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Changes in regional cerebral blood flow (rCBF) and glucose metabolism are commonly associated with traumatic brain injury (TBI). Reactive oxygen species (ROS) have been implicated as key contributors to the secondary injury process after TBI. Here, pretreatment with the nitrone radical scavengers (alpha-phenyl-N-tert-butyl nitrone (PBN) or its sulfonated analogue sodium 2-sulfophenyl-N-tert-butyl nitrone (S-PBN) were used as tools to study the effects of ROS on rCBF and glucose metabolism after moderate (2.4-2.6 atm) lateral fluid percussion injury (FPI) in rats. S-PBN has a half-life in plasma of 9 min and does not penetrate the blood-brain barrier (BBB). In contrast, PBN has a half-life of 3 h and readily penetrates the BBB. Regional cerebral blood flow (rCBF) and glucose metabolism was estimated by using (99m)Tc-HMPAO and [(18)F]Fluoro-2-deoxyglucose (FDG) autoradiography, respectively, at 42 min (n = 37) and 12 h (n = 34) after the injury. Regions of interest were the parietal cortex and hippocampus bilaterally. As expected, FPI produced an early (42-min) hypoperfusion in ipsilateral cortex and an increase in glucose metabolism in both cortex and hippocampus, giving way to a state of hypoperfusion and decreased glucose metabolism at 12 h postinjury. On the contralateral side, a hypoperfusion in the cortex and hippocampus was seen at 12 h only, but no significant changes in glucose metabolism. Both S-PBN and PBN attenuated the trauma-induced changes in rCBF and glucose metabolism. Thus, the early improvement in rCBF and glucose metabolism correlates with and may partly mediate the improved functional and morphological outcome after TBI in nitrone-treated rats.

Adult rats were subjected to a moderate lateral fluid percussion injury (FPI), followed by survival periods of 2 and 12 h. Regional NMDA subtype glutamate, muscarinic acetylcholine and GABA(A) receptor binding in various brain regions was analysed by quantitative in vitro autoradiography and short-lived positron emission tomography tracers [11C]cyano-dizocilpine, 4-N-[11C]methylpiperidylbenzilate (4-N-[11C]MPB), and [11C]flumazenil, respectively. The binding potential (BP, Bmax/KD) was calculated. The data with [11C]cyano-dizocilpine showed a significant decrease in BP bilaterally for the frontoparietal cortex and hippocampus at both time points, in comparison with that of the sham-operated controls. At 12 h the decrease was significantly more prominent for the ipsilateral cortex and hippocampus than for the contralateral side. The BP of 4-N-[11C]MPB was significantly decreased after 2 h for the trauma-side hippocampus, and after 12 h it had decreased for the trauma-site cortex and the bilateral hippocampus. The [11C]flumazenil exhibited a significant decrease in BP for the trauma-site cortex and the underlying hippocampus by 2 h after the traumatic brain injury. After 12 h a significantly decreased BP was observed only for the trauma-site cortex. The finding of a decreased BP demonstrates the involvement of these receptor systems in the development of cellular dysfunction, which is widespread and not limited to the site of lateral FPI.

A one-pot synthesis of (+)-3-[11C]cyano-MK-801 was performed using a palladium-promoted reaction of hydrogen [11C]cyanide with (+)-3-iodo-MK-801. The synthesis time was 32 min counted from end of bombardment, affording (+)-3-[11C]cyano-MK-801 in 37% decay-corrected radiochemical yield based on hydrogen [11C]cyanide. The radiochemical purity was higher than 95% and the specific radioactivity was in the range of 220–600 GBq/μmol. In vitro investigations in homogenate and frozen sections of rat brain indicated that (+)-3-[11C]cyano-MK-801 may be used as tracer in studies of the NMDA receptor. © 1998 John Wiley & Sons, Ltd.

(+)-3-[11C]Cyano-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine ([11C]MKC) was successfully synthesized as a potential radiotracer for PET studies on the NMDA receptor channel complex. In vitro binding properties of [11C]MKC were investigated with newly developed techniques for efficient evaluation of 11C-labeled compounds. The association curve of [11C]MKC binding in rat forebrain membranes showed that the specific binding reached an equilibrium within 30 min. Specific binding was saturable with affinity constant KD = 8.2 ± 0.4 nM and Bmax = 1.62 ± 0.04 pmol/mg protein with glutamate and glycine included in the incubation medium. The binding of [11C]MKC was decreased by extensive washing of the membrane preparation. (+)- and (−)-Dizocilpine, 3-cyano-dizocilpine, and ketamine inhibited the specific binding of [11C]MKC with IC50 values of 37.3, 445.0, 65.8 nM and 3.91 μM, respectively. High specific binding in in vitro autoradiography was distributed predominantly in telencephalic regions (the hippocampus, cerebral cortex, and striatum) followed by thalamus. PET studies using rhesus monkeys under anesthesia showed high uptake of [11C]MKC in the temporoparietal and frontal cerebral cortices, striatum, and thalamic regions, although it is problematic to verify the specific binding in vivo by PET.

Recently we developed a novel imaging technique using positron emitter-labeled compounds as probes and a storage phosphor screen as a detector. This approach makes it possible to follow a variety of biochemical processes with spatial information in living brain slices. Further technical development is reported here in terms of time-resolved imaging and receptor characterization in a real equilibrium state. The method was validated by use of [ 11 C]Ro15-1788, a benzodiazepine receptor antagonist. Fresh brain slices were incubated with [ 11 C]Ro15-1788 in oxygenated Krebs-Ringer solution at 37°C, in a specially designed chamber. By placing the chamber on a storage phosphor screen, we could obtain two-dimensional images of radioactivity in the slices. Time-resolved imaging was made at 5 min intervals, revealing that it took 60 min to reach equilibrium binding. The dissociation process was observed by adding an excess amount of unlabeled Ro15-1788 to the chamber, 25 min was required for the full dissociation. In the equilibrium state, i.e. in the presence of free radio-ligand, Scatchard plot analysis was performed on the cerebral cortex (K d = 7.4 nM, B max = 146 fmol/mg tissue) and striatum (K d = 7.5 nM, B max = 107 fmol/mg tissue), suggesting the presence of a single component of binding site in these two regions. The present method, for the first time, made it possible to study a ligand-receptor interaction in living brain slices with temporal and spatial resolutions. This technique should prove useful for studies of receptor function under physiological conditions.

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.

As a complement to in vivo studies with positron emission tomography (PET), it is desirable to perform in vitro characterization of newly developed 11C tracers. In this report we describe the technique for determination of receptor-ligand kinetics utilizing ligands labeled with the short-lived radionuclide 11C. The limitations and advantages are discussed. The benzodiazepine antagonist [11C]Ro 15-1788 was used as a model substance, and the use of storage phosphor plates for quantification of radioactivity was validated. Storage phosphor plates showed an excellent linear range (approximately 10[3]) and acceptable resolution (approximately 0.5 mm). Receptor-ligand kinetics, including depletion, association and dissociation, saturation and displacement were evaluated with good results through the use of short-lived radiotracers and storage phosphor plates.

Following experimental and clinical traumatic brain injury (TBI), the local cerebral metabolic rate of glucose (lCMR(Glc)) is commonly estimated using the 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG) method. The adequate estimation of lCMR(Glc) using FDG requires a correction factor, the lumped constant (LC), to convert FDG net uptake into lCMR(Glc). The LC, and thus lCMR(Glc) calculations, require a steady-state that may be disrupted following TBI. In the present report, we hypothesized that [1-(14)C]glucose uptake would accurately reflect glucose dynamics early post-injury, and was compared to the regional uptake of FDG in 44 rats subjected to moderate (2.4-2.6 atm) lateral fluid percussion brain injury (FPI) or sham injury. Cortical energy state and adenylate (ATP, ADP, and AMP) levels were also measured. Early (7-42 min) after FPI, FDG uptake was increased in the ipsilateral cortex and hippocampus (p < 0.05). In contrast, no change in [1-(14)C]glucose uptake (7 and 17 min post-injury) or cortical adenylate content (42 min post-injury) was observed. At 12 h following FPI, the ipsilateral FDG and [1-(14)C]glucose uptake were decreased in the cortex and hippocampus, and the ipsilateral cortical ATP concentration was decreased in comparison to sham-injured controls (p < 0.05). Under the present experimental conditions, the rate of cerebral uptake of FDG and of [1-(14)C]glucose differed, and indicated that following TBI, regional changes in the LC may occur in the immediate, but not in the late, post-injury phase. These results should be considered when interpreting results obtained using FDG for the estimation of lCMR(Glc) early following experimental TBI.