Miklós Tóth

The gut microbiota influences blood-brain barrier permeability in mice

The intestinal microbiota helps to maintain the integrity of the blood-brain barrier in fetal and adult mice. The Gut Microbiota and the Blood-Brain Barrier The blood-brain barrier is an important gateway that controls the passage of molecules and nutrients in and out of the brain. An intact blood-brain barrier is a crucial checkpoint for appropriate development and function of the brain. Braniste et al. now show that germ-free pregnant dams, devoid of maternal microbes, have offspring that show increased permeability of the blood-brain barrier. This elevated permeability was also observed in adult germ-free mice. However, fecal transplants from mice exposed to bacteria into adult germ-free mice reduced blood-brain barrier permeability, possibly through the regulation of tight junction proteins. These findings suggest that crosstalk between the gut microbiota and the brain, initiated during the intrauterine period, is perpetuated throughout life. Pivotal to brain development and function is an intact blood-brain barrier (BBB), which acts as a gatekeeper to control the passage and exchange of molecules and nutrients between the circulatory system and the brain parenchyma. The BBB also ensures homeostasis of the central nervous system (CNS). We report that germ-free mice, beginning with intrauterine life, displayed increased BBB permeability compared to pathogen-free mice with a normal gut flora. The increased BBB permeability was maintained in germ-free mice after birth and during adulthood and was associated with reduced expression of the tight junction proteins occludin and claudin-5, which are known to regulate barrier function in endothelial tissues. Exposure of germ-free adult mice to a pathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junction proteins. Our results suggest that gut microbiota–BBB communication is initiated during gestation and propagated throughout life.

Performance Evaluation of the Small-Animal nanoScan PET/MRI System

nanoScan is a high-resolution integrated system for consecutive PET and MR imaging of small laboratory animals. We evaluated the performance of the system, using the NEMA NU 4-2008 protocol for the PET component and the NEMA MS 1-2007, MS 2-2008, and MS 3-2007 standards for the MR imaging component. Methods: The imaging system uses magnetically shielded position-sensitive photomultiplier tubes and a compact 1-T permanent-magnet MR imaging platform. Spatial resolution, sensitivity, counting rate capabilities, and image quality parameters were evaluated in accordance with the aforementioned NEMA standards. Further in vivo evaluation experiments complement the physical validation results. Results: The spatial resolution of the PET system enabled the 0.8-mm rods of a Derenzo phantom to be resolved. With point source and 2-dimensional filtered backprojection reconstruction, the resolution varied from 1.50 to 2.01 mm in full width at half maximum in the radial direction and from 1.32 to 1.65 mm in the tangential direction within the radius of 25 mm. Peak absolute sensitivity was 8.41%. Scatter fraction was 17.3% and 34.0%, and maximum noise-equivalent counting rate was 406 and 119 kcps in the mouselike and ratlike phantom, respectively. The image quality test found a nonuniformity of 3.52% and a spillover ratio of 6.2% and 5.8% in water and air, respectively. In testing of the MR imaging component, artifact-free images with high signal-to-noise ratio were recorded. Geometric distortion was below 5%, and image uniformity was at least 94.5% and 96.6% for the 60- and 35-mm radiofrequency coils, respectively. Conclusion: The nanoScan integrated small-animal PET/MR imaging system has excellent spatial resolution and sensitivity. The performance characteristics of the PET and the MR imaging components are not compromised as a result of their integration onto a single platform. Because of its combination of features and performance parameters, the system provides crucial advantages for preclinical imaging studies over existing PET/CT systems, especially in neurologic and oncologic research.

Age and disease related changes in the translocator protein (TSPO) system in the human brain: Positron emission tomography measurements with [11C]vinpocetine

BACKGROUNDS AND PURPOSE The main objectives of the present study were (i) to measure density changes of activated microglia and the peripheral benzodiazepine receptor/translocator protein (TSPO) system during normal ageing in the human brain with positron emission tomography (PET) using the TSPO molecular imaging biomarker [(11)C]vinpocetine and (ii) to compare the level and pattern of TSPO in Alzheimer (AD) patients with age matched healthy subjects, in order to assess the biomarkers usefulness as a diagnostic imaging marker in normal (ageing) and pathological (AD) up-regulation of microglia. METHODS AND SUBJECTS PET measurements were made in healthy volunteers, aged between 25 and 78 years, and AD patients, aged between 67 and 82 years, using [(11)C]vinpocetine as the tracer. Global and regional quantitative parameters of tracer uptake and binding, including time activity curves (TAC) of standard uptake values (%SUV), binding affinity parameters, intensity spectrum and homogeneity of the uptake distribution were measured and analysed. RESULTS Both %SUV and binding values increased with age linearly in the whole brain and in all brain regions. There were no significant differences between the %SUV values of the AD patients and age matched control subjects. There were, however, significant differences in %SUV values in a large number of brain regions between young subjects and old subjects, as well as young subjects and AD patients. The intensity spectrum analysis and homogeneity analysis of the voxel data show that the homogeneity of the %SUV values decreases with ageing and during the disease, whereas the centre of the intensity spectrum is shifted to higher %SUV values. These data indicate an inhomogeneous up-regulation of the TSPO system during ageing and AD. These changes were significant between the group of young subjects and old subjects, as well as young subjects and AD patients, but not between old subjects and AD patients. CONCLUSIONS The present data indicate that [(11)C]vinpocetine may serve as a molecular imaging biomarker of the activity of the TSPO system and, consequently, of the up-regulation of microglia during ageing and in neuroinflammatory diseases. However, the global and regional brain %SUV values between AD patients and age matched controls are not different from each other. The disease specific changes, measured with [(11)C]vinpocetine in AD, are significantly different from those measured in age matched controls only if the inhomogeneities in the uptake pattern are explored with advanced mathematical techniques. For this reason, PET studies using [(11)C]vinpocetine, as molecular imaging biomarker, can efficiently visualise the activation of microglia and the up-regulation of TSPO during ageing and in diseased brains with the help of an appropriate inhomogeneity analysis of the radioligands brain uptake pattern.

Quantification of serotonin transporter availability with [11C]MADAM--a comparison between the ECAT HRRT and HR systems.

UNLABELLED The High Resolution Research Tomograph (HRRT) is the PET system providing the highest resolution for imaging of the human brain. In this study, the improved quantitative performance of the HRRT was evaluated in comparison with a previously developed lower resolution PET system, the ECAT HR. The radioligand [(11)C]MADAM was chosen for the purpose since it provides a signal for serotonin transporter (5-HTT) binding in cortical and sub-cortical brain regions of different sizes and expressing different 5-HTT densities. A secondary objective was to assess the effect of partial volume effect (PVE) correction on the cross-comparability between the two systems. METHOD Six male control subjects (ages 20-35 yr) were examined twice using the HRRT and the HR system, respectively. Regions of interest (ROIs) included cortical regions (frontal cortex, temporal cortex, insula, anterior cingulate cortex, and hippocampus), sub-cortical regions (caudate, putamen, thalamus, dorsal brainstem and ventral midbrain) and cerebellum. The ROIs were manually delineated on T1-weighted MRI-images and subsequently applied to both HRRT and HR images. Regional binding potential (BP(ND)) values were calculated with the simplified reference tissue model (SRTM) using cerebellum as the reference region. The percent difference in BP(ND) between the systems was calculated for each ROI. In addition, both HRRT and HR data were corrected for PVE using established MRI-based methods described by Meltzer and Müller-Gärtner. The effect of PVE correction (PVEc) on the agreement between the systems was assessed via percent difference calculation and linear regression analysis. RESULTS Quantification with SRTM showed that regional BP(ND) values for [(11)C]MADAM were on average 23% higher for the HRRT than those obtained by the HR system. More specifically, BP(ND) measured with HRRT was 31.1±48.1% higher in neocortical/limbic regions and 14.6±20.9% higher in sub-cortical regions. The effect of PVEc varied between regions. After correction according to Müller-Gärtner, the agreement between systems was best in the neocortical/limbic regions (3.7±22.5%). With the exception of the caudate, in which the agreement was improved by approximately 17% using the Meltzer method, the effect of PVEc in sub-cortical regions was less pronounced. Linear regression analysis showed improved correlation between the two systems after PVEc, particularly in the neocortical/limbic regions. CONCLUSION As expected, BP(ND) values measured with the HRRT were higher than those measured with the HR due to higher resolution and recovery. The difference in BP(ND) between the two systems was approximately 30% in the neocortical/limbic regions. PVEc improved the agreement between the systems in particular for the neocortical/limbic regions. In these regions, the best agreement was found after applying Müller-Gärtners PVEc. The demonstrated agreement provides an opportunity for combining data between the two systems in clinical studies aimed at evaluating receptor/transporter availability in cortical brain regions.

Visualising Neuroinflammation in Post-Stroke Patients: A Comparative PET Study with the TSPO Molecular Imaging Biomarkers [ 11 C]PK11195 and [ 11 C]vinpocetine

With the main objective of comparing the prospective diagnostic power of two 11C-labelled molecular imaging biomarkers with affinity for TSPO and used for the visualisation of activated microglia after a stroke, we measured with positron emission tomography (PET) in four post-stroke patients the regional brain uptake and binding potential of [11C]vinpocetine and [11C]PK11195. Percentage standard uptake values (%SUV) and binding potential (BPND) were used as outcome measures. The total peak brain uptake value and average global brain uptake value were higher for [11C]vinpocetine than for [11C]PK11195. The regional %SUV values were significantly higher for [11C]vinpocetine than for [11C]PK11195 in the hemispheres as well as in almost all standard brain regions. The %SUV values of [11C]vinpocetine were higher in the peri-infarct zone than in the ischaemic core, however, the difference did not prove to be significant. There was basically no difference in %SUV values between the ischaemic core and the peri-infarct zone for [11C]PK11195. The BPND values for [11C]vinpocetine were higher in all standard regions than those for [11C]PK11195, but the difference was not significant between them. The BPND values of [11C]vinpocetine were higher in the peri-infarct zone than in the ischaemic core, however, the difference did not prove to be significant. A comparative analysis of the two ligands indicates that [11C]vinpocetine shows a number of favourable characteristics over [11C]PK11195, but to demonstrate that it may serve as a prospective molecular imaging biomarker of microglia activation in post-stroke patients, further studies are required.

Kinetic Analysis and Quantification of the Dopamine Transporter in the Nonhuman Primate Brain with 11C-PE2I and 18F-FE-PE2I

18F-(E)-N-(3-iodoprop-2-enyl)-2β-carbofluoroethoxy-3β- (4′-methyl-phenyl)nortropane (18F-FE-PE2I) is a novel radioligand for dopamine transporter (DAT) PET. As compared with 11C-N-(3-iodoprop-2E-enyl)-2β-carbomethoxy-3β-(4-methylphenyl)nortropane (11C-PE2I), 18F-FE-PE2I shows faster kinetics and more favorable metabolism, with less production of a radiometabolite with intermediate lipophilicity (M1), which—in the case of 11C-PE2I—has been shown to enter the rat brain. In this study, we compared DAT quantification with 11C-PE2I and 18F-FE-PE2I in nonhuman primates, using kinetic and graphical analysis with the input function of both the parent and the radiometabolite, to assess the potential contribution of the radiometabolite. Methods: Three rhesus monkeys were examined with 11C-PE2I and 18F-FE-PE2I using the HRRT system. Arterial input functions of the parent and radiometabolite M1 were measured. Kinetic and graphical analyses were applied using either the parent input (methods 1 and 3) or the parent plus radiometabolite input (methods 2 and 4). Outcome measures were distribution volumes (VT and VND), specific-to-nondisplaceable tissue radioactivity ratio at equilibrium (BPND; parent input), and specific–to–nondisplaceable tissue radioactivity ratio at equilibrium in the presence of metabolites (RT; parent plus radiometabolite input). Results: 11C-PE2I showed higher distribution volumes than 18F-FE-PE2I calculated with methods 1 and 3 (striatal VT, ∼300%; VND in cerebellum, ∼30%). With methods 2 and 4, VT in the striatum was approximately 60% higher in the case of 11C-PE2I, whereas no difference in VND was found in the cerebellum. For each radioligand, BPND estimated with methods 1 and 3 tended to be higher than RT estimated with methods 2 and 4. However, the bias of BPND, compared with RT, was much larger for 11C-PE2I (40%–60% in the caudate and putamen) than for 18F-FE-PE2I (<10% in the caudate and putamen). Conclusion: The direct comparison between the radioligands confirmed that 18F-FE-PE2I shows faster kinetics and more favorable metabolism than 11C-PE2I. The kinetic and graphical analyses with the input function of the parent and radiometabolite showed that the bias in BPND was much lower for 18F-FE-PE2I than for 11C-PE2I and suggested that the lower production of the radiometabolite M1 would make 18F-FE-PE2I more suitable for the DAT quantification. Further studies in humans are necessary to confirm these findings.

Development of a novel fluorine-18 labeled deuterated fluororasagiline ([18F]fluororasagiline-D2) radioligand for PET studies of monoamino oxidase B (MAO-B)

The objective of this study was to synthesize and evaluate a novel fluorine-18 labeled deuterium substituted analogue of rasagiline (9, [(18)F]fluororasagiline-D2) as a potential PET radioligand for studies of monoamine oxidase B (MAO-B). The precursor compound (6) and reference standard (7) were synthesized in multi-step syntheses. Radiolabeling of 9 was accomplished by a two-step synthesis, compromising a nucleophilic substitution followed by hydrolysis of the sulfamidate group. The incorporation radiochemical yield from fluorine-18 fluoride was higher than 30%, the radiochemical purity was >99% and the specific radioactivity was >160GBq/μmol at the time of administration. In vitro compound 7 inhibited the MAO-B activity with an IC50 of 173.0±13.6nM. The MAO-A activity was inhibited with an IC50 of 9.9±1.1μM. The fluorine-18 version 9 was characterized in the cynomolgus monkey brain where a high brain uptake was found (275% SUV at 4min). There was a higher uptake in the striatum and thalamus compared to the cortex and cerebellum. A pronounced blocking effect (50% decrease) was observed in the specific brain regions after administration of l-deprenyl (0.5mg/kg) 30min prior to the administration of 9. Radiometabolite studies demonstrated 40% of unchanged radioligand at 90min post injection. An efficient radiolabeling of 9 was successfully established and in the monkey brain 9 binds to MAO-B rich regions and its binding is blocked by the selective MAO-B compound l-deprenyl. The radioligand 9 is a potential candidate for human PET studies.

Preclinical Characterization of a Novel Class of 18F-Labeled PET Tracers for Amyloid-β

Imaging of amyloid-β (Aβ) plaques by PET is more and more integrated into concepts for Alzheimer disease (AD) diagnosis and drug development. The objective of this study was to find novel chemical entities that can be transformed into 18F-labeled Aβ tracers with favorable brain washout kinetics and low background signal. Methods: High-throughput screening of a large chemical library was used to identify new ligands for fibrillar aggregates of Aβ1–42 peptide. Thirty-two fluorinated derivatives were synthesized and tested for their affinity toward AD brain homogenate. Twelve ligands have been radiolabeled with 18F. The pharmacokinetic properties of the radioligands were investigated in mouse and monkey biodistribution studies. Binding characteristics were determined by autoradiography of AD brain sections in vitro and using amyloid precursor protein transgenic mice in vivo. Results: The systematic search for Aβ imaging agents revealed several fluorinated derivatives with nanomolar affinity for Aβ. The fluoropyridyl derivative BAY 1008472 showed a high initial brain uptake (6.45 percentage injected dose per gram at 2 min) and rapid brain washout (ratio of percentage of injected dose per gram of tissue at 2 and 30 min after injection, 9.2) in mice. PET studies of healthy rhesus monkeys confirmed the high initial brain uptake of BAY 1008472 (2.52 standardized uptake value at peak) and a fast elimination of total radioactivity from gray and white matter areas (ratio of standardized uptake value at peak uptake and 60 min 11.0). In autoradiographic analysis, BAY 1008472 selectively detected Aβ deposits in human AD brain sections with high contrast and did not bind to τ- or α-synuclein pathologies. Finally, ex vivo autoradiography of brain sections from amyloid precursor protein-transgenic mice confirmed that BAY 1008472 is indeed suitable for the in vivo detection of Aβ plaques. Conclusion: A new chemical class of Aβ tracers has been identified by high-throughput screening. The fluoropyridyl derivative BAY 1008472 shows a favorable preclinical profile including low background binding in gray and white matter. These properties might qualify this new tracer, in particular, to detect subtle amounts or changes of Aβ burden in presymptomatic AD and during therapy.

In vivo evaluation in cynomolgus monkey brain and metabolism of [18F]fluorodeprenyl: A new MAO-B pet radioligand

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO‐B) radioligand, [18F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [18F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [11C]deprenyl, an established MAO‐B radioligand. The highest uptake was observed in the striatum, one of the MAO‐B‐rich regions, with a peak at approximately 2–3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [18F]fluorodeprenyl was largely inhibited by preadministration of the MAO‐B inhibitor, L‐deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO‐B. [18F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [11C]deprenyl. These results suggest that [18F]fluorodeprenyl is a potential PET radioligand for visualization of MAO‐B activity. Synapse, 2012.

Molecular Imaging of PDE10A Knockout Mice with a Novel PET Radiotracer: [ 11 C]T-773

Purpose[11C]T-773 is a new radioligand for positron emission tomography (PET) targeting the phosphodiesterase 10A enzyme (PDE10A). PDE10A is highly expressed in the striatum by medium spiny neurons, and it has been demonstrated to be involved in the regulation of striatal signaling through the reduction of medium spiny neuronal sensitivity towards glutamatergic excitation. PDE10A is associated with Parkinson’s disease and different neuropsychiatric disorders such as Huntington’s disease, obsessive-compulsive disorders (OCD) and schizophrenia. Studies have indicated that the inhibition of PDE10A may represent a novel therapeutic approach to the treatment of the aforementioned diseases characterized by the reduced activity of medium spiny neurons. An appropriate PET radioligand for PDE10A would help to facilitate drug development and drug evaluation.ProceduresWe have evaluated the [11C]T-773 ligand in PDE10A knockout mice (heterozygous [HET] and homozygous [HOM]) as well as in normal control animals (WILD) with PET.ResultsThe regional percent standardized uptake values (%SUV; mean ± SD) in the striatum were 48.2 ± 1.0 (HOM), 63.6 ± 5.3 (HET) and 85.1 ± 6.3 (WILD). Between each animal group the striatal %SUV values were significantly different (p < 0.0001). The striatal BPND values (mean ± SD) were 0.0 ± 0.0 (HOM), 0.14 ± 0.07 (HET) and 0.56 ± 0.15 (WILD). The BPND values were significantly lower in homozygous and heterozygous animals compared to wild type (p < 0.0001).ConclusionsThe novel PDE10A radioligand [11C]T-773 shows increased signals with higher levels of PDE10A and acceptable binding in the striatum in control animals compared to knockout mice.