A Novel Acetyl-CoA Carboxylase 2 Selective Inhibitor Improves Whole-Body Insulin Resistance and Hyperglycemia in Diabetic Mice through Target-Dependent Pathways.

Excess intramyocellular lipid (IMCL) deposition in skeletal muscle is closely associated with insulin resistance. Pharmacological inhibition of acetyl-CoA carboxylase (ACC) 2 offers a promising approach to treat insulin resistance through stimulation of mitochondrial fatty acid oxidation (FAO) and reduction of IMCL deposition. Previously reported experimental ACC2 inhibitors exhibited plasma glucose-lowering effects in diabetic rodents. However, their antidiabetic action may be potentially biased by off-target effects on triglyceride metabolism or by neurologic side effects. In this study, we investigated a safety profile, target dependency of its action, and antidiabetic efficacy of compound 2e, a novel olefin derivative potent ACC2 selective inhibitor. Four-day administration of suprapharmacological dose of compound 2e did not exhibit any obvious side effects in Sprague-Dawley rats. In db/db mice, single administration of compound 2e led to significantly elevated FAO and reduced IMCL deposition in skeletal muscle. In ACC2 knockout mice, treatment with pharmacological doses of compound 2e did not reduce plasma triglyceride levels, whereas A-908292, a previously reported ACC2 inhibitor, caused a significant triglyceride reduction, showing that compound 2e was devoid of off-target triglyceride-lowering activity. Chronic treatment of db/db mice with compound 2e improved hyperglycemia but did not decrease plasma triglyceride levels. Additionally, compound 2e showed significant improvements of whole-body insulin resistance in the clamp study and insulin tolerance test. Collectively, compound 2e demonstrated a good safety profile and significant antidiabetic effects through inhibition of ACC2-dependent pathways. These findings provide further evidence that selective inhibition of ACC2 is an attractive strategy against insulin resistance and type 2 diabetes. SIGNIFICANCE STATEMENT: This study shows that pharmacological inhibition of acetyl-CoA carboxylase (ACC) 2 leads to significant improvements in whole-body glucose homeostasis, independently of off-target metabolic pathways and toxicity, which were observed in previously reported ACC2 inhibitors. These findings support the concept that ACC2-selective inhibitors will be a novel remedy for treatment of type 2 diabetes.

Detection of neuroinflammation before selective neuronal loss appearance after mild focal ischemia using [18F]DPA-714 imaging.

BACKGROUND: Translocator protein (TSPO) imaging can be used to detect neuroinflammation (including microglial activation) after acute cerebral infarction. However, longitudinal changes of TSPO binding after mild ischemia that induces selective neuronal loss (SNL) without acute infarction are not well understood. Here, we performed TSPO imaging with [18F]DPA-714 to determine the time course of neuroinflammation and SNL after mild focal ischemia. RESULTS: Mild focal ischemia was induced by middle cerebral artery occlusion (MCAO) for 20 min. In MCAO rats without acute infarction investigated by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, in vitro ARG revealed a significant increase of [18F]DPA-714 binding in the ipsilateral striatum compared with that in the contralateral side at 1, 2, 3, and 7 days after MCAO. Increased [18F]DPA-714 binding was observed in the cerebral cortex penumbra, reaching maximal values at 7 days after MCAO. Activation of striatal microglia and astrocytes was observed with immunohistochemistry of ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) at 2, 3, and 7 days after MCAO. SNL was investigated with Nissl staining and neuronal nuclei (NeuN) immunostaining and observed in the ischemic core region of the striatum on days 3 and 7 after MCAO. We confirmed that total distribution volume of [18F]DPA-714 in the ipsilateral striatum was significantly increased at 2 and 7 days after MCAO using positron emission tomography (PET). CONCLUSIONS: [18F]DPA-714 binding measured with in vitro ARG was increased before SNL appeared, and this change was detected by in vivo PET. These findings suggest that TSPO PET imaging might be useful for detection of neuroinflammation leading to SNL after focal ischemia.

[18F]-BMS-747158-02PET imaging for evaluating hepatic mitochondrial complex 1dysfunction in a mouse model of non-alcoholic fatty liver disease.

BACKGROUND: Mitochondrial dysfunction is one of the main causes of non-alcohol fatty liver disease (NAFLD). [18F]-BMS-747158-02 (18F-BMS) which was originally developed as a myocardial perfusion imaging agent was reported to bind mitochondrial complex-1 (MC-1). The aim of this study was to investigate the potential use of 18F-BMS for evaluating hepatic MC-1 activity in mice fed a methionine- and choline-deficient (MCD) diet. Male C57BL/6J mice were fed a MCD diet for up to 2 weeks. PET scans with 18F-BMS were performed after 1 and 2 weeks of the MCD diet. 18F-BMS was intravenously injected into mice, and the uptake (standardized uptake value (SUV)) in the liver was determined. The binding specificity for MC-1 was assessed by pre-administration of rotenone, a specific MC-1 inhibitor. Hepatic MC-1 activity was measured using liver homogenates generated after each positron emission tomography (PET) scan. Blood biochemistry and histopathology were also assessed. RESULTS: In control mice, hepatic 18F-BMS uptake was significantly inhibited by the pre-injection of rotenone. The uptake of 18F-BMS was significantly decreased after 2 weeks of the MCD diet. The SUV at 30-60 min was well correlated with hepatic MC-1 activity (r = 0.73, p < 0.05). Increases in plasma ALT and AST levels were also noted at 1 and 2 weeks. Mild hepatic steatosis with or without minimal inflammation was histopathologically observed at 1 and 2 weeks in mice liver on the MCD diet. However, inflammation was observed only at 2 weeks in mice on the MCD diet. CONCLUSIONS: The present study demonstrated that 18F-BMS is a potential PET probe for quantitative imaging of hepatic MC-1 activity and its mitochondrial dysfunction induced by steatosis and inflammation, such as in NAFLD.

Longitudinal imaging of the availability of dopamine transporter and D2 receptor in rat striatum following mild ischemia.

The changes in the availability of striatal dopamine transporter and dopamine D2 receptor after mild focal ischemia in rats were measured using a small animal positron emission tomography system. Mild focal ischemia was induced by 20-minute middle cerebral artery occlusion. [11C]PE2I binding to dopamine transporter was transiently increased on the ipsilateral side of the striatum at 2 days after middle cerebral artery occlusion. On day 7 and 14 after middle cerebral artery occlusion, [11C]PE2I binding levels were decreased. In contrast, [11C]raclopride binding to dopamine D2 receptor in the ipsilateral striatum had not changed at 2 days after middle cerebral artery occlusion. [11C]Raclopride binding was significantly decreased on the ischemic side of the striatum at 7 and 14 days after middle cerebral artery occlusion. Moreover, on day 1 and 2 after middle cerebral artery occlusion, significant circling behavior to the contralateral direction was induced by amphetamine challenge. This behavior disappeared at 7 days after middle cerebral artery occlusion. At 14 days, circling behavior to the ipsilateral direction (middle cerebral artery occlusion side) was significantly increased, and that to the contralateral direction also appeared again. The present study suggested that amphetamine-induced circling behavior indicated striatal dopaminergic alterations and that dopamine transporter and dopamine D2 receptor binding could be key markers for predicting motor dysfunction after mild focal ischemia.

Imaging of reactive oxygen species in focal ischemic mouse brain using a radical trapping tracer [(3)H]hydromethidine.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in the pathophysiology of the brain after ischemic stroke. In this study, we investigate the generation of brain ROS after transient focal ischemia in mice using a radical trapping radiotracer, [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]hydromethidine), which we recently reported as a ROS imaging probe. We also examined the effect of dimethylthiourea (DMTU), a hydroxyl radical scavenger, on brain ROS generation and infarct volume after transient focal ischemia in mice. METHODS: [(3)H]Hydromethidine was intravenously injected into mice at 1, 2, 5, and 7 h after transient middle cerebral artery occlusion (tMCAO), and then, the brain autoradiogram was acquired at 60 min after tracer injection. Brain infarct volumes at 24 h after tMCAO were assessed by 2,3,5-triphenyltetrazolium chloride staining. RESULTS: Accumulation of radioactivity was observed in the ipsilateral striatum and cortex at 1 h after tMCAO. The increase of radioactivity was attenuated at 2 h after tMCAO and then became maximized at 5 h. The high accumulation of radioactivity remained until 7 h after tMCAO. DMTU treatment significantly attenuated the accumulation of radioactivity in the ipsilateral hemisphere at 1, 5, and 7 h after tMCAO. Brain infarct volumes were also significantly reduced in DMTU-treated mice at 24 h after tMCAO. CONCLUSIONS: These results indicated that [(3)H]hydromethidine is a useful radiotracer for detecting in vivo brain ROS generation such as hydroxyl radical after ischemic injury.

Imaging of reactive oxygen species using [(3)H]hydromethidine in mice with cisplatin-induced nephrotoxicity.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in cisplatin-induced nephrotoxicity. The aim of this study was to investigate the potential of using [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]hydromethidine) for ex vivo imaging of regional ROS overproduction in mouse kidney induced by cisplatin. METHODS: Male C57BL/6 J mice were intraperitoneally administered with a single dose of cisplatin (30 mg/kg). Renal function was assessed by measuring serum creatinine and blood urea nitrogen (BUN) levels and morphology by histological examination. Renal malondialdehyde levels were measured as a lipid peroxidation marker. Autoradiographic studies were performed with kidney sections from mice at 60 min after [(3)H]hydromethidine injection. RESULTS: Radioactivity accumulation after [(3)H]hydromethidine injection was observed in the renal corticomedullary area of cisplatin-treated mice and was attenuated by pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger. Cisplatin administration significantly elevated serum creatinine and BUN levels, caused renal tissue damage, and promoted renal lipid peroxidation. These changes were significantly suppressed by DMTU pretreatment. CONCLUSIONS: The present study showed that [(3)H]hydromethidine was rapidly distributed to the kidney after its injection and trapped there in the presence of ROS such as hydroxyl radicals, suggesting that [(3)H]hydromethidine is useful for assessment of the renal ROS amount in cisplatin-induced nephrotoxicity.

Assessment of glutamine synthetase activity by [13N]ammonia uptake in living rat brain.

Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [(13) N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [(13) N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [(13) N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [(13) N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [(15) O]H2 O. The reduction of [(13) N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [(13) N]ammonia may enable measurement of GS activity in the living brain.

Improvement of brain uptake for in vivo PET imaging of astrocytic oxidative metabolism using benzyl [1-(11)C]acetate.

Brain uptake of acetate is insufficient for obtaining a quantitative image of astrocytic oxidative metabolism. To improve the brain uptake of [1-(11)C]acetate, we synthesized benzyl [1-(11)C]acetate ([1-(11)C]BA) and conducted a positron emission tomography (PET) study assessing astrocytic oxidative metabolism. The brain uptake of [1-(11)C]BA was markedly higher compared with [1-(11)C]acetate, and disappeared with a half-life of 20 min in all regions studied. The brain uptake of [1-(11)C]BA was significantly decreased by fluorocitrate. The results indicate that [1-(11)C]BA could be a useful PET probe for assessing astrocytic oxidative metabolism.

[11C]PK11195 PET imaging of spinal glial activation after nerve injury in rats.

The role of glial activation has been implicated in the development and persistence of neuropathic pain after nerve injury by recent studies. PK11195 binding to the translocator protein 18kDa (TSPO) has been shown to be enhanced in activated microglia. This study was designed to assess PK11195 imaging in spinal microglia during activation after nerve injury. The development of neuropathic pain was induced by partial sciatic nerve ligation (PSL). PSL rats on days 7 and 14 after nerve injury were subjected to imaging with a small-animal positron emission tomography/computed tomography (PET/CT) scanner using [(11)C]PK11195 to detect spinal microglial activation by means of noninvasive in vivo imaging. Spinal [(3)H]PK11195 autoradiography was performed to confirm the results of [(11)C]PK11195 PET in PSL rats. Quantitative RT-PCR of CD11b and GFAP mRNA, and the immunohistochemistry of Iba1 and GFAP were investigated to detect activated microglia and astrocytes. Mechanical allodynia was observed in the ipsilateral paw of PSL rats from day 3 after nerve injury and stably persisted from days 7 to 14. PET/CT fusion images clearly showed large amounts of accumulation of [(11)C]PK11195 in the lumbar spinal cord on days 7 and 14 after nerve injury. [(11)C]PK11195 enhanced images were restricted to the L3-L6 area of the spinal cord. The standardized uptake value (SUV) of [(11)C]PK11195 was significantly increased in the lumbar spinal cord compared to that of the thoracic region. Increased specific binding of [(11)C]PK11195 to TSPO in the spinal cord of PSL rats was confirmed by competition studies using unlabeled (R, S)-PK11195. Increased [(3)H]PK11195 binding was also observed in the ipsilateral dorsal horn of the L3-L6 spinal cord on days 7 and 14 after nerve injury. CD11b mRNA and Iba1 immunoreactive cells increased significantly on days 7 and 14 after nerve injury by PSL. However, changes in GFAP mRNA and immunoreactivity were slight in the ipsilateral side of PSL rats. In the present study, we showed that glial activation could be quantitatively imaged in the spinal cord of neuropathic pain rats using [(11)C]PK11195 PET, suggesting that high resolution PET using TSPO-specific radioligands might be useful for imaging to assess the role of glial activation, including neuroinflammatory processes, in neuropathic pain patients.

In vivo imaging and quantitative analysis of TSPO in rat peripheral tissues using small-animal PET with [18F]FEDAC.

INTRODUCTION: The translocator protein (18 kDa) (TSPO) is widely expressed in peripheral tissues, including the heart, lung, and kidney. Our laboratory developed N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[(18)F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide ([(18)F]FEDAC) as a TSPO positron emission tomography (PET) ligand. Here, using small-animal PET with [(18)F]FEDAC, we performed TSPO imaging and quantitative analysis of TSPO binding in rat peripheral tissues. METHODS: The in vivo distribution and kinetics of [(18)F]FEDAC were measured in rat peripheral tissues (heart, lung and kidney). Using the in vivo pseudo-equilibrium method, TSPO binding parameters [TSPO density (B(max)), dissociation constant (K(D))] and receptor occupancy were estimated in these peripheral tissues. RESULTS: [(18)F]FEDAC was highly distributed in the lung, heart and kidney, and these TSPO-enriched tissues could be clearly visualized. The kinetics of this radioligand in these tissues was rapid, which is suitable for the determination of in vivo TSPO binding parameters and receptor occupancy. The B(max) value of TSPO in the heart, lung, and kidney was 393, 141, and 158 pmol/ml, respectively. The K(D) value of the radioligand in the heart, lung, and kidney was 119, 36 and 123 nM, respectively. By pretreatment with 5 mg/kg Ro 5-4864 (a TSPO ligand), about 90% of binding sites for TSPO in the heart and lung were occupied. In the kidney, the binding sites were completely occupied by 5 mg/kg Ro 5-4864. CONCLUSIONS: [(18)F]FEDAC is a suitable PET ligand for TSPO imaging and quantitative analysis of TSPO binding in rat peripheral tissues. The utilization of [(18)F]FEDAC-PET and the pseudo-equilibrium method can contribute to the study of the TSPO function and evaluate the in vivo binding parameters and receptor occupancy of TSPO therapeutic compounds.

PK11195 might selectively suppress the quinolinic acid-induced enhancement of anaerobic glycolysis in glial cells.

PK11195 was previously reported to attenuate the quinolinic acid (QUIN)-induced enhancement of glucose metabolism in rat brain. In the present study, the effect of PK11195 or anesthesia on [(14)C]2-deoxyglucose ([(14)C]DG) uptake was investigated in order to determine whether the QUIN-induced enhancement of glucose metabolism occurred in glial cells or neurons. We confirmed that the microinjection of QUIN caused a significant enhancement of [(14)C]DG uptake at 2h after the infusion, while the co-injection of PK11195 and QUIN almost completely suppressed this enhancement of [(14)C]DG uptake. No effect of chloral hydrate anesthesia on the QUIN-induced enhancement of [(14)C]DG uptake was observed. In contrast to rats treated with QUIN, PK11195 did not affect the enhancement of [(14)C]DG uptake induced by fluorocitrate (FC); however, chloral hydrate anesthesia completely suppressed the FC-induced increase in [(14)C]DG uptake. These results indicated that the enhancement of glucose metabolism induced by QUIN mainly occurred in glial cells, and the neuroprotective effect of PK11195 in rats injected with QUIN might be related to the suppression of anaerobic glycolysis in glial cells.

Remarkable selectivity of the in vivo binding of [3H]Ro15-4513 to α5 subtype of benzodiazepine receptor in the living mouse brain.

To evaluate the binding characteristics of [(3)H]Ro15-4513 with the central benzodiazepine (BZ) receptor, inhibition experiments of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 were performed both in vitro and in vivo, using two BZ ligands, flunitrazepam (FNP), and ethyl-ß-carboline-3-carboxylate (ß-CCE). FNP inhibited the binding of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 in a dose-dependent manner in the mouse cerebral cortex, hippocampus, and cerebellum, both in vitro and in vivo. ß-CCE also inhibited the binding of [(3)H]Ro15-1788 and [(3)H]Ro15-4513 in all the aforementioned brain regions in vitro. However, in vivo, ß-CCE inhibited the binding of [(3)H]Ro15-4513 in the cerebral cortex and cerebellum, but not in the hippocampus, even at an injected dose of up to 1mg/kg. In contrast, more than 50% of the in vivo binding of [(3)H]Ro15-1788 was inhibited by 1 mg/kg of ß-CCE in all regions. The time-activity curve of [(3)H]Ro15-4513 in the hippocampus also showed no alteration of the peak uptake between the control group and 0.3 mg/kg of ß-CCE coinjected group. These results indicated that the binding characteristics of [(3)H]Ro15-4513 with the BZ receptor differed markedly between the in vitro and in vivo condition, and the selectivity of [(3)H]Ro15-4513 binding to α5 subtype of BZ receptor in the mouse brain seemed to be remarkable under the in vivo condition.

Remarkable increase in 14C-acetate uptake in an epilepsy model rat brain induced by lithium-pilocarpine.

The present study demonstrates changes in rat brain glial metabolism during the acute phase of epilepsy. Status epilepticus (SE) was induced using the lithium-pilocarpine model. Glial metabolism was measured with (14)C-acetate. Local cerebral blood flow and glucose metabolism were also measured using (14)C-N-isopropyl-p-iodoamphetamine (IMP) and (14)C-2-deoxyglucose (2DG), respectively. At the initiation of the seizure, (14)C-acetate uptake did not change significantly. However, a marked increase was observed 2 h after the pilocarpine injection in all brain regions studied. The increase of brain uptake was transient, and the maximum enhancement was seen at 2 h after the pilocarpine injection. The increase of (14)C-acetate uptake was almost to the same degree in all regions, whereas (14)C-IMP and (14)C-2DG uptakes showed a heterogeneous increase. In the case of (14)C-IMP, the highest increase was observed in the thalamus (280%), and a moderate increase (120 to 150%) was seen in the orbital cortex, cingulate cortex and pyriform cortex. (14)C-2DG uptake increased by 130 to 240% in most regions of the brain, however, an increase of only 40 and 20% was observed in the cerebellum and pons-medulla, respectively. These results demonstrated that glial energy metabolism was markedly enhanced during a prolonged seizure. To our knowledge, this study is the first observation showing large and widespread glial metabolic increases in the rat brain during status epilepticus.

Glucose utilization in the brain during acute seizure is a useful biomarker for the evaluation of anticonvulsants: effect of methyl ethyl ketone in lithium-pilocarpine status epilepticus rats.

Enhancement of glucose utilization in the brain has been well known during acute seizure in various kinds of animal model of epilepsy. This enhancement of glucose utilization might be related to neural damage in these animal models. Recently, we found that methyl ethyl ketone (MEK) had both anticonvulsive and neuroprotective effects in lithium-pilocapine (Li-pilo) status epilepticus (SE) rat. In this article, we measured the uptake of [(14)C]2-deoxyglucose ([(14)C]DG) in the Li-pilo SE and Li-pilo SE with MEK rat brain in order to assess whether the glucose utilization was a useful biomarker for the detection of efficacy of anticonvulsive compounds. Significant increase of [(14)C]DG uptake (45 min after the injection) in the cerebral cortex, hippocampus, amygdala and thalamus during acute seizure induced by Li-pilo were observed. On the other hand, the initial uptake of [(14)C]DG (1 min after the injection) in the Li-pilo SE rats was not different from the control rats. Therefore, the enhancement of glucose metabolism during acute seizure was due to the facilitation of the rate of phosphorylation process of [(14)C]DG in the brain. Pretreatment with MEK (8 mmol/kg) completely abolished the enhancement of glucose utilization in the Li-pilo SE rats. The present results indicated that glucose utilization in the brain during acute seizure might be a useful biomarker for the evaluation of efficacy of anticonvulsive compounds.

Lactate is an alternative energy fuel to glucose in neurons under anesthesia.

The uptake of [14C]lactate was measured in the brains of mice anesthetized with pentobarbital or chloral hydrate. The results showed significant increase of the [14C]lactate uptake in the brain under both anesthesia. Despite energy metabolism in the brain being suppressed by both pentobarbital and chloral hydrate, the [14C]lactate uptake was unexpectedly increased under anesthesia. [14C]Lactate uptake in rat brain injured by infusion of quinolic acid was significantly decreased, and the reduction of [14C]lactate uptake was parallel to neural cell death, suggesting that exogenous lactate might be selectively taken up by neuron. These results indicated that lactate rather than glucose might serve as an energy substrate for neuron in intact brain under anesthesia.

Inhibitory effect of methyl ethyl ketone upon the enhancement of cerebral blood flow during status epilepticus induced by lithium-pilocarpine.

Significant increases in local cerebral blood flow during lithium-pilocarpine (Li-P) induced seizure have been reported. We recently found that both acetone and methyl ethyl ketone (MEK) showed anticonvulsive effects in status epilepticus induced by Li-P in rats. In this study, we examined whether MEK also suppressed the enhancement of local cerebral blood flow induced by Li-P with a simplified autoradiographic method using [(14)C]-para-iodo-N-isopropyl amphetamine ([(14)C]-IMP). Significant increases in local cerebral blood flow in the thalamus, hypothalamus, hippocampus and cerebellum were observed in Li-P induced status epilepticus rats. Pretreatment with MEK (8 mmol/kg) completely suppressed the enhancement of local cerebral blood flow to or below the control level in all regions.

De-coupling of blood flow and metabolism in the rat brain induced by glutamate.

OBJECTIVE: Glutamate plays an essential role in neuronal cell death in many neurological disorders. In this study, we examined both glucose metabolism and cerebral blood flow in the same rat following infusion of glutamate or ibotenic acid using the dual-tracer technique. The effects of MK-801, an NMDA receptor antagonist, and NBQX, an AMPA-kainate receptor antagonist, on the changes in the glucose metabolism and cerebral blood flow induced by glutamate were also examined. METHODS: The rats were microinjected with glutamate (1 micromol/microl, 2 microl) or ibotenic acid (10 microg/microl, 1 microl) into the right striatum, and dual-tracer autoradiograms of [(18)F]FDG and [(14)C]IMP were obtained. MK-801 and NBQX were injected intravenously about 45 and 30 min, respectively, after the infusion of glutamate. RESULTS: De-coupling of blood flow and metabolism was noted in the glutamate-infused hemisphere (as assessed by no alteration of [(18)F]FDG uptake and significant decrease of [(14)C]IMP uptake). Pretreatments with MK-801, NBQX, or combined use of MK-801 and NBQX did not affect the de-coupling of the blood flow and metabolism induced by glutamate. A histochemical study revealed that about 20% neuronal cell death had occurred in the striatum at 105 min after the infusion of glutamate. In addition, a significant increase of the [(18)F]FDG uptake and decrease of [(14)C]IMP uptake were also seen in the rat brain infused with ibotenic acid. CONCLUSION: These results indicate that glutamate and ibotenic acid caused a significant de-coupling of blood flow and glucose metabolism in the intact rat brain during the early phase of neurodegeneration. It is necessary to evaluate the relation between metabotropic glutamate receptors and de-coupling of blood flow and metabolism.

Role of NMDA receptor upon [14C]acetate uptake into intact rat brain.

OBJECTIVE: To clarify the role of N-methyl-D: -aspartate (NMDA) receptors upon [(14)C]acetate uptake in the rodent central nervous system (CNS), ibotenic acid (IBO) was infused into the right striatum of the rat brain. METHODS: Autoradiograms of [(14)C]acetate uptake in the brain for 2 h following the infusion of IBO (10 microg/microl) were obtained in both non-treated and MK-801 (1 mg/kg, i.v.) pretreated rats. The effect of MK-801 on [(14)C]acetate uptake in the normal rat brain was also studied. RESULTS: Infusion of IBO significantly decreased [(14)C]acetate uptake in the infused side of the striatum. The expression of monocarboxylate transporter-1 was not altered, suggesting that the activity of tricarboxylic acid (TCA) cycle in glial cells might be depressed. Pretreatment with MK-801 completely blocked the decreasing effect of IBO on [(14)C]acetate uptake. MK-801 also increased [(14)C]acetate uptake in the whole brain of normal rats. CONCLUSIONS: These results indicate the important roles of NMDA receptors on [(14)C]acetate uptake in the intact rat brain.

In vivo imaging of microglial activation using a peripheral benzodiazepine receptor ligand: [11C]PK-11195 and animal PET following ethanol injury in rat striatum.

OBJECTIVE: To investigate whether [(11)C]PK-11195, a specific peripheral benzodiazepine receptors (PBRs) ligand for positron emission tomography (PET), can show activated microglia in a rat brain injury model. METHODS: On day 1, ethanol was injected into the rat's right striatum (ST) using a stereotaxic operative procedure. On day 3, head magnetic resonance imaging (MRI) scans for surgically treated rats were performed to evaluate ethanol injury morphologically. On day 4, dynamic PET scans (17 injured rats and 7 non-injured controls) were performed for 60 min with an animal PET scanner under chloral hydrate anesthesia following a bolus injection of [(11)C]PK-11195 through tail vein. Because PBRs are present throughout the brain, there is no suitable receptor-free reference region. The reference tissue model may not be applicable because of low target to background ratio for low affinity of [(11)C]PK-11195 to PBRs. We evaluated the PBRs binding with regions of interest (ROIs)-based approach to estimate total distribution volume (V). We used an integral from 0 min to 60 min (V (60)) as an estimate of V. On the coronal PET image, ROIs were placed on bilateral ST. Differences in right/left ST V (60) ratios between lesioned and unlesioned control rats were compared using unpaired t tests. Immunohistochemical staining was performed for confirming the presence of activated microglia following decapitation on the PET experiment day. RESULTS: The right/left ST V (60) ratios in lesioned rats (1.07 +/- 0.08) were significantly higher than those in unlesioned control rats (1.00 +/- 0.06, P < 0.05). On immunohistochemical staining, activated microglia were exclusively observed in the injured right ST but not in the noninjured left ST of the injury rats and the bilateral ST of the non-injured control rats. CONCLUSIONS: These results suggest that [(11)C]PK-11195 PET imaging would be a useful tool for evaluating microglial activation in a rat brain injury model.

[14C]Benzyl acetate is a potential radiotracer for the measurement of glial metabolism in the rat brain.

In order to develop a suitable radiotracer for the measurement of glial metabolism, we synthesized four different types of ester derivatives of [14C]acetate, namely, [14C]phenyl acetate, [14C]para-nitrophenyl acetate, [14C]2,4-dinitrophenyl acetate and [14C]benzyl acetate ([14C]BA), and evaluated their potencies in rats. Among the derivatives, the highest brain uptake at 30 s postinjection was observed for [14C]BA, which was more than 23 times higher than that of [14C]acetate itself. A long-term retention of [14C]BA radioactivity in the brain was observed, whereas rapid clearance of radioactivity was seen in the heart. [14C]BA was rapidly hydrolyzed in the intact rat brain, and less than 5% of radiolabeled parent was observed 1 min after the injection. Radiochemical analysis using thin-layer chromatography revealed that [14C]BA was rapidly converted to [14C]glutamine and [14C]glutamate in the cortex within 10 min after injection. Furthermore, the uptake of [14C]BA was significantly decreased following microinjection of fluorocitrate, a selective glial toxin. These results strongly suggest that [14C]BA may be a useful radiotracer for the measurement of glial metabolism in the intact rat brain.