Oxidative-nitrosative stress and systemic vascular function in highlanders with and without exaggerated hypoxemia.

BACKGROUND: Acute exposure to high altitude stimulates free radical formation in lowlanders, yet whether this persists during chronic exposure in healthy, well-adapted and maladapted highlanders suffering from chronic mountain sickness (CMS) remains to be established. METHODS: Oxidative-nitrosative stress (as determined by the presence of the biomarkers ascorbate radical [A •- ], via electron paramagnetic resonance spectroscopy, and nitrite [NO 2 2 ], via ozone-based chemiluminescence) was assessed in venous blood of 25 male highlanders in Bolivia living at 3,600 m with CMS (n 5 13, CMS 1 ) and without CMS (n 5 12, CMS 2 ). Twelve age- and activity-matched, healthy, male lowlanders were examined at sea level and during acute hypoxia. We also measured fl ow-mediated dilatation (FMD), arterial stiffness defined by augmentation index normalized for a heart rate of 75 beats/min (AIx-75), and carotid intima-media thickness (IMT). RESULTS: Compared with normoxic lowlanders, oxidative-nitrosative stress was moderately increased in the CMS 2 group ( P , .05), as indicated by elevated A •- (3,191 457 arbitrary units [AU] vs 2,640 445 AU) and lower NO 2 2 (206 55 nM vs 420 128 nM), whereas vascular function remained preserved. This was comparable to that observed during acute hypoxia in lowlanders in whom vascular dysfunction is typically observed. In contrast, this response was markedly exaggerated in CMS 1 group (A •- , 3,765 429 AU; NO 2 2 , 148 50 nM) compared with both the CMS 2 group and lowlanders ( P , .05). This was associated with systemic vascular dysfunction as indicated by lower ( P , .05 vs CMS 2 ) FMD (4.2% 0.7% vs 7.6% 1.7%) and increased AIx-75 (23% 8% vs 12% 7%) and carotid IMT (714 127 m M vs 588 94 m M). CONCLUSIONS: Healthy highlanders display a moderate, sustained elevation in oxidative-nitrosative stress that, unlike the equivalent increase evoked by acute hypoxia in healthy lowlanders, failed to affect vascular function. Its more marked elevation in patients with CMS may contribute to systemic vascular dysfunction.

The origin of black colouration in onyx agate from Mali

Natural onyx agate from Mali was investigated in an integrated mineralogical and chemical study to reveal the origin of the unusual black colouration. Detailed studies by polarizing microscopy, scanning electron microscopy and micro-Raman spectroscopy showed that the colour of the dark bands is related to the incorporation of small particles of carbon (low-crystalline graphite) up to 200 nm in size into the cryptocrystalline silica matrix. The dark bands have carbon contents of 1.88 wt.%. The location of the graphite particles is closely related to the primary structural banding in the chalcedony. Cathodoluminescence data shows that the banding is interrupted by small fissures containing secondary hydrothermal quartz. The carbon isotope composition (delta C-13 value of -31.1+/-0.2 parts per thousand) of the carbonaceous material points to an organic precursor. Both the direct hydrothermal formation of graphite from methane under elevated temperature and the graphitization of organic precursors by secondary hydrothermal or metamorphic overprint are possible explanations for the colour of the dark bands. The graphitization of organic precursors results in an intense electron spin resonance line at g(eff) = 2.0026.

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy.

Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-kappa B and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38 alpha) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-alpha, markers of fibrosis (transforming growth factor-beta, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-kappa B activation, and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis. (J Am Coll Cardiol 2010;56:2115-25) (C) 2010 by the American College of Cardiology Foundation.

Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.3% by irradiating the 94 GHz electron spin resonance of admixed TEMPO nitroxide radicals in a magnetic field of 3.35 T during ca. 3 h at 1.2 K. The sample was subsequently transferred to a high-resolution magnet, and the enhanced polarization was converted from (15)N to methyl- and methylene protons, using the small (2,3)J((1)H,(15)N) couplings in choline. The room-temperature lifetime of nitrogen polarization in choline, T(1)((15)N) approximately 200 s, could be considerably increased by partial deuteration of the molecule. This procedure enables studies of choline metabolites in vitro and in vivo using DNP-enhanced proton NMR.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study.

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using (13)C- and (31)P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N(2) fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: An electron microscopy and P-31 and C-13 nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using C-13- and P-31-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N-2 fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.

Coronary magnetic resonance angiography.

Coronary magnetic resonance angiography (MRA) is a powerful noninvasive technique with high soft-tissue contrast for the visualization of the coronary anatomy without X-ray exposure. Due to the small dimensions and tortuous nature of the coronary arteries, a high spatial resolution and sufficient volumetric coverage have to be obtained. However, this necessitates scanning times that are typically much longer than one cardiac cycle. By collecting image data during multiple RR intervals, one can successfully acquire coronary MR angiograms. However, constant cardiac contraction and relaxation, as well as respiratory motion, adversely affect image quality. Therefore, sophisticated motion-compensation strategies are needed. Furthermore, a high contrast between the coronary arteries and the surrounding tissue is mandatory. In the present article, challenges and solutions of coronary imaging are discussed, and results obtained in both healthy and diseased states are reviewed. This includes preliminary data obtained with state-of-the-art techniques such as steady-state free precession (SSFP), whole-heart imaging, intravascular contrast agents, coronary vessel wall imaging, and high-field imaging. Simultaneously, the utility of electron beam computed tomography (EBCT) and multidetector computed tomography (MDCT) for the visualization of the coronary arteries is discussed.

On the dual contrast enhancement mechanism in frequency-selective inversion-recovery magnetic resonance angiography (IRON-MRA)

The susceptibility of blood changes after administration of a paramagnetic contrast agent that shortens T(1). Concomitantly, the resonance frequency of the blood vessels shifts in a geometry-dependent way. This frequency change may be exploited for incremental contrast generation by applying a frequency-selective saturation prepulse prior to the imaging sequence. The dual origin of vascular enhancement depending first on off-resonance and second on T(1) lowering was investigated in vitro, together with the geometry dependence of the signal at 3T. First results obtained in an in vivo rabbit model are presented.

Improved three-dimensional free-breathing coronary magnetic resonance angiography using gadocoletic acid (B-22956) for intravascular contrast enhancement.

PURPOSE: To evaluate gadocoletic acid (B-22956), a gadolinium-based paramagnetic blood pool agent, for contrast-enhanced coronary magnetic resonance angiography (MRA) in a Phase I clinical trial, and to compare the findings with those obtained using a standard noncontrast T2 preparation sequence. MATERIALS AND METHODS: The left coronary system was imaged in 12 healthy volunteers before B-22956 application and 5 (N = 11) and 45 (N = 7) minutes after application of 0.075 mmol/kg of body weight (BW) of B-22956. Additionally, imaging of the right coronary system was performed 23 minutes after B-22956 application (N = 6). A three-dimensional gradient echo sequence with T2 preparation (precontrast) or inversion recovery (IR) pulse (postcontrast) with real-time navigator correction was used. Assessment of the left and right coronary systems was performed qualitatively (a 4-point visual score for image quality) and quantitatively in terms of signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), vessel sharpness, visible vessel length, maximal luminal diameter, and the number of visible side branches. RESULTS: Significant (P < 0.01) increases in SNR (+42%) and CNR (+86%) were noted five minutes after B-22956 application, compared to precontrast T2 preparation values. A significant increase in CNR (+40%, P < 0.05) was also noted 45 minutes postcontrast. Vessels (left anterior descending artery (LAD), left coronary circumflex (LCx), and right coronary artery (RCA)) were also significantly (P < 0.05) sharper on postcontrast images. Significant increases in vessel length were noted for the LAD (P < 0.05) and LCx and RCA (both P < 0.01), while significantly more side branches were noted for the LAD and RCA (both P < 0.05) when compared to precontrast T2 preparation values. CONCLUSION: The use of the intravascular contrast agent B-22956 substantially improves both objective and subjective parameters of image quality on high-resolution three-dimensional coronary MRA. The increase in SNR, CNR, and vessel sharpness minimizes current limitations of coronary artery visualization with high-resolution coronary MRA.

High-resolution magnetic resonance imaging quantitatively detects individual pancreatic islets.

OBJECTIVE-We studied whether manganese-enhanced high-field magnetic resonance (MR) imaging (MEHFMRI) could quantitatively detect individual islets in situ and in vivo and evaluate changes in a model of experimental diabetes.RESEARCH DESIGN AND METHODS-Whole pancreata from untreated (n = 3), MnCl(2) and glucose-injected mice (n = 6), and mice injected with either streptozotocin (STZ; n = 4) or citrate buffer (n = 4) were imaged ex vivo for unambiguous evaluation of islets. Exteriorized pancreata of MnCl(2) and glucose-injected mice (n = 6) were imaged in vivo to directly visualize the gland and minimize movements. In all cases, MR images were acquired in a 14.1 Testa scanner and correlated with the corresponding (immuno)histological sections.RESULTS-In ex vivo experiments, MEHFMRI distinguished different pancreatic tissues and evaluated the relative abundance of islets in the pancreata of normoglycemic mice. MEHFMRI also detected a significant decrease in the numerical and volume density of islets in STZ-injected mice. However, in the latter measurements the loss of beta-cells was undervalued under the conditions tested. The experiments on the externalized pancreata confirmed that MEHFMRI could visualize native individual islets in living, anesthetized mice.CONCLUSIONS-Data show that MEHFMRI quantitatively visualizes individual islets in the intact mouse pancreas, both ex vivo and in vivo. Diabetes 60:2853-2860, 2011

Distal dendrites of frog motor neurons: a computer-aided electron microscopic study of cobalt-filled cells.

With the aid of the cobalt labelling technique, frog spinal cord motor neuron dendrites of the subpial dendritic plexus have been identified in serial electron micrographs. Computer reconstructions of various lengths (2.5-9.8 micron) of dendritic segments showed the contours of these dendrites to be highly irregular, and to present many thorn-like projections 0.4-1.8 micron long. Number, size and distribution of synaptic contacts were also determined. Almost half of the synapses occurred at the origins of the thorns and these synapses had the largest contact areas. Only 8 out of 54 synapses analysed were found on thorns and these were the smallest. For the total length of reconstructed dendrites there was, on average, one synapse per 1.2 micron, while 4.4% of the total dendritic surface was covered with synaptic contacts. The functional significance of these distal dendrites and their capacity to influence the soma membrane potential is discussed.

Reproducibility of free-breathing cardiovascular magnetic resonance coronary angiography.

OBJECTIVE: Contemporary free-breathing non contrast enhanced cardiovascular magnetic resonance angiography (CMRA) was qualitatively and quantitatively evaluated to ascertain the reproducibility of the method for coronary artery luminal dimension measurements. SUBJECTS AND METHODS: Twenty-two healthy volunteers (mean age 32 +/- 7 years, 12 males) without coronary artery disease were imaged at 2 centers (1 each in Europe and North America) using navigator-gated and corrected SSFP CMRA on a commercial whole body 1.5T System. Repeat images of right (RCA, n = 21), left anterior descending (LAD, n = 14) and left circumflex (LCX, n = 14) coronary arteries were obtained in separate sessions using identical scan protocol and imaging parameters. True visible vessel length, signal-to-noise (SNR), contrast-to-noise ratios (CNR) and the average luminal diameter over the first 4 cm of the vessel were measured. Intra-observer, inter-observer and inter-scan reproducibility of coronary artery luminal diameter were determined using Pearson's correlation, Bland-Altman analysis and intraclass correlation coefficients (ICC). RESULTS: CNR, SNR and the mean length of the RCA, LAD and LCX imaged for original and repeat scans were not significantly different (all p > 0.30). There was a high degree of intra-observer, inter-observer and inter-scan agreements for RCA, LAD and LCX luminal diameter respectively on Bland-Altman and ICC analysis (ICC's for RCA: 0.98. 0.98 and 0.86; LAD: 0.89, 0.89 and 0.63; LCX: 0.95, 0.94 and 0.79). CONCLUSION: In a 2-center study, we demonstrate that free-breathing 3D SSFP CMRA can visualize long continuous segments of coronary vessels with highly reproducible measurements of luminal diameter.

Quantification of aortic flow by phase-contrast magnetic resonance in patients with bicuspid aortic valve.

AIMS: Bicuspid aortic valve (BAV) causes complex flow patterns in the ascending aorta (AAo), which may compromise the accuracy of flow measurement by phase-contrast magnetic resonance (PC-MR). Therefore, we aimed to assess and compare the accuracy of forward flow measurement in the AAo, where complex flow is more dominant in BAV patients, with flow quantification in the left ventricular outflow tract (LVOT) and the aortic valve orifice (AV), where complex flow is less important, in BAV patients and controls. METHODS AND RESULTS: Flow was measured by PC-MR in 22 BAV patients and 20 controls at the following positions: (i) LVOT, (ii) AV, and (iii) AAo, and compared with the left ventricular stroke volume (LVSV). The correlation between the LVSV and the forward flow in the LVOT, the AV, and the AAo was good in BAV patients (r = 0.97/0.96/0.93; P < 0.01) and controls (r = 0.96/0.93/0.93; P < 0.01). However, in relation with the LVSV, the forward flow in the AAo was mildly underestimated in controls and much more in BAV patients [median (inter-quartile range): 9% (4%/15%) vs. 22% (8%/30%); P < 0.01]. This was not the case in the LVOT and the AV. The severity of flow underestimation in the AAo was associated with flow eccentricity. CONCLUSION: Flow measurement in the AAo leads to an underestimation of the forward flow in BAV patients. Measurement in the LVOT or the AV, where complex flow is less prominent, is an alternative means for quantifying the systolic forward flow in BAV patients.

Coronary magnetic resonance angiography for assessment of the stent lumen: a phantom study.

PURPOSE: To evaluate the feasibility of visualizing the stent lumen using coronary magnetic resonance angiography in vitro. MATERIAL AND METHODS: Nineteen different coronary stents were implanted in plastic tubes with an inner diameter of 3 mm. The tubes were positioned in a plastic container filled with gel and included in a closed flow circuit (constant flow 18 cm/sec). The magnetic resonance images were obtained with a dual inversion fast spin-echo sequence. For intraluminal stent imaging, subtraction images were calculated from scans with and without flow. Subsequently, intraluminal signal properties were objectively assessed and compared. RESULTS: As a function of the stent type, various degrees of in-stent signal attenuation were observed. Tantalum stents demonstrated minimal intraluminal signal attenuation. For nitinol stents, the stent lumen could be identified, but the intraluminal signal was markedly reduced. Steel stents resulted in the most pronounced intraluminal signal voids. CONCLUSIONS: With the present technique, radiofrequency penetration into the stents is strongly influenced by the stent material. Thesefindings may have important implicationsforfuture stent design and stent imaging strategies.

Skeletal Muscle Mitochondrial and Lipid Droplet Volume Density: Validity of Electron Microscopy Point-counting Measurements

Mitochondrial (M) and lipid droplet (L) volume density (vd) are often used in exercise research. Vd is the volume of muscle occupied by M and L. The means of calculating these percents are accomplished by applying a grid to a 2D image taken with transmission electron microscopy; however, it is not known which grid best predicts these values. PURPOSE: To determine the grid with the least variability of Mvd and Lvd in human skeletal muscle. METHODS: Muscle biopsies were taken from vastus lateralis of 10 healthy adults, trained (N=6) and untrained (N=4). Samples of 5-10mg were fixed in 2.5% glutaraldehyde and embedded in EPON. Longitudinal sections of 60 nm were cut and 20 images were taken at random at 33,000x magnification. Vd was calculated as the number of times M or L touched two intersecting grid lines (called a point) divided by the total number of points using 3 different sizes of grids with squares of 1000x1000nm sides (corresponding to 1µm2), 500x500nm (0.25µm2) and 250x250nm (0.0625µm2). Statistics included coefficient of variation (CV), 1 way-BS ANOVA and spearman correlations. RESULTS: Mean age was 67 ± 4 yo, mean VO2peak 2.29 ± 0.70 L/min and mean BMI 25.1 ± 3.7 kg/m2. Mean Mvd was 6.39% ± 0.71 for the 1000nm squares, 6.01% ± 0.70 for the 500nm and 6.37% ± 0.80 for the 250nm. Lvd was 1.28% ± 0.03 for the 1000nm, 1.41% ± 0.02 for the 500nm and 1.38% ± 0.02 for the 250nm. The mean CV of the three grids was 6.65% ±1.15 for Mvd with no significant differences between grids (P>0.05). Mean CV for Lvd was 13.83% ± 3.51, with a significant difference between the 1000nm squares and the two other grids (P<0.05). The 500nm squares grid showed the least variability between subjects. Mvd showed a positive correlation with VO2peak (r = 0.89, p < 0.05) but not with weight, height, or age. No correlations were found with Lvd. CONCLUSION: Different size grids have different variability in assessing skeletal muscle Mvd and Lvd. The grid size of 500x500nm (240 points) was more reliable than 1000x1000nm (56 points). 250x250nm (1023 points) did not show better reliability compared with the 500x500nm, but was more time consuming. Thus, choosing a grid with square size of 500x500nm seems the best option. This is particularly relevant as most grids used in the literature are either 100 points or 400 points without clear information on their square size.