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Neuroinflammation and oxidative stress are hallmarks of neurodegenerative diseases. Microglia, the major important regulators of neuroinflammation, are activated in response to excessive generation of reactive oxygen species (ROS) from damaged cells and resulting in elevated and sustained damages. However, the relationship between microglia and ROS-regulatory system in the early stages of neuroinflammation prior to the appearance of neuronal damages have not been elucidated in detail. In this study, we analyzed the time-dependent changes in ROS generation during acute neuroinflammation in rats that were given an intrastriatal injection of lipopolysaccharide (LPS). We evaluated the effects of minocycline, an anti-inflammatory antibiotic, and N,N′-dimethylthiourea (DMTU), a radical scavenger, to understand the correlation between activated microglia and ROS generation. Ex vivo fluorescence imaging using dihydroethidium (DHE) clearly demonstrated an increased ROS level in the infused side of striatum in the rats treated with LPS. The level of ROS was changed in time-dependent manner, and the highest level of ROS was observed on day 3 after the infusion of LPS. Immunohistochemical studies revealed that time-dependent changes in ROS generation were well correlated to the presence of activated microglia. The inhibition of microglial activation by minocycline remarkably reduced ROS levels in the LPS-injected striatum, which indicated that the increased ROS generation caused by LPS was induced by activated microglia. DMTU decreased ROS generation and resulted in remarkable inhibitory effect on microglial activation. This study demonstrated that ROS generation during acute neuroinflammation induced by LPS was considerably associated with microglial activation, in an intact rat brain. The results provides a basis for understanding the interaction of ROS-regulatory system and activated microglia during neuroinflammation underlying neurodegenerative diseases.

Introduction Significant discrepancies between in vitro and in vivo binding of the muscarinic receptor ligand – 3H-labeled Quinuclidinyl Benzilate (QNB) – have been well documented. Discernable in vivo cerebellar [3H]QNB binding has been observed in mouse brain, despite the maximum number of binding sites (Bmax) being low. In order to understand this unique in vivo binding phenomenon, the binding of two muscarinic receptor ligands – [3H]QNB and N-[11C]methylpiperidyl Benzilate ([11C]NMPB) – were compared in vivo and in vitro in 3- and 8-week-old mice. Method In vitro binding parameters of [3H]QNB were determined using brain homogenates. The time course of radioactivity concentration (TACs) in the cerebral cortex and cerebellum was measured following injection of [3H]QNB and [11C]NMPB with or without 3 mg/kg of carrier QNB in 3- and 8 week old mice using a dual tracer administration technique. A graphical method was employed for the quantitative analysis of in vivo binding of these radioligands. Results In vitro, the available number of binding sites for cerebral cortical muscarinic receptors increased by 17% during the developmental period studied. Paradoxically, in vivo, we observed a decrease of [3H]QNB binding in the cerebral cortex, while [11C]NMPB binding was markedly increased. In vivo saturation analysis of [3H]QNB in 3-week-old mice revealed an apparent positive cooperativity of binding in the cerebral cortex. Conclusions Our results support the hypothesis that microenvironmental factors proximal to muscarinic receptors cause a local decrease in the cortical free-ligand concentration of [3H]QNB and that this ‘ligand barrier’ is modulated during brain development. Advances in knowledge The present study demonstrates that the combined use of radiolabeled QNB and NMPB has the potential to reveal the important effects of receptor microenvironmental factors on receptor function in the living brain.

Objective: Oxidative stress plays an important role in the onset of many neuronal and peripheral disorders. We examined the feasibility of obtaining semiquantitative fluorescent images of reactive oxygen species (ROS) generation in mouse brain and kidney utilizing a planar laser scanner and dihydroethidium (DHE). Methods: To investigate ROS generation in brain, sodium nitroprusside was injected into the striatum. Dihydroethidium was injected into the tail vein. After DHE injection, tissue slices were analyzed utilizing a planar laser scanner. For kidney study, cis-diamminedichloroplatinum [II] (cisplatin) was intraperitoneally administrated into mice. Results: Clear and semiquantitative fluorescent images of ROS generation in the mouse brain and kidney were obtained. Furthermore, the fluorescence intensity was stable and not affected by fading. Sodium nitroprusside induced approximately 6 times the fluorescence accumulation in the brain. Cisplatin caused renal injury in all mice, and in comparison with control mice, more than 10 times fluorescence accumulation was observed in the renal medulla with tubular necrosis and vacuolization. Conclusions: We successfully obtained ex vivo semiquantitative fluorescent images of ROS generation utilizing a planar laser scanner and DHE. This simple method is useful for ROS detection in several ROS-related animal models and would be applicable to a variety of biochemical processes.

Cyclooxygenase (COX), a prostanoid-synthesizing enzyme, is considered to be involved in the neuroinflammatory process of neurodegenerative diseases. However, the role of COX in the progression of neurodegeneration is not well understood. We hypothesized that in vivo imaging of COX by PET will contribute to elucidation of the function of COX during the neurodegenerative process in Alzheimer’s disease (AD). 11C-labeled ketoprofen methyl ester (racemic (RS)-11C-KTP-Me) developed recently by our group is a useful PET probe for in vivo imaging of COX-1 during neuroinflammation. The (S)-enantiomer of ketoprofen is known to be pharmacologically more active than the (R)-enantiomer. We thus synthesized 11C-labeled (S)-ketoprofen methyl ester ((S)-11C-KTP-Me) as an improved PET probe specific for COX-1 and applied it for investigation of the changes in COX-1 during the progression of AD in a mouse model. Methods: The specificity of (S)-11C-KTP-Me for COXs was examined in PET studies with rats that had intrastriatal injection of lipopolysaccharide. To determine the details of changes in COX-1 during progression of amyloid-β (Aβ) plaque formation in amyloid precursor protein transgenic (APP-Tg) mice, we performed immunohistochemical studies and ex vivo autoradiography with (S)-11C-KTP-Me. Results: PET studies using hemispheric lipopolysaccharide injection into rats revealed that the sensitivity of (S)-11C-KTP-Me in neuroinflammation was much higher than that of (RS)-11C-KTP-Me and (R)-11C-KTP-Me; these results closely corresponded to the inhibitory activities of each enantiomer against COX-1 estimated by an in vitro assay. In APP-Tg mice, (S)-11C-KTP-Me administration resulted in progressive and significant increases in accumulation of radioactivity in the brain from 16 to 24 mo old in accordance with the histopathologic appearance of abundant Aβ plaques and activated microglia, whereas few changes in radioactivity accumulation and few Aβ plaques were seen in age-matched wild-type control mice. High-radioactivity accumulation by (S)-11C-KTP-Me was markedly observed in the frontal cortex and hippocampus in which COX-1–expressing activated microglia tightly surrounded and enclosed large and more intensely stained Aβ plaques, indicating neuroinflammation that originated with Aβ. Conclusion: (S)-11C-KTP-Me is a potent PET probe that is highly selective for COX-1. Studies using APP-Tg mice demonstrated that (S)-11C-KTP-Me could detect activated microglia that are associated with amyloid plaque progression, suggesting the involvement of COX-1 in the neuroinflammatory process in AD.

Introduction Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease (AD). As a biomarker of neuroinflammatory processes, we designed 11 C-labeled ketoprofen methyl ester ([ 11 C]KTP-Me) to increase the blood–brain barrier permeability of ketoprofen (KTP), a selective cyclooxygenase-1 (COX-1) inhibitor. Animal studies indicated that [ 11 C]KTP-Me enters the brain and accumulates in activated microglia of inflammatory lesions. In a first-in-human study, we reported that [ 11 C]KTP-Me is a safe positron emission tomography (PET) tracer and enters the brain; the radioactivity is washed out from normal cerebral tissue. Here we explored the efficacy of [ 11 C]KTP-Me as a diagnostic biomarker of neuroinflammatory processes in AD. Methods [ 11 C]KTP-Me was synthesized by rapid C -[ 11 C]methylation of [ 11 C]CH 3 I and the corresponding arylacetate precursor. Nine subjects (four healthy subjects, two Pittsburgh compound-B (PiB)-positive patients with mild cognitive impairment (MCI), and three PiB-positive AD patients) underwent a dynamic brain PET scan for 70min after injection. We evaluated differences in cortical retention and washout rate in the brain between healthy subjects and MCI/AD patients. Results A brain distribution pattern reflecting blood flow in the early-phase image was seen in both healthy subjects and MCI/AD patients. Cortical activity gradually cleared in all groups. However, we observed no obvious difference in the washout rate between healthy subjects and MCI/AD patients or between MCI and AD patients. Conclusions [ 11 C]KTP-Me cannot be useful as a potential diagnostic biomarker for MCI/AD. Further improvements in binding affinity and specificity, etc., are needed to be a diagnostic biomarker of neuroinflammation in AD. Advances in knowledge and implications for patient care [ 11 C]KTP-Me is a new tracer that targets COX-1. [ 11 C]KTP-Me is expected to be a diagnostic biomarker of neuroinflammation in AD in the future. The effectiveness was limited in a small number of AD patients. Therefore, further studies are needed to clarify the usefulness of [ 11 C]KTP-Me.

Cyclooxygenase (COX)-1 and -2 are prostanoid-synthesizing enzymes that play important roles in the regulation of neuroinflammation and in the development of neurodegenerative disorders. However, the specific functions of these isoforms are still unclear. We recently developed 11C-labeled ketoprofen methyl ester as a PET probe that targets the COXs for imaging neuroinflammation, though its responsible isoform is yet to be determined. In the present study, we performed ex vivo and in vivo imaging studies with 11C-ketoprofen methyl ester and determined the contributions of the COX isoforms during the neuroinflammatory process. Methods: To identify the COX isoform responsible for 11C-ketoprofen methyl ester in the brain, we examined the ex vivo autoradiography of 11C-ketoprofen methyl ester using COX-deficient mice. Time-dependent changes in accumulation of 11C-ketoprofen methyl ester during the neuroinflammatory process were evaluated by PET in rats with hemispheric neuroinflammation induced by intrastriatal injection of lipopolysaccharide or quinolinic acid. In both rat models, cell-type specificity of COX isoform expression during neuroinflammation was identified immunohistochemically. Results: Ex vivo autoradiographic analysis of COX-deficient mice revealed a significant reduction of 11C-ketoprofen methyl ester accumulation only in COX-1–deficient mice, not COX-2–deficient mice. PET of rats after intrastriatal injection of lipopolysaccharide showed a significant increase in accumulation of 11C-ketoprofen methyl ester in the inflamed area. This increase was evident at the early phase of 6 h, peaked at day 1, and then returned to basal levels by day 7. In addition, immunohistochemical analysis revealed that the population of activated microglia and macrophages was elevated at the early phase with COX-1 expression but not COX-2. A significant increase in 11C-ketoprofen methyl ester accumulation was also observed at day 1 after intrastriatal injection of quinolinic acid, with increased COX-1–expressing activated microglia and macrophages. Conclusion: We have identified 11C-ketoprofen methyl ester as a COX-1–selective PET probe, and using this, we have also demonstrated a time-dependent expression of COX-1 in activated microglia and macrophages during the neuroinflammatory process in the living brain. Thus, COX-1 may play a crucial role in the pathology of neuroinflammation and might be a critical target for the diagnosis and therapy of neurodegenerative disorders.

Neurogenic inflammation triggered by extravasation of plasma protein has been hypothesized as a key factor in the generation of the pain sensation associated with migraine. The principal immune cell that responds to this inflammation is the parenchymal microglia of the central nervous system. Methods: Using a PET technique with 11C-(R)-[1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide] (11C-PK11195), a PET ligand for peripheral type–benzodiazepine receptor, we evaluated the microglial activation in the rat brain after generation of unilateral cortical spreading depression, a stimulation used to bring up an experimental animal model of migraine. Results: We found a significant increase in the brain uptake of 11C-PK11195, which was completely displaceable by the excess amounts of unlabeled ligands, in the ipsilateral hemisphere of the spreading depression–generated rats. Moreover, the binding potential of 11C-PK11195 in the spreading depression–generated rats was significantly higher than that in the sham-operated control rats. Conclusion: These results suggest that as an inflammatory reaction, microglial cells are activated in response to the nociceptive stimuli induced by cortical spreading depression in the rat brain. Therefore, the 11C-PK11195 PET technique could have a potential for diagnostic and therapeutic monitoring of neurologic disorders related to neuroinflammation such as migraine.

18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) has become a useful imaging tool for inflammatory diseases. In this study we investigated the effects of rolipram, a selective phosphodiesterase type 4 inhibitor, on 14C-deoxyglucose (DG) uptake in inflammatory lesions and other normal tissues, and attempted to improve the inflammation/muscle ratio.To induce inflammation, mice were inoculated with turpentine oil. Inflammation-bearing mice were pretreated with rolipram (3 mg/kg i.p. or i.v.), and the effect on 14C-DG uptake was measured using a tissue dissection method and autoradiography. The inflammatory tissue samples were stained with haematoxylin and eosin.Rolipram (3 mg/kg i.p.) significantly decreased 14C-DG uptake in normal tissues like brain, heart and skeletal muscle (brain 31%, heart 60%, skeletal muscle 61%). On the other hand, 14C-DG uptake in inflammatory lesions was not significantly altered by pretreatment with rolipram. The inflammation/muscle ratio of 14C-DG uptake (30 min after tracer injection) was enhanced from 1.1 to 2.8 by rolipram. An autoradiographic study revealed heterogeneous distributions of 14C-DG in the inflammatory lesions and skeletal muscle of animals that were not treated with rolipram. Pretreatment with rolipram significantly attenuated the intramuscular distribution of 14C-DG, producing a relatively homogeneous distribution of radioactivity.These results indicate that rolipram decreased 14C-DG uptake in skeletal muscle by activation of the adenosine 3',5'-cyclic monophosphate system, whereas 14C-DG uptake in inflammatory lesions was not significantly altered. Therefore, rolipram may be a valuable tool for improving the visualisation of inflammatory lesions in clinical PET studies employing FDG.

Introduction Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease and other brain disorders, and nonsteroidal anti-inflammatory drugs (NSAIDs) are considered therapeutic candidates. As a biomarker of neuroinflammatory processes, 11 C-labeled ketoprofen methyl ester ([ 11 C]KTP-Me) was designed to allow cerebral penetration of ketoprofen (KTP), an active form of a selective cyclooxygenase-1 inhibitor that acts as an NSAID. Rat neuroinflammation models indicate that [ 11 C]KTP-Me enters the brain and is retained in inflammatory lesions, accumulating in activated microglia. [ 11 C]KTP-Me is washed out from normal tissues, leading to the present first-in-human exploratory study. Methods [ 11 C]KTP-Me was synthesized by rapid C -[ 11 C]methylation of [ 11 C]CH 3 I and the corresponding arylacetate precursor, purified with high-performance liquid chromatography, and prepared as an injectable solution including PEG400, providing radiochemical purity of >99% and specific activity of >25GBq/μmol at injection. Six young healthy male humans were injected with [ 11 C]KTP-Me and scanned with PET camera to determine the early-phase brain time course followed by three whole-body scans starting 8, 20, and 40min post-injection, together with sequential blood sampling and labeled metabolite analysis. Results No adverse effects were observed during PET scanning after [ 11 C]KTP-Me injection. [ 11 C]KTP-Me was rapidly metabolized to 11 C-labeled ketoprofen ([ 11 C]KTP) within 2–3min and was gradually cleared from blood. The radioactivity entered the brain with an average peak cortical SUV of 1.5 at 2min. The cortical activity was gradually washed out. Whole-body images indicated that the urinary bladder was the major excretory pathway. The organ with the highest radiation dose was the urinary bladder (average dose of 41μGy/MBq, respectively). The mean effective dose was 4.7μSv/MBq, which was comparable to other 11 C-labeled radiopharmaceuticals. Conclusion [ 11 C]KTP-Me demonstrated a favorable dosimetry, biodistribution, and safety profile. [ 11 C]KTP-Me entered the human brain, and the radioactivity was washed out from cerebral tissue. These data warrant further exploratory studies on patients with neuroinflammation.