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). (11)C-labeled ketoprofen methyl ester (racemic (RS)-(11)C-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 (11)C-labeled (S)-ketoprofen methyl ester ((S)-(11)C-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.
The specificity of (S)-(11)C-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)-(11)C-KTP-Me.
PET studies using hemispheric lipopolysaccharide injection into rats revealed that the sensitivity of (S)-(11)C-KTP-Me in neuroinflammation was much higher than that of (RS)-(11)C-KTP-Me and (R)-(11)C-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)-(11)C-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)-(11)C-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.
(S)-(11)C-KTP-Me is a potent PET probe that is highly selective for COX-1. Studies using APP-Tg mice demonstrated that (S)-(11)C-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.
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.
Bradykinin B1 receptor (B1R) has garnered attention as a cancer therapeutic and diagnostic target. Several reports on radiolabelled derivatives of B1R antagonists have shown favourable properties as ...imaging agents in cells highly expressing hB1R following transfection. In the present study, we assessed whether radiolabelled probes can detect B1R endogenously expressed in cancer cells. To this end, we evaluated 111In-labelled derivatives of a B1R antagonist (111InIn-DOTA-Ahx-R954) using glioblastoma cell lines (U87MG and U251MG) with different B1R expression levels. Cellular uptake studies showed that the specific accumulation of 111InIn-DOTA-Ahx-R954 in U87MG was higher than that in U251MG, which correlated with B1R expression levels. Tissue distribution in U87MG-bearing mice revealed approximately 2-fold higher radioactivity in tumours than in the muscle in the contralateral leg. The specific accumulation of 111InIn-DOTA-Ahx-R954 in the tumour was demonstrated by the reduction in the tumour-to-plasma ratios in nonlabelled R954-treated mice. Moreover, ex vivo autoradiographic images revealed that the intratumoural distribution of 111InIn-DOTA-Ahx-R954 correlated with the localisation of B1R-expressing glioblastoma cells. In conclusion, we demonstrated that 111InIn-DOTA-Ahx-R954 radioactivity correlated with B1R expression in glioblastoma cells, indicating that radiolabelled derivatives of the B1R antagonist could serve as promising tools for elucidating the involvement of B1R in cancer.
In vivo imaging, such as PET, requires restriction of body movements and is generally conducted under sedation by anesthetic agents in studies using laboratory animals. Because anesthetics reduce ...neural activity and metabolism, physiologic neural functions are difficult to assess in animal PET studies. Therefore, use of an appropriate method in conscious animals is important and is a practical requirement for physiologic in vivo brain imaging studies. Here, we established an in vivo imaging system for conscious mice to reveal the physiologic regional cerebral glucose metabolic rate (rCMRglu) with (18)F-FDG PET.
We first developed a head holder to enable brain PET of a conscious mouse. To obtain optimal rCMRglu, we examined the effects of physical and psychologic stresses caused by ambient temperature, intravenous injection, and acclimation to the apparatus and immobile state. Finally, quantitative kinetic analysis was performed for rCMRglu based on a 2-tissue-compartment model with an input function of arterial blood sampling under both conscious and anesthetized (1.5% isoflurane) conditions.
Increasing the ambient temperature increased uptake of (18)F-FDG in the brain significantly while reducing the uptake in skeletal muscle and brown adipose tissue that was caused by shivering. The reduction of brain (18)F-FDG uptake caused by tail holding and manual injection was significantly ameliorated by the use of an automated slow injection. Although brain uptake of (18)F-FDG varied at the first session of PET, uptake at the second and subsequent sessions was stable, even after long-term acclimation. After these beneficial changes, brain uptake of (18)F-FDG improved significantly, to approximately 260% above the preconditioned state, which is comparable with that obtained in mice that have been allowed to move freely about their home cages. Quantitative kinetic analyses revealed that isoflurane anesthesia lowered rCMRglu in the cerebral cortex, striatum, thalamus, and cerebellum by 66%, 59%, 62%, and 22%, respectively, mainly by reducing the k(3) value, a rate constant for phosphorylation by hexokinase.
To our knowledge, this is the first study to report quantitative kinetic analysis of rCMRglu in mice that have been conscious throughout PET. This investigation will open avenues for research into in vivo functional brain molecular imaging in both normal and genetically manipulated mice.
Purpose
Our study aimed to elucidate the intracellular processes associated with quinolinic acid (QA)-induced brain injury by acquiring semiquantitative fluorescent images of reactive oxygen species ...(ROS) generation and positron emission tomography (PET) images of mitochondrial complex I (MC-I) activity.
Methods
Ex vivo fluorescent imaging with dihydroethidium (DHE) and PET scans with
18
F-BCPP-EF were conducted at 3 h and 24 h after QA injection into the rat striatum. Immunohistochemical studies were performed 24 h after QA injection into the rat brain using monoclonal antibodies against neuronal nuclei (NeuN) and CD11b.
Results
A strong DHE-derived fluorescent signal was detected in a focal area within the QA-injected striatum 3 h after QA injection, and increased fluorescent signal spread throughout the striatum and parts of the cerebral cortex after 24 h. By contrast,
18
F-BCPP-EF uptake in the QA-injected rat brain was unchanged after 3 h and markedly decreased after 24 h, not only in the striatum but also in the cerebral hemisphere. The fluorescent signal in the striatum 24 h after QA injection colocalised with microglial marker expression.
Conclusions
We successfully obtained functional images of focal ROS generation during the early period of excitotoxic injury, and microglial ROS generation and mitochondrial dysfunction were observed during the progression of the inflammatory response. Both ex vivo DHE imaging and in vivo
18
F-BCPP-EF-PET were sufficiently sensitive to detect the respective processes of QA-induced brain damage. Our study contributes to the functional imaging of multiple events during the pathological process.
Abstract 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 CKTP-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 CKTP-Me enters the brain and is retained in inflammatory lesions, accumulating in activated microglia. 11 CKTP-Me is washed out from normal tissues, leading to the present first-in-human exploratory study. Methods 11 CKTP-Me was synthesized by rapid C -11 Cmethylation of 11 CCH3 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 > 25 GBq/μmol at injection. Six young healthy male humans were injected with 11 CKTP-Me and scanned with PET camera to determine the early-phase brain time course followed by three whole-body scans starting 8, 20, and 40 min post-injection, together with sequential blood sampling and labeled metabolite analysis. Results No adverse effects were observed during PET scanning after 11 CKTP-Me injection. 11 CKTP-Me was rapidly metabolized to11 C-labeled ketoprofen (11 CKTP) within 2–3 min and was gradually cleared from blood. The radioactivity entered the brain with an average peak cortical SUV of 1.5 at 2 min. 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 4.7 μSv/MBq, which was comparable to other11 C-labeled radiopharmaceuticals. Conclusion 11 CKTP-Me demonstrated a favorable dosimetry, biodistribution, and safety profile. 11 CKTP-Me entered the human brain, and the radioactivity was washed out from cerebral tissue. These data warrant further exploratory studies on patients with neuroinflammation.
► The food reaching test (FRT) is an ideal task for experiments with restrained monkeys. ► FRT time is an objective index for evaluating bradykinesia. ► The success rate in FRT is an index for ...overall motor impairments. ► FRT time is more sensitive than PDRS scores.
We modified an objective behavioral test, namely the food reaching test (FRT), for quantitative assessment of motor performance improved by deep brain stimulation (DBS) of the subthalamic nucleus (STN) in the Parkinsonian monkeys. The symptomatic features and their severity in 3 monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were evaluated with a subjective monkey Parkinson's disease rating scale (PDRS). We then performed STN-DBS with the minimum current intensity that stopped the tremor. The time required for the monkeys to pick up all 5 pieces of potato (FRT time) was measured as a major index to evaluate bradykinesia. The success rate was adopted as another index for assessing overall motor impairments. Although both FRT time and PDRS score were similarly improved by STN-DBS, change of FRT time appeared more sensitive than that of PDRS scores. FRT is an easily trained behavioral test with high objectivity and sensitivity that can be applied for assessing motor performance in MPTP-treated monkeys during experiments in a restrained condition such as functional imaging of the brain.
Neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease (AD). As a biomarker of neuroinflammatory processes, we designed 11C-labeled ketoprofen methyl ester ...(11CKTP-Me) to increase the blood–brain barrier permeability of ketoprofen (KTP), a selective cyclooxygenase-1 (COX-1) inhibitor. Animal studies indicated that 11CKTP-Me enters the brain and accumulates in activated microglia of inflammatory lesions. In a first-in-human study, we reported that 11CKTP-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 11CKTP-Me as a diagnostic biomarker of neuroinflammatory processes in AD.
11CKTP-Me was synthesized by rapid C-11Cmethylation of 11CCH3I 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.
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.
11CKTP-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.
11CKTP-Me is a new tracer that targets COX-1. 11CKTP-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 11CKTP-Me.
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.
•Ex vivo imaging technique by using DHE is useful for ROS detection in rat brain.•DHE visualized ROS upregulation induced by intrastriatal injection of LPS.•The increased ROS are well correlated to microglial activation.•Activation of microglia and ROS-regulatory system are in a mutual relationship.
Multivalent RGD peptides have been used as an excellent targeting vector to integrin αvβ3-positive tumors. However, little attention has been paid to the influence of linker molecules in multivalent ...RGD peptides on their dissociation kinetics from tumor cells. In this study, we evaluated the dissociation kinetics of 99mTc-labeled hexavalent RGD peptides which have (CH2-CH2-O) n (n = 4, 99mTcTc(L1)6+ and n = 12, 99mTcTc(L2)6+) or (DPro-Gly) n (n = 1, 99mTcTc(L3)6+; n = 6, 99mTcTc(L4)6+; and n = 9, 99mTcTc(L5)6+) as a linker molecule. The results showed that 99mTcTc(L4)6+ and 99mTcTc(L5)6+ displayed slower dissociation kinetics and 99mTcTc(L4)6+ showed exceptionally high in vitro cellular uptake (203.1 ± 16.7% dose/mg protein) and the highest tumor to blood ratio (138.1 ± 26.3 at 4 h p.i.) in tumor bearing nude mice. These findings indicate that the use of appropriate length of (DPro-Gly) n would maximize the binding of multivalent RGD peptides to clustered integrin αvβ3.
PDF
