Animals
Six beagle dogs (two males and four females; DooYeol biotech, Seoul, Republic of Korea), weighing 8.72 ± 1.05 [mean ± standard deviation (SD)] kg and ranging in age from 3 to 4 years (mean ± SD, 3.19 ± 0.24 years) were used in this study. All dogs were healthy without a history of neurological disorder; they had no abnormal signs in physical and neurological examinations. They were also tested for metabolic diseases by establishing a complete blood count and serum chemistry profile. The dogs were acclimated for more than 2 weeks and housed under the following conditions: an ambient temperature of 20 ± 2 °C, relative humidity of 50 ± 10%, air ventilation rate of 10 cycles per h, and a 12:12 h light:dark cycle. The dogs were fed a commercial dry food (L.I.D. Limited Ingredient Diets® Potato & Duck Dry Dog Formula, Natural balance, SanFrancisco, CA, USA) twice a day, and fresh water was supplied continuously throughout the experimental period. This experimental protocol was approved by the Institutional Animal Care and Use Committee (CBNUA-1168-18-01) of the Laboratory Animal Research Center of Chungbuk National University. After the experiment, all dogs were adopted as companion animals.
Animal preparation and anesthesia for PET/CT scanning
All dogs were fasted for at least 12 h before the induction of anesthesia, but had free access to drinking water. After intravenous (IV) catheter placement at the saphenous vein, anesthesia was induced with propofol (6–8 mg/kg IV; Provive, Myungmoon Pharm, Seoul, Republic of Korea). Endotracheal intubation was performed, and anesthesia was maintained with isoflurane (Terrell; Piramal Critical Care, Bethlehem, PA, USA) at 2.5 to 3% of the inspired volume during scanning, in a circle rebreathing system. Intermittent positive pressure ventilation was applied and tidal volume for ventilation was 10–20 mL/kg body weight with a respiratory frequency of 10–15 breaths per minute. A urethral catheter was inserted under sterile conditions, which included wearing sterile gloves. Dogs received normal saline (0.9% NaCl) solution (5 mL/kg/h) during anesthesia. They were positioned in sternal recumbency within the PET/CT gantry. Vital signs, such as heart rate, oxygen saturation (SPO2), end-tidal CO2-concentration, and blood pressure were continuously monitored, and the dogs were warmed with a warming pad (Equator; SurgiVet, Saint Paul, MN, USA).
18F-flutemetamol PET/CT scanning
The PET/CT system used in this study was a Discovery-STE (General Electric Medical Systems, Waukesha, WI, USA). An 8-slice helical CT scanner and a cylindrical PET device with 13,440 bismuth germanium oxide crystals arranged in 24 rings were included in the Discovery-STE system. The crystals, which have a dimension of 4.7 × 6.3 × 30 mm3, are organized in blocks of 8 × 6, coupled to a single photomultiplier with four anodes. The PET system provides 47 images at 3.27-mm intervals covering an axial field-of-view of 256 mm.
The injection dose (3.083 MBq/kg) of 18F-flutemetamol (Vizamyl, GE Healthcare, Arlington Heights, IL, USA) was calculated based on the dosing scheme of 18F-fluorodeoxyglucose in pediatric patient published by the 2010 North American Consensus Guidelines [36].
For static emission acquisition, 18F-flutemetamol was injected IV into a saphenous vein as a slow bolus, followed by flushing with 5 ml of 0.9% normal saline. A low-dose CT scan was performed prior to each PET scan. Twenty-minute PET images were acquired three times, from 30 to 50 min, from 60 to 80, and from 90 to 110 min after tracer injection.
For dynamic emission acquisition, images were obtained 1 week after obtaining static acquisitions. A transmission scan of 1-min duration was acquired first in the two-dimensional mode. Subsequently, a low-dose CT scan was performed and 18F-flutemetamol (3.083 MBq/kg) was administered intravenously. Dynamic three-dimensional PET images were acquired for 120 min starting at 2 min after the 18F-flutemetamol injection. A total of 31 frames were acquired at 6 × 30 s, 6 × 60 s, 7 × 180 s, 6 × 300 s, and 6 × 600 s.
The images were reconstructed with iterative techniques (four iterations with 70 subsets) with a slice thickness of 3.27 mm, matrix size of 128 × 128, with pixel sizes of 2 mm for the emission scan. Corrections for attenuation and scatter were applied. A Gaussian post-reconstructing smoothing filter with a 5-mm full-width at half-maximum was used to achieve uniform image resolution across sites.
Image analysis
OsiriX MD v10.0 (Pixmeo Sarl, Geneva, Switzerland) was used to analyze PET images. The regions of interest (ROIs) for each brain area were drawn manually on transverse (frontal cortex, parietal cortex, temporal cortex, occipital cortex, anterior cingulate cortex, posterior cingulate cortex, cerebellar cortex, and cerebral white matter) and midsagittal (midbrain, pons, and medulla oblongata) CT images (Fig. 3). For semiquantitative image analysis, ROIs were drawn over three consecutive slices except for the cerebellum (two slices) and brainstem (one slice). All PET analyses were performed using the same standardized ROI in the brain. The ROIs were then transferred to the corresponding region of PET/CT fusion images, and the SUV (average tissue concentration of 18F-flutemetamol [kBq/ml] / total injected dose [kBq] / body weight [g]) was calculated for each ROI. The SUVRs were also calculated for each ROI, to quantify brain uptake of the tracer. Dividing the SUVs of the individual target areas by that of the reference region (cerebellar cortex) yielded regional SUVRs. In addition to the regional SUVs and SUVRs, composite SUVs and SUVRs were obtained by calculating the mean SUV and SUVR from the frontal, temporal, parietal, occipital, anterior cingulate, and posterior cingulate cortices. Time–activity curves were generated over the entire acquisition period to determine the temporal course of regional brain 18F-flutemetamol uptake.
Statistical analysis
Data were analyzed using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA). The Friedman test was employed to test data within one group for changes over time, and, where overall significance was found, Dunn’s multiple-comparison post-hoc test was used to determine the origin of differences. Differences between groups were assessed using the Kruskal–Wallis test. If differences were significant, Dunn’s test was used for comparisons between groups. All values in each table were expressed as means ± SD. Differences were considered significant at P < 0.05.