633. Excessive superoxide production and endothelial dysfunction in cerebral arteries of atherosclerotic mice are due to enhanced activity of Nox2-containing NADPH-Oxidase
A.A. Miller, T.M. De Silva, C.P. Judkins, H. Diep, G.R. Drummond and C.G. Sobey
Department of Pharmacology, Monash University, Melbourne, VIC, Australia
Objectives: Despite the absence of atherosclerotic lesions, endothelial dysfunction occurs in cerebral arteries of atherosclerotic mice. Although such changes are proposed to result from oxidative stress, the contributing molecular mechanisms are unknown. Here, we have tested the hypothesis that enhanced superoxide (O2−) production by Nox2-containing NADPH oxidase leads to impaired endothelial function in cerebral arteries of high fat-fed apolipoprotein E-deficient (ApoE−/−) mice.
Methods: All mice were fed a high fat diet (21% fat and 0.15% cholesterol) from 5 weeks of age for 7–14 weeks. Cerebral arteries (pooled middle cerebral and basilar) were isolated from C57Bl6/J wild-type (n = 13), ApoE−/−(n = 26), Nox2 deficient (Nox2−/−; n = 10) and novel Nox2−/−/ApoE−/− (n = 23) mice. Basal O2− production by cerebral arteries was measured using L-012 (100 μmol/L)-enhanced chemiluminescence. Endothelial function was assessed in isolated cannulated middle cerebral arteries using a perfusion myograph via the vasoconstrictor response to the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME; 100 μmol/L).
Results: Plasma total cholesterol levels of wild-type (n = 6), ApoE−/− (n = 9) and Nox2−/−/ApoE−/− (n = 10) mice were 13±0.2, 83±16, and 97±12 mmol/L, respectively. En face oil red O staining showed that there were no atherosclerotic lesions in aortae of wild-type mice (n = 13). In ApoE−/− mice, aortic lesions were prominent (∼9% of total aortic surface, n = 13) but no lesions were observed in the cerebral arteries. O2− production was elevated in cerebral arteries from ApoE−/− (35±5 × 103 counts/mg; n = 14, P<0.05) versus wild-type (16±2 × 103 counts/mg; n = 11) and Nox2−/− (10±2 × 103 counts/mg; n = 6) mice. However, in Nox2−/−/ApoE−/− mice, cerebral artery O2− production was not elevated (11±1 × 103 counts/mg; n = 17). The magnitude of L-NAME-induced contractions of isolated middle cerebral arteries from ApoE−/− mice (Δ diameter = −13%±4%; n = 6, P<0.05) was <50% of that in wild-type mice (−34%±2%; n = 6), whereas in Nox2−/−/ApoE−/− (−33%±10%; n = 6) mice endothelial function was comparable to that in wild-types. By contrast, similar constrictor responses to high K+ (124 mmol/L) were observed in wild-type (−59%±4%), ApoE−/− (−60%±5%), and Nox2−/−/ApoE−/− (−65%±3%) mice. In the presence of the O2− scavenger, tempol (1 mmol/L), endothelial function was similar in ApoE−/− (−25%±5%) and wild-type (−24%±3%) mice.
Conclusions: In summary, excessive O2− production and endothelial dysfunction occur in cerebral arteries of atherosclerotic mice even in the absence of lesions. Furthermore, we report for the first time that these changes appear to be exclusively due to increased activity of Nox2-containing NADPH oxidase.
220. Functional and structural synergy for resolution recovery and partial volume correction in brain pet
M. Shidahara1, C. Tsoumpas2, A. Hammers2, N. Boussion3, D. Visvikis3, H. Ito1, Y. Kimura1, T. Suhara1, I. Kanno1 and F Turkheimer2
1Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan; 2MRC Clinical Sciences Centre, Hammersmith Hospital, Imperial College London, London, UK; 3INSERM, U650, Laboratoire de Traitement de l′Information Medicale (LaTIM), Brest, France
Objectives: Positron Emission Tomography (PET) has the unique capability of measuring brain function but its clinical potential is affected by low resolution and lack of morphological detail. The purpose of this study is to evaluate a wavelet synergistic approach that combines functional and structural information from a number of sources (CT, MRI and anatomical frequency-based atlases1) for the accurate quantitative recovery in PET imaging. When the method is combined with anatomical frequency-based atlases, the outcome is a functional volume corrected for partial volume effects of all the selected regions.
Methods: The proposed method is based on the multiresolution property of the wavelet transform.2 First, the target PET image and the corresponding anatomical image are decomposed into several resolution elements. Secondly, high-resolution components of the PET image are replaced, in part, with those of the anatomical image after appropriate scaling. The amount of structural input is weighted by the relative high frequency signal content of the two modalities. Anatomical information was provided either by CT and MRI volumes or by a frequency-based anatomical atlas1 whose regional intensity values were obtained by averaging the PET data for each ROI. Accurate simulations of a brain [18F]FDG dataset based on Zubal brain phantom3 were conducted to evaluate the accuracy of ROI values after resolution recovery. As anatomical information, three images (the measured MRI, CT and the segmented image) were used. The resolution-recovered images were compared with the true image of 10 ROIs. The proposed method was also applied to two clinical datasets from an Alzheimer disease patient with moderate atrophy ([18F]FDG) and a young normal volunteer ([11C]raclopride). Static images of these datasets were processed using both MRI and the Hammersmith frequency-based Atlas,1 and the results were compared.
Results: Simulation studies showed that resolution recovery with CT images, which contain minimum amount of tissue anatomy, did not improve resolution. The use of MRI images brought significant improvements in PET image resolution but improvements were maximized when atlas-based segmented images as anatomical references were used (Figure 1A). These results were replicated in the clinical data sets (Figure 1B).
(A) ROI comparisons of the simulated FDG images against true image. (B) Original and resolution recovered images of a [18F]FDG and [11C]raclopride studies.
Conclusions: The synergistic use of functional and structural data, and the incorporation of anatomical information in particular, generate morphologically corrected PET images of exquisite quality.
617. Hyperperfusion and changes in vasoreactivity after ischemic stroke: implications for tissue recovery
S. Wegener1, M. Weller1 and E. Wong2
1Neurology, University Zürich, Zürich, Switzerland; 2Radiology and Psychiatry, University California San Diego, La Jolla, California, USA
Objectives: Hyperperfusion (or ‘luxury perfusion’) after stroke has been observed early after reperfusion in patients as well as experimental models. While restoration of blood flow is the prerequisite for tissue recovery after stroke, it can have deleterious effects leading to reperfusion injury. There is still uncertainty about the phenomenon of post-stroke hyperperfusion: is it an indicator of a favorable outcome for the affected tissue, or is it part of the reperfusion injury cascade?
Methods: 8 Wistar rats were subjected to transient (60 min) middle cerebral artery occlusion (MCAO). MR images were acquired during the occlusion (day 0) and after reperfusion (days 1, 4, 14). Quantitative cerebral blood flow (CBF) maps were obtained using flow-sensitive alternating inversion recovery (FAIR) arterial spin labeling with the QUIPSSII modification. In order to get an estimate of vasoreactivity, CBF was measured in room air or in 5%CO2 added to the anesthetic gas on days 4 and 14. In addition, diffusion-weighted (DWI) images, T1 and T2 maps and anatomical T2w images were obtained.
Results: During the vascular occlusion, CBF was significantly reduced in the ipsilateral MCA territory. On day 1, CBF had completely recovered, and most animals had a slight increase in CBF compared to the contralateral side in the formerly ischemic area. Hyperperfusion was much more pronounced and more localized on days 4 and 14. On vasoreactivity maps (CBFCo2–CBFair), the signal was low on day 4, while vasoreactivity had returned to baseline values or was even overshooting on day 14 (Figure 1). On individual images co-registered to the T2w images on day 14, the MR areas with hyperperfusion on days 4 and 14 could be mapped to the infarct border zone, while changes in vasoreactivity took place in more widespread areas throughout infarct core and border. In infarcts with cystic transformation on day 14, vasoreactivity remained depressed.
Conclusions: In a longitudinal MRI series after MCAO in rats, we observed late (>24 h) hyperperfusion in the reperfused tissue, with a maximum on day 4. Hyperperfusion occurred mainly in tissue voxels in the infarct border. This could indicate the beginning of vascular adaptation processes in the infarct periphery. Vasoreactivity was depressed on day 4 in the ischemic area, but recovered towards day 14. Interestingly, there were areas of increased vasoreactivity on day 14 in almost all animals. Depressed vasoreactivity on day 4 could reflect the long lasting impact of stroke on endothelial function; furthermore, immature blood vessels are known to have less vasodilatory capacity. The reason for the overshooting vasoreactivity on day 14 remains open. We are currently analyzing brain slices for changes in the density and distribution of blood vessels, tissue damage and inflammatory infiltrate in the areas indicated on MRI.
A, B: Averaged CBF maps acquired on days 4 and 14. C, D: Averaged Vasoreacivity (VR) maps (CBFCo2–CBFair) from days 4 and 14. On the right panel, voxels with CBF values below 30 ml/100 g per mins are overlaid onto the averaged d0 CBF map. Below is the averaged T2 map from d14.
59. GABA, glutamate and astroglial mediators in the cortical neurovascular coupling response to basal forebrain cholinergic input
C. Lecrux1, A. Kocharyan1, P. Fernandes1, E. Vaucher2 and E. Hamel1
1Laboratory of Cerebrovascular Research, Montréal Neurological Institute, McGill University; 2Ecole d'Optométrie, Université de Montréal, Montréal, QC, Canada
Background and aims: Neurovascular coupling or the tight adjustment of cerebral blood flow (CBF) to neurons displaying increased activity is widely used in functional neuroimaging techniques to map changes in neuronal activity. GABA interneurons contribute to the cortical CBF response to basal forebrain (BF) stimulation (Kocharyan et al, JCBFM 2008; 28:221–31). However, little is known about their interactions with other cortical neurons and astrocytes in this response. Hence, we investigated the neuroglial components involved in the CBF response to BF stimulation by blocking GABA and/or glutamatergic receptors, astroglial function or vasoactive mediators.
Methods: Bilateral cortical CBF responses to electrical BF stimulation were measured in urethane-anesthetised rats by Laser-Doppler flowmetry at baseline and after intracisternal (3 μl, 10−4 mol/L, pH 7.4 buffered solution) injection of vehicles, antagonists of NMDA (MK-801), AMPA (CNQX), metabotropic glutamate (mGluR) (MPEP+LY367385), GABA-A (picrotoxin), epoxyeicosatrienoic acids (EETs) (14,15-EEZE), muscarinic (scopolamine) receptors, blockers of astroglial metabolism (fluorocitrate), or inhibitors of EET-producing P450-epoxygenase (MS-PPOH) or prostaglandin-synthesizing cyclooxygenase-1 and 2 (COX-1 and 2, SC-560 and NS-398, respectively). Body temperature, blood gases and arterial blood pressure were stable throughout the experiments. Changes in CBF, expressed as mean±SEM, were compared by repeated-measures analysis of variance (ANOVA) or by one-way ANOVA for three group comparisons.
Results: The role of glutamate and GABA was evidenced by the significant decrease in the CBF response to BF stimulation after MK-801 (−32.2%±5.3%, P<0.01), CNQX (−30.2%±5.5%, P<0.01), LY367385+MPEP (−19.6%±4.5%, P<0.05) and picrotoxin (−24.5%±4.1%, P<0.05). MK-801 and picrotoxin administered together had an additive inhibitory effect on this response (−45.9%±6.2%, P<0.01). As previously reported, scopolamine reduced the CBF response (−55.5%±4.2%, P<0.01), but no further decrease was found when combined with MK-801 (−54.7%±9.5%, P<0.01), suggesting that the glutamate effect is downstream of muscarinic receptor activation. Inhibition of astroglial metabolism reduced (−43.6%±5.9%, P<0.001) the CBF response, as did blockade of EET synthesis (MS-PPOH, −44.2%±6.2%, P<0.01) or receptors (14,15-EEZE, −52.5%±3.6%, P<0.01). The combined administration of MS-PPOH with MK-801 or picrotoxin had no significant additive effect as compared to MS-PPOH alone (−34.5%±3.5%, P<0.01 and −35.1%±4.3%, P<0.001), suggesting that glutamate and GABA act partly through the astroglial EET cascade. In contrast, COX-1 or COX-2 inhibition had no effect on the evoked CBF response (−9.8%±10.2%, ns and +6.9%±9.5%, ns).
Conclusion: Our results demonstrate that (i) specific networks of cortical GABA and glutamate neurons are involved in the cortical CBF response induced by BF stimulation, (ii) these cortical neurons act, at least in part, through vasoactive EETs derived from astroglial arachidonic acid metabolism and (iii) COX-2 derivatives are not involved in the perfusion response to BF stimulation, in contrast to the thalamocortical pathway (Niwa et al, J Neurosci 2000; 20:763–70; Fernandes et al, Brain07, Abstract #BO12–3, and this meeting). Together with previous studies, these results suggest that neurovascular coupling in the cerebral cortex is input-specific, and that different vasoactive mediators are involved depending on the neurons recruited locally by a given afferent pathway.
Supported by CIHR (MOP-84209, EH) and Heart & Stroke Foundation of Canada/Canadian Stroke Network fellowship (CL).
