Evaluation of PET imaging as a tool for detecting neonatal hypoxic-ischemic encephalopathy in a preclinical animal model.

Hypoxic-ischemic encephalopathy due to insufficient oxygen delivery to brain tissue is a leading cause of death or severe morbidity in neonates. The early recognition of the most severely affected individuals remains a clinical challenge. We hypothesized that hypoxic-ischemic injury can be detected using PET radiotracers for hypoxia ([18F]EF5), glucose metabolism ([18F]FDG), and inflammation ([18F]F-DPA). METHODS: A preclinical model of neonatal hypoxic-ischemic brain injury was made in 9-d-old rat pups by permanent ligation of the left common carotid artery followed by hypoxia (8% oxygen and 92% nitrogen) for 120 min. In vivo PET imaging was performed immediately after injury induction or at different timepoints up to 21 d later. After imaging, ex vivo brain autoradiography was performed. Brain sections were stained with cresyl violet to evaluate the extent of the brain injury and to correlate it with [18F]FDG uptake. RESULTS: PET imaging revealed that all three of the radiotracers tested had significant uptake in the injured brain hemisphere. Ex vivo autoradiography revealed high [18F]EF5 uptake in the hypoxic hemisphere immediately after the injury (P < 0.0001), decreasing to baseline even 1 d postinjury. [18F]FDG uptake was highest in the injured hemisphere on the day of injury (P < 0.0001), whereas [18F]F-DPA uptake was evident after 4 d (P = 0.029), peaking 7 d postinjury (P < 0.0001), and remained significant 21 d after the injury. Targeted evaluation demonstrated that [18F]FDG uptake measured by in vivo imaging 1 d postinjury correlated positively with the brain volume loss detected 21 d later (r = 0.72, P = 0.028). CONCLUSION: Neonatal hypoxic-ischemic brain injury can be detected using PET imaging. Different types of radiotracers illustrate distinct phases of hypoxic brain damage. PET may be a new useful technique, worthy of being explored for clinical use, to predict and evaluate the course of the injury.

Segmentation of Dynamic Total-Body [18F]-FDG PET Images Using Unsupervised Clustering.

Clustering time activity curves of PET images have been used to separate clinically relevant areas of the brain or tumours. However, PET image segmentation in multiorgan level is much less studied due to the available total-body data being limited to animal studies. Now, the new PET scanners providing the opportunity to acquire total-body PET scans also from humans are becoming more common, which opens plenty of new clinically interesting opportunities. Therefore, organ-level segmentation of PET images has important applications, yet it lacks sufficient research. In this proof of concept study, we evaluate if the previously used segmentation approaches are suitable for segmenting dynamic human total-body PET images in organ level. Our focus is on general-purpose unsupervised methods that are independent of external data and can be used for all tracers, organisms, and health conditions. Additional anatomical image modalities, such as CT or MRI, are not used, but the segmentation is done purely based on the dynamic PET images. The tested methods are commonly used building blocks of the more sophisticated methods rather than final methods as such, and our goal is to evaluate if these basic tools are suited for the arising human total-body PET image segmentation. First, we excluded methods that were computationally too demanding for the large datasets from human total-body PET scanners. These criteria filtered out most of the commonly used approaches, leaving only two clustering methods, k-means and Gaussian mixture model (GMM), for further analyses. We combined k-means with two different preprocessing approaches, namely, principal component analysis (PCA) and independent component analysis (ICA). Then, we selected a suitable number of clusters using 10 images. Finally, we tested how well the usable approaches segment the remaining PET images in organ level, highlight the best approaches together with their limitations, and discuss how further research could tackle the observed shortcomings. In this study, we utilised 40 total-body [18F] fluorodeoxyglucose PET images of rats to mimic the coming large human PET images and a few actual human total-body images to ensure that our conclusions from the rat data generalise to the human data. Our results show that ICA combined with k-means has weaker performance than the other two computationally usable approaches and that certain organs are easier to segment than others. While GMM performed sufficiently, it was by far the slowest one among the tested approaches, making k-means combined with PCA the most promising candidate for further development. However, even with the best methods, the mean Jaccard index was slightly below 0.5 for the easiest tested organ and below 0.2 for the most challenging organ. Thus, we conclude that there is a lack of accurate and computationally light general-purpose segmentation method that can analyse dynamic total-body PET images.

ImmunoPET Directed to the Brain: A New Tool for Preclinical and Clinical Neuroscience.

Immuno-positron emission tomography (immunoPET) is a non-invasive in vivo imaging method based on tracking and quantifying radiolabeled monoclonal antibodies (mAbs) and other related molecules, such as antibody fragments, nanobodies, or affibodies. However, the success of immunoPET in neuroimaging is limited because intact antibodies cannot penetrate the blood-brain barrier (BBB). In neuro-oncology, immunoPET has been successfully applied to brain tumors because of the compromised BBB. Different strategies, such as changes in antibody properties, use of physiological mechanisms in the BBB, or induced changes to BBB permeability, have been developed to deliver antibodies to the brain. These approaches have recently started to be applied in preclinical central nervous system PET studies. Therefore, immunoPET could be a new approach for developing more specific PET probes directed to different brain targets.

Evaluation of [18F]FMTEB in Sprague Dawley rats as a PET tracer for metabotropic glutamate receptor 5.

INTRODUCTION: [18F]FMTEB, along with other tracers, was developed as a promising PET radioligand for imaging metabotropic glutamate receptor subtype 5 (mGluR5). Despite favorable preliminary results, it has not been used further for studies of mGluR5. This paper presents an in-depth preclinical evaluation of [18F]FMTEB in healthy Sprague Dawley rats. METHODS: [18F]FMTEB was synthesized from a boronic ester precursor using copper-mediated fluorination. In vivo PET imaging was performed on six rats, of which three were pre-treated with a high affinity mGluR5 receptor antagonist. An additional 18 rats were used for ex vivo experiments for metabolite analyses in plasma, brain and urine, and for biodistribution and ex vivo brain autoradiography at different time points. RESULTS: [18F]FMTEB was synthesized in adequate radiochemical yield and a molar activity of 154 ± 64 GBq/µmol. Both in vivo imaging and ex vivo brain autoradiography showed high specificity for mGluR5, and the blocking experiments showed a clear decrease in radioactivity in mGluR5-rich brain areas. Metabolite analyses confirmed fast metabolism of the tracer in plasma. The percentage of parent compound in brain tissue exceeded 90 % up to 90 min after injection. CONCLUSION: [18F]FMTEB produced via copper-mediated 18F-fluorination fulfilled the requirements for preclinical evaluation in rats. The absence of specific uptake in cerebellum and absence of defluorination of the tracer allowed cerebellum to be used as a reference tissue. Due to the fast kinetics in rats, the region-to-cerebellum ratios equilibrated within 30 min. These results prove [18F]FMTEB to be a good candidate for mapping mGluR5 in rat brain and a suitable alternative to [18F]FPEB.

ImmunoPET imaging of amyloid-beta in a rat model of Alzheimer's disease with a bispecific, brain-penetrating fusion protein.

BACKGROUND: Hijacking the transferrin receptor (TfR) is an effective strategy to transport amyloid-beta (Aß) immuno-positron emission tomography (immunoPET) ligands across the blood-brain barrier (BBB). Such ligands are more sensitive and specific than small-molecule ligands at detecting Aß pathology in mouse models of Alzheimer's disease (AD). This study aimed to determine if this strategy would be as sensitive in rats and to assess how TfR affinity affects BBB transport of bispecific immunoPET radioligands. METHODS: Two affinity variants of the rat TfR antibody, OX26, were chemically conjugated to a F(ab')2 fragment of the anti-Aß antibody, bapineuzumab (Bapi), to generate two bispecific fusion proteins: OX265-F(ab')2-Bapi and OX2676-F(ab')2-Bapi. Pharmacokinetic analyses were performed 4 h and 70 h post-injection of radioiodinated fusion proteins in wild-type (WT) rats. [124I]I-OX265-F(ab')2-Bapi was administered to TgF344-AD and WT rats for in vivo PET imaging. Ex vivo distribution of injected [124I]I-OX265-F(ab')2-Bapi and Aß pathology were assessed. RESULTS: More [125I]I-OX265-F(ab')2-Bapi was taken up into the brain 4 h post-administration than [124I]I-OX2676-F(ab')2-Bapi. [124I]I-OX265-F(ab')2-Bapi PET visualized Aß pathology with significantly higher signals in the TgF344-AD rats than in the WT littermates without Aß pathology. The PET signals significantly correlated with Aß levels in AD animals. CONCLUSION: Affinity to TfR affects how efficiently a TfR-targeting bispecific fusion protein will cross the BBB, such that the higher-affinity bispecific fusion protein crossed the BBB more efficiently. Furthermore, bispecific immunoPET imaging of brain Aß pathology using TfR-mediated transport provides good imaging contrast between TgF344-AD and WT rats, suggesting that this immunoPET strategy has the potential to be translated to higher species.

Biomarkers of the Endocannabinoid System in Substance Use Disorders.

Despite substance use disorders (SUD) being one of the leading causes of disability and mortality globally, available therapeutic approaches remain ineffective. The difficulty in accurately characterizing the neurobiological mechanisms involved with a purely qualitative diagnosis is an obstacle to improving the classification and treatment of SUD. In this regard, identifying central and peripheral biomarkers is essential to diagnosing the severity of drug dependence, monitoring therapeutic efficacy, predicting treatment response, and enhancing the development of safer and more effective pharmacological tools. In recent years, the crucial role that the endocannabinoid system (ECS) plays in regulating the reinforcing and motivational properties of drugs of abuse has been described. This has led to studies characterizing ECS alterations after exposure to various substances to identify biomarkers with potential diagnostic, prognostic, or therapeutic utility. This review aims to compile the primary evidence available from rodent and clinical studies on how the ECS components are modified in the context of different substance-related disorders, gathering data from genetic, molecular, functional, and neuroimaging experimental approaches. Finally, this report concludes that additional translational research is needed to further characterize the modifications of the ECS in the context of SUD, and their potential usefulness in the necessary search for biomarkers.

Direct Comparison of [18F]F-DPA with [18F]DPA-714 and [11C]PBR28 for Neuroinflammation Imaging in the same Alzheimer's Disease Model Mice and Healthy Controls.

PURPOSE: In this study we compared the recently developed TSPO tracer [18F]F-DPA, with [18F]DPA-714 and [11C]PBR28 by performing in vivo PET imaging on the same Alzheimer's disease mouse model APP/PS1-21 (TG) and wild-type (WT) mice with all three radiotracers. PROCEDURES: To compare the radiotracer uptake, percentage of injected dose/mL (%ID/mL), standardized uptake value ratios to cerebellum (SUVRCB), and voxel-wise analyses were performed. RESULTS: The peak uptake of [18F]F-DPA was higher than 4.3% ID/mL, while [18F]DPA-714 reached just over 3% ID/mL, and [11C]PBR28 was over 4% ID/mL in only one brain region in the WT mice. The peak/60-min uptake ratios of [18F]F-DPA were significantly higher (p < 0.001) than those of [18F]DPA-714 and [11C]PBR28. The differences in [18F]F-DPA SUVRCB between WT and TG mice were highly significant (p < 0.001) in the three studied time periods after injection. [18F]DPA-714 uptake was significantly higher in TG mice starting in the 20-40-min timeframe and increased thereafter, whereas [11C]PBR28 uptake became significant at 10-20 min (p < 0.05). The voxel-wise analysis confirmed the differences between the radiotracers. CONCLUSIONS: [18F]F-DPA displays higher brain uptake, higher TG-to-WT SUVRCB ratios, and faster clearance than [18F]DPA-714 and [11C]PBR28, and could prove useful for detecting low levels of inflammation and allow for shorter dynamic PET scans.

(S)-[18F]THK5117 brain uptake is associated with Aß plaques and MAO-B enzyme in a mouse model of Alzheimer's disease.

The mouse model of beta-amyloid (Aß) deposition, APP/PS1-21, exhibits high brain uptake of the tau-tracer (S)-[18F]THK5117, although no neurofibrillary tangles are present in this mouse model. For this reason we investigated (S)-[18F]THK5117 off-target binding to Aß plaques and MAO-B enzyme in APP/PS1-21 transgenic (TG) mouse model of Aß deposition. APP/PS1-21 TG and wild-type (WT) control mice in four different age groups (2-26 months) were imaged antemortem by positron emission tomography with (S)-[18F]THK5117, and then brain autoradiography. Additional animals were used for immunohistochemical staining and MAO-B enzyme blocking study with deprenyl pre-treatment. Regional standardized uptake value ratios for the cerebellum revealed a significant temporal increase in (S)-[18F]THK5117 uptake in aged TG, but not WT, brain. Immunohistochemical staining revealed a similar increase in Aß plaques but not endogenous hyper-phosphorylated tau or MAO-B enzyme, and ex vivo autography showed that uptake of (S)-[18F]THK5117 co-localized with the amyloid pathology. Deprenyl hydrochloride pre-treatment reduced the binding of (S)-[18F]THK5117 in the neocortex, hippocampus, and thalamus. This study's findings suggest that increased (S)-[18F]THK5117 binding in aging APP/PS1-21 TG mice is mainly due to increasing Aß deposition, and to a lesser extent binding to MAO-B enzyme, but not hyper-phosphorylated tau.

Prodromal neuroinflammatory, cholinergic and metabolite dysfunction detected by PET and MRS in the TgF344-AD transgenic rat model of AD: a collaborative multi-modal study.

Mouse models of Alzheimer's disease (AD) are valuable but do not fully recapitulate human AD pathology, such as spontaneous Tau fibril accumulation and neuronal loss, necessitating the development of new AD models. The transgenic (TG) TgF344-AD rat has been reported to develop age-dependent AD features including neuronal loss and neurofibrillary tangles, despite only expressing APP and PSEN1 mutations, suggesting an improved modelling of AD hallmarks. Alterations in neuronal networks as well as learning performance and cognition tasks have been reported in this model, but none have combined a longitudinal, multimodal approach across multiple centres, which mimics the approaches commonly taken in clinical studies. We therefore aimed to further characterise the progression of AD-like pathology and cognition in the TgF344-AD rat from young-adults (6 months (m)) to mid- (12 m) and advanced-stage (18 m, 25 m) of the disease. Methods: TgF344-AD rats and wild-type (WT) littermates were imaged at 6 m, 12 m and 18 m with [18F]DPA-714 (TSPO, neuroinflammation), [18F]Florbetaben (Aß) and [18F]ASEM (α7-nicotinic acetylcholine receptor) and with magnetic resonance spectroscopy (MRS) and with (S)-[18F]THK5117 (Tau) at 15 and 25 m. Behaviour tests were also performed at 6 m, 12 m and 18 m. Immunohistochemistry (CD11b, GFAP, Aß, NeuN, NeuroChrom) and Tau (S)-[18F]THK5117 autoradiography, immunohistochemistry and Western blot were also performed. Results: [18F]DPA-714 positron emission tomography (PET) showed an increase in neuroinflammation in TG vs wildtype animals from 12 m in the hippocampus (+11%), and at the advanced-stage AD in the hippocampus (+12%), the thalamus (+11%) and frontal cortex (+14%). This finding coincided with strong increases in brain microgliosis (CD11b) and astrogliosis (GFAP) at these time-points as assessed by immunohistochemistry. In vivo [18F]ASEM PET revealed an age-dependent increase uptake in the striatum and pallidum/nucleus basalis of Meynert in WT only, similar to that observed with this tracer in humans, resulting in TG being significantly lower than WT by 18 m. In vivo [18F]Florbetaben PET scanning detected Aß accumulation at 18 m, and (S)-[18F]THK5117 PET revealed subsequent Tau accumulation at 25m in hippocampal and cortical regions. Aß plaques were low but detectable by immunohistochemistry from 6 m, increasing further at 12 and 18 m with Tau-positive neurons adjacent to Aß plaques at 18 m. NeuroChrom (a pan neuronal marker) immunohistochemistry revealed a loss of neuronal staining at the Aß plaques locations, while NeuN labelling revealed an age-dependent decrease in hippocampal neuron number in both genotypes. Behavioural assessment using the novel object recognition task revealed that both WT & TgF344-AD animals discriminated the novel from familiar object at 3 m and 6 m of age. However, low levels of exploration observed in both genotypes at later time-points resulted in neither genotype successfully completing the task. Deficits in social interaction were only observed at 3 m in the TgF344-AD animals. By in vivo MRS, we showed a decrease in neuronal marker N-acetyl-aspartate in the hippocampus at 18 m (-18% vs age-matched WT, and -31% vs 6 m TG) and increased Taurine in the cortex of TG (+35% vs age-matched WT, and +55% vs 6 m TG). Conclusions: This multi-centre multi-modal study demonstrates, for the first time, alterations in brain metabolites, cholinergic receptors and neuroinflammation in vivo in this model, validated by robust ex vivo approaches. Our data confirm that, unlike mouse models, the TgF344-AD express Tau pathology that can be detected via PET, albeit later than by ex vivo techniques, and is a useful model to assess and longitudinally monitor early neurotransmission dysfunction and neuroinflammation in AD.

Longitudinal [18F]FDG and [13N]NH3 PET/CT imaging of brain and spinal cord in a canine hemisection spinal cord injury model.

To further understand the neurological changes induced by spinal cord injury (SCI) in its acute and subacute stages, we evaluated longitudinal changes in glucose and glutamate metabolism in the spinal cord and brain regions of a canine hemisection SCI model. [18F]FDG and [13N]NH3 positron-emission tomography (PET) with computed tomography (CT) was performed before SCI and at 1, 3, 7, 14, and 21 days after SCI. Spinal cord [18F]FDG uptake increased and peaked at 3 days post SCI. Similar changes were observed in the brain regions but were not statistically significant. Compared to the acute phase of SCI, [13N]NH3 uptake increased in the subacute stage and peaked at 7 days post SCI in all analyzed brain regions. But in spinal cord, no [13N]NH3 uptake was detected before SCI when the blood-spinal cord barrier (BSCB) was intact, then gradually increased when the BSCB was damaged after SCI. [13N]NH3 uptake was significantly correlated with plasma levels of the BSCB disruption marker, monocyte chemoattractant protein-1 (MCP-1). Overall, we showed that SCI induced in vivo changes in glucose uptake in both the spinal cord and the examined brain regions, and changes in glutamine synthetase activity in the latter. Moreover, our results suggest that [13N]NH3 PET may serve as a potential method for assessing BSCB permeability in vivo.

Intravenous transplantation of olfactory ensheathing cells reduces neuroinflammation after spinal cord injury via interleukin-1 receptor antagonist.

Rationale: Olfactory ensheathing cell (OEC) transplantation has emerged as a promising therapy for spinal cord injury (SCI) repair. In the present study, we explored the possible mechanisms of OECs transplantation underlying neuroinflammation modulation. Methods: Spinal cord inflammation after intravenous OEC transplantation was detected in vivo and ex vivo by translocator protein PET tracer [18F]F-DPA. To track transplanted cells, OECs were transduced with enhanced green fluorescent protein (eGFP) and HSV1-39tk using lentiviral vector and were monitored by fluorescence imaging and [18F]FHBG study. Protein microarray analysis and ELISA studies were employed to analyze differential proteins in the injured spinal cord after OEC transplantation. The anti-inflammation function of the upregulated protein was also proved by in vitro gene knocking down experiments and OECs/microglia co-culture experiment. Results: The inflammation in the spinal cord was decreased after OEC intravenous transplantation. The HSV1-39tk-eGFP-transduced OECs showed no accumulation in major organs and were found at the injury site. After OEC transplantation, in the spinal cord tissues, the interleukin-1 receptor antagonist (IL-1Ra) was highly upregulated while many chemokines, including pro-inflammatory chemokines IL-1α, IL-1ß were downregulated. In vitro studies confirmed that lipopolysaccharide (LPS) stimulus triggered OECs to secrete IL-1Ra. OECs significantly suppressed LPS-stimulated microglial activity, whereas IL-1Ra gene knockdown significantly reduced their ability to modulate microglial activity. Conclusion: The OECs that reached the lesion site were activated by the release of pro-inflammatory cytokines from activated microglia in the lesion site and secreted IL-1Ra to reduce neuroinflammation. Intravenous transplantation of OECs has high therapeutic effectiveness for the treatment of SCI via the secretion of IL-1Ra to reduce neuroinflammation.

Evaluation of [18F]F-DPA as a target for TSPO in head and neck cancer under normal conditions and after radiotherapy.

BACKGROUND: Many malignant tumours have increased TSPO expression, which has been related to a poor prognosis. TSPO-PET tracers have not comprehensively been evaluated in peripherally located tumours. This study aimed to evaluate whether N,N-diethyl-2-(2-(4-([18F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([18F]F-DPA) can reflect radiotherapy (RT)-induced changes in TSPO activity in head and neck squamous cell carcinoma (HNSCC). METHODS: RT was used to induce inflammatory responses in HNSCC xenografts and cells. [18F]F-DPA uptake was measured in vivo in non-irradiated and irradiated tumours, followed by ex vivo biodistribution, autoradiography, and radiometabolite analysis. In vitro studies were performed in parental and TSPO-silenced (TSPO siRNA) cells. TSPO protein and mRNA expression, as well as tumour-associated macrophages (TAMs), were also assessed. RESULTS: In vivo imaging and ex vivo measurement revealed significantly higher [18F]F-DPA uptake in irradiated, compared to non-irradiated tumours. In vitro labelling studies with cells confirmed this finding, whereas no effect of RT on [18F]F-DPA uptake was detected in TSPO siRNA cells. Radiometabolite analysis showed that the amount of unchanged [18F]F-DPA in tumours was 95%, also after irradiation. PK11195 pre-treatment reduced the tumour-to-blood ratio of [18F]F-DPA by 73% in xenografts and by 88% in cells. TSPO protein and mRNA levels increased after RT, but were highly variable. The proportion of M1/M2 TAMs decreased after RT, whereas the proportion of monocytes and migratory monocytes/macrophages increased. CONCLUSIONS: [18F]F-DPA can detect changes in TSPO expression levels after RT in HNSCC, which does not seem to reflect inflammation. Further studies are however needed to clarify the physiological mechanisms regulated by TSPO after RT.

Ruthenium-Mediated 18F-Fluorination and Preclinical Evaluation of a New CB1 Receptor Imaging Agent [18F]FPATPP.

Cannabinoid receptor 1 (CB1R) controls various physiological and pathological conditions, including memory, motivation, and inflammation, and is thus an interesting target for positron emission tomography (PET). Herein, we report a ruthenium-mediated radiolabeling synthesis and preclinical evaluation of a new CB1R specific radiotracer, [18F]FPATPP. [18F]FPATPP was produced with 16.7 ± 5.7% decay-corrected radiochemical yield and >95 GBq/µmol molar activity. The tracer showed high stability, low defluorination, and high specific binding to CB1Rs in mouse brain.

Comparison of high and low molar activity TSPO tracer [18F]F-DPA in a mouse model of Alzheimer's disease.

[18F]F-DPA, a novel translocator protein 18 kDa (TSPO)-specific radioligand for imaging neuroinflammation, has to date been synthesized with low to moderate molar activities (Am's). In certain cases, low Am can skew the estimation of specific binding. The high proportion of the non-radioactive component can reduce the apparent-specific binding by competitively binding to receptors. We developed a nucleophilic synthesis of [18F]F-DPA resulting in high Am (990 ± 150 GBq/µmol) and performed in vivo comparison with low Am (9.0 ± 2.9 GBq/µmol) [18F]F-DPA in the same APP/PS1-21 and wild-type mice (injected masses: 0.34 ± 0.13 µg/kg and 38 ± 15 µg/kg, respectively). The high level of microgliosis in the APP/PS1-21 mouse model enables good differentiation between diseased and healthy animals and serves better to distinguish the effect of differing Am on specific binding. The differing injected masses affect the washout profile and shape of the time-activity curves. Ratios of standardized uptake values obtained with high and low Am [18F]F-DPA demonstrate that there is a 1.5-fold higher uptake of radioactivity in the brains of APP/PS1-21 animals when imaging is carried out with high Am [18F]F-DPA. The differences between APP/PS1-21 and wild-type animals showed higher significance when high Am was used.

Effect of genotype and age on cerebral [18F]FDG uptake varies between transgenic APPSwe-PS1dE9 and Tg2576 mouse models of Alzheimer's disease.

Back-translation of clinical imaging biomarkers of Alzheimer's disease (AD), such as alterations in cerebral glucose metabolism detected by [18F]FDG positron emission tomography (PET), would be valuable for preclinical studies evaluating new disease-modifying drugs for AD. However, previous confounding results have been difficult to interpret due to differences in mouse models and imaging protocols between studies. We used an equivalent study design and [18F]FDG µPET imaging protocol to compare changes in cerebral glucose metabolism in commercial transgenic APPSwe-PS1dE9 (n = 12), Tg2576 (n = 15), and wild-type mice (n = 15 and 9). Dynamic [18F]FDG scans were performed in young (6 months) and aged (12 or 17 months) mice and the results verified by ex vivo methods (i.e., tissue counting, digital autoradiography, and beta-amyloid and Iba-1 immunohistochemistry). [18F]FDG uptake exhibited significant regional differences between genotypes (TG < WT) and ages (6 months <12 months) in the APPSwe-PS1dE9 model, whereas similar differences were not present in Tg2576 mice. In both models, only weak correlations were detected between regional beta-amyloid deposition or microgliosis and [18F]FDG uptake. By using equivalent methodology, this study demonstrated differences in cerebral glucose metabolism dysfunction detected with [18F]FDG PET between two widely used commercial AD mouse models.

Radiosynthesis and Preclinical Evaluation of an α2A-Adrenoceptor Tracer Candidate, 6-[18F]Fluoro-marsanidine.

PURPOSE: The α2-adrenoceptors mediate many effects of norepinephrine and epinephrine, and participate in the regulation of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. Of the three receptor subtypes, only α2A and α2C are found in the brain in significant amounts. Subtype-selective positron emission tomography (PET) imaging of α2-adrenoceptors has been limited to the α2C subtype. Here, we report the synthesis of 6-[18F]fluoro-marsanidine, a subtype-selective PET tracer candidate for α2A-adrenoceptors, and its preclinical evaluation in rats and mice. PROCEDURES: 6-[18F]Fluoro-marsanidine was synthesized using electrophilic F-18 fluorination with [18F]Selectfluor bis(triflate). The tracer was evaluated in Sprague Dawley rats and in α2A-knockout (KO) and wild-type (WT) mice for subtype selectivity. In vivo PET imaging and ex vivo brain autoradiography were performed to determine the tracer distribution in the brain. The specificity of the tracer for the target was determined by pretreatment with the subtype-non-selective α2-agonist medetomidine. The peripheral biodistribution and extent of metabolism of 6-[18F]fluoro-marsanidine were also analyzed. RESULTS: 6-[18F]Fluoro-marsanidine was synthesized with [18F]Selectfluor bis(triflate) in a radiochemical yield of 6.4 ± 1.7 %. The molar activity was 3.1 to 26.6 GBq/µmol, and the radiochemical purity was > 99 %. In vivo studies in mice revealed lower uptake in the brains of α2A-KO mice compared to WT mice. The results for selectivity were confirmed by ex vivo brain autoradiography. Blocking studies revealed reduced uptake in α2A-adrenoceptor-rich brain regions in pretreated animals, demonstrating the specificity of the tracer. Metabolite analyses revealed very rapid metabolism of 6-[18F]fluoro-marsanidine with blood-brain barrier-permeable metabolites in both rats and mice. CONCLUSION: 6-[18F]Fluoro-marsanidine was synthesized and evaluated as a PET tracer candidate for brain α2A-adrenoceptors. However, rapid metabolism, extensive presence of labeled metabolites in the brain, and high non-specific uptake in mouse and rat brain make 6-[18F]fluoro-marsanidine unsuitable for α2A-adrenoceptor targeting in rodents in vivo.

In vivo characterization of a novel norepinephrine transporter PET tracer [18F]NS12137 in adult and immature Sprague-Dawley rats.

Norepinephrine modulates cognitive processes such as working and episodic memory. Pathological changes in norepinephrine and norepinephrine transporter (NET) function and degeneration of the locus coeruleus produce irreversible impairments within the whole norepinephrine system, disrupting cognitive processes. Monitoring these changes could enhance diagnostic accuracy and support development of novel therapeutic components for several neurodegenerative diseases. Thus, we aimed to develop a straightforward nucleophilic fluorination method with high molar activity for the novel NET radiotracer [18F]NS12137 and to demonstrate the ability of [18F]NS12137 to quantify changes in NET expression. Methods: We applied an 18F-radiolabeling method in which a brominated precursor was debrominated by nucleophilic 18F-fluorination in dimethyl sulfoxide. Radiolabeling was followed by a deprotection step, purification, and formulation of the radiotracer. The [18F]NS12137 brain uptake and distribution were studied with in vivo PET/CT and ex vivo autoradiography using both adult and immature Sprague-Dawley rats because postnatal NET expression peaks at 10-20 days post birth. The NET specificity for the tracer was demonstrated by pretreatment of the animals with nisoxetine, which is well-known to have a high affinity for NET. Results: [18F]NS12137 was successfully synthesized with radiochemical yields of 18.6±5.6%, radiochemical purity of >99%, and molar activity of >500 GBq/µmol at the end of synthesis. The in vivo [18F]NS12137 uptake showed peak standard uptake values (SUV) of over 1.5 (adult) and 2.2 (immature) in the different brain regions. Peak SUV/30 min and peak SUV/60 min ratios were calculated for the different brain regions of the adult and immature rats, with a peak SUV/60 min ratio of more than 4.5 in the striatum of adult rats. As expected, in vivo studies demonstrated uptake of the tracer in brain areas rich in NET, particularly thalamus, neocortex, and striatum, and remarkably also in the locus coeruleus, a quite small volume for imaging with PET. The uptake was significantly higher in immature rats compared to the adult animals. Ex vivo studies using autoradiography showed very strong specific binding in NET-rich areas such as the locus coeruleus and the bed nucleus of the stria terminalis, and high binding in larger grey matter areas such as the neocortex and striatum. The uptake of [18F]NS12137 was dramatically reduced both in vivo and ex vivo by pretreatment with nisoxetine, demonstrating the specificity of binding. Conclusions: [18F]NS12137 was synthesized in good yield and high molar activity and demonstrated the characteristics of a good radiotracer, such as good brain penetration, fast washout, and high specific binding to NET.

[18F]F-DPA for the detection of activated microglia in a mouse model of Alzheimer's disease.

INTRODUCTION: Neuroinflammation is associated with several neurological disorders, including Alzheimer's disease (AD). The translocator protein 18 kDa (TSPO), due to its overexpression during microglial activation and relatively low levels in the brain under normal neurophysiological conditions, is commonly used as an in vivo biomarker for neuroinflammation. Reported here is the preclinical evaluation of [18F]F-DPA, a promising new TSPO-specific radioligand, as a tool for the detection of activated microglia at different ages in the APP/PS1-21 mouse model of AD and a blocking study to determine the specificity of the tracer. METHODS: [18F]F-DPA was synthesised by the previously reported electrophilic 18F-fluorination methodology. In vivo PET and ex vivo brain autoradiography were used to observe the tracer distribution in the brains of APP/PS1-21 and wildtype mice at different ages (4.5-24 months). The biodistribution and degree of metabolism of [18F]F-DPA were analysed and the specificity of [18F]F-DPA for its target was determined by pre-treatment with PK11195. RESULTS: The in vivo PET imaging and ex vivo brain autoradiography data showed that [18F]F-DPA uptake in the brains of the transgenic animals increased with age, however, there was a drop in the tracer uptake at 19 mo. Despite the slight increase in [18F]F-DPA uptake with age in healthy animal brains, significant differences between wildtype and transgenic animals were seen in vivo at 9 months and ex vivo already at 4.5 months. The specificity study demonstrated that PK11195 can be used to significantly block [18F]F-DPA uptake in all the brain regions studied. CONCLUSIONS: In vivo time activity curves plateaued at approximately 20-40 min suggesting that this is the optimal imaging time. Significant differences in vivo are seen at 9 and 12 mo. Due to the higher resolution, ex vivo autoradiography with [18F]F-DPA can be used to visualise activated microglia at an early stage of AD pathology.

[18F]FMPEP-d2 PET imaging shows age- and genotype-dependent impairments in the availability of cannabinoid receptor 1 in a mouse model of Alzheimer's disease.

Contradictory findings on the role of the type 1 cannabinoid receptor (CB1R) during the pathogenesis of Alzheimer's disease (AD) have been reported. Here, we evaluated the CB1R brain profile in an AD mouse model using longitudinal positron emission tomography with an inverse agonist for CB1R, [18F]FMPEP-d2. APP/PS1-21 and wild-type (n = 8 in each group) mice were repeatedly imaged between 6 to 15 months of age, accompanied by brain autoradiography, western blot, and CB1R immunohistochemistry with additional mice. [18F]FMPEP-d2 positron emission tomography demonstrated lower (p < 0.05) binding ratios in the parietotemporal cortex and hippocampus of APP/PS1-21 mice compared with age-matched wild-type mice. Western blot demonstrated no differences between APP/PS1-21 and wild-type mice in the CB1R abundance, whereas significantly lower (p < 0.05) receptor expression was observed in male than female mice. The results provide the first demonstration that [18F]FMPEP-d2 is a promising imaging tool for AD research in terms of CB1R availability, but not expression. This finding may further facilitate the development of novel therapeutic approaches based on endocannabinoid regulation.

18F-labeled norepinephrine transporter tracer [18F]NS12137: radiosynthesis and preclinical evaluation.

INTRODUCTION: Several psychiatric and neurodegenerative diseases are associated with malfunction of brain norepinephrine transporter (NET). However, current clinical evaluations of NET function are limited by the lack of sufficiently sensitive methods of detection. To this end, we have synthesized exo-3-[(6-[18F]fluoro-2-pyridyl)oxy]-8-azabicyclo[3.2.1]-octane ([18F]NS12137) as a radiotracer for positron emission tomography (PET) and have demonstrated that it is highly specific for in vivo detection of NET-rich regions of rat brain tissue. METHODS: We applied two methods of electrophilic, aromatic radiofluorination of the precursor molecule, exo-3-[(6-trimethylstannyl-2-pyridyl)oxy]-8-azabicyclo-[3.2.1]octane-8-carboxylate: (1) direct labeling with [18F]F2, and (2) labeling with [18F]Selectfluor, a derivative of [18F]F2, using post-target produced [18F]F2. The time-dependent distribution of [18F]NS12137 in brain tissue of healthy, adult Sprague-Dawley rats was determined by ex vivo autoradiography. The specificity of [18F]NS12137 binding was demonstrated on the basis of competitive binding by nisoxetine, a known NET antagonist of high specificity. RESULTS: [18F]NS12137 was successfully synthesized with radiochemical yields of 3.9% ± 0.3% when labeled with [18F]F2 and 10.2% ± 2.7% when labeled with [18F]Selectfluor. The molar activity of radiotracer was 8.8 ± 0.7 GBq/µmol with [18F]F2 labeling and 6.9 ± 0.4 GBq/µmol with [18F]Selectfluor labeling at the end of synthesis of [18F]NS12137. Uptake of [18F]NS12137 in NET-rich areas in rat brain was demonstrated with the locus coeruleus (LCoe) having the highest regional uptake. Prior treatment of rats with nisoxetine showed no detectable [18F]NS12137 in the LCoe. Analyses of whole brain samples for radiometabolites showed only the parent compound [18F]NS12137. Uptake of 18F-radioactivity in bone increased with time. CONCLUSIONS: The two electrophilic 18F-labeling methods proved to be suitable for synthesis of [18F]NS12137 with the [18F]Selectfluor method providing an approximate three-fold higher yield than the [18F]F2 method. As an electrostatically neutral radiotracer [18F]NS12137 crosses the blood-brain barrier and enabled specific labeling of NET-rich regions of rat brain tissue with the highest concentration in the LCoe.