General
Fluorine-18 was produced by 18O(p, n)18F nuclear reaction using a 12 MeV cyclotron (HM-12, Sumitomo Heavy Industry, Tokyo, Japan). [18F]FDG was radiosynthesized using a [18F]FDG synthesizer F200 (Sumitomo Heavy Industries). The radiochemical purity of [18F]FDG, determined by thin-layer chromatography (Tec-Control chromatography system, Biodex, NY, USA), was higher than 95%. [125I]IMP was supplied by Nihon Medi-Physics Co., Ltd. (Tokyo, Japan). Male Sprague–Dawley rats (Slc:SD) were supplied by Japan SLC Co. (Hamamatsu, Japan) and housed under a 12-h light/12-h dark cycle with free access to food and water. The animal experiments were performed in accordance with institutional and national guidelines regarding animal care, and were approved by the Animal Care and Use Committee of the Hamamatsu University School of Medicine.
Animal preparation
Rats aged 8–9 weeks old were used for the experiments, and their weights ranged from 233.8 to 339.4 g (277.2 ± 24.4 g). Rats (n = 6 or 7) were fasted for 15 h from the evening before and then anesthetized with the anesthetic agents described below. A polyethylene catheter (i.d. 0.5 mm, o.d. 0.8 mm) was inserted into the femoral artery for blood sampling and filled with heparin saline solution (10 IU/mL), and an indwelling needle (Surflo I.V. Catheter 24 G, SR-OT2419C, Terumo Co., Tokyo, Japan) was inserted into the tail vein for the administration of radiotracers and continuous infusion of propofol. These cannulas were in place until euthanasia. Body temperature was maintained at approximately 37 °C by a heating pad during the preparation. Rats were restrained on the bed and a head CT scan was performed using a small animal PET/SPECT/CT system (FLEX; Gamma Medica Ideas, Northridge, CA, USA). Blood gas profiles and glucose levels in arterial blood were measured using an i-STAT analyzer with an i-STAT CG8 + cartridge test (Abbot Point of Care, Illinois, USA) 5 min before [18F]FDG injection.
Handmade restrainer
Two square pieces of denim fabric (200 mm × 200 mm and 160 mm × 160 mm) were sewn together and folded to make a hood. A small cut was made in the hood to create an air vent. Several hook-and-loop fasteners were attached to hold the head, neck, and abdomen of the rats (Fig. 1a).
Anesthetic conditions
Conscious (CONS): To allow the rats to acclimate to the restrainer, the restrainer was applied before being used for the experiments, as described below. Rats were anesthetized with an inhalation of 3% isoflurane (ISOFLURANE Inhalation Solution, Mylan, PA, USA) in 50% oxygen (1 L/min), and wrapped in the handmade restrainer (Fig. 1b) within 5 min. The rats were then restrained on a bed for 75 min without isoflurane inhalation. This acclimation was repeated once daily every weekday at least nine times.
On the experiment day, rats were anesthetized with an inhalation of 3% isoflurane in 50% oxygen (1 L/min) during arterial and venous cannulation (< 30 min), and lidocaine hydrochloride (Xylocaine Jelly 2%, Aspen Japan KK, Tokyo, Japan) was applied to the incision. After cannulation, rats were wrapped in the restrainer, and restrained for 30 min to induce emergence from isoflurane.
Medetomidine, midazolam, and butorphanol (MMB) [12]: Rats were intraperitoneally injected with a mixture of 0.375 mg/kg medetomidine hydrochloride (Domitol, Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), 2 mg/kg midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan), and 2.5 mg/kg butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan).
Ketamine + xylazine (KX): Rats were intraperitoneally injected with a mixture of 80 mg/kg ketamine hydrochloride (Ketalar, Sankyo Lifetech Co., Ltd., Tokyo, Japan) and 8 mg/kg xylazine hydrochloride (Celactar, Bayer, Ltd., Tokyo, Japan).
Chloral hydrate (Chloral): Rats were intraperitoneally injected with 3.5% chloral hydrate (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) solution in saline (400 mg/kg).
Pentobarbital (PTB): Rats were intraperitoneally injected with 50 mg/kg sodium pentobarbital (Somnopentyl, Kyoritsu Seiyaku Co., Tokyo, Japan).
Propofol (PF): Anesthesia was initially induced by intravenous injection of 10 mg/kg propofol (1%, Maruishi Pharmaceutical. Co., Ltd. Osaka, Japan), and then maintained by intravenous infusion of 20 mg/kg/h propofol via the intravenous cannula, except during radiotracer injection (~ 30 s each).
Isoflurane (IFL): Anesthesia was initially induced by inhalation of 4% isoflurane in 50% oxygen (1 L/min), and then maintained by inhalation of 1.5% isoflurane in 50% oxygen (1 L/min).
Experimental protocol
[18F]FDG (~ 37 MBq) dissolved in 0.5 mL of saline was injected intravenously into the rats. Immediately after the [18F]FDG injection, dynamic PET scans were performed for 45 min (6 × 30 s and 42 × 60 s) using a PET scanner HITS-655 K (Hamamatsu Photonics KK, Hamamatsu, Japan) [13]. Arterial blood (approximately 100 μL per sample) was collected at 5, 15, 25, 35, 45, 55, 90, 120, 300, 450, 600, 1500, 2100, and 2700 s post-injection of [18F]FDG, and radioactivity concentration in the plasma was measured using an auto-well γ counter (1480 WIZARD2 3, PerkinElmer, Waltham, MA, USA). Heparinized saline was injected via the arterial cannula to avoid hypovolemia and cannula embolization at the interval of arterial blood sampling. PET images were reconstructed by list-mode dynamic row action maximum likelihood algorithm (LM-DRAMA) with four iterations without attenuation and scatter corrections [13]. The ROIs were manually drawn over the whole brain and over several brain regions (cerebellum, hippocampus, striatum, cortex, thalamus, and hypothalamus) by referring to normal rat brain MRI images, and radioactivity in the ROIs was quantified as the mean concentration. [18F]FDG brain accumulation was presented as standardized uptake value (SUV): SUV = tissue radioactivity concentration (Bq/mL)/injected dose (Bq) × body weight (g). Index values, attained by multiplying the brain/plasma ratio by blood glucose levels (BPG), were calculated as follows: BPG (mmol/L) = brain radioactivity concentration (Bq/mL) at 44–45 min post-injection of [18F]FDG/plasma radioactivity concentration (Bq/mL) at 45 min post-injection of [18F]FDG × blood glucose levels (mmol/L). [18F]FDG kinetics were analyzed by the 2-tissue-3-compartment model using arterial plasma 18F radioactivity concentration as an input function to obtain uptake rate constants (K1; mL/min/g), efflux rate constants (k2; /min), phosphorylation reaction constants (k3; /min), and dephosphorylation reaction constants (k4; /min) using Microsoft Excel Solver (Microsoft Corp., Redmond, WA) [14]. We used a single lumped constant (0.625 [15]) for the different anesthetized groups for CMRglu calculation as the detailed effects of anesthetic agents on the lumped constant have not been elucidated.
After PET scanning, the rats were intravenously injected with [125I]IMP (~ 185 kBq), and arterial blood (approximately 50 µL per sample) was collected at 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 100, 120, 150, 180, 210, 240, and 300 s post-injection of [125I]IMP. At 5-min post-injection of [125I]IMP, the rats were immediately sacrificed by decapitation, and the brain was removed and weighed. 125I radioactivity concentrations in the arterial blood samples and 18F and 125I radioactivity concentrations in the brain tissues were measured with an auto-well γ counter. The CBF value was calculated as previously described [16] using 0.92 [17] as the first-pass extraction fraction of IMP and 1.0 (0–26.5 s), 0.8 (26.5–51.5 s), and 0.75 (51.5–300 s) as the octanol extraction ratios, indicating the ratio of intact [125I]IMP in each blood sample [18].
Corticosterone assay
To monitor the stress caused by restraining and/or blood sampling during PET acquisition, plasma corticosterone levels were measured. Arterial blood (200 µL) was collected for corticosterone assay from conscious rats and MMB- and IFL-anesthetized rats (before [18F]FDG injection, n = 4). Five-minutes later, these rats were intravenously injected with 0.5 mL of saline instead of [18F]FDG, and arterial blood sampling was performed as described above. After arterial blood sampling at 2700 s post-injection, additional arterial blood (200 µL) was collected for the corticosterone assay (after [18F]FDG-PET). Next, 0.5 mL of saline instead of [125I]IMP was intravenously injected and arterial blood sampling was performed as described above. After arterial blood sampling at 300 s post-injection, additional arterial blood (200 µL) was collected for the corticosterone assay (after [125I]IMP assay). Arterial blood samples for the corticosterone assay were centrifuged at 1500×g at 4 °C for 5 min to prepare plasma, which was stored at − 80 °C until the assay. The plasma corticosterone levels were measured using an ELISA kit (Enzo Life Science, ADI-900-097, PA).
Statistical analysis
Data are expressed as mean ± SD. The statistical significance of the differences between the conscious and anesthetized rats was determined using non-repeated ANOVA with Dunnett’s multiple comparison test. The statistical significance of the differences in plasma corticosterone levels between before [18F]FDG injection, after [18F]FDG-PET, and after [125I]IMP assay was determined using repeated ANOVA with Tukey’s multiple comparison test. P values of less than 0.05 were considered statistically significant. The association between SUV determined by PET and that determined by γ counter was calculated by Pearson’s product moment correlation coefficient. Differences between regression coefficients of the regression lines were determined by testing the t-value. The association between CMRglu and brain SUV or BPG were also calculated by Pearson’s product moment correlation coefficient.