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Background Transcranial direct current stimulation (tDCS) promotes recovery after stroke in humans. The underlying mechanisms, however, remain to be elucidated. Animal models suggest tDCS effects on neuroinflammation, stem cell proliferation, neurogenesis, and neural plasticity. Objective In a longitudinal study, we employed tDCS in the subacute and chronic phase after experimental focal cerebral ischemia in mice to explore the relationship between functional recovery and cellular processes. Methods Mice received photothrombosis in the right motor cortex, verified by Magnetic Resonance Imaging. A composite neuroscore quantified subsequent functional deficits. Mice received tDCS daily: either 5 sessions from day 5 to 9, or 10 sessions with days 12 to 16 in addition. TDCS with anodal or cathodal polarity was compared to sham stimulation. Further imaging to assess proliferation and neuroinflammation was performed by immunohistochemistry at different time points and Positron Emission Tomography at the end of the observation time of 3 weeks. Results Cathodal tDCS at 198 kC/m2 (220 A/m2) between days 5 and 9 accelerated functional recovery, increased neurogenesis, decreased microglial activation, and mitigated CD16/32-expression associated with M1-phenotype. Anodal tDCS exerted similar effects on neurogenesis and microglial polarization but not on recovery of function or microglial activation. TDCS on days 12 to 16 after stroke did not induce any further effects, suggesting that the therapeutic time window was closed by then. Conclusion Overall, data suggest that non-invasive neuromodulation by tDCS impacts neurogenesis and microglial activation as critical cellular processes influencing functional recovery during the early phase of regeneration from focal cerebral ischemia.

Background Treatment of patients with solid tumors and KRAS mutations remains disappointing. One option is the combined inhibition of pathways involved in RAF‐MEK‐ERK and PI3K‐AKT‐mTOR. Methods Patients with relapsed solid tumors were treated with escalating doses of everolimus (E) 2.5‐10.0 mg/d in a 14‐day run‐in phase followed by combination therapy with sorafenib (S) 800 mg/d from day 15. KRAS mutational status was assessed retrospectively in the escalation phase. Extension phase included KRAS‐mutated non–small‐cell lung cancer (NSCLC) only. Pharmacokinetic analyses were accompanied by pharmacodynamics assessment of E by FDG‐PET. Efficacy was assessed by CT scans every 6 weeks of combination. Results Of 31 evaluable patients, 15 had KRAS mutation, 4 patients were negative for KRAS mutation, and the KRAS status remained unknown in 12 patients. Dose‐limiting toxicity (DLT) was not reached. The maximum tolerated dose (MTD) was defined as 7.5 mg/d E + 800 mg/d S due to toxicities at previous dose level (10 mg/d E + 800 mg/d S) including leucopenia/thrombopenia III° and pneumonia III° occurring after the DLT interval. The metabolic response rate in FDG‐PET was 17% on day 5 and 20% on day 14. No patient reached partial response in CT scan. Median progression free survival (PFS) and overall survival (OS) were 3.25 and 5.85 months, respectively. Conclusions Treatment of patients with relapsed solid tumors with 7.5 mg/d E and 800 mg/d S is safe and feasible. Early metabolic response in FDG‐PET was not confirmed in CT scan several weeks later. The combination of S and E is obviously not sufficient to induce durable responses in patients with KRAS‐mutant solid tumors., The study investigated the combination of sorafenib and everolimus in patients with solid tumors (dose escalation part) and KRAS mutated NSCLC (expansion part). Although the trial showed manageable safety profile of sorafenib and everolimus, it failed in providing long terms responses for patients with solid tumors and KRAS mutated NSCLC. The novelty of the study was the early pharmacodynamics assessment using FDG‐PET implemented in a phase‐I.

Brains are among the most energetically costly tissues in the mammalian body.1 This is predominantly caused by expensive neurons with high glucose demands.2 Across mammals, the neuronal energy budget appears to be fixed, possibly posing an evolutionary constraint on brain growth.3-6 Compared to similarly sized mammals, birds have higher numbers of neurons, and this advantage conceivably contributes to their cognitive prowess.7 We set out to determine the neuronal energy budget of birds to elucidate how they can metabolically support such high numbers of neurons. We estimated glucose metabolism using positron emission tomography (PET) and 2-[18F]fluoro-2-deoxyglucose ([18F]FDG) as the radiotracer in awake and anesthetized pigeons. Combined with kinetic modeling, this is the gold standard to quantify cerebral metabolic rate of glucose consumption (CMRglc).8 We found that neural tissue in the pigeon consumes 27.29 ± 1.57 μmol glucose per 100 g per min in an awake state, which translates into a surprisingly low neuronal energy budget of 1.86 × 10-9 ± 0.2 × 10-9 μmol glucose per neuron per minute. This is approximately 3 times lower than the rate in the average mammalian neuron.3 The remarkably low neuronal energy budget explains how pigeons, and possibly other avian species, can support such high numbers of neurons without associated metabolic costs or compromising neuronal signaling. The advantage in neuronal processing of information at a higher efficiency possibly emerged during the distinct evolution of the avian brain.

Summary Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control., Graphical abstract, Highlights • Intersectional mapping of sensory neurons identifies distinct gut innervation patterns • Gut-innervating GLP1R+ vagal afferents relay anorexigenic signals to brainstem neurons • Gut-innervating GPR65+ vagal afferent stimulation increases hepatic glucose production • GLP1R+ vagal afferent activity is required to control glycemia during feeding, Borgmann et al. devise an intersectional genetic approach to probe the contribution of molecularly defined sensory neurons in gut-brain communication. They identify that distinct gut-innervating vagal afferents differentially control food intake and peripheral glucose metabolism, and engage distinct downstream circuits in the brain.

Summary Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia., Graphical Abstract, Highlights • Acute high-fat-diet feeding activates PNOC neurons in the arcuate nucleus (ARC) • GABAergic PNOCARC neurons inhibit anorexigenic POMC neurons • Optogenetic activation of PNOCARC neurons promotes feeding • Ablation of PNOCARC neurons protects from obesity, Calorie-rich diets induce hyperphagia and promote obesity. Here, Jais et al. report that short-term high-fat-diet (HFD) feeding in mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). They characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia.

Regulation of body temperature critically depends on thyroid hormone (TH). Recent studies revealed that TH induces browning of white adipose tissue, possibly contributing to the observed hyperthermia in hyperthyroid patients and potentially providing metabolic benefits. Here, we show that browning by TH requires TH-receptor β and occurs independently of the sympathetic nervous system. The beige fat, however, lacks sufficient adrenergic stimulation and is not metabolically activated despite high levels of uncoupling protein 1 (UCP1). Studies at different environmental temperatures reveal that TH instead causes hyperthermia by actions in skeletal muscle combined with a central body temperature set-point elevation. Consequently, the metabolic and thermogenic effects of systemic hyperthyroidism were maintained in UCP1 knockout mice, demonstrating that neither beige nor brown fat contributes to the TH-induced hyperthermia and elevated glucose consumption, and underlining that the mere presence of UCP1 is insufficient to draw conclusions on the therapeutic potential of browning agents This work was supported by grants of the Deutsche Forschungsgemeinschaft (MI1242/2-2 and 3-2 Heisenberg Program and MI1242/4-1 and 5-1 SPP1629 “Thyroid TransAct” to J.M.; AS547-1/1 to M.A.; GRK1957 “Adipocyte-Brain-Crosstalk”), Young Active Research in Endocrinology, Bioscientifica Trust (BT-000011), and European Society for Endocrinology to K.J. A.V.-P. and S.V. were funded by the BHF (RG/18/7/33636). M.L. was funded by Xunta de Galicia (2016-PG068) and Ministerio de Economía y Competitividad (MINECO) (SAF2015-71026-R). H.O. is a Lichtenberg Fellow of the Volkswagen Stiftung SI

To date, the spatiotemporal release of specific neurotransmitters at physiological levels in the human brain cannot be detected. Here, we present a method that relates minute-by-minute fluctuations of the positron emission tomography (PET) radioligand [11C]raclopride directly to subsecond dopamine release events. We show theoretically that synaptic dopamine release induces low frequency temporal variations of extrasynaptic extracellular dopamine levels, at time scales of one minute, that can evoke detectable temporal variations in the [11C]raclopride signal. Hence, dopaminergic activity can be monitored via temporal fluctuations in the [11C]raclopride PET signal. We validate this theory using fast-scan cyclic voltammetry and [11C]raclopride PET in mice during chemogenetic activation of dopaminergic neurons. We then apply the method to data from human subjects given a palatable milkshake and discover immediate and—for the first time—delayed food-induced dopamine release. This method enables time-dependent regional monitoring of stimulus-evoked dopamine release at physiological levels., It has proven difficult to measure the release of neurotransmitters, such as dopamine, in the human brain. Here, the authors introduce and validate a new method that infers dopamine release based on minute-by-minute fluctuations of the positron emission tomography (PET) radioligand [11C]raclopride.

High-fat diet (HFD) feeding induces rapid reprogramming of systemic metabolism. Here, we demonstrate that HFD feeding of mice downregulates glucose transporter (GLUT)-1 expression in blood-brain barrier (BBB) vascular endothelial cells (BECs) and reduces brain glucose uptake. Upon prolonged HFD feeding, GLUT1 expression is restored, which is paralleled by increased expression of vascular endothelial growth factor (VEGF) in macrophages at the BBB. In turn, inducible reduction of GLUT1 expression specifically in BECs reduces brain glucose uptake and increases VEGF serum concentrations in lean mice. Conversely, myeloid-cell-specific deletion of VEGF in VEGF(Δmyel) mice impairs BBB-GLUT1 expression, brain glucose uptake, and memory formation in obese, but not inlean mice. Moreover, obese VEGF(Δmyel) mice exhibit exaggerated progression of cognitive decline and neuroinflammation on an Alzheimer's disease background. These experiments reveal that transient, HFD-elicited reduction of brain glucose uptake initiates a compensatory increase of VEGF production and assign obesity-associatedmacrophage activation a homeostatic role torestore cerebral glucose metabolism, preserve cognitive function, and limit neurodegeneration in obesity.

Activation of Agouti-related peptide (AgRP) neurons potently promotes feeding, and chronically altering their activity also affects peripheral glucose homeostasis. We demonstrate that acute activation of AgRP neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue (BAT). AgRP neuron activation acutely reprograms gene expression in BAT toward a myogenic signature, including increased expression of myostatin. Interference with myostatin activity improves insulin sensitivity that was impaired by AgRP neurons activation. Optogenetic circuitry mapping reveals that feeding and insulin sensitivity are controlled by both distinct and overlapping projections. Stimulation of AgRP → LHA projections impairs insulin sensitivity and promotes feeding while activation of AgRP → anterior bed nucleus of the stria terminalis (aBNST)vl projections, distinct from AgRP → aBNSTdm projections controlling feeding, mediate the effect of AgRP neuron activation on BAT-myostatin expression and insulin sensitivity. Collectively, our results suggest that AgRP neurons in mice induce not only eating, but also insulin resistance by stimulating expression of muscle-related genes in BAT, revealing a mechanism by which these neurons rapidly coordinate hunger states with glucose homeostasis.

Calorie-rich diets induce hyperphagia and promote obesity, although the underlying mechanisms remain poorly defined. We find that short-term high-fat-diet (HFD) feeding of mice activates prepronociceptin (PNOC)-expressing neurons in the arcuate nucleus of the hypothalamus (ARC). PNOCARC neurons represent a previously unrecognized GABAergic population of ARC neurons distinct from well-defined feeding regulatory AgRP or POMC neurons. PNOCARC neurons arborize densely in the ARC and provide inhibitory synaptic input to nearby anorexigenic POMC neurons. Optogenetic activation of PNOCARC neurons in the ARC and their projections to the bed nucleus of the stria terminalis promotes feeding. Selective ablation of these cells promotes the activation of POMC neurons upon HFD exposure, reduces feeding, and protects from obesity, but it does not affect food intake or body weight under normal chow consumption. We characterize PNOCARC neurons as a novel ARC neuron population activated upon palatable food consumption to promote hyperphagia. ISSN:0896-6273 ISSN:1097-4199

We analyzed the metabolic response to cortical spreading depression that drastically increases local energy demand to restore ion homeostasis. During single and multiple cortical spreading depressions in the rat cortex, we simultaneously monitored extracellular levels of glucose and lactate using rapid sampling microdialysis and glucose influx using 18 F-fluorodeoxyglucose positron emission tomography while tracking cortical spreading depression using laser speckle imaging. Combining the acquired data with steady-state requirements we developed a mass-conserving compartment model including neurons and glia that was consistent with the observed data. In summary, our findings are: (1) Early breakdown of glial glycogen provides a major source of energy during increased energy demand and leaves 80% of blood-borne glucose to neurons. (2) Lactate is used solely by neurons and only if extracellular lactate levels are >80% above normal. (3) Although the ratio of oxygen and glucose consumption transiently reaches levels 90%) of the overall energy supply is from oxidative metabolism. (4) During cortical spreading depression, brain release of lactate exceeds its consumption suggesting that lactate is only a circumstantial energy substrate. Our findings provide a general scenario for the metabolic response to increased cerebral energy demand.

Background and purpose: In vivo imaging of inflammatory processes is a valuable tool in stroke research. We here investigated the combination of two imaging modalities in the chronic phase after cerebral ischemia: magnetic resonance imaging (MRI) using intravenously applied ultra small supraparamagnetic iron oxide particles (USPIO), and positron emission tomography (PET) with the tracer [11C]PK11195. Methods: Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) by the macrosphere model and monitored by MRI and PET for 28 or 56 days, followed by immunohistochemical endpoint analysis. To our knowledge, this is the first study providing USPIO-MRI data in the chronic phase up to 8 weeks after stroke. Results: Phagocytes with internalized USPIOs induced MRI-T2∗ signal alterations in the brain. Combined analysis with [11C]PK11195-PET allowed quantification of phagocytic activity and other neuroinflammatory processes. From 4 weeks after induction of ischemia, inflammation was dominated by phagocytes. Immunohistochemistry revealed colocalization of Iba1+ microglia with [11C]PK11195 and ED1/CD68 with USPIOs. USPIO-related iron was distinguished from alternatively deposited iron by assessing MRI before and after USPIO application. Tissue affected by non-phagocytic inflammation during the first week mostly remained in a viably vital but remodeled state after 4 or 8 weeks, while phagocytic activity was associated with severe injury and necrosis accordingly. Conclusions: We conclude that the combined approach of USPIO-MRI and [11C]PK11195-PET allows to observe post-stroke inflammatory processes in the living animal in an intraindividual and longitudinal fashion, predicting long-term tissue fate. The non-invasive imaging methods do not affect the immune system and have been applied to human subjects before. Translation into clinical applications is therefore feasible.

Regional cerebral blood flow ( rCBF) is spatially and temporally adjusted to local energy needs. This coupling involves dilation of vessels both at the site of metabolite exchange and upstream of the activated region. Deficits in upstream blood supply limit the ‘capacity to raise rCBF’ in response to functional activation and therefore compromise brain function. We here demonstrate in rats that the ‘capacity to raise rCBF’ can be determined from real-time measurements of rCBF using laser speckle imaging during an energy challenge induced by cortical spreading depolarizations (CSDs). Cortical spreading depolarizations (CSDs) occur with high incidence in stroke and various other brain injuries and cause large metabolic changes. Various conditions of cerebral perfusion were induced, either by modifying microvascular tone, or by altering upstream blood supply independently. The increase in rCBF per unit of time in response to CSD was linearly correlated to the upstream blood supply. In an experimental model of stroke, we found that this marker of the capacity to raise rCBF which, in pathologic tissue may be additionally limited by impaired vasoactive signaling, was a better indicator of the functional status of cerebral tissue than local rCBF levels.

Inflammatory cells such as microglia need energy to exert their functions and to maintain their cellular integrity and membrane potential. Subsequent to cerebral ischemia, inflammatory cells infiltrate tissue with limited blood flow where neurons and astrocytes died due to insufficient supply with oxygen and glucose. Using dual tracer positron emission tomography (PET), we found that concomitant with the presence of inflammatory cells, transport and consumption of glucose increased up to normal levels but returned to pathological levels as soon as inflammatory cells disappeared. Thus, inflammatory cells established sufficient glucose supply to satisfy their energy demands even in regions with insufficient supply for neurons and astrocytes to survive. Our data suggest that neurons and astrocytes died from oxygen deficiency and inflammatory cells metabolized glucose non-oxidatively in regions with residual availability. As a consequence, glucose metabolism of inflammatory cells can mask metabolic deficits in neurodegenerative diseases. We further found that the PET tracer did not bind to inflammatory cells in severely hypoperfused regions and thus only a part of the inflammation was detected. We conclude that glucose consumption of inflammatory cells should be taken into account when analyzing disease-related alterations of local cerebral metabolism., Highlights • Inflammatory cells consume high amounts of glucose in supply-limited brain regions. • Glucose metabolism of inflammatory cells masks metabolic deficits in the brain. • In vivo markers only reach inflammatory cells in regions with residual blood supply. • Measuring inflammation and metabolism provide complementary information.

Minocycline has been reported to reduce infarct size after focal cerebral ischemia, due to an attenuation of microglia activation and prevention of secondary damage from stroke-induced neuroinflammation. We here investigated the effects of minocycline on endogenous neural stem cells (NSCs) in vitro and in a rat stroke model. Primary cultures of fetal rat NSCs were exposed to minocycline to characterize its effects on cell survival and proliferation. To assess these effects in vivo, permanent cerebral ischemia was induced in adult rats, treated systemically with minocycline or placebo. Imaging 7 days after ischemia comprised (i) Magnetic Resonance Imaging (MRI), assessing the extent of infarcts, (ii) Positron Emission Tomography (PET) with [(11)C]PK11195, characterizing neuroinflammation, and (iii) PET with 3'-deoxy-3'-[(18)F]fluoro-L-thymidine ([(18)F]FLT), detecting proliferating endogenous NSCs. Immunohistochemistry was used to verify ischemic damage and characterize cellular inflammatory and repair processes in more detail. In vitro, specific concentrations of minocycline significantly increased NSC numbers without increasing their proliferation, indicating a positive effect of minocycline on NSC survival. In vivo, endogenous NSC activation in the subventricular zone (SVZ) measured by [(18)F]FLT PET correlated well with infarct volumes. Similar to in vitro findings, minocycline led to a specific increase in endogenous NSC activity in both the SVZ as well as the hippocampus. [(11)C]PK11195 PET detected neuroinflammation in the infarct core as well as in peri-infarct regions, with both its extent and location independent of the infarct size. The data did not reveal an effect of minocycline on stroke-induced neuroinflammation. We show that multimodal PET imaging can be used to characterize and quantify complex cellular processes occurring after stroke, as well as their modulation by therapeutic agents. We found minocycline, previously implied in attenuating microglial activation, to have positive effects on endogenous NSC survival. These findings hold promise for the development of novel treatments in stroke therapy.

Background and Purpose— Experimental stroke models are essential to study in vivo pathophysiological processes of focal cerebral ischemia. In this study, an embolic stroke model in rats was applied (1) to characterize early development of regional cerebral blood flow and metabolism with positron emission tomography (PET) using [ 15 O]H 2 O and [ 18 F]-2-fluoro-2-deoxy-D-glucose (FDG); and (2) to identify potential parameters for predicting tissue fate. Methods— Remote occlusion of the middle cerebral artery was induced in 10 Wistar rats by injection of 4 TiO 2 macrospheres. Sequential [ 15 O]H 2 O-PET (baseline, 5, 30, 60 minutes after middle cerebral artery occlusion) and FDG-PET measurements (75 minutes after middle cerebral artery occlusion) were performed. [ 15 O]H 2 O-PET data and FDG kinetic parameters were compared with MRIs and histology at 24 hours. Results— Regional cerebral blood flow decreased substantially within 30 minutes after middle cerebral artery occlusion (41% to 58% of baseline regional cerebral blood flow; P K1 of FDG in all animals ( r =0.86±0.09; P K1 and net influx rate constant Ki of FDG. The infarct volume predicted by FDG-PET (375.8±102.3 mm 3 ) correlated significantly with the infarct size determined by MRI after 24 hours (360.8±93.7 mm 3 ; r =0.85). Conclusions— Hypoperfused tissue can be identified by decreased K1 of FDG. Acute ischemic tissue can be well characterized using K1 and Ki allowing for discrimination between infarct core and early viable tissue. Because FDG-PET is widely spread, our findings can be easily translated into clinical application for early diagnoses of ischemia.

The hypothesis of this study was that distinct experimental glioblastoma phenotypes resembling human disease can be noninvasively distinguished at various disease stages by imaging in vivo. Methods: Cultured spheroids from 2 human glioblastomas were implanted into the brains of nude rats. Glioblastoma growth dynamics were followed by PET using 18F-FDG, 11C-methyl-l-methionine (11C-MET), and 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and by MRI at 3–6 wk after implantation. For image validation, parameters were coregistered with immunohistochemical analysis. Results: Two tumor phenotypes (angiogenic and infiltrative) were obtained. The angiogenic phenotype showed high uptake of 11C-MET and 18F-FLT and relatively low uptake of 18F-FDG. 11C-MET was an early indicator of vessel remodeling and tumor proliferation. 18F-FLT uptake correlated to positive Ki67 staining at 6 wk. T1- and T2-weighted MR images displayed clear tumor delineation with strong gadolinium enhancement at 6 wk. The infiltrative phenotype did not accumulate 11C-MET and 18F-FLT and impaired the 18F-FDG uptake. In contrast, the Ki67 index showed a high proliferation rate. The extent of the infiltrative tumors could be observed by MRI but with low contrast. Conclusion: For angiogenic glioblastomas, noninvasive assessment of tumor activity corresponds well to immunohistochemical markers, and 11C-MET was more sensitive than 18F-FLT at detecting early tumor development. In contrast, infiltrative glioblastoma growth in the absence of blood–brain barrier breakdown is difficult to noninvasively follow by existing imaging techniques, and a negative 18F-FLT PET result does not exclude the presence of proliferating glioma tissue. The angiogenic model may serve as an advanced system to study imaging-guided antiangiogenic and antiproliferative therapies.

INTRODUCTION: Locally administered agents against malignant gliomas demand sophisticated metabolic imaging to monitor local and systemic treatment effects in animal models. The purpose of this feasibility study was to create an animal model suitable to represent the effect of locally delivered, soluble temozolomide in orthotopically implanted gliomas using micro-[18F] FLT-PET for high quality metabolic information with a distinct resolution for observation of treatment effects. METHODS: U87 glioma cells were stereotactically implanted in 48 female T-cell deficient athymic nude rats (RNU). Tumor existence and proliferation was proven by [18F] FLT-PET and microMRI. Rats were allocated to four treatment groups. Group one and two received a one-time intratumoral stereotactic injection of soluble temozolomide (TMZ) (group 1: 0.03mg; group 2: 0.015mg). Group three was administered systemic TMZ for 2 days (5mg). Group four served as untreated control group. Treatment was performed on day 15 post implantation. FLT-PET and MRI were performed 7 and 14 days after treatment. Animals showing signs of neurological impairment or reduced general health were immediately examined by MRI and sacrificed according to the animal ethical guidelines. RESULTS: The tumor take rate was 100 percent. Characteristically, U87 tumor cell formation was rather solid. Tumor size (MRI) and proliferation rate (PET) were congruent in all groups prior to treatment. Post treatment, FLT-PET was more specific for tumor growth compared to MRI. Tumor proliferation rates were significantly lower in the local treatment groups. MRI confirmed intratumoral TMZ fluid distribution which decreased within 7 days. CONCLUSION: Our orthotopic rat glioma model proved to be feasible and efficient for the performance of local therapy studies. Sophisticated Micro-PET offers distinct monitoring of tumor proliferation and supplements MR tumor imaging very well. The FLT-PET tracer is most suitable for the monitoring of local treatment effects of TMZ.

Decision making refers to the process by which subjects choose between competing courses of action based on the expected costs and benefits of their consequences. Lesion studies in rats suggest that the anterior cingulate cortex and the nucleus accumbens are key structures of a neural system that subserves effort-based decision making. Little is known about brain activation associated with effort-based decisions in intact rats. Using an open hypothesis approach, we used 2-deoxy-2[(18)F]fluoro-d-glucose positron emission tomography (FDG-PET) to assess regional metabolic changes in two conditions of an effort-based decision making task. In the “same effort” condition, male rats could choose between two response options associated with the same effort but different reward sizes, i.e., decision making was simply a function of reward size. By contrast, in the “different effort” condition, an integration of different efforts and reward sizes associated with the two response options was necessary before making a decision. Separate PET scans were performed from each condition. Subtractive analysis revealed that metabolic activity was increased in the different effort relative to the same effort condition in the left anterior cingulate, left orbitofrontal and prelimbic cortex region. Metabolic activity was decreased in the infralimbic cortex and septum region. Our findings suggest that making decisions on how much effort to invest to obtain greater rewards evokes changes of metabolic activity in multiple brain areas associated with cognitive, limbic, motor and autonomic functions. This study demonstrates that FDG-PET provides a tool to determine in rats regional brain metabolic activity in cognitive tasks.

Purpose Compartmental modelling of 3′-deoxy-3′-[18F]-fluorothymidine (18F-FLT) PET-derived kinetics provides a method for noninvasive assessment of the proliferation rate of gliomas. Such analyses, however, require an input function generally derived by serial blood sampling and counting. In the current study, 18F-FLT kinetic parameters obtained from image-derived input functions were compared with those from input functions derived from arterialized blood samples. Methods Based on the analysis of 11 patients with glioma (WHO grade II–IV) a procedure for the automated extraction of an input function from 18F-FLT brain PET data was derived. The time–activity curve of the volume of interest with the maximum difference in 18F-FLT uptake during the first 5 min after injection and the period from 60 to 90 min was corrected for partial-volume effects and in vivo metabolism of 18F-FLT. For each patient a two-compartment kinetic model was applied to the tumour tissue using the image-derived input function. The resulting kinetic rate constants K1 (transport across the blood–brain barrier) and Ki (metabolic rate constant or net influx constant) were compared with those obtained from the same data using the input function derived from blood samples. Additionally, the metabolic rate constant was correlated with the frequency of tumour cells stained with Ki-67, a widely used immunohistochemical marker of cell proliferation. Results The rate constants from kinetic modelling were comparable when the blood sample-derived input functions were replaced by the image-derived functions (K1,img and K1,sample, r = 0.95, p

Purpose: The aim of this study was to investigate the relationship between the in vivo derived kinetic parameters of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) and the proliferation rate measured in vitro by Ki-67 staining in patients with newly diagnosed high-grade gliomas. Experimental Design: Thirteen patients with newly diagnosed high-grade gliomas were investigated with 18F-FLT and methyl-11C- l-methionine (11C-MET) positron emission tomography (PET) and T1-, Gd-T1–, and T2-weighted magnetic resonance imaging on consecutive days. Tracer kinetic parameters of 18F-FLT as well as the standardized uptake value and the tumor-to-background (T/B) ratio of 18F-FLT and 11C-MET were determined. Data of kinetic modeling, standardized uptake value, and T/B values derived from 18F-FLT-PET were compared with T/B values derived from 11C-MET-PET and to the in vitro proliferation marker Ki-67. Results: A significant correlation was observed between the metabolic rate constant Ki and the proliferation index as measured by Ki-67 immunostaining [Ki, r = 0.79 (P = 0.004)]. Also, the phosphorylation rate constant k3 correlated with Ki-67 [k3, r = 0.76 (P = 0.006)], whereas the rate constant for transport through the blood brain barrier K1 showed a weaker correlation with Ki-67 [K1, r = 0.62 (P = 0.044)]. No significant correlation between 11C-MET and 18F-FLT uptake ratios and Ki-67 was observed. Conclusions: This study shows that kinetic analysis of 18F-FLT tracer uptake is essential for the in vivo assessment of tumor proliferation in high-grade gliomas, whereas uptake ratios of 11C-MET and 18F-FLT failed to correlate with the in vitro determined proliferation marker. Thus, kinetic analysis of 18F-FLT might provide an accurate method for the assessment of early response to glioma treatment in the future.

BACKGROUND: Stroke patients suffering from occlusion of the anterior cerebral artery (ACAo) develop cognitive and executive deficits. Experimental models to investigate such functional impairments and recovery are rare and not satisfyingly validated. NEW METHOD: We stereotactically injected the vasoconstrictor endothelin-1 (ET-1) close to the ACA of rats and assessed magnitude and course of CBF reduction using [(14)C]iodoantipyrine autoradiography and [(15)O]H2O-PET. [(18)F]FDG-PET and T2-weighted MRI determined regional metabolic and structural alterations. To test cognitive and executive functions, we analyzed decision-making in a food-carrying task, spatial working memory in a spontaneous alternation task and anxiety in an elevated plus maze test before and 1 month after ACAo. RESULTS: CBF decreased immediately after ET-1 injection, started to recover 1-2h and returned to control 4h thereafter. Metabolic and structural lesions developed permanently in the ACA territory. Hypometabolism occurring bilaterally in the piriform region may reflect diaschisis. Behavioral testing after ACAo revealed context-dependent changes in decision making, exploratory activity and walking speed, as well as decreased anxiety and spatial working memory. COMPARISON WITH EXISTING METHOD(S): Aside from modeling a known entity of stroke patients, ACAo in rats allows to longitudinally study deterioration of cognitive and executive function without major interference by disturbed primary motor function. It complements therefore stroke research since common models using middle cerebral artery occlusion (MCAo) all affect motor function severely. CONCLUSION: The established ACAo model in rats effectively reflects deficits characteristic for ACA stroke in humans. It is furthermore highly suitable for longitudinal assessment of cognitive and executive functions. publisher: Elsevier articletitle: Longitudinal assessment of infarct progression, brain metabolism and behavior following anterior cerebral artery occlusion in rats journaltitle: Journal of Neuroscience Methods articlelink: http://dx.doi.org/10.1016/j.jneumeth.2014.11.003 content_type: article copyright: Copyright © 2014 The Authors. Published by Elsevier B.V. ispartof: Journal of Neuroscience Methods vol:253 pages:279-291 ispartof: location:Netherlands status: published

The magnetic field signature obtained by Cassini during its first close encounter with Titan on 26 October 2004 is presented and explained in terms of an advanced model. Titan was inside the saturnian magnetosphere. A magnetic field minimum before closest approach marked Cassini's entry into the magnetic ionopause layer. Cassini then left the northern and entered the southern magnetic tail lobe. The magnetic field before and after the encounter was approximately constant for ∼20 Titan radii, but the field orientation changed exactly at the location of Titan's orbit. No evidence of an internal magnetic field at Titan was detected.

Neuroinflammation with microglia activation (MA) constitutes a key tissue response in acute stroke. Until now, its course in the chronic stage is less well defined. Here, we investigated (i) neuroinflammation in the chronic stage of a rat model of embolic stroke (n=6), and (ii) whether this process can be visualized in vivo by multimodal imaging using Magnetic Resonance Imaging (MRI) and Positron-Emission-Tomography (PET). Imaging data were verified using histology and immunohistochemistry. Repetitive PET studies until week 6 after stroke reveal poststroke inflammation as a dynamic process that involved the infarct, the surrounding tissue and secondary degenerating areas in a complex fashion. At the end, 7 months after stroke, neuroinflammation had almost completely vanished at the lesion side. In contrast, remote from the primarily infarcted areas, a marked T2(*)- hypointensity was detected in the ipsilateral thalamus. In the corresponding area, [(11)C]PK11195-PET detected microglia activation. Immunohistochemistry confirmed activated microglia in the ipsilateral thalamus with signs of extensive phagocytosis and iron deposition around plaque-like amyloid deposition. Neuronal staining (NeuN) revealed pronounced neuronal loss as an endpoint of neurodegeneration in these areas. In conclusion, the data demonstrate not only ongoing thalamic neuroinflammation but also marked neurodegeneration remote from the lesion site in the chronic phase after stroke in rats. Both, neuroinflammation and neurodegeneration were accessible to (immuno-) histochemical methods as well as to in vivo methods using [(11)C]PK11195-PET and T2(*)-weighted MRI. Although the functional roles of these dynamic processes remain to be elucidated, ongoing destruction of neuronal tissue is conceivable. Its inhibition using anti-inflammatory substances may be beneficial in chronic post-stroke conditions, while multimodal imaging can be used to evaluate putative therapeutic effects in vivo.

Brain function critically relies on the supply with energy substrates (oxygen and glucose) via blood flow. Alterations in energy demand as during neuronal activation induce dynamic changes in substrate fluxes and blood flow. To study the complex system that regulates cerebral metabolism requires the combination of methods for the simultaneous assessment of multiple parameters. We developed a multimodal imaging device to combine positron emission tomography (PET) with laser speckle imaging (LSI) and RGB reflectometry (RGBR). Depending on the radiotracer, PET provides 3-dimensional quantitative information of specific molecular processes, while LSI and RGBR measure cerebral blood flow (CBF) and hemoglobin oxygenation at high temporal and spatial resolution. We first tested the functional capability of each modality within our system and showed that interference between the modalities is negligible. We then cross-calibrated the system by simultaneously measuring absolute CBF using (15)O-H2O PET (CBF(PET)) and the inverse correlation time (ICT), the LSI surrogate for CBF. ICT and CBF(PET) correlated in multiple measurements in individuals as well as across different animals (R(2)=0.87, n=44 measurements) indicating that ICT can be used for absolute quantitative assessment of CBF. To demonstrate the potential of the combined system, we applied it to cortical spreading depression (CSD), a wave of transient cellular depolarization that served here as a model system for neurovascular and neurometabolic coupling. We analyzed time courses of hemoglobin oxygenation and CBF alterations coupled to CSD, and simultaneously measured regional uptake of (18)F-2-fluoro-2-deoxy-D-glucose ((18)F-FDG) used as a radiotracer for regional glucose metabolism, in response to a single CSD and to a cluster of CSD waves. With this unique combination, we characterized the changes in cerebral metabolic rate of oxygen (CMRO2) in real-time and showed a correlation between (18)F-FDG uptake and the number of CSD waves that passed the local tissue. Finally, we examined CSD spontaneously occurring during focal ischemia also referred to as peri-infarct depolarization (PID). In the vicinity of the ischemic territory, we observed PIDs that were characterized by reduced CMRO2 and increased oxygen extraction fraction (OEF), indicating a limitation of oxygen supply. Simultaneously measured PET showed an increased (18)F-FDG uptake in these regions. Our combined system proved to be a novel tool for the simultaneous study of dynamic spatiotemporal alterations of cortical blood flow, oxygen metabolism and glucose consumption under normal and pathologic conditions.

Complex biological systems often require measurements of multiple parameters with high temporal and spatial resolution. Multimodal approaches and the combination of methods are therefore a powerful tool to address such scientific questions. Laser speckle imaging (LSI) is an optical method that monitors dynamic changes in cortical blood flow (CBF) with high temporal resolution. Positron emission tomography (PET) allows for quantitative imaging of physiological processes and is a gold standard method to determine absolute cerebral blood flow. We developed a setup that allows simultaneous measurement with both modalities. Here, we simultaneously measured CBF with PET and LSI in rats and analyzed how the correlation of PET and LSI is modified when (1) different methods are used for the calculation of speckle inverse correlation time (ICT), (2) speckle data is acquired through thinned or craniectomized skull, (3) influence of surface vessels is removed from the speckle data. For the latter, a method for automated vessel segmentation from LSI data was developed. We obtained the best correlation (R2 = 0.890, p

In Alzheimer's disease (AD), persistent microglial activation as sign of chronic neuroinflammation contributes to disease progression. Our study aimed to in vivo visualize and quantify microglial activation in 13- to 15-month-old AD mice using [C-11]-(R)-PK11195 and positron emission tomography (PET). We attempted to modulate neuroinflammation by subjecting the animals to an anti-inflammatory treatment with pioglitazone (5-weeks' treatment, 5-week wash-out period). [C-11]-(R)-PK11195 distribution volume values in AD mice were significantly higher compared with control mice after the wash-out period at 15 months, which was supported by immunohistochemistry data. However, [C-11]-(R)-PK11195 mu PET could not demonstrate genotype-or treatment-dependent differences in the 13- to 14-month-old animals, suggesting that microglial activation in AD mice at this age and disease stage is too mild to be detected by this imaging method. (C) 2013 Elsevier Inc. All rights reserved.

Neural stem cells reside in two major niches in the adult brain [i.e., the subventricular zone (SVZ) and the dentate gyrus of the hippocampus]. Insults to the brain such as cerebral ischemia result in a physiological mobilization of endogenous neural stem cells. Since recent studies showed that pharmacological stimulation can be used to expand the endogenous neural stem cell niche, hope has been raised to enhance the brain's own regenerative capacity. For the evaluation of such novel therapeutic approaches, longitudinal and intraindividual monitoring of the endogenous neural stem cell niche would be required. However, to date no conclusive imaging technique has been established. We used positron emission tomography (PET) and the radiotracer 3′-deoxy-3′-[18F]fluoro-l-thymidine ([18F]FLT) that enables imaging and measuring of proliferation to noninvasively detect endogenous neural stem cells in the normal and diseased adult rat brainin vivo. This method indeed visualized neural stem cell niches in the living rat brain, identified as increased [18F]FLT-binding in the SVZ and the hippocampus. Focal cerebral ischemia and subsequent damage of the blood–brain barrier did not interfere with the capability of [18F]FLT-PET to visualize neural stem cell mobilization. Moreover, [18F]FLT-PET allowed for anin vivoquantification of increased neural stem cell mobilization caused by pharmacological stimulation or by focal cerebral ischemia. The data suggest that noninvasive longitudinal monitoring and quantification of endogenous neural stem cell activation in the brain is feasible and that [18F]FLT-PET could be used to monitor the effects of drugs aimed at expanding the neural stem cell niche.

Diseases and dysfunction of the central nervous system are often associated with regional changes in cerebral glucose metabolism, which can be measured in vivo by PET using 18F-FDG as the tracer. For quantification, the arterial tracer input function must be determined. For rodents in particular, direct measurement of blood radioactivity concentration is scarcely feasible for follow-up of individual animals because of the invasiveness of blood sampling. We show that the whiskers area of the rat9s muzzle serves as an extracerebral reference region. The derived model also takes into account local variations of the lumped constant, which is crucial in pathologic tissue. Methods: In 11 rats, the reference tissue kinetic parameters were determined from PET data and measured whole blood radioactivity concentration. Parametric images of cerebral kinetic rate constants were calculated using the directly measured input function, the reference tissue time–activity curve with individually fitted reference kinetic parameters, and the reference time–activity curve with fixed reference kinetic parameters calculated from the fitted parameters averaged over all animals. The need for kinetic modeling in disease models is demonstrated in 5 rats subjected to acute focal cerebral ischemia. 18F-FDG metabolism and transport rate constants and local cerebral glucose metabolic rates were calculated. Results: Cerebral kinetic constants derived from the 3 methods corresponded closely. The maximum difference in whole-brain kinetic parameters observed between the directly measured input function and the reference tissue time–activity curve with individually fitted reference kinetic parameters was less than 5%. Taking fixed reference parameters (the reference time–activity curve with fixed reference kinetic parameters calculated from the fitted parameters averaged over all animals) still provided whole-brain kinetic parameters with an accuracy of approximately 90%. In the rats subjected to focal cerebral ischemia, 18F-FDG kinetic parameters in healthy tissue were not significantly different from whole-brain kinetic parameters in naive rats. The ischemic region was characterized by preserved glucose metabolism, although 18F-FDG uptake was elevated significantly—that is, the lumped constant in the ischemic region was different from that of healthy brain tissue. Conclusion: The method presented here allows for the quantitative noninvasive determination of cerebral glucose consumption in rats, takes into account local variations of the lumped constant, and is suitable for follow-up measurements of individuals.

We present two patients with glioblastoma with an unusually stable clinical course and long-term survival who were treated after surgery and radiotherapy with adjuvant temozolomide (TMZ) chemotherapy for 17 and 20 cycles, respectively. Afterward, adjuvant TMZ chemotherapy was discontinued in one patient and the dosage of TMZ was reduced in the other. In addition to clinical status and magnetic resonance imaging, the biologic activity of the tumors was monitored by repeated methyl- 11 C-l-methionine (MET) and 3′-deoxy-3′- 18 F-fluorothymidine (FLT) positron emission tomography (PET) studies in these patients. In these patients, repeated MET-and FLT-PET imaging documented complete response to the initial treatment regimen, including resection, radiation, and TMZ, and during the course of the disease, recurrent, uncontrollable tumor activity. Continuation or dose escalation of TMZ in both patients was shown to be ineffective to overcome the metabolic activity of the tumor. Our data suggest that repeated MET- and FLT-PET imaging provide information on the biologic activity of a tumor that is highly useful to monitor and detect changes in activity.

Focal cerebral ischemia elicits strong inflammatory responses involving activation of resident microglia and recruitment of monocytes/macrophages. These cells express peripheral benzodiazepine receptors (PBRs) and can be visualized by positron emission tomography (PET) using [11C]PK11195 that selectively binds to PBRs. Earlier research suggests that transient ischemia in rats induces increased [11C]PK11195 binding within the infarct core. In this study, we investigated the expression of PBRs during permanent ischemia in rats. Permanent cerebral ischemia was induced by injection of macrospheres into the middle cerebral artery. Multimodal imaging 7 days after ischemia comprised (1) magnetic resonance imaging that assessed the extent of infarcts; (2) [18F]-2-fluoro-2-deoxy-d-glucose ([18F]FDG)-PET characterizing cerebral glucose transport and metabolism; and (3) [11C]PK11195-PET detecting neuroinflammation. Immunohistochemistry verified ischemic damage and neuroinflammatory processes. Contrasting with earlier data for transient ischemia, no [11C]PK11195 binding was found in the infarct core. Rather, permanent ischemia caused increased [11C]PK11195 binding in the normoperfused peri-infarct zone (mean standard uptake value (SUV): 1.93 ± 0.49), colocalizing with a 60% increase in the [18F]FDG metabolic rate constant with accumulated activated microglia and macrophages. These results suggest that after permanent focal ischemia, neuroinflammation occurring in the normoperfused peri-infarct zone goes along with increased energy demand, therefore extending the tissue at risk to areas adjacent to the infarct.

Radiolabelled epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitors potentially facilitate the assessment of EGFR overexpression in tumors. Since elaborate multi-step radiosyntheses are required for (18)F-labelling of EGFR-specific anilinoquinazolines we report on the development of a two-step click labelling approach that was adapted to a fully automated synthesis module. 6-(4-N,N-Dimethylaminocrotonyl)amido-4-(3-chloro-4-fluoro)phenylamino-7-{3-[4-(2-[(18)F]fluoroethyl)-2,3,4-triazol-1-yl]propoxy}quinazoline ([(18)F]6) was synthesized via Huisgen 1,3-dipolar cycloaddition between 2-[(18)F]fluoroethylazide ([(18)F]4) and the alkyne modified anilinoquinazoline precursor 5. PET images of PC9 tumor xenograft using the novel biomarker showed promising results to visualize EGFR overexpression.

Introduction: 18F-Fluoro-L-thymidine (18F-FLT) has been established as a non-invasive marker for tumor proliferation by positron emission tomography (PET). It has been shown that the uptake of 18F-FLT might correlate to increased transport as well as to S-phase specific thymidine kinase 1 (TK1) activity (Jacobs et al. 2005). Aim of the study was to further characterize 18F-FLT as marker for tumor activity and proliferation in patients with newly diagnosed gliomas. Material and methods: 13 patients (age: from 35 to 71y) with newly diagnosed primary brain tumors underwent 18F-FLT- and 11C-MET-PET, as well as T1- and T2-weighted MRI with and without contrast media on consecutive days. Tracer kinetics (k1, k2, k3, ki) were determined in tumor and in contralateral grey matter. The uptake of 18F-FLT was calculated by the standardized uptake value (SUV) and the tumor-to-background (T/B) ratio. Data of kinetic modelling, SUV and T/B values derived from 18F-FLT-PET were compared to T/B values derived from 11C-MET-PET and histology. Results: In 12 patients the diagnosis of a glioma was confirmed by histology (WHO III: n=6; WHO IV: n=6. In 4/6 patients with anaplastic gliomas no significant k3 value was measured. In contrast, in all patients with glioblastomas (n=6) k3 values ranged between 0,02/min and 0,015/min. The net influx constant ki correlated significantly with the FLT uptake ratio (p=0,002, r=0,78). Moreover, a significant correlation was observed between the SUV and metabolic rate constants ki and k3 with the proliferation index as measured by Ki67 immunostaining (p=0,038, r=0,604; p=0,023, r=0,65; p=0,043, r=0,60). A positive correlation was found between 11C-MET uptake and 18F-FLT uptake indicating the relationship between the increased transport of amino acids and nucleosides into the tumor (p=0,016, r=0,65). Conclusion: FLT uptake is a measure of increased uptake as measured by Ki and increased trapping in proliferating tumor cells as measured by k3at least in patients with glioblastoma. The significant correlation between ki and k3 with the histological proliferation index ki67 demonstrates that FLT uptake might be an indirect measure of tumor proliferation. Moreover, the positive correlation between 11C-MET uptake and k3 indicates, that 18F-FLT uptake is related to cell proliferation in areas with an increased microvessel density.

Titan's upper atmosphere is ionized by solar radiation and particle bombardment from Saturn's magnetosphere. The induced ionosphere plays a key role in the coupling of Titan's atmosphere with the Kronian environment. It also provides unique signatures for identifying energy sources upon Titan's upper atmosphere. Here we focus on observations from the first, close flyby by the Cassini spacecraft and assess the ionization and electron heating sources in Titan's sunlit ionosphere. We compare CAPS electron spectra with spectra produced by an electron transport model based on the INMS neutral densities and a MHD interaction model. In addition, we compare RPWS electron temperature against the models. The important terms in the electron energy equation include loss through excitation of vibrational states of N-2 and CH4, Coulomb collisions with suprathermal electrons, and thermal conduction. Our analysis highlights the important role of the magnetic field line configuration for aeronomic studies at Titan.