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Automatic analysis toolboxes are popular in brain image analysis, both in clinical and in preclinical practices. In this regard, we proposed a new toolbox for mouse PET–CT brain image analysis including a new Statistical Parametric Mapping-based template and a pipeline for image registration of PET–CT images based on CT images. The new templates is compatible with the common coordinate framework (CCFv3) of the Allen Reference Atlas (ARA) while the CT based registration step allows to facilitate the analysis of mouse PET–CT brain images. From the ARA template, we identified 27 volumes of interest that are relevant for in vivo imaging studies and provided binary atlas to describe them. We acquired 20 C57BL/6 mice with [18F]FDG PET–CT, and 12 of them underwent 3D T2-weighted high-resolution MR scans. All images were elastically registered to the ARA atlas and then averaged. High-resolution MR images were used to validate a CT-based registration pipeline. The resulting method was applied to a mouse model of Parkinson’s disease subjected to a test–retest study (n = 6) with the TSPO-specific radioligand [18F]VC701. The identification of regions of microglia/macrophage activation was performed in comparison to the Ma and Mirrione template. The new toolbox identified 11 (6 after false discovery rate adjustment, FDR) brain sub-areas of significant [18F]VC701 uptake increase versus the 4 (3 after FDR) macro-regions identified by the Ma and Mirrione template. Moreover, these 11 areas are functionally connected as found by applying the Mouse Connectivity tool of ARA. In conclusion, we developed a mouse brain atlas tool optimized for PET–CT imaging analysis that does not require MR. This tool conforms to the CCFv3 of ARA and could be applied to the analysis of mouse brain disease models.

Brain energy metabolism actively regulates synaptic transmission and activity. We have previously shown that acute footshock (FS)-stress induces fast and long-lasting functional and morphological changes at excitatory synapses in prefrontal cortex (PFC). Here, we asked whether FS-stress increased energy metabolism in PFC, and modified related cognitive functions. Using positron emission tomography (PET), we found that FS-stress induced a redistribution of glucose metabolism in the brain, with relative decrease of [18F]FDG uptake in ventro-caudal regions and increase in dorso-rostral ones. Absolute [18F]FDG uptake was inversely correlated with serum corticosterone. Increased specific hexokinase activity was also measured in purified PFC synaptosomes (but not in total extract) of FS-stressed rats, which positively correlated with 2-Deoxy [3H] glucose uptake by synaptosomes. In line with increased synaptic energy demand, using an electron microscopy-based stereological approach, we found that acute stress induced a redistribution of mitochondria at excitatory synapses, together with an increase in their volume. The fast functional and metabolic activation of PFC induced by acute stress, was accompanied by rapid and sustained alterations of working memory performance in delayed response to T-maze test. Taken together, the present data suggest that acute stress increases energy consumption at PFC synaptic terminals and alters working memory.

Cyclooxygenase-2 (COX-2) is involved in the inflammatory response, and its recurrent overexpression in cancers as well as in neurodegenerative disorders has made it an important target for therapy. For this reason, noninvasive imaging of COX-2 expression may represent an important diagnostic tool. In this work, a COX-2 inhibitor analogue, VA426 [1-(4-fluorophenyl)-3-(2-methoxyethyl)-2-methyl-5-(4-(methylsulfonil)phenyl)-1H-pyrrole], was synthesized and radiolabelled with the 11C radioisotope. The ex vivo biodistribution profile of 11C-VA426 was evaluated in the brain and periphery of healthy rats and mice and in brain and periphery of inflammation models, based on the administration of LPS. 11C-VA426 synthesis with the tBuOK base showed optimal radiochemical yield (15 ± 2%) based on triflate activity, molar activity (range 37–148 GBq/μmol), and radiochemical purity (>95%). Ex vivo biodistribution studies showed a fast uptake of radioactivity but a rapid washout, except in regions expressing COX-2 (lungs, liver, and kidney) both in rats and in mice, with maximum values at 30 and 10 minutes p.i., respectively. LPS administration did not show significant effect on radioactivity accumulation. Celecoxib competition experiments performed in rats and mice treated with LPS produced a general target unrelated reduction of radioactivity concentration in all peripheral tissues and brain areas examined. Finally, in agreement with the negative results obtained from biodistribution experiments, radiometabolites analysis revealed that 11C-VA426 is highly unstable in vivo. This study indicates that the compound 11C-VA426 is not currently suitable to be used as radiopharmaceutical for PET imaging. This family of compounds needs further implementation in order to improve in vivo stability.

The aim of this paper is to evaluate the risks and doses for the internal contamination of the radiochemistry staff in a high workload medical cyclotron facility. The doses from internal contamination derive from the inhalation of radioactive gas leakage from the cells by personnel involved in the synthesis processes and are calculated from urine sample measurements. Various models are considered for the calculation of the effective committed dose from the analysis of these urine samples, and the results are compared with data obtained from local environmental measurement of the radioactivity released inside the lab.

Microglia activation and neuroinflammation play a pivotal role in the pathogenesis of lysosomal storage disorders (LSD) affecting the central nervous system (CNS), which are amenable to treatment by hematopoietic stem cell transplantation (HSCT). HSCT efficacy relies on replacing the intra- and extra-vascular hematopoietic cell compartments, including CNS microglia, with a cell population expressing the functional enzyme. Non-invasive and quantitative assessment of microglia activation and of its reduction upon HSCT might allow for evaluation of disease evolution and response to treatment in LSD. We here demonstrate that microglia activation can be quantified ex vivo and in vivo by PET using the peripheral benzodiazepine receptor ligand PK11195 in two models of LSD. Furthermore, we show a differential PBR binding following microglia replacement by donor cells in mice undergoing HSCT. Our data indicates that PBR ligands constitute valuable tools for monitoring the evolution and the response to treatment of LSD with CNS involvement, and enable us to evaluate whether the turnover between endogenous and donor microglia following HSCT could be adequate enough to delay disease progression.

// Daniela Gaglio 1, 2, 6, 8 , Silvia Valtorta 1, 2, 3, 4 , Marilena Ripamonti 1, 2 , Marcella Bonanomi 2 , Chiara Damiani 2 , Sergio Todde 5 , Alfredo Simone Negri 6 , Francesca Sanvito 7 , Fabrizia Mastroianni 2 , Antonella Di Campli 8 , Gabriele Turacchio 8 , Giuseppe Di Grigoli 1, 2, 3 , Sara Belloli 1, 2, 3 , Alberto Luini 8 , Maria Carla Gilardi 1, 2 , Anna Maria Colangelo 2, 9 , Lilia Alberghina 2, 9, * , Rosa Maria Moresco 2, 3, 4, * 1 Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Segrate, Italy 2 SYSBIO.IT, Centre of Systems Biology, Milano, Italy 3 Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy 4 Department of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy 5 Tecnomed Foundation of University of Milano-Bicocca, Monza, Italy 6 Department of Agricultural and Environmental Sciences - Production, Landscape, Agroenergy University of Milan, Milan, Italy 7 Mouse Histopathology Unit, Department of Pathology, IRCCS San Raffaele Scientific Institute, Milan, Italy 8 Institute of Protein Biochemistry, National Research Council, Naples, Italy 9 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy * These authors have contributed equally to this work Correspondence to: Daniela Gaglio, email: daniela.gaglio@ibfm.cnr.it Rosa Maria Moresco, email: moresco.rosamaria@hsr.it Keywords: tumor, metabolic rewiring, PET-imaging, metabolomics-mass-spectrometry, oncogenic-K-ras Received: February 02, 2016 Accepted: June 17, 2016 Published: July 07, 2016 ABSTRACT Oncogenic K-ras is capable to control tumor growth and progression by rewiring cancer metabolism. In vitro NIH-Ras cells convert glucose to lactate and use glutamine to sustain anabolic processes, but their in vivo environmental adaptation and multiple metabolic pathways activation ability is poorly understood. Here, we show that NIH-Ras cancer cells and tumors are able to coordinate nutrient utilization to support aggressive cell proliferation and survival. Using PET imaging and metabolomics-mass spectrometry, we identified the activation of multiple metabolic pathways such as: glycolysis, autophagy recycling mechanism, glutamine and serine/glycine metabolism, both under physiological and under stress conditions. Finally, differential responses between in vitro and in vivo systems emphasize the advantageous and uncontrolled nature of the in vivo environment, which has a pivotal role in controlling the responses to therapy.

The selective dopamine D(3) receptor ligands N-4-[4-[(2,3-dichlorophenyl)piperazin-1-yl]butyl]1-methoxy-2-naphthalencarboxamide (1) and N-4-[4-[(2,3-dichlorophenyl)piperazin-1-yl]butyl]-7-methoxy-2-benzofurancarboxamide (2) were labeled with (11)C (t(1/2) = 20.4 min) as potential radioligands for the noninvasive assessment of the dopamine D(3) neurotransmission system in vivo with positron emission tomography (PET). The radiosynthesis consisted in an O-methylation of the des-methyl precursors N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-1-hydroxy-2-naphthalenecarboxamide (3) and N-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl]-7-hydroxy-2-benzofurancarboxamide (4) with [(11)C]methyl iodide using tBuOK/HMPA and KOH/DMSO, respectively. The radiotracers [(11)C]1 and [(11)C]2 were obtained in 35 min with over 99% radiochemical purity, 74 +/- 37 GBq/mumol of specific radioactivity, 13% and 26% radiochemical yield (EOB, decay-corrected). Distribution studies in rats demonstrated that the new tracers [(11)C]1 and [(11)C]2 cross the blood-brain barrier and localize in the brain. However, the kinetics of cerebral uptake did not reflect the regional expression of the D(3) receptors. Despite their in vitro pharmacological profile, [(11)C]1 and [(11)C]2 do not display an in vivo behavior suitable to image D(3) receptor expression using PET.

PURPOSE: The aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1? activity, in the context of hypoxia, by means of different imaging techniques. PROCEDURES: Engineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1? activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI). RESULTS: In vitro analyses highlighted the relationship between HIF-1? and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI. CONCLUSIONS: The findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1? activity, using both molecular and preclinical non-invasive imaging techniques.

PET is widely used at the clinical and preclinical levels for tumor assessment and evaluation of treatment efficacy. Here, we established and took advantage of a preclinical model of peritoneal carcinomatosis to evaluate the contribution of inflammatory infiltrating macrophages in tumor progression that was followed using 18F-FDG PET. Methods: Groups of mice with peritoneal carcinomatosis were longitudinally evaluated with 18F-FDG PET. Intraperitoneal depletion of macrophages was achieved by an approach (i.e., administration of clodronate encapsulated into liposomes) that proved to be safe and effective. Sham liposomes were used in control animal cohorts. Results: 18F-FDG PET allowed us to detect and monitor peritoneal lesion growth and diffusion. Macrophage-depleted animals showed a substantial reduction in tumor burden paralleled by a decrement in the extent of radioactivity distribution. A significant correlation between lesion dimension and metabolic volume was observed not only in macrophage-depleted but also in sham-treated mice. Conclusion: 18F-FDG PET allowed a noninvasive detection of peritoneal carcinomatosis lesions. Although macrophages play a key role in the early growth and spreading of lesions in the peritoneal cavity, neoplastic cells apparently represent the major player in this system in the uptake of 18F-FDG.

PURPOSE: Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice spontaneously develop hormone-dependent and hormone-independent prostate cancer (PC) that potentially resembles the human pathological condition. The aim of the study was to validate PET imaging as a reliable tool for in vivo assessment of disease biology and progression in TRAMP mice using radioligands routinely applied in clinical practice: [(18)F]FDG and [(11)C]choline. METHODS: Six TRAMP mice were longitudinally evaluated starting at week 11 of age to visualize PC development and progression. The time frame and imaging pattern of PC lesions were subsequently confirmed on an additional group of five mice. RESULTS: PET and [(18)F]FDG allowed detection of PC lesions starting from 23 weeks of age. [(11)C]Choline was clearly taken up only by TRAMP mice carrying neuroendocrine lesions, as revealed by post-mortem histological evaluation. CONCLUSION: PET-based molecular imaging represents a state-of-the-art tool for the in vivo monitoring and metabolic characterization of PC development, progression and differentiation in the TRAMP model

The previous exploration of the structure-affinity relationships concerning 4-phenyl-2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands 6 showed as an interesting result the importance of the presence of a chlorine atom in the methylene carbon at position 3 of the quinoline nucleus. The subnanomolar PBR affinity shown by N-benzyl-3-chloromethyl-N-methyl-4-phenyl-2-quinolinecarboxamide (6b) suggested its chlorine atom to be replaced with other halogens in order to optimize the interaction of the quinolinecarboxamide derivatives with PBR and to develop suitable candidates for positron emission tomography (PET) or single photon emission computed tomography (SPECT) studies. The binding studies led to the discovery of fluoromethyl derivative 6a, which showed an IC(50) value of 0.11nM and is, therefore, one of the most potent PBR ligands so far described. Fluoromethyl derivative 6a has been labeled with (11)C (t(1/2)=20.4min, beta(+)=99.8%) starting from the corresponding des-methyl precursor (14) using [(11)C]CH(3)I in the presence of tetrabutylammonium hydroxide in DMF with a 35-40% radiochemical yield (corrected for decay) and 1.5Ci/mumol of specific radioactivity. Ex vivo rat biodistribution and inhibition (following intravenous pre-administration of PK11195) studies showed that [(11)C]6a rapidly and specifically accumulated in PBR-rich tissues such as heart, lung, kidney, spleen, and adrenal, and at a lower level in other peripheral organs and in the brain. The images obtained in mouse with small animal YAP-(S)PET essentially confirmed the result of the ex vivo biodistribution experiments. The biological data suggest that [(11)C]6a is a promising radioligand for peripheral benzodiazepine receptor PET imaging in vivo.