The ins and outs of the BCCAo model for chronic hypoperfusion: a multimodal and longitudinal MRI approach.

Cerebral hypoperfusion induced by bilateral common carotid artery occlusion (BCCAo) in rodents has been proposed as an experimental model of white matter damage and vascular dementia. However, the histopathological and behavioral alterations reported in this model are variable and a full characterization of the dynamic alterations is not available. Here we implemented a longitudinal multimodal magnetic resonance imaging (MRI) design, including time-of-flight angiography, high resolution T1-weighted images, T2 relaxometry mapping, diffusion tensor imaging, and cerebral blood flow measurements up to 12 weeks after BCCAo or sham-operation in Wistar rats. Changes in MRI were related to behavioral performance in executive function tasks and histopathological alterations in the same animals. MRI frequently (70%) showed various degrees of acute ischemic lesions, ranging from very small to large subcortical infarctions. Independently, delayed MRI changes were also apparent. The patterns of MRI alterations were related to either ischemic necrosis or gliosis. Progressive microstructural changes revealed by diffusion tensor imaging in white matter were confirmed by observation of myelinated fiber degeneration, including severe optic tract degeneration. The latter interfered with the visually cued learning paradigms used to test executive functions. Independently of brain damage, BCCAo induced progressive arteriogenesis in the vertebrobasilar tree, a process that was associated with blood flow recovery after 12 weeks. The structural alterations found in the basilar artery were compatible with compensatory adaptive changes driven by shear stress. In summary, BCCAo in rats induces specific signatures in multimodal MRI that are compatible with various types of histological lesion and with marked adaptive arteriogenesis.

Middle cerebral artery alterations in a rat chronic hypoperfusion model.

Chronic cerebral hypoperfusion (CHP) induces microvascular changes that could contribute to the progression of vascular cognitive impairment and dementia in the aging brain. This study aimed to analyze the effects of CHP on structural, mechanical, and myogenic properties of the middle cerebral artery (MCA) after bilateral common carotid artery occlusion (BCCAO) in adult male Wistar rats. Sham animals underwent a similar surgical procedure without carotid artery (CA) ligation. After 15 days of occlusion, MCA and CA were dissected and MCA structural, mechanical, and myogenic properties were assessed by pressure myography. Collagen I/III expression was determined by immunofluorescence in MCA and CA and by Western blot in CA. mRNA levels for 1A1, 1A2, and 3A1 collagen subunits were quantified by quantitative real-time PCR in CA. Matrix metalloproteinase (MMP-1, MMP-2, MMP-9, and MMP-13) and hypoxia-inducible factor-1α (HIF-1α) protein expression were determined in CA by Western blot. BCCAO diminished cross-sectional area, wall thickness, and wall-to-lumen ratio. Nevertheless, whereas wall stress was increased, stiffness was not modified and myogenic response was diminished. Hypoperfusion triggered HIF-1α expression. Collagen I/III protein expression diminished in MCA and CA after BCCAO, despite increased mRNA levels for 1A1 and 3A1 collagen subunits. Therefore, the reduced collagen expression might be due to proteolytic degradation, since the expression of MMP-1 and MMP-9 increased in the CA. These data suggest that BCCAO induces hypotrophic remodeling by a mechanism that involves a reduction of collagen I/III in association with increased MMP-1 and MMP-9 and that decreases myogenic tone in major arteries supplying the brain.

Transient mesenteric ischemia leads to remodeling of rat mesenteric resistance arteries.

Mesenteric ischemia/reperfusion (I/R) is associated with high rates of morbidity and mortality. We studied the effect of mesenteric I/R on structural and mechanical properties of rat mesenteric resistance artery (MRA) that, once disrupted, might impact the outcome of this devastating clinical condition. Superior mesenteric artery from Wistar-Kyoto rats was occluded (90 min) and reperfused (24 h). The effect of tezosentan, a dual endothelin (ET)-receptor antagonist, was studied in ischemic (IO) and sham-operated (SO) animals. MRA structure and mechanics were assessed by pressure myography. Nuclei distribution, elastin content and organization, collagen I/III and ET-1 expression, ET-1 plasma levels, superoxide anion ([Formula: see text]) production, and mRNA levels of NAD(P)H-oxidase subunits were measured. To assess ET-1 effects on [Formula: see text] production, MRA from non-operated rats were incubated in culture medium with ET-1. Mesenteric I/R increased MRA wall thickness (P < 0.05) and cross-sectional area (P < 0.05) but decreased wall stiffness (P < 0.05). Arterial remodeling was paralleled by enhancement of: (i) collagen I/III expression (P < 0.01), ET-1 expression (P < 0.05), and [Formula: see text] formation (P < 0.01) in the vessel wall; (ii) number of internal elastic lamina (IEL) fenestrae (P < 0.05); and (iii) plasma levels of ET-1 (P < 0.05). Moreover, ET-1 increased [Formula: see text] (P < 0.05) production in cultured MRA. Tezosentan prevented hypertrophic remodeling and collagen I/III deposition, and enhanced [Formula: see text] production, but it did not affect the decreased wall stiffness after mesenteric I/R. These results indicate that 90 min occlusion/24 h reperfusion induces hypertrophic remodeling of MRA linked to ET-1-mediated increase of collagen and [Formula: see text]. Decreased stiffness may be associated with increased number of IEL fenestrae. The resulting MRA remodeling, initially adaptive, might become maladaptive contributing to the pathology and poor outcome of mesenteric I/R, and might be a valuable treatment target for mesenteric I/R.

Influence of dietary fat on oxidative stress and inflammation in murine macrophages.

OBJECTIVE: Many studies have shown that the nature of the lipid consumed in the diet significantly affects the development of inflammatory diseases. In this study, we compared the effect of diets supplemented with 15% by weight of fish oil (FO), refined olive oil (ROO), and pomace olive oil (POO) with that of a low-fat diet, 2% by weight of corn oil, considered as the basal diet (BD), on the ability to modify reactive oxidative species and proinflammatory mediator generation by stimulated murine macrophages. METHODS: Mice were fed the different oil-enriched diets for 8 wk. Peritoneal macrophages were isolated from these mice and subsequently stimulated. Reactive oxygen species and proinflammatory mediators were measured in the corresponding supernatants. Data were statistically treated by one-way analysis of variance and Tukey's multiple comparison post hoc test. RESULTS: The ROO and POO significantly reduced the hydrogen peroxide production compared with BD, whereas FO stimulated its production. Moreover, the generation of nitric oxide was significantly prevented in all the experimental oil-enriched dietary groups. The ROO and FO groups showed significantly reduced cytokine (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6) and prostaglandin E(2) production. CONCLUSION: These results confirm the prevention action on proinflammatory mediator generation exerted by FO and demonstrate the protective antioxidant properties not only of olive oil but also of POO. The consumption of these olive oils may help to prevent cellular oxidative stress and inflammation.

Long-chain fatty alcohols from pomace olive oil modulate the release of proinflammatory mediators.

Pomace olive oil is a by-product of olive oil extraction that is traditionally produced and consumed in Spain. The nonglyceride matter of this oil is a good source of interesting minor compounds, like long-chain fatty alcohols, which are present free or as part of waxes. In the present study, long-chain fatty alcohols were isolated from the nonglyceride fraction of pomace olive oil, and the composition was identified and quantified. The major components of long-chain fatty alcohols were tetracosanol, hexacosanol and octacosanol. We investigated the ability of long-chain fatty alcohols from pomace olive oil to inhibit the release of different proinflammatory mediators in vitro by cells involved in inflammatory processes. Long-chain fatty alcohols significantly and dose-dependently decreased nitric oxide production by RAW 264.7 murine macrophages stimulated with lipopolysaccharide. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression. Long-chain fatty alcohols also reduced tumor necrosis factor-alpha and prostaglandin E(2) production, although the potency of inhibition for the latter was lower. On the other hand, long-chain fatty alcohols significantly reduced thromboxane A(2) production in rat peritoneal neutrophils stimulated with the calcium ionophore A-23187. The reduction of eicosanoid release was related to the inhibition of phospholipase A(2) enzyme activity by long-chain fatty alcohols, reaching an inhibitory concentration 50% value of 6.2 microg/ml. These results showed that long-chain fatty alcohols may have a protective effect on some mediators involved in the inflammatory damage development, suggesting its potential value as a putative functional component of pomace olive oil.

Supressive effect of maslinic acid from pomace olive oil on oxidative stress and cytokine production in stimulated murine macrophages.

The pentacyclic triterpene maslinic acid (MA) is a natural compound present in the non glyceride fraction of pomace olive oil, also called orujo olive oil. This compound has previously demonstrated antioxidant properties against lipid peroxidation in vitro, but its effects on reactive oxygen and nitrogen-derived species and pro-inflammatory cytokines generated by a cell system have not yet been investigated. In this study, we have tested the effect of MA upon oxidative stress and cytokine production using peritoneal murine macrophages. MA significantly inhibited the enhanced production of nitric oxide (NO) induced by lypopolysaccharide (LPS) when it was measured by the nitrite production with an inhibitory concentration 50% value (IC(50)) of 25.4 microM. This inhibiting effect seems to be consequence of an action at the level of the LPS-induction of the inducible nitric oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The secretion of the inflammatory cytokines interleukine-6 and TNF-a from LPS-stimulated murine macrophages was also significantly reduced (p < 0.05 and 0.01) by 50 and 100 microM of MA. In addition, reactive oxygen species were measured after stimulation with phorbol-12-myristate-13-acetate (PMA). Thus, pre-treatment with MA reduced the generation of hydrogen peroxide from stimulated macrophages in a dose-dependent manner (IC(50): 43.6 microM) as assayed by the oxidation of the peroxidase enzyme. However, no inhibitory effect on superoxide release, measured by the reduction of ferricytochrome c, was observed after the pretreatment with MA in the culture medium. These results suggest a potential biopharmaceutical use of this hydroxy-pentacyclic triterpene derivative, present in orujo olive oil, on preventing oxidative stress and pro-inflammatory cytokine generation.

Modulation of cytokine secretion by pentacyclic triterpenes from olive pomace oil in human mononuclear cells.

Olive pomace oil, also known as "orujo" olive oil, is a blend of refined-pomace oil and virgin olive oil, fit for human consumption. Maslinic acid, oleanolic acid, erythrodiol, and uvaol are pentacyclic triterpenes, found in the non-glyceride fraction of orujo oil, which have previously been reported to have anti-inflammatory properties. In the present work, we investigated the effect of these minor components on pro-inflammatory cytokine production by human peripheral blood mononuclear cells in six different samples. Uvaol, erythrodiol, and oleanolic acid significantly decreased IL-1beta and IL-6 production in a dose-dependent manner. All three compounds significantly reduced TNF-alpha production at 100microM; however, at 10microM, uvaol and oleanolic acid enhanced the generation of TNF-alpha. In contrast, maslinic acid did not significantly alter the concentration of those cytokines, with the exception of a slight inhibitory effect at 100microM. All four triterpenes inhibited production of I-309, at 50microM and 100microM. However, uvaol enhanced I-309 production at 10microM. The triterpenic dialcohols had a similar effect on MIG production. In conclusion, this study demonstrates that pentacyclic triterpenes in orujo oil exhibit pro- and anti-inflammatory properties depending on chemical structure and dose, and may be useful in modulating the immune response.

Regulation of vascular tone from spontaneously hypertensive rats by the HMG-CoA reductase inhibitor, simvastatin.

The acute effect of simvastatin on aortic rings from spontaneously hypertensive rats (SHRs) was identified. Simvastatin-evoked relaxations of both depolarized and phenylephrine-precontracted arteries were independent of the presence of endothelium. This effect was inhibited by diltiazem and mevalonate, but not by the Rho-kinase inhibitor, Y-27632. Simvastatin prevented contraction induced by phenylephrine, calcium ionophore A-23187 and CaCl2 in Ca2+-free medium. Y-27632 decreased the effect of simvastatin. On the contrary, contraction induced by noradrenaline in Ca2+-free medium was not affected. These results suggest that simvastatin elicited an effect on vascular smooth muscle cells from SHRs that may involve blockade of extracellular calcium entry and decrease vascular contraction by affecting Rho-kinase.