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Tumor spheroids are promising three-dimensional (3D) in vitro tumor models for the evaluation of drug delivery methods. The design of noninvasive and targeted drug methods is required to improve the intratumoral bioavailability of chemotherapeutic drugs and reduce their adverse off-target effects. Among such methods, microbubble-assisted ultrasound (MB-assisted US) is an innovative modality for noninvasive targeted drug delivery. The aim of the present study is to evaluate the efficacy of this US modality for the delivery of bleomycin, doxorubicin, and irinotecan in colorectal cancer (CRC) spheroids. MB-assisted US permeabilized the CRC spheroids to propidium iodide, which was used as a drug model without affecting their growth and viability. Histological analysis and electron microscopy revealed that MB-assisted US affected only the peripheral layer of the CRC spheroids. The acoustically mediated bleomycin delivery induced a significant decrease in CRC spheroid growth in comparison to spheroids treated with bleomycin alone. However, this US modality did not improve the therapeutic efficacy of doxorubicin and irinotecan on CRC spheroids. In conclusion, this study demonstrates that tumor spheroids are a relevant approach to evaluate the efficacy of MB-assisted US for the delivery of chemotherapeutics.
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Antineoplásicos , Neoplasias , Humanos , Irinotecano , Microbolhas , Doxorrubicina/farmacologia , Bleomicina , Esferoides Celulares , Linhagem Celular TumoralRESUMO
We synthesized 5-[18 F]-fluoro-1H-indol-2-yl)(4-methyl-1-piperazinyl)methanone ([18 F]5) via a Suzuki approach starting from a protected pinacol borane precursor followed by acidic hydrolysis of the t-Boc protecting group. The non-optimized radiochemical yield was 5.7 ± 1.35%, radiochemical purity was over 99%, and molar activity was 100.7 ± 34.5 GBq/µmol (n = 3). [18 F]5 was stable in rat plasma for at least 4 h and was evaluated by µPET imaging and biodistribution using a unilateral quinolinic acid rat model of neuroinflammation. The time-activity curve showed that [18 F]5 entered the brain immediately after intravenous injection and then left it progressively with a very low level reached from 30 min after injection. The biodistribution study showed no difference in the accumulation of [18 F]5 between the lesioned and intact side of the brain and between control rats and animals pretreated with a saturating dose of JNJ-7777120 as a specific H4R antagonist. Hence, despite its in vitro nanomolar affinity for H4R, and its ability to cross the blood-brain barrier in rats, [18 F]5 does not appear suitable to image in vivo the receptor by PET.
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Receptores Histamínicos H4RESUMO
Parkinson's disease (PD) is characterized by the degeneration of dopaminergic (DA) neurons constituting the nigrostriatal pathway. Neuroinflammation, related to microglial activation, plays an important role in this process. Exploration of animal models of PD using neuroimaging modalities allows to better understand the pathophysiology of the disease. Here, we fully explored a moderate lesion model in the rat in which 6-hydroxydopamine was unilaterally delivered in three sites along the striatum. The degenerative process was assessed through in vivo Positron Emission Tomography (PET) imaging and in vitro autoradiographic quantitation of the striatal dopamine transporter (DAT) and immunostaining of tyrosine hydroxylase (TH). The microglial activation was studied through in vitro autoradiographic quantitation of the 18 kDa translocator protein (TSPO) in the striatum and CD11b staining in the SN. In addition, a targeted metabolomics exploration was performed in both these structures using mass spectrometry coupled to HPLC. Our results showed a reproducible decrease in the striatal DAT density associated with a reduction in the number of TH-positive cells in the SN and striatum, reflecting a robust moderate degeneration of nigrostriatal DA neurons. In addition, we observed strong microglia activation in both the striatum and SN ipsilateral to the lesion, highlighting that this moderate degeneration of DA neurons was associated with a marked neuroinflammation. Our metabolomics studies revealed alterations of specific metabolites and metabolic pathways such as carnitine, arginine/proline, and histidine metabolisms. These results bring new insights in the PD mechanism knowledge and new potential targets for future therapeutic strategies.
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Corpo Estriado/patologia , Neurônios Dopaminérgicos/patologia , Oxidopamina/toxicidade , Doença de Parkinson/patologia , Animais , Proteínas de Transporte/metabolismo , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Masculino , Metaboloma , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Doença de Parkinson/etiologia , Tomografia por Emissão de Pósitrons , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Subarachnoid hemorrhage (SAH) can lead to delayed cerebral ischemia, which increases the rate of morbidity and mortality. The detection of microglial activation may serve as a biomarker for the identification of patients at risk of this deleterious consequence. We assessed this hypothesis in a rat model of SAH in which the exploration of neuroinflammation related to microglial activation was correlated with the degree of bleeding. We used the rat filament model and evaluated (at 48 hours postsurgery) the intensity of neuroinflammation using positron emission tomography (PET) imaging with the 18-kDa translocator protein (TSPO) tracer [(18)F]DPA-714, quantitative autoradiography with [(3)H]PK-11195, and SAH grade by postmortem brain picture. High SAH grades were strongly and positively correlated with in vivo PET imaging of TSPO in the cortex and striatum. In addition, a positive correlation was found in the cortex in TSPO, with densities determined by imaging and autoradiographic approaches. Qualitative immunofluorescence studies indicated that overexpression of TSPO was linked to astrocytic/microglial activation. In this model, PET imaging of TSPO using [(18)F]DPA-714 appeared to be a relevant index of the degree of bleeding, indicating that this imaging method could be used in human patients to improve the management of patients with SAH.
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Radioisótopos de Flúor/farmacologia , Microglia/patologia , Tomografia por Emissão de Pósitrons/métodos , Pirazóis/química , Pirimidinas/química , Hemorragia Subaracnóidea/diagnóstico por imagem , Animais , Modelos Animais de Doenças , Diagnóstico Precoce , Radioisótopos de Flúor/química , Humanos , Masculino , Microglia/metabolismo , Ratos , Receptores de GABA/metabolismo , Hemorragia Subaracnóidea/metabolismo , Hemorragia Subaracnóidea/patologiaRESUMO
Tumor spheroids as well as multicellular tumor spheroids (MCTSs) are promising 3D in vitro tumor models for drug screening, drug design, drug targeting, drug toxicity, and validation of drug delivery methods. These models partly reflect the tridimensional architecture of tumors, their heterogeneity and their microenvironment, which can alter the intratumoral biodistribution, pharmacokinetics, and pharmacodynamics of drugs. The present review first focuses on current spheroid formation methods and then on in vitro investigations exploiting spheroids and MCTS for designing and validating acoustically mediated drug therapies. We discuss the limitations of the current studies and future perspectives. Various spheroid formation methods enable the easy and reproducible generation of spheroids and MCTSs. The development and assessment of acoustically mediated drug therapies have been mainly demonstrated in spheroids made up of tumor cells only. Despite the promising results obtained with these spheroids, the successful evaluation of these therapies will need to be addressed in more relevant 3D vascular MCTS models using MCTS-on-chip platforms. These MTCSs will be generated from patient-derived cancer cells and nontumor cells, such as fibroblasts, adipocytes, and immune cells.
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The metabotropic glutamate receptor subtype 5 (mGluR5) is a class C G-protein-coupled receptor (GPCR) that has been implicated in various neuronal processes and, consequently, in several neuropsychiatric or neurodevelopmental disorders. Over the past few decades, mGluR5 has become a major focus for pharmaceutical companies, as an attractive target for drug development, particularly through the therapeutic potential of its modulators. In particular, allosteric binding sites have been targeted for better specificity and efficacy. In this context, Positron Emission Tomography (PET) appears as a useful tool for making decisions along a drug candidate's development process, saving time and money. Thus, PET provides quantitative information about a potential drug candidate and its target at the molecular level. However, in this area, particular attention has to be given to the interpretation of the PET signal and its conclusions. Indeed, the complex pharmacology of both mGluR5 and radioligands, allosterism, the influence of endogenous glutamate and the choice of pharmacokinetic model are all factors that may influence the PET signal. This review focuses on mGluR5 PET radioligands used at several stages of central nervous system drug development, highlighting advances and setbacks related to the complex pharmacology of these radiotracers.
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New ultrasound parameters, potentially predictive of tumor response to chemotherapy, were sought after analyzing details of vascular architecture of mammary tumors during chemotherapy. Tumor-bearing rats were separated into untreated or docetaxel-treated group (6 mg/kg/week). Power Doppler Index and vascular contrast-enhanced ultrasound (CEUS) reference endpoints (Peak, area under the curve (AUC), blood flow) were evaluated at the beginning (W (0)), and after 2 and 6 weeks of docetaxel treatment (W (+2) and W (+6)). An improved CEUS image analysis, taking advantage of individual pixel intensity, was developed to quantify large, medium, and small vessels of tumors. Standard immunohistochemistry validated this new methodology analyzing tumor vascular architecture. In rats, there was an enrichment of vascularization with large vessels during tumor growth indicative of a vascular adjustment to tumor size. Docetaxel stopped tumor growth, and showed a sequential effect on vascular parameters. After an initial enrichment in larger vessels (by threefold) at W (+2), docetaxel led to a diminution of vascular parameters at W (+6) (-46 % for peak, -55 % for AUC -31 % compared to W (0)) and a vascular remodeling in favor of small vessels. One of the CEUS parameters measured before chemotherapy, the so-called global contrast-enhanced pixels density, was predictive of rat tumor response to treatment (r = 0.80; p < 0.01). The method was then applied in a clinical setting to detect changes of vascular architecture during chemotherapy of human breast carcinoma. The docetaxel chemotherapy of breast carcinomas induced a similar sequential effect, with vessel enlargement after two cycles of docetaxel treatment and an antiangiogenic effect after six cycles. Such vascular remodeling was not noticed when patients were treated with 5-fluorouracil-epirubicin-cyclophosphamide. Taken together, the sharpened analysis of CEUS pixel intensity presented here strengthened the monitoring of breast tumor vasculature with the potential to improve the prediction of docetaxel efficacy.
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Antineoplásicos/administração & dosagem , Neoplasias Mamárias Experimentais/irrigação sanguínea , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Taxoides/administração & dosagem , Adulto , Idoso , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Docetaxel , Feminino , Humanos , Neoplasias Mamárias Experimentais/patologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Ratos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Taxoides/farmacologia , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , UltrassonografiaRESUMO
The different depressive disorders that exist can take root at adolescence. For instance, some functional and structural changes in several brain regions have been observed from adolescence in subjects that display either high vulnerability to depressive symptoms or subthreshold depression. For instance, adolescents with depressive disorder have been shown to exhibit hyperactivity in hippocampus, amygdala and prefrontal cortex as well as volume reductions in hippocampus and amygdala (prefrontal cortex showing more variable results). However, no animal model of adolescent subthreshold depression has been developed so far. Our objective was to design an animal model of adolescent subthreshold depression and to characterize the neural changes associated to this phenotype. For this purpose, we used adolescent Swiss mice that were evaluated on 4 tests assessing cognitive abilities (Morris water maze), anhedonia (sucrose preference), anxiety (open-field) and stress-coping strategies (forced swim test) at postnatal day (PND) 28-35. In order to identify neural alterations associated to behavioral profiles, we assessed brain resting state metabolic activity in vivo using 18F-FDG PET imaging at PND 37. We selected three profiles of mice distinguished in a composite Z-score computed from performances in the behavioral tests: High, Intermediate and Low Depressive Risk (HDR, IDR and LDR). Compared to both IDR and LDR, HDR mice were characterized by passive stress-coping behaviors, low cognition and high anhedonia and anxiety and were associated with significant changes of 18F-FDG uptakes in several cortical and subcortical areas including prelimbic cortex, infralimbic cortex, nucleus accumbens, amygdala, periaqueductal gray and superior colliculus, all displaying higher metabolic activity, while only the thalamus was associated with lower metabolic activity (compared to IDR). LDR displayed an opposing behavioral phenotype and were associated with significant changes of 18F-FDG uptakes in the dorsal striatum and thalamus that both exhibited markedly lower metabolic activity in LDR. In conclusion, our study revealed changes in metabolic activities that can represent neural signatures for behavioral profiles predicting subthreshold depression at adolescence in a mouse model.
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Depressão , Fluordesoxiglucose F18 , Anedonia , Animais , Ansiedade/diagnóstico por imagem , Modelos Animais de Doenças , Humanos , Camundongos , Tomografia por Emissão de PósitronsRESUMO
BACKGROUND: Non-human primates (NHP) are critical in biomedical research to better understand the pathophysiology of diseases and develop new therapies. Based on its translational and longitudinal abilities along with its non-invasiveness, PET/CT systems dedicated to non-human primates can play an important role for future discoveries in medical research. The aim of this study was to evaluate the performance of a new PET/CT system dedicated to NHP imaging, the IRIS XL-220 developed by Inviscan SAS. This was performed based on the National Electrical Manufacturers Association (NEMA) NU 4-2008 standard recommendations (NEMA) to characterize the spatial resolution, the scatter fraction, the sensitivity, the count rate, and the image quality of the system. Besides, the system was evaluated in real conditions with two NHP with 18F-FDG and (-)-[18F]FEOBV which targets the vesicular acetylcholine transporter, and one rat using 18F-FDG. RESULTS: The full width at half maximum obtained with the 3D OSEM algorithm ranged between 0.89 and 2.11 mm in the field of view. Maximum sensitivity in the 400-620 keV and 250-750 keV energy windows were 2.37% (22 cps/kBq) and 2.81% (25 cps/kBq), respectively. The maximum noise equivalent count rate (NEC) for a rat phantom was 82 kcps at 75 MBq and 88 kcps at 75 MBq for energy window of 250-750 and 400-620 keV, respectively. For the monkey phantom, the maximum NEC was 18 kcps at 126 MBq and 19 kcps at 126 MBq for energy window of 250-750 and 400-620 keV, respectively. The IRIS XL provided an excellent quality of images in non-human primates and rats using 18F-FDG. The images acquired using (-)-[18F]FEOBV were consistent with those previously reported in non-human primates. CONCLUSIONS: Taken together, these results showed that the IRIS XL-220 is a high-resolution system well suited for PET/CT imaging in non-human primates.
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Age-related neurodegenerative diseases have in common the occurrence of cognitive impairment, a highly incapacitating process that involves the cholinergic neurotransmission system. The vesicular acetylcholine transporter (VAChT) positron emission tomography (PET) tracer [18F]fluoroethoxybenzovesamicol ((-)-[18F]FEOBV) has recently demonstrated its high value to detect alterations of the cholinergic system in Alzheimer's disease, Parkinson's disease and dementia with Lewy body. We present here the development of the new vesamicol derivative tracer (-)-(R,R)-5-[18F]fluorobenzovesamicol ((-)[18F]FBVM) that we compared to (-)[18F]FEOBV in the same experimental conditions. We show that: i) in vitro affinity for the VAChT was 50-fold higher for (-)FBVM (Ki = 0.9 ± 0.3 nM) than for (-)FEOBV (Ki = 61 ± 2.8 nM); ii) in vivo in rats, a higher signal-to-noise specific brain uptake and a lower binding to plasma proteins and peripheral defluorination were obtained for (-)[18F]FBVM compared to (-)[18F]FEOBV. Our findings demonstrate that (-)[18F]FBVM is a highly promising PET imaging tracer which could be sufficiently sensitive to detect in humans the cholinergic denervation that occurs in brain areas having a low density of VAChT such as the cortex and hippocampus.
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Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Humanos , Animais , Ratos , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , ColinérgicosRESUMO
Despite an apparently silent imaging, some patients with mild traumatic brain injury (TBI) experience cognitive dysfunctions, which may persist chronically. Brain changes responsible for these dysfunctions are unclear and commonly overlooked. It is thus crucial to increase our understanding of the mechanisms linking the initial event to the functional deficits, and to provide objective evidence of brain tissue alterations underpinning these deficits. We first set up a murine model of closed-head controlled cortical impact, which provoked persistent cognitive and sensorimotor deficits, despite no evidence of brain contusion or bleeding on MRI, thus recapitulating features of mild TBI. Molecular MRI for P-selectin, a key adhesion molecule, detected no sign of cerebrovascular inflammation after mild TBI, as confirmed by immunostainings. By contrast, in vivo PET imaging with the TSPO ligand [18F]DPA-714 demonstrated persisting signs of neuroinflammation in the ipsilateral cortex and hippocampus after mild TBI. Interestingly, immunohistochemical analyses confirmed these spatio-temporal profiles, showing a robust parenchymal astrogliosis and microgliosis, at least up to 3 weeks post-injury in both the cortex and hippocampus. In conclusion, we show that even one single mild TBI induces long-term behavioural deficits, associated with a persistent neuro-inflammatory status that can be detected by PET imaging.
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Concussão Encefálica , Lesões Encefálicas Traumáticas , Animais , Humanos , Camundongos , Encéfalo , Concussão Encefálica/complicações , Concussão Encefálica/diagnóstico por imagem , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/diagnóstico por imagem , Modelos Animais de Doenças , Doenças Neuroinflamatórias , Tomografia por Emissão de Pósitrons/métodos , Receptores de GABARESUMO
OBJECT: The impact of inflammation in utero on amniotic fluid composition, the delivery term and the number of newborn rats per litter was investigated. The growth of newborns during the first fourteen days of life was analysed. MATERIALS AND METHODS: Changes in the metabolome were evaluated using (1)H NMR spectroscopy combined with multivariate analysis. NMR spectra were segmented and principal component analysis was performed. Three groups were compared: a control group that received saline solution, a hyperthermic group (HYP) and a group that received injections of lipopolysaccharides (LPS) (350 µg/kg/day). RESULTS: The most discriminating metabolites in the three profiles were identified, highlighting different metabolic pathways for providing glucose and energy to the foetus. The LPS group was characterized by glycolysis under anaerobic conditions, while the HYP group was characterized by a gluconeogenic amino acid pathway. These metabolic changes in amniotic fluid were accompanied by changes in the gestation outcome, the main differences concerning the mean number of pups per litter (control 9.74 ± 0.6, HYP 6.81 ± 0. and LPS 4.85 ± 1.11) and the biometric growth of the pups. CONCLUSION: Some consistent metabolic changes, observable by (1)H NMR spectroscopy, occurred in amniotic fluid during prenatal stress caused by hyperthermia and LPS-induced inflammation and had an impact on the gestation outcome.
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Líquido Amniótico/metabolismo , Febre/embriologia , Febre/metabolismo , Inflamação/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Efeitos Tardios da Exposição Pré-Natal , Estresse Fisiológico , Líquido Amniótico/química , Animais , Animais Recém-Nascidos , Deutério , Feminino , Febre/induzido quimicamente , Glicólise , Inflamação/induzido quimicamente , Lipopolissacarídeos , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Metaboloma , Gravidez , Resultado da Gravidez , Análise de Componente Principal/métodos , Ratos , Ratos Sprague-DawleyRESUMO
In a previous study, we showed that viniferin decreased amyloid deposits and reduced neuroinflammation in APPswePS1dE9 transgenic mice between 3 and 6 months of age. In the present study, wild type and APPswePS1dE9 transgenic mice were treated from 7 to 11 or from 3 to 12 months by a weekly intraperitoneal injection of either 20 mg/kg viniferin or resveratrol or their vehicle, the polyethylene glycol 200 (PEG 200). The cognitive status of the mice was evaluated by the Morris water maze test. Then, amyloid burden and neuroinflammation were quantified by western-blot, Enzyme-Linked ImmunoSorbent Assay (ELISA), immunofluorescence, and in vivo micro-Positon Emission Tomography (PET) imaging. Viniferin decreased hippocampal amyloid load and deposits with greater efficiency than resveratrol, and both treatments partially prevented the cognitive decline. Furthermore, a significant decrease in brain uptake of the TSPO PET tracer [18F]DPA-714 was observed with viniferin compared to resveratrol. Expression of GFAP, IBA1, and IL-1ß were decreased by viniferin but PEG 200, which was very recently shown to be a neuroinflammatory inducer, masked the neuroprotective power of viniferin.
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To date there is no treatment able to stop or slow down the loss of dopaminergic neurons that characterizes Parkinson's disease. It was recently observed in a rodent model of Alzheimer's disease that the interaction between the a7 subtype of nicotinic acetylcholine receptor (a7-nAChR) and sigma-1 receptor (s1-R) could exert neuroprotective effects through the modulation of neuroinflammation which is one of the key components of the pathophysiology of Parkinson's disease. In this context, the aim of the present study was to assess the effects of the concomitant administration of N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide (PHA) 543613 as an a7-nAChR agonist and 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate (PRE)-084 as a s1-R agonist in a well-characterized 6-hydroxydopamine rat model of Parkinson's disease. The animals received either vehicle separately or the dual therapy PHA/PRE once a day until day 14 post-lesion. Although no effect was noticed in the amphetamine-induced rotation test, our data has shown that the PHA/PRE treatment induced partial protection of the dopaminergic neurons (15-20%), assessed by the dopamine transporter density in the striatum and immunoreactive tyrosine hydroxylase in the substantia nigra. Furthermore, this dual therapy reduced the degree of glial activation consecutive to the 6-hydroxydopamine lesion, i.e, the 18 kDa translocation protein density and glial fibrillary acidic protein staining in the striatum, and the CD11b and glial fibrillary acidic protein staining in the substantia nigra. Hence, this study reports for the first time that concomitant activation of a7-nAChR and s1-R can provide a partial recovery of the nigro-striatal dopaminergic neurons through the modulation of microglial activation. The study was approved by the Regional Ethics Committee (CEEA Val de Loire n°19) validated this protocol (Authorization N°00434.02) on May 15, 2014.
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Altered glutamate signaling is thought to be involved in a myriad of psychiatric disorders. Positron emission tomography (PET) imaging with [18F]FPEB allows assessing dynamic changes in metabotropic glutamate receptor 5 (mGluR5) availability underlying neuropathological conditions. The influence of endogenous glutamatergic levels into receptor binding has not been well established yet. The purpose of this study was to explore the [18F]FPEB binding regarding to physiological fluctuations or acute changes of glutamate synaptic concentrations by a translational approach; a PET/MRS imaging study in 12 healthy human volunteers combined to a PET imaging after an N-acetylcysteine (NAc) pharmacological challenge in rodents. No significant differences were observed with small-animal PET in the test and retest conditions on the one hand and the NAc condition on the other hand for any regions. To test for an interaction of mGuR5 density and glutamatergic concentrations in healthy subjects, we correlated the [18F]FPEB BPND with Glu/Cr, Gln/Cr, Glx/Cr ratios in the anterior cingulate cortex VOI; respectively, no significance correlation has been revealed (Glu/Cr: r = 0.51, p = 0.09; Gln/Cr: r = -0.46, p = 0.13; Glx/Cr: r = -0.035, p = 0.92).These data suggest that the in vivo binding of [18F]FPEB to an allosteric site of the mGluR5 is not modulated by endogenous glutamate in vivo. Thus, [18F]FPEB appears unable to measure acute fluctuations in endogenous levels of glutamate.
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Acetilcisteína , Receptor de Glutamato Metabotrópico 5 , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Voluntários Saudáveis , Humanos , Tomografia por Emissão de Pósitrons , Piridinas , Compostos Radiofarmacêuticos , Ratos , Receptor de Glutamato Metabotrópico 5/metabolismoRESUMO
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.
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Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Espectroscopia de Ressonância Magnética , Placa Amiloide/metabolismo , Tomografia por Emissão de Pósitrons , Proteínas tau/metabolismo , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Doença de Alzheimer/patologia , Animais , Escala de Avaliação Comportamental , Disfunção Cognitiva/genética , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Feminino , Radioisótopos de Flúor , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Gliose/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Imuno-Histoquímica , Inflamação/metabolismo , Locomoção/genética , Locomoção/fisiologia , Masculino , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Transgênicos , Receptores Colinérgicos/metabolismo , Tálamo/metabolismo , Tálamo/patologiaRESUMO
PURPOSE: The nicotinic acetylcholine alpha-7 receptors (α7R) are involved in a number of neuropsychiatric and neurodegenerative brain disorders such as Parkinson's disease (PD). However, their specific pathophysiologic roles are still unclear. In this context, we studied the evolution of these receptors in vivo by positron emission tomography (PET) imaging using the recently developed tracer 3-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-6-[18F]fluorodibenzo[b,d]thiophene-5,5-dioxide) in a rat model mimicking early stages of PD. PROCEDURES: PET imaging of α7R was performed at 3, 7, and 14 days following a partial striatal unilateral lesion with 6-hydroxydopamine in adult rats. After the last imaging experiments, the status of nigro-striatal dopamine neurons as well as different markers of neuroinflammation was evaluated on brain sections by autoradiographic and immunofluorescent experiments. RESULTS: We showed an early and transitory rise in α7R expression in the lesioned striatum and substantia nigra, followed by over-expression of several gliosis activation markers in these regions of interest. CONCLUSIONS: These findings support a longitudinally follow-up of α7R in animal models of PD and highlight the requirement to use a potential neuroprotective approach through α7R ligands at the early stages of PD.
Assuntos
Encéfalo/diagnóstico por imagem , Doença de Parkinson/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Anfetaminas/farmacologia , Animais , Autorradiografia , Compostos Azabicíclicos , Mapeamento Encefálico/métodos , Óxidos S-Cíclicos , Modelos Animais de Doenças , Radioisótopos de Flúor , Masculino , Neuroproteção , Compostos Radiofarmacêuticos , Ratos , Ratos WistarRESUMO
In this paper we describe the design and synthesis of bis(Het)Aryl-1,2,3-triazole quinuclidine α7R ligands using an efficient three-step sequence including a Suzuki-Miyaura cross coupling reaction with commercially available and home-made boron derivatives. The exploration of SAR required the preparation of uncommon boron derivatives. Forty final drugs were tested for their ability to bind the target and nine of them exhibited Ki values below nanomolar concentrations. The best scores were always obtained when the 5-phenyl-2-thiophenyl core was attached to the triazole. The selectivity of these compounds towards the nicotinic α4ß2 and serotoninergic 5HT3 receptors was assessed and their brain penetration was quantified by the preparation and in vivo evaluation of two [18F] radiolabelled derivatives. It can be expected from our results that some of these compounds will be suitable for further developments and will have effects on cognitive disorders.
Assuntos
Encéfalo/diagnóstico por imagem , Transtornos Cognitivos/tratamento farmacológico , Radioisótopos de Flúor/química , Marcação por Isótopo , Agonistas Nicotínicos/farmacologia , Tomografia por Emissão de Pósitrons , Quinuclidinas/farmacologia , Triazóis/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Transtornos Cognitivos/metabolismo , Relação Dose-Resposta a Droga , Ligantes , Estrutura Molecular , Agonistas Nicotínicos/síntese química , Agonistas Nicotínicos/química , Quinuclidinas/síntese química , Quinuclidinas/química , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , Células Tumorais Cultivadas , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
We studied concomitantly the level of neuroinflammation and ß-amyloid (Aß) load in the APPswePS1dE9 transgenic mouse model of Alzheimer's disease using positron emission tomography. The translocator protein 18 kDa (TSPO) tracer [(18)F]DPA-714 was used to measure neuroinflammation and [(18)F]AV-45 for Aß load in mice at 6, 9, 12, 15, and 19 months of age. At 19 months, we also analyzed the neuroinflammatory and neuroanatomic status of mice brains. The main affected brain areas were the cortex and hippocampus, with a concomitant progression of neuroinflammation with increased amyloid burden. At 19 months, no increase in TSPO binding was observed in the cerebellum; immunostaining revealed W0-2-positive plaques, indicating that the amyloid deposits seemed not stimulate inflammation. This finding was in agreement with the observed level of microglia and astrocytes staining. Our findings provide a better understanding of the relationships between neuroinflammation and plaque accumulation in the course of the disease in this mouse model. The monitoring of both processes should be of value to validate potential therapeutic approaches.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Receptores de GABA/metabolismo , Doença de Alzheimer/diagnóstico por imagem , Animais , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Estudos Longitudinais , Masculino , Camundongos Transgênicos , Inflamação Neurogênica/diagnóstico por imagem , Inflamação Neurogênica/metabolismo , Inflamação Neurogênica/patologia , Tomografia por Emissão de Pósitrons , Ligação ProteicaRESUMO
The inverse association between nicotine intake and Parkinson's disease (PD) is well established and suggests that this molecule could be neuroprotective through anti-inflammatory action mediated by nicotinic receptors, including the α7-subtype (α7R). The objective of this study was to evaluate the effects of an agonist of α7R, PHA 543613, on striatal dopaminergic neurodegeneration and neuroinflammation in a rat model of PD induced by 6-hydroxydopamine (6-OHDA) lesion. Adult male Wistar rats were lesioned in the right striatum and assigned to either the PHA group (n = 7) or the Sham group (n = 5). PHA 543613 hydrochloride at the concentration of 6 mg/kg (PHA group) or vehicle (Sham group) was intra-peritoneally injected 2 h before 6-OHDA lesioning and then at days 2, 4, and 6 post-lesion. Positron emission tomography (PET) imaging was performed at 7 days post-lesion using [(18)F]LBT-999 to quantify the striatal dopamine transporter (DAT). After PET imaging, neuroinflammation was evaluated in same animals in vitro through the measurement of the microglial activation marker 18 kDa translocator protein (TSPO) by quantitative autoradiography with [(3)H]PK-11195. The DAT density reflecting the integrity of dopaminergic neurons was significantly decreased while the intensity of neuroinflammation measured by TSPO density was significantly increased in the lesioned compared to intact striatum in both groups. However, these both modifications were partially reversed in the PHA group compared to Sham. In addition, a significant positive correlation between the degree of lesion and the intensity of neuroinflammation was evidenced. These findings indicate that PHA 543613 exerts neuroprotective effects on the striatal dopaminergic neurons associated with a reduction in microglial activation in this model of PD. This reinforces the hypothesis that an α7R agonist could provide beneficial effects for the treatment of PD.