycloheximide treatment resulted in 10%–16% positive nuclei. Panels show data from study in Figure 2A (2-h animal was not imaged) (A), data from separate study (B), and data from study in Figure 2B (90-min animal was not imaged) (C). Error bars refer to SD of mean.

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FIGURE 5. Regression analysis of ¹⁸F-annexin V uptake in liver tissue versus percentage of TUNEL-positive nuclei in liver tissue slices (r² = 0.89). Analysis included data from the 3 studies presented in Figure 4, as well as other studies (n = 13 animals total; each data point represents 1 animal). Error bars refer to SD of mean for percentage of TUNEL-positive values.

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FIGURE 6. Time–activity curves of ¹⁸F-annexin V uptake in heart (A), liver (B), kidney (C), and urine/urinary bladder (D) in normal rats. Uptake is measured as radioactivity (in becquerels) per organ per kilobecquerel injected. These time–activity curves were generated from imaging study used to calculate dosimetry data in Table 2 and are based on ROIs drawn over most central (highest uptake) transaxial slice of organ in PET images, except for urinary bladder. Curves begin at time zero with injection of radiotracer. Data points represent values for each of 3 animals, and dotted line represents average of those values.