Non-invasive assessment of microvascular dysfunction in patients with microvascular angina.

BACKGROUND: We aimed to evaluate the microvascular function in patients with microvascular angina (MVA) by assessing 1) the endothelial glycocalyx barrier properties using sublingual microscopy, and 2) the myocardial perfusion reserve using cardiovascular magnetic resonance (CMR) imaging. METHODS: Sublingual microscopy was performed in 13 MVA patients (angina pectoris, ST-depression on treadmill testing, normal coronary angiogram) and compared with 2 control groups of 13 volunteers and 14 patients with known obstructive coronary artery disease (CAD). To test the glycocalyx-mediated microvascular responsiveness, the erythrocyte perfused boundary region (PBR) was assessed at baseline and after nitroglycerin challenge. RESULTS: The baseline PBR of MVA patients was similar to controls with CAD (p=0.72), and larger than in volunteers (p=0.02). Only the volunteers demonstrated a significant increase in PBR after nitroglycerin (p=0.03). In the 13 MVA patients, adenosine stress CMR perfusion imaging was performed. Although a significant increase in myocardial perfusion was observed in both the subendocardium and subepicardium during stress, the subendocardial perfusion reserve was significantly lower (p=0.02). The PBR responsiveness of the sublingual microvasculature showed a strong correlation with the transmural myocardial perfusion reserve (r=0.86, p<0.001). CONCLUSIONS: Patients with MVA can be characterized by microvascular glycocalyx dysfunction using sublingual microscopy. The strong correlation between sublingual PBR responsiveness and myocardial perfusion reserve suggests that the glycocalyx may play an important role in the regulation of microvascular volume for myocardial perfusion and supports the concept of impaired glycocalyx barrier properties in MVA.

Efficacy of radiation safety glasses in interventional radiology.

PURPOSE: This study was designed to evaluate the reduction of the eye lens dose when wearing protective eyewear in interventional radiology and to identify conditions that optimize the efficacy of radiation safety glasses. METHODS: The dose reduction provided by different models of radiation safety glasses was measured on an anthropomorphic phantom head. The influence of the orientation of the phantom head on the dose reduction was studied in detail. The dose reduction in interventional radiological practice was assessed by dose measurements on radiologists wearing either leaded or no glasses or using a ceiling suspended screen. RESULTS: The different models of radiation safety glasses provided a dose reduction in the range of a factor of 7.9-10.0 for frontal exposure of the phantom. The dose reduction was strongly reduced when the head is turned to the side relative to the irradiated volume. The eye closest to the tube was better protected due to side shielding and eyewear curvature. In clinical practice, the mean dose reduction was a factor of 2.1. Using a ceiling suspended lead glass shield resulted in a mean dose reduction of a factor of 5.7. CONCLUSIONS: The efficacy of radiation protection glasses depends on the orientation of the operator's head relative to the irradiated volume. Glasses can offer good protection to the eye under clinically relevant conditions. However, the performance in clinical practice in our study was lower than expected. This is likely related to nonoptimized room geometry and training of the staff as well as measurement methodology.

Brain imaging in chronic epilepsy patients after depth electrode (stereoelectroencephalography) implantation: magnetic resonance imaging or computed tomography?

BACKGROUND: The accurate localization of depth electrodes in epilepsy surgery is important for correct interpretation of stereoelectroencephalography recordings and neurosurgical resection. Unfortunately, image quality in postimplantation magnetic resonance imaging (MRI) is degraded by metal artifacts. The registration of postimplantation computed tomography (CT) or MRI to preimplantation (artifact-free) MRI facilitates electrode imaging and optimal visualization of brain anatomy. However, registration errors negatively affect electrode localization accuracy. OBJECTIVE: To compare the relative registration deviation between postimplantation CT and MRI with preimplantation MRI. METHODS: Retrospectively, 14 pharmacoresistant epilepsy patients were included who underwent stereotactic insertion of multiple depth electrodes and preimplantation and postimplantation MRI and postimplantation CT. Postimplantation MRI and CT image sets were registered to preimplantation MRI. The registration error between the registered postimplantation MRI and CT was quantified by measuring the geometrical distance between the electrodes of the registered postimplantation CT and the postimplantation MRI. RESULTS: The registration error of postimplantation imaging to preimplantation MRI was dependent on the algorithm used. After optimization, the smallest registration error was 1.22 ± 0.29 mm (mean ± SD) at the tip and 2.25 ± 1.18 mm at the base of the electrode. CONCLUSION: The good correspondence between the CT/MRI and the MRI/MRI registration suggests that either postimplantation MRI or CT is sufficient for accurate electrode localization. In case of postoperative morphological brain deformations, postimplantation MRI is still recommended.

Imaging cold-activated brown adipose tissue using dynamic T2*-weighted magnetic resonance imaging and 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography.

OBJECTIVES: The objective of this study was to explore the use of magnetic resonance imaging (MRI) to identify and quantify active brown adipose tissue (BAT) in adult humans. 2-Deoxy-2-[F]fluoro-D-glucose (FDG) positron emission tomography (PET) combined with computed tomography was used as a reference method to identify active BAT depots and to guide the MRI data analysis. MATERIALS AND METHODS: The ethics committee of the institute approved the protocol, and all participants provided written informed consent before participation. Both PET combined with computed tomography and MRI of BAT were performed in 11 healthy volunteers. Brown adipose tissue was activated by cooling the participants using a dedicated water-perfused suit. For the MRI examination of BAT, water-fat imaging and dynamic T2* imaging were performed at an effective temporal resolution of 2 minutes per volume. Water-fat images were derived from a multiecho MRI sequence using the Dixon technique. RESULTS: 2-Deoxy-2-[F]fluoro-D-glucose-PET identified active BAT in 8 of the 11 participants. Water-fat MRI showed that BAT depots had a fat fraction of 65.2% (7.0%) compared with 81.5% (5.4%) for the subcutaneous white adipose tissue (paired difference of 16.3% [4.9%]; P < 0.05). Dynamic T2* imaging during cold stimulation revealed signal fluctuations that were sensitive to BAT activation. The presence of these components correlated with BAT activation quantified from FDG-PET (r = 0.63; P < 0.05). CONCLUSIONS: Although FDG-PET has superior contrast for identifying active BAT, the MRI temporal resolution provides insight in activation dynamics. In addition, the flexibility of MRI allows for simultaneous mapping of tissue fat content and functional responses. The results indicate that MRI is a promising addition to PET for the identification of BAT and its activity responses to stimulation. An MRI-based methodology to quantify BAT activity is a highly desirable step in addressing the role of BAT in obesity disorders.

Extracting atomic numbers and electron densities from a dual source dual energy CT scanner: experiments and a simulation model.

BACKGROUND AND PURPOSE: Dual energy CT (DECT) imaging can provide both the electron density ρ(e) and effective atomic number Z(eff), thus facilitating tissue type identification. This paper investigates the accuracy of a dual source DECT scanner by means of measurements and simulations. Previous simulation work suggested improved Monte Carlo dose calculation accuracy when compared to single energy CT for low energy photon brachytherapy, but lacked validation. As such, we aim to validate our DECT simulation model in this work. MATERIALS AND METHODS: A cylindrical phantom containing tissue mimicking inserts was scanned with a second generation dual source scanner (SOMATOM Definition FLASH) to obtain Z(eff) and ρ(e). A model of the scanner was designed in ImaSim, a CT simulation program, and was used to simulate the experiment. RESULTS: Accuracy of measured Z(eff) (labelled Z) was found to vary from -10% to 10% from low to high Z tissue substitutes while the accuracy on ρ(e) from DECT was about 2.5%. Our simulation reproduced the experiments within ±5% for both Z and ρ(e). CONCLUSIONS: A clinical DECT scanner was able to extract Z and ρ(e) of tissue substitutes. Our simulation tool replicates the experiments within a reasonable accuracy.

Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism.

BACKGROUND: The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory. METHODOLOGY/PRINCIPAL FINDINGS: Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general. CONCLUSIONS/SIGNIFICANCE: The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.

Membrane interactions of oligomeric alpha-synuclein: potential role in Parkinson's disease.

alpha-Synuclein is a small neuronal protein that has been implicated to play an important role in Parkinson's disease. Genetic mutations and multiplications in the alpha-synuclein gene can cause familial forms of the disease. In aggregated fibrillar form, alpha-synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinson's disease. The loss of functional dopaminergic neurons in Parkinson's disease may be caused by a gain in toxic function of the protein. Elucidating if this gain of toxic function is related to the aggregation of alpha-synuclein may be vital in understanding Parkinson's disease. Although there are many ideas on how alpha-synuclein could be involved in the disease, this review will focus on the amyloid pore hypothesis. This hypothesis assumes that aggregation intermediates or oligomers are more likely to be toxic than monomeric or fibrillar forms of the protein. Oligomeric species are thought to exercise their toxicity through permeabilization of cellular membranes. Membrane pore formation by an oligomeric intermediate might play a role in other neurodegenerative disorders in which protein aggregation and amyloid formation play a role, such as Alzheimer's disease. We will discuss the role of this hypothesis in Parkinson's disease.

A stable lipid-induced aggregate of alpha-synuclein.

The Parkinson's disease-related protein alpha-Synuclein (alphaS) is a 140 residue intrinsically disordered protein. Its membrane-binding properties are thought to be relevant for its physiological or pathologic activity. Here, the interaction of alphaS with POPG [1-Palmitoyl-2-Oleoyl-sn-Glycero-3-(Phosphorac-(1-glycerol))] small unilamellar vesicles (SUVs) is investigated by spin-label EPR using double electron-electron resonance (DEER). Intermolecular distances between four single mutants reveal that well-defined aggregates are formed. The data suggest a coexistence of two dimer structures with main interactions in the helix 2, encompassing residues 50-100. Previously, the horseshoe conformation was detected by intramolecular restraints obtained by DEER on alphaS double mutants (Drescher et al. J. Am. Chem. Soc. 2008, 130, 7796). The present study suggests that interdigitation of two monomers in the aggregate fills the void between the two helices of each of the monomers thus providing a rationale for the horseshoe structure. This aggregate is lipid induced and affects the structure of the POPG SUVs, which become leaky and diminish in size upon contact with alphaS suggesting a possible origin of conflicting results in the recent literature (Jao et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (50), 19666; Georgieva et al. J. Am. Chem. Soc. 2008, 130 (39), 12856; Bortolus et al. J. Am. Chem. Soc. 2008, 130, 6690).

Lipid bilayer disruption by oligomeric alpha-synuclein depends on bilayer charge and accessibility of the hydrophobic core.

Soluble oligomeric aggregates of alpha-synuclein have been implicated to play a central role in the pathogenesis of Parkinson's disease. Disruption and permeabilization of lipid bilayers by alpha-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer-membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged alpha-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer-membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer-membrane interactions.

Membrane binding of oligomeric alpha-synuclein depends on bilayer charge and packing.

Membrane disruption by oligomeric alpha-synuclein (alphaS) is considered a likely mechanism of cytotoxicity in Parkinson's disease (PD). However, the mechanism of oligomer binding and the relation between binding and membrane disruption is not known. We have visualized alphaS oligomer-lipid binding by fluorescence microscopy and have measured membrane disruption using a dye release assay. The data reveal that oligomeric alphaS selectively binds to membranes containing anionic lipids and preferentially accumulates into liquid disordered (Ld) domains. Furthermore, we show that binding of oligomers to the membrane and disruption of the membrane require different lipid properties. Thus membrane-bound oligomeric alphaS does not always cause bilayer disruption.

Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices.

The putative function of the Parkinson's disease-related protein alpha-Synuclein (alphaS) is thought to involve membrane binding. Therefore, the interaction of alphaS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of alphaS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but alphaS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative-charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of alphaS. Thus, the interaction of alphaS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of alphaS to membranes. These findings contribute to our understanding of the details and structural consequences of alphaS-membrane interactions.

Antiparallel arrangement of the helices of vesicle-bound alpha-synuclein.

alpha-Synuclein (alphaS) is the main component of Lewy bodies from Parkinson's disease. That alphaS binds to membranes is known, but the conformation it adopts is still unclear. Pulsed EPR on doubly spin-labeled variants of alphaS sheds light on the most likely structure. For alphaS bound to vesicles large enough to accommodate also the extended conformation, an antiparallel helix conformation is found. This suggests that the bent structure shown is the preferred conformation of alphaS on membranes.

Tissue transglutaminase modulates alpha-synuclein oligomerization.

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated alpha-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant alpha-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective alpha-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/alpha-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all alpha-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal alpha-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal beta-sheet-containing oligomers, the tTG/alpha-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant alpha-synuclein variants. We propose that tTG cross-linking imposes structural constraints on alpha-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.