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Aim: K-ras proteins have been found mutated in about 35% of human tumors and appear to be an important factor for tumorigenesis. Its expression correlates with metabolic alterations such as increased glycolysis and glutamine consumption. This ability of cancer cells to decouple glucose and glutamine uptake reprogramming their metabolism leads to a more efficient use of nutrients in order to support cell proliferation and represents an interesting target for cancer therapy. Aim of this study is to investigate the metabolic alterations occurring in k-ras transformed fibroblasts combining in vivo and in vitro studies. Materials and methods: nu/nu mice were subcutaneously implanted with 2.5 x 105 k-ras transformed murine NIH3T3 fibroblasts (oncogenic k-ras) or with 2.5 x 105 murine fibroblasts with the dominant negative mutation on GEF protein that attenuates k-ras activation reverting to the wild type phenotype (reverted). Lesions size was constantly monitored with calliper and volumes calculated as (L x l2)/2 mm3. Animals performed [18F]FDG- and [18F]FLTPET studies at several time points starting with a tumor dimension consistent with animal PET spatial resolution (approximately 2mm). Images were calibrated, corrected for isotope half-life and elaborated with PMOD software to calculate Standardized Uptake Value (SUVmax). Finally, animals were sacrificed and tumor collected for metabolomic analysis. Results: All animals injected with oncogenic k-ras fibroblasts develop in few days fast growing, aggressive and highly glycolytic tumors that appear homogeneous for both [18F]FDG and [18F]FLT SUV values. On the contrary, 40% of k-ras reverted animals develop heterogeneous tumors at later time. Among k-ras reverted animals, two distinct tumor phenotypes can be observed: small, slow growing and poor glycolitic tumors with low uptake of both tracers, and small, slow growing but highly glycolytic and proliferating tumors that present SUVmax values comparable to those of k-ras oncogenic tumors. A good correlation, even if not significant, between tumor volume and [18F]FLT SUVmax has been observed for oncogenic k-ras animals. Conclusions:PET imaging is an accurate in vivo technique able to visualize and monitor tumor development in the k-Ras fibroblasts mouse model. K-Ras transformed fibroblasts give rise to aggressive and fast-growing tumors that represent a good model to study the efficacy of cell metabolism based therapy. Finally, K-Ras reverted tumors need further investigations to understand the interaction among tumor microenvironment, metabolic alterations and genetic component that may trigger tumor development.

PURPOSE: This study investigates whether positron emission tomography (PET) can be used to monitor the inflammatory response and its correlation with the later fibroproliferative phase in an experimental model of acute lung injury. METHODS: Hydrochloric acid (0.1 N, pH 1, 1.5 ml/kg) was instilled into the right bronchus of mice. A group of mice underwent a micro-computed tomography (CT) scan 1 h after lung injury and a series of 2-[(18)F]fluorine-2-deoxy-D: -glucose (FDG)-PET scans (6, 24 and 48 h and 7 days after surgery). After 21 days respiratory static compliance was assessed and lung tissue was collected in order to measure the hydroxy (OH)-proline content. Other groups of mice underwent micro-CT and micro-PET scans at the same time points, and then were immediately killed to assess arterial blood gases and histology. RESULTS: Histological analysis showed the recruitment of neutrophils and macrophages into the damaged lung, reaching the peak at 24 and 48 h, respectively. The time course of the [(18)F]FDG signal, used as a marker of inflammation, correlated with that of recruited inflammatory cells. In mice killed 21 days after the surgery, a correlation was found between reduced respiratory static compliance and high PET signal 7 days after lung injury. The PET signal also correlated with the OH-proline content. CONCLUSIONS: This study demonstrated that PET imaging is a valid means of tracking the inflammatory response, also in longitudinal studies. Moreover, a correlation was found between persistence of the inflammatory response and fibrotic evolution of the injury.

Introduction: The aim of our study is to analyze the relationship between tumor growth and tumor hypoxia in an orthotopic glioma murine model by using a multimodal procedure, and to compare the features of the different imaging techniques in revealing hypoxia induction. Materials and methods: Engineered U251 cells were gently provided by Dr. Giovanni Melillo, National Cancer Institute, Frederick (MD). These cells express the luciferase reporter gene under control of a constitutive promoter (U251-LUC) or under control of three copies of a Hypoxic Responsive Element (U251-HRE). Cells has been analyzed by means of three different approaches: 1) In vitro evaluation of transcriptional activation of HIF-1? at different times after deferoxamine (DFX) treatment by immunocytochemistry (ICC). 2) In vivo analysis of tumoral progression in orthotopic murine models obtained by stereotaxic injection of 10^5 glioma cells; animals were monitored weekly with BLI (CCD camera), PET ([18F]FDG,[18F]FAZA,[18F]FLT) and MRI to evaluate tumoral progression and hypoxia activation. 3)Ex vivo analysis by H&E staining and hypoxia markers (HIF-1? and CAIX) were performed. Results: In vitro studies showed no differences among the two cell lines as regards to HIF-1? activation kinetic with a peak of activation between 3 and 6h and a decrease at 20h. In vivo, U251-HRE model showed a detectable and progressive luciferase activity induction starting at 18 days from injection demonstrating that luminescence expression is dependent on HRE-mediated activation. On the other hand, in U251-LUC model luciferase activity was detectable immediately after injection and remained proportional to tumor growth. Lesions were highly proliferative ( [18F]FLT) but hypo-metabolic ([18F]FDG). In partial agreement with the results of U251-HRE, a hypoxia dependent [18F]FAZA uptake was observed at later times (30 days). MRI provided morphological characterization and diffusion studies showing hypoxic necrotic areas only at later days. Ex vivo H&E staining and immunohistochemistry (HIF-1? and CAIX) performed after 30 days confirmed in vivo results including the presence of hypoxic regions. Conclusions: This study demonstrates that the U251-HRE orthotopic murine model may be proposed as a predictive and reliable tool to evaluate hypoxia dependent processes of human glioma in preclinical studies by BLI. Differences among the three imaging techniques may be related to methods sensitivity. Further ex vivo post mortem measurements of HIF-1 and CAIX at earlier times will be performed. Additional studies will be conducted to understand the clinical meaning of early or delayed hypoxia identification in terms of response to treatment.

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.

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

Small-animal positron emission tomography (PET) and single photon emission tomography (SPECT) are invaluable tools in drug discovery and in assessing novel therapeutic agents. In a typical small-animal PET or SPECT imaging session, a radiopharmaceutical is administered and the kinetics of its distribution tracked. Whereas the PET and SPECT protocols for the radiotraceras used in clinical practice are well established, at least for the radiotracers that are used in clinical practice, the acquisition, reconstruction and data analysis procedures applied in small rodents are heterogeneous among centres, often being determined by the scanner characteristics and by the animal models and anaesthesia procedures used in the laboratory. This review, intended to serve as a guide to preclinical PET and SPECT experiments, aims to familiarise the reader with the aspects of PET and SPECT that pertain to in vivo preclinical acquisitions and, in particular, with the technical and methodological know-how that is needed for better application of radionuclide imaging techniques in preclinical cancer research.

Purpose. Huntington’s disease (HD) is a progressive neurodegenerative disorder, which is characterised by prominent neuronal cell loss in the basal ganglia with motor and cognitive disturbances. One of the most well-studied pharmacological models of HD is produced by local injection in the rat brain striatum of the excitotoxin quinolinic acid (QA), which produces many of the distinctive features of this human neurodegenerative disorder. Here, we report a detailed analysis, obtained both in vivo and in vitro of this pharmacological model of HD. Materials and methods. By combining emission tomography (PET) with autoradiographic and immunocytochemical confocal laser techniques, we quantified in the QA-injected striatum the temporal behavior (from 1 to 60 days from the excitotoxic insult) of neuronal cell density and receptor availability (adenosine A2A and dopamine D2 receptors) together with the degree of microglia activation. Results. Both approaches showed a loss of adenosine A2A and dopamine D2 receptors paralleled by an increase of microglial activation. Conclusion. This combined longitudinal analysis of the disease progression, which suggested an impairment of neurotransmission, neuronal integrity and a reversible activation of brain inflammatory processes, might represent a more quantitative approach to compare the differential effects of treatments in slowing down or reversing HD in rodent models with potential applications to human patients.

PURPOSE: The aim of this study was to evaluate the suitability of [11C]SCH442416 for the in vivo imaging of adenosine A2A receptors. METHODS: In rats and Macaca nemestrina, we evaluated the time course of the cerebral distribution of [11C]SCH442416. Furthermore, in rats we investigated the rate of metabolic degradation, the inhibitory effects of different drugs acting on adenosine or dopamine receptors and the modification induced by the intrastriatal administration of quinolinic acid (QA). RESULTS: The rate of metabolic degradation of [11C]SCH442416 in rats was slow; 60 min after tracer injection, more than 40% of total plasma activity was due to unmetabolised [11C]SCH442416. At the time of maximum uptake, radioactive metabolites represented only 6% of total extractable activity in the cerebellum and less than 1% in the striatum. In the striatum, the region with the highest expression of A2A receptors, the in vivo uptake of [11C]SCH442416 was significantly reduced only by drugs acting on A2A receptors or by QA, a neurotoxin that selectively reduces the number of intrastriatal GABAergic neurons. Position emission tomography (PET) studies in monkeys indicated that the tracer rapidly accumulates in brain, reaching maximum uptake between 5 and 10 min. Twenty minutes after the injection, radioactivity concentration in the striatum was two times that in the cerebellum. CONCLUSION: The specificity of binding, the rank order of regional distribution in the brain of rats and M. nemestrina, the good signal to noise ratios and the low amount of radioactive metabolites in brain and periphery indicate that [11C]SCH442416 is a promising tracer for the in vivo imaging of A2A adenosine receptors using PET.

The radiolabelling with the positron-emitter Carbon-11 and the biological evaluation in rats of3-[2-[4-(2-[11C]methoxyphenyl)piperazin-1-yl]ethyl] pyrimido[5,4-b]indole-2,4-dione([11C]RN5), a1-adrenoceptor antagonist (Ki= 0.21 nM), as a putative radioligand for the non-invasive assessment of alpha1-adrenoceptors with Positron Emission Tomography (PET) is reported. The radiosynthesis procedure consisted of O-methylation of des-methyl precursor with [11C]methyl iodide in the presence of potassium hydroxide in dimethylformamide (DMF) at 80°C. [11C]RN5 was obtained in >99% radiochemical purity in 25 min with a radiochemical yield in the 20-30% range, end of synthesis (EOS) (non-decay corrected) and a specific radioactivity of 92.5±18.5 GBq/mmol. Pre-clinical studies in rats showed a high uptake of [11C]RN5 in heart, spleen, adrenal gland, lung and kidney but not in the brain. Inhibition studies with high doses of different adrenergic antagonists indicate that more than 70% of myocardial uptake of [11C]RN5 is due to specific binding to alpha1-adrenoceptors. Our results indicate that [11C]RN5 is suitable to be further developed as a potential radioligand for the in vivo PET imaging of myocardial alpha1-adrenoceptors in humans.

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A simple method for the quantitative description of sleep microstructure is proposed. The application of this method makes it possible to recognize epochs characterized by the increase in the amplitude of some band activity. The length of these epochs and the time distance between two successive epochs are measured. The method is based on the consideration that the values of the band activity amplitudes, when averaged over time intervals in the order of approximately 1 minute, are generally related to the macrostructure, but, when averaged over shorter time intervals, tend to concern both the macrostructure and microstructure. From the calculation of these two different kinds of averages, it is possible to derive an adimensional descriptor closely related to the microstructure. The method was applied examining 4 frequency bands (1 to 4 Hz, 4 to 8 Hz, 8 to 12 Hz and 12.5 to 14.5 Hz), choosing the shorter time interval equal to 2 s and the longer equal to 64 s, and putting the threshold for the descriptors equal to 1. The results obtained automatically with this method were in close agreement with those provided by visual analysis.

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Steady-state evoked responses combined with Fourier analysis permit a faster collection and evaluation of data than transient evoked potentials in all the three sensory modalities. Steady-State Visual Evoked Potentials (SS-VEPs) induced by applying flickering flashes of light were recorded in a group of young and a group of elderly normal volunteers. The aim of the study was twofold: a) to implement a method for a quick routine analysis of the visual function; b) to verify the effects of ageing on different parameters of SS-VEPs. The 14 subjects were divided into a younger group (mean age 21.5 ± 2.6 years) and an elderly group (mean age 64.6 ± 5.1 years). Each of the subjects had a normal or corrected visual acuity of or better. The flashes, which lasted 0.1 ms, were presented binocularly on a stroboscope in a partially darkened room, at the frequencies of 4, 6, 8, 12 and 16 Hz, for epochs of 10 s. The EEG was recorded from 20 electrodes (10-20 International System) referred to linked mastoids. The signals were analysed off-line by means of a software package developed in our laboratory for the Fourier analysis of biological signals. The following parameters were calculated: spectral moments of orders 0, 2 and 4; barycentric frequency; Hjorth's parameters; intra- and inter-hemispheric correlations and coherences; mean deviations of the amplitudes of the various Fourier harmonics over the 10 s epochs.

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