Vascular Reactivity In Rats Research Articles

Reduction of vascular reactivity in rat aortas following pilocarpine-induced status epilepticus

ObjectiveThe authors investigated changes in vascular reactivity in rats following pilocarpine-induced status epilepticus. MethodMale Wistar rats weighing between 250g and 300g were used. Status epilepticus was induced using 385 mg/kg i.p. pilocarpine. After 40 days the thoracic aorta was dissected and divided into 4 mm rings and the vascular smooth muscle reactivity to phenylephrine was evaluated. ResultsEpilepsy decreased the contractile responses of the aortic rings to phenylephrine (0.1 nM–300 mM). To investigate if this reduction was induced by increasing NO production with/or hydrogen peroxide L-NAME and Catalase were used. L-NAME (N-nitro-L arginine methyl ester) increased vascular reactivity but the contractile response to phenylephrine increased in the epileptic group. Catalase administration decreased the contractile responses only in the rings of rats with epilepsy. ConclusionsOur findings demonstrated for the first time that epilepsy is capable of causing a reduction of vascular reactivity in rat aortas. These results suggest that vascular reactivity reduction is associated with increased production of Nitric Oxide (NO) as an organic attempt to avoid hypertension produced by excessive sympathetic activation.

Periodontal status, vascular reactivity, and platelet aggregation changes in rats submitted to hypercholesterolemic diet and periodontitis

Periodontitis can corroborate with development and progression of atherosclerosis and a possible bidirectional interaction between both pathologies has been hypothesized. The aim of this work was to study the interactions between diet-induced hypercholesterolemia and ligature-induced periodontitis in Wistar rats submitted to both conditions. Animals were divided into four experimental groups: C (control: standard diet without periodontitis), Perio (periodontitis plus standard diet), HC (high cholesterol diet without periodontitis), and HC+Perio (high cholesterol diet plus periodontitis). The diets were offered for 45days and a silk ligature was applied in the lower first molars of Perio and HC-Perio animals on day 34 and maintained for 11days until euthanasia. The mandibles were excised, and alveolar bone loss was determined by macroscopic and micro-tomographic (µ-CT) imaging. Blood samples were obtained, and platelet aggregation was induced in plasma rich in platelets by adenosine diphosphate (ADP) and collagen. Endothelium-dependent vascular reactivity and protein expression of endothelial (eNOS), phosphorylated endothelial (peNOS), and inducible (iNOS) nitric oxide synthases were evaluated in aorta samples. The HC diet combined with periodontitis (HC+Perio group) was associated with an increased alveolar bone loss, when compared to the other groups. Both in Perio and HC groups, platelet aggregation induced by ADP or collagen was increased, while maximum aortic relaxation induced by acetylcholine was decreased. Periodontitis or HC diet alone decreased the expression of peNOS and HC diet increased the expression of iNOS. In contrast, no additive or synergistic effects were found in vascular reactivity or in platelet aggregation when the two conditions were associated (HC+Perio group). Hypercholesterolemia accelerated the process of bone loss induced by periodontitis while a high cholesterol diet or periodontitis individually increased platelet aggregation and vascular reactivity in rats without additive or synergistic effects, when associated.

Abstract P3046: Elevated Free Fatty Acids During Pregnancy Promote Vascular Impairment, Uteroplacental Dysfunction And Angiogenic Imbalance In Rats

Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-8% of pregnancies and is associated with vascular dysfunction and an increase in anti-angiogenic factors. Recent studies have shown that obese women are 4 times more likely to develop preeclampsia, and obesity/preeclampsia has been shown to increase circulating free fatty acids (FA). However, the role of FA on preeclamptic characteristics, such as uteroplacental and vascular dysfunction, as well as angiogenic imbalance has yet to be determined. To test the hypothesis that FAs induce a placental ischemic-like phenotype, pregnant rats were infused daily (IV) from gestational days (GD) 13-18 with specific free FA: 10mM Oleate and Palmitate, 4mM Linoleic Acid, and 0.8mM Arachadonic Acid. On GD 19, MAP, blood and tissues were collected. Third order mesentery arteries and aortas were used for vascular reactivity studies. Placental villi from normal pregnant rats exposed to FA ex vivo were examined for production of angiogenic factors. While infusion of FA resulted in no change in MAP, pup or placental weight, there was a trend for an increase in fetal reabsorption (9.7±3.3% vs 2.4±1.3%; p=0.07) and decrease in plasma PlGF in FA-infused rats (506±22vs 583±35 pg/mL; p=0.07), indicating moderate impairment in uteroplacental function in FA-infused pregnant rats compared to vehicle normal pregnant rats. Aortas of FA-infused pregnant rats revealed impaired Phe-induced vasoconstriction beginning at 3E -6.5 M of Phe (31.3±7.5 vs 70.8±11.4; p=<0.02), while vasorelaxation was attenuated in mesenteric arteries of FA-infused pregnant rats compared to normal pregnant rats (p<0.01). Moreover, FA caused an increase in sFlt-1 (533±34 vs 181±7 pg/mL; p<0.01) and sFlt-1:PlGF ratio (4.8±.1 vs 3.2±.2; p<0.01) in placental explants compared to vehicle. These data suggest that elevated FA during pregnancy, at levels comparable to those observed in preeclampsia, promote uteroplacental dysfunction, angiogenic imbalance, and impaired vascular reactivity in rats. Further studies to examine the mechanisms associated with FA-induced uteroplacental outcomes in pregnancy could elucidate the role of elevated FA as a risk factor for placental ischemia.

Vardenafil and cilostazol can improve vascular reactivity in rats with diabetes mellitus and rheumatoid arthritis co-morbidity

Endothelial dysfunction and vascular reactivity defects secondary to metabolic and immunological disorders carry risk of serious cardiovascular complications. Here, the effects of the phosphodiesterase (PDE) inhibitors vardenafil and cilostazol were examined against rheumatoid arthritis (RA)/diabetes mellitus (DM)-co-morbidity-induced endothelial dysfunction and vascular reactivity defects. After setting of RA/DM-co-morbidity model, rats were divided into a normal control group, an RA/DM-co-morbidity group, and two treatment groups receiving oral vardenafil (10 mg/kg/day) and cilostazol (30 mg/kg/day) for 21 days after RA/DM-co-morbidity induction. Aorta was isolated for biochemical estimations of the pro-inflammatory vasoconstrictor molecules angiotensin-II (Ang-II) and endothelin-1 (ET-1), the adhesion molecules P-selectin and vascular cell adhesion molecule-1 (VCAM-1), the energy sensor adenosine-5′-monophosphate-activated protein kinase (AMPK), and the vasodilator anti-inflammatory molecule vasoactive intestinal peptide (VIP) using enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Immunohistochemical estimations of endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 were performed coupled with histopathological examination using routine hematoxylin and eosin (H&E) and special Masson trichrome staining. The in vitro study was conducted using aortic strips where cumulative concentration response curves were done for the endothelium-dependent relaxing factor acetylcholine and the endothelium-independent relaxing factor sodium nitroprusside after submaximal contraction with phenylephrine. Vardenafil and cilostazol significantly improved endothelial integrity biomarkers in vivo supported with histopathological findings in addition to improved vasorelaxation in vitro. Apart from their known PDE inhibition, up-regulation of vascular AMPK and eNOS coupled with down-regulation of Ang-II, ET-1, P-selectin, VCAM-1 and MMP-2 may explain vardenafil and cilostazol protective effect against RA/DM-co-morbidity-induced endothelial dysfunction and vascular reactivity defects.

Extract Stem Bark of Hymenaea Rubriflora D: A New Perspective of Vasorelaxant Agent

In this study, was investigated the action of the hydroalcoholic extract of the stem bark of Hymenaea rubriflora Ducke (HR-HAc) on the vascular reactivity of rats in vitro and administered in vivo. Cumulative doses of HR-HAc promoted dose-dependent relaxation aortic rings of Wistar rats with functional endothelium, which was inhibited in the presence of L-NAME. After, animals (n=5) were treated with 25, 50 and 100 mg / kg or saline for four weeks for determination of reactivity to acetylcholine, nitroprusside and phenylephrine. Was observed increased acetylcholine relaxing potency at all doses, with a greater effect on G50 (pD2 = 7.8 ± 0.1, Emax = 95.6 ± 1.1) and a decreased contractile potency to phenylephrine in G25 (pD2 = 6.9 ± 0.06, Emax = 61.5 ± 6.0%) and G50 (pD2 = 6.6 ± 0.06, Emax = 71.0 ± 8.5%) when compared to GC in the presence and absence of endothelium (pD2 = 6.4 ± 0.1, 6.4 ± 0.1 and 6.9 ± 0.1, respectively). Cumulative doses of nitroprusside resulted in increased relaxing potency in all supplemented groups and maintained Emax at 100%. Concludes that HR-HAc has vasorelaxant capacity and endothelium-dependent contractile inhibitor, either applied directly to aortic rings or after treatment with in vivo supplementation, which places this extract as a potential antihypertensive agent for future studies.

Arginine and aerobic training prevent endothelial and metabolic alterations in rats at high risk for the development of the metabolic syndrome - CORRIGENDUM.

Endothelial function is a key mechanism in the development of CVD. Arginine and exercise are important non-pharmacological strategies for mitigating the impact of metabolic changes in the metabolic syndrome, but the effect of their combined administration is unknown. Thus, the aim of this study was to investigate the isolated and combined effects of aerobic training and arginine supplementation on metabolic variables and vascular reactivity in rats at high risk for developing the metabolic syndrome. Wistar rats were divided into two groups: control and fructose (F - water with 10 % fructose). After 2 weeks, the F group was divided into four groups: F, fructose+arginine (FA, 880 mg/kg per d of l-arginine), fructose+training (FT) and fructose+arginine+training (FTA); treatments lasted for 8 weeks, and no difference was observed in body mass gain. Arginine did not improve the body protein content, and both the FA and FT groups show a reversal of the increase in adipose tissue. Insulin increase was prevented by training and arginine, without additive effect, and the increase in serum TAG was prevented only by training. The F group showed impaired endothelium-dependent vasodilation and hyperreactivity to phenylephrine, but arginine and training were capable of preventing these effects, even separately. Higher nitric oxide level was observed in the FA and FT groups, and no potentiating effect was detected. Thus, only training was able to prevent the increase in TAG and improve the protein mass, and training and arginine exert similar effects on fat content, insulin and endothelial function, but these effects are not additive.

Abstract P524: Vasoprotective Effect of Hesperidin in the Onset of Arterial Hypertension Induced by L-NAME

Hesperidin has been used to manage venous disease and has been shown to have vasodilator, antiinflammatory and antioxidant properties. The aim of this work was to evaluate the efects of acute treatment with hesperidin (HD) on the vascular reactivity of rats in the onset of arterial hypertension induced by L-NAME. Male Wistar rats were divided into 3 groups as follow: 1- Control (CTL); 2- L-NAME (LN); 3- L-NAME + Hesperidin (LNHD). LN was given by gavage at the dose of 75mg/Kg, concomitantly with HD at the dose of 0.5 mmol/Kg, 24 hours previously to the sacrifice. After sacrifice, the blood was collected and the aorta removed and mounted in a wire myograph to assess concentration response curves to acetylcholine (ACh) and sodium nitroprusside (SNP). ACh elicited concentration dependent relaxation in aorta of CTL rats, reaching a maximum relaxation of 89 ± 3%, which was significantly reduced in LN group (37 ± 7%). Treatment with HD partially prevented this decrease, reaching the maximum relaxation of 72 ± 2%. On the other hand, SNP induced relaxation was more potent on the aorta of LN (pEC 50 9.67 ± 0.11) when compared to CTL (pEC 50 8.51 ± 0.13) and LNHD rats (pEC 50 8.90 ± 0.09). The oxidative status was not changed by any treatment since SOD activity was 11 ± 1, 9 ± 2 and 12 ± 0.5 U/mL, for CTL, LN and LNHD, respectively and lipid peroxidation measured by TBARS assay showed a MDA concentration of 29 ± 8, 35 ± 7 and 23 ± 3 μM for CTL, LN and LNHD, respectively. Altogether, these data suggest that that Hesperidin prevents the impairment of endothelium-dependent vascular relaxation, through a mechanism not related to its antioxidant property.

Arginine and aerobic training prevent endothelial and metabolic alterations in rats at high risk for the development of the metabolic syndrome.

Endothelial function is a key mechanism in the development of CVD. Arginine and exercise are important non-pharmacological strategies for mitigating the impact of metabolic changes in the metabolic syndrome, but the effect of their combined administration is unknown. Thus, the aim of this study was to investigate the isolated and combined effects of aerobic training and arginine supplementation on metabolic variables and vascular reactivity in rats at high risk for developing the metabolic syndrome. Wistar rats were divided into two groups: control and fructose (F - water with 10 % fructose). After 2 weeks, the F group was divided into four groups: F, fructose+arginine (FA, 880 mg/kg per d of l-arginine), fructose+training (FT) and fructose+arginine+training (FTA); treatments lasted for 8 weeks, and no difference was observed in body mass gain. Arginine did not improve the body protein content, and both the FA and FT groups show a reversal of the increase in adipose tissue. Insulin increase was prevented by training and arginine, without additive effect, and the increase in serum TAG was prevented only by training. The F group showed impaired endothelium-dependent vasodilation and hyperreactivity to phenylephrine, but arginine and training were capable of preventing these effects, even separately. Higher nitric oxide level was observed in the FA and FT groups, and no potentiating effect was detected. Thus, only training was able to prevent the increase in TAG and improve the protein mass, and training and arginine exert similar effects on fat content, insulin and endothelial function, but these effects are not additive.

Aerobic training prevents oxidative profile and improves nitric oxide and vascular reactivity in rats with cardiometabolic alteration.

Cardiovascular disease is the major cause of death worldwide; therefore it is important to understand the natural history of the pathophysiologic process and develop strategies to halt its progression. Thus this study investigated the protective effect of aerobic training on pathophysiological mechanisms involved in subclinical cardiometabolic alterations in a model with constant exposure to a prejudicial agent. Male Wistar rats were divided into a control group (C), which received drinking water, fructose group (F), which was fed 10% fructose in drinking water for 10 wk, and control training (CT) and fructose training groups (FT), in which moderate aerobic training was added in the last 8 wk of the study. Insulin, triacylglycerol, and isoprostane were higher and superoxide dismutase (SOD) was lower in the F group. There was no difference in thoracic aorta histology, but a decreased vascularization was seen in the F group, avoided by training in left ventricle. Regarding vascular function, the F group exhibited increased vasoconstrictory reactivity to phenylephrine. The F group presented impaired vasodilation to acetylcholine. Regarding endothelial nitric oxide synthase (eNOS), the F group presented a lower expression, and phosphorylated eNOS was higher in the trained groups than in their respective control groups. This same pattern was observed for nitric oxide bioavailability, antioxidant protein expression in aorta, left ventricle, and muscle (catalase, SOD, and glutathione peroxidase), serum SOD activity, and muscle mass. These results suggest that exercise training enhanced the antioxidant pathway and, as a consequence, the eNOS pathway, preventing an impairment in vascular vasodilatory capacity.

Arginine and Aerobic Training Prevent Impairment in Endothelial Function in Rats Under High Risk to Develop Metabolic Syndrome

It is not elucidated if aerobic training and arginine supplementation can have similar protective effects in individuals with constant exposure to the triggering agent of the metabolic syndrome, with the presence of subclinical alterations, but without the presence of cardiovascular disease. So the purpose of this study was to investigate the effect of arginine supplementation and aerobic training on vascular reactivity of rats under high risk of metabolic syndrome development. Wistar rats, males, adults were, initially, divided in two groups Control group (C; drinking water) and Fructose group (F; water with 10% fructose), after two weeks the F group was divided into four others groups: Fructose group (F), that continued with the same treatment, Fructose Arginine group (FA), Fructose Training group (FT) and Fructose Training Arginine group (FTA) same as F group, adding aerobic training on a treadmill and/or arginine supplementation by gavage (800mg/kg BM/day), in the last 8 weeks. There were no differences in body mass gain and caloric intake between groups (P>0.05). It was observed higher serum insulin in F group compared to control group after two (P=0.046) and ten weeks (P=0.014), while it was not observed any difference between the other groups compared to C group. In relation to serum triacylglycerol levels, after two weeks, there was no difference (P=0.154) between C and F groups. After ten weeks the F group presented higher triacylglycerol concentration (P<0.01) compared to C group and the training normalized this increased, however arginine supplementation had no effect on this parameter. The malondialdehyde serum concentration, an oxidative stress marked, had the same pattern as the triacylglycerol. Concerning vascular reactivity, there was no difference after two weeks of treatment. While at the end of protocol, the F group exhibited increased vasoconstrictory reactivity (P<0.01) to phenylephrine when compared to C and to trained groups (FT and FTA). The F group also presented an impaired endothelial vasodilator response to acetylcholine compared to other groups and the arginine supplementation and the aerobic training were able to prevent this impaired response. Thus aerobic training prevents the metabolic alterations, the hypereactivity to phenylephrine and the endothelial damage of rats with high risk to develop metabolic syndrome, while arginine supplementation was capable only to prevent the endothelial damage in this experimental model, with no potentiating effect.Support or Funding InformationCAPES, CNPq, FAPERJ e FINEP.

Tonic Effects and Vascular Reactivity in Rats Submitted to Parkinsonism by Infusion of Bilateral 6‐OHDA in Substantia Nigra

We aimed to investigate the cardiovascular changes in an animal model of Parkinsonism. Adult male Wistar rats were anesthetized to receive bilateral microinfusion of 6-hydroxy dopamine (6-OHDA) or ...

Aerobic Training Prevents Impairment in Vascular Reactivity in Rats Under High Risk of Metabolic Syndrome

It is still unknown the whole mechanisms through which the aerobic training could prevent the development of metabolic syndrome. So the aim of this study was to investigate the vascular mechanism of aerobic training on vascular reactivity of rats under high risk of metabolic syndrome. Wistar rats (n=7/group), males, adults, were divided in Control group (C; drinking water), Fructose group (F; water with 10% fructose), both for 10 weeks; Control Training group (CT) and Fructose Training group (FT), both same as C and F groups, respectively, adding moderated aerobic training on a treadmill (04 times/week) in the last 8 weeks. There were no differences in body mass gain, caloric intake, glycaemia, blood pressure and heart rate between groups. Triglycerides, insulin and isoprostane were higher only in F group and was similar between FT, CT, and C groups. Nitric oxide was higher in trained groups when compared to sedentary groups. In addition, the F group exhibited increased vasoconstrictory reactivity to phenylephrine when compared to C and FT. The F group also presented an impaired endothelial vasodilator response to acetylcholine compared to C and FT vasodilatory response was similar to C and CT groups. F group had lower eNOS expression when compared to C group. The groups that trained had higher values when compared to their sedentary controls; there was no difference between CT and FT groups. In relation to eNOS (phospho S1177) it was observed that the trained groups had higher values when compared to sedentary groups. In conclusion aerobic exercise prevents damage in vascular reactivity, increases eNOS and peNOS expression in this model.

Unsaturated fatty acids supplementation reduces blood lead level in rats.

Some dietary factors could inhibit lead toxicity. The aim of this study was to evaluate the effect of dietary compounds rich in unsaturated fatty acids (FA) on blood lead level, lipid metabolism, and vascular reactivity in rats. Serum metallothionein and organs' lead level were evaluated with the aim of assessing the possible mechanism of unsaturated FA impact on blood lead level. For three months, male Wistar rats that were receiving drinking water with (100 ppm Pb) or without lead acetate were supplemented per os daily with virgin olive oil or linseed oil (0.2 mL/kg b.w.) or egg derived lecithin fraction: “super lecithin” (50 g/kg b.w.). Mesenteric artery was stimulated ex vivo by norepinephrine (NE) administered at six different doses. Lecithin supplementation slightly reduced pressor responses of artery to NE. Lead administered to rats attenuated the beneficial effect of unsaturated FA on lipid metabolism and vascular reactivity to adrenergic stimulation. On the other hand, the super lecithin and linseed oil that were characterized by low omega-6 to omega-3 ratio (about 1) reduced the blood lead concentration. This effect was observed in lead poisoned rats (p < 0.0001) and also in rats nonpoisoned with lead (p < 0.05).

Ryanodine receptor 2 contributes to hemorrhagic shock-induced bi-phasic vascular reactivity in rats.

Ryanodine receptor 2 (RyR2) is a critical component of intracellular Ca(2+) signaling in vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the role of RyR2 in abnormal vascular reactivity after hemorrhagic shock in rats. SD rats were hemorrhaged and maintained mean arterial pressure (MAP) at 40 mmHg for 30 min or 2 h, and then superior mesenteric arteries (SMA) rings were prepared to measure the vascular reactivity. In other experiments, SMA rings of normal rats and rat VSMCs were exposed to a hypoxic medium for 10 min or 3 h. SMA rings of normal rats and VSMCs were transfected with siRNA against RyR2. Intracellular Ca(2+) release in VSMCs was assessed using Fura-2/AM. The vascular reactivity of the SMA rings from hemorrhagic rats was significantly increased in the early stage (30 min), but decreased in the late stage (2 h) of hemorrhagic shock. Similar results were observed in the SMA rings exposed to hypoxia for 10 min or 3 h. The enhanced vascular reactivity of the SMA rings exposed to hypoxia for 10 min was partly attenuated by transfection with RyR2 siRNA, whereas the blunted vascular reactivity of the SMA rings exposed to hypoxia for 3 h was partly restored by transfection with RyR2 siRNA. Treatment with the RyR agonist caffeine (1 mmol/L) significantly increased Ca(2+) release in VSMCs exposed to hypoxia for 10 min or 3 h, which was partially antagonized by transfection with RyR2 siRNA. RyR2-mediated Ca(2+) release contributes to the development of bi-phasic vascular reactivity induced by hemorrhagic shock or hypoxia.

Antibodies against the second extracellular loop of β1-adrenergic receptors induce endothelial dysfunction in conductance and resistance arteries of the Wistar rat

Autoantibodies against β₁-adrenoceptors (β₁-ARs) have been detected in the serum of patients with various cardiac diseases; however, the pathological impact of these autoantibodies (β₁-AABs) has only been evaluated in cardiac tissue. The purpose of the present study was to evaluate whether β₁-AABs have deleterious effects on vascular reactivity in rats. An enzyme-linked immunosorbent assay was used to detect β₁-AABs in sera from immunized rats over a period of 1-3 months using the peptidic sequence of the second extracellular loop of human β₁-AR. Functional studies were performed in thoracic aortic (TA) and small mesenteric artery (SMA) rings from immunized rats. Following pre-contraction with phenylephrine (0.3 μM and 3 μM for the TA and SMA respectively), cumulative concentration-response curves (CCRCs) to various β-AR agonists (isoproterenol, dobutamine, salbutamol, SR 58611A), acetylcholine, A23187, and sodium nitroprusside (SNP) were then plotted. The relaxations induced by dobutamine, SR 58611A, and acetylcholine were significantly impaired, but salbutamol-induced relaxations were not affected, in both vessels from immunized rats. A significant impairment of isoproterenol-induced relaxation was only observed in SMA. CCRCs to SNP were not modified in either of the vessels. A23187-induced relaxation was impaired in immunized rats. Following pretreatment with L-arginine, vasorelaxation to acetylcholine and SR 58611A was restored in immunized rats. This study demonstrates that immunization against the second extracellular loop of β₁-ARs has a deleterious impact on vasorelaxations in the TA and SMA of rats, involving alterations in endothelium-dependent NO signaling pathways.

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.

New method for isolation of both kidneys for studies of vascular reactivity in rats

The isolated perfused rat kidney technique is one of the most widely used methods in renal research. It has proven useful to investigate biochemical, physiological, pharmacological and pathophysiological aspects of renal function, allowing variables to be changed in a controlled manner and eliminating systemic influences. This study was designed to test a new surgical procedure to isolate both kidneys for their utilization in this technique, mainly as a vascular preparation. The viability of the resulting preparations was compared with those obtained by the classical method, analyzing the responsiveness to the vasoconstrictor phenylephrine and to the endothelium-dependent vasodilator acetylcholine. Vascular reactivity was evaluated under normal conditions and in endothelium-denuded preparations. The dose response curves to phenylephrine and to acetylcholine were similar in the three experimental groups, regardless of the procedure used for kidney isolation, in both endothelium-intact and endothelium-denuded preparations. We give a step-by-step description of the isolation method and key points for the success of the technique. In conclusion, the surgical procedure proposed in this paper reduces the number of animals required by half and hence reduces the cost of the experiments. This novel procedure is of special interest for acute experiments to test new vasoactive drugs and for analysis of the action mechanisms of these drugs. It could also be used in chronic studies or in genetically modified animals when different experimental protocols are performed on the left and right kidneys and to improve the accuracy of the results by analyzing cases in duplicate.

Blueberry intervention improves vascular reactivity and lowers blood pressure in high-fat-, high-cholesterol-fed rats

Growing evidence suggests that intake of flavonoid-containing foods may exert cardiovascular benefits in human subjects. We have investigated the effects of a 10-week blueberry (BB) supplementation on blood pressure (BP) and vascular reactivity in rats fed a high-fat/high-cholesterol diet, known to induce endothelial dysfunction. Rats were randomly assigned to follow a control chow diet, a chow diet supplemented with 2 % (w/w) BB, a high-fat diet (10 % lard; 0·5 % cholesterol) or the high fat plus BB for 10 weeks. Rats supplemented with BB showed significant reductions in systolic BP (SBP) of 11 and 14 %, at weeks 8 and 10, respectively, relative to rats fed the control chow diet (week 8 SBP: 107·5 (SEM 4·7) v. 122·2 (SEM 2·1) mmHg, P= 0·018; week 10 SBP: 115·0 (SEM 3·1) v. 132·7 (SEM 1·5) mmHg, P< 0·0001). Furthermore, SBP was reduced by 14 % in rats fed with the high fat plus 2 % BB diet at week 10, compared to those on the high-fat diet only (SBP: 118·2 (SEM 3·6) v. 139·5 (SEM 4·5) mmHg, P< 0·0001). Aortas harvested from BB-fed animals exhibited significantly reduced contractile responses (to L-phenylephrine) compared to those fed the control chow or high-fat diets. Furthermore, in rats fed with high fat supplemented with BB, aorta relaxation was significantly greater in response to acetylcholine compared to animals fed with the fat diet. These data suggest that BB consumption can lower BP and improve endothelial dysfunction induced by a high fat, high cholesterol containing diet.

Altered sympathetic reflexes and vascular reactivity in rats after exposure to chronic intermittent hypoxia

Exposure to chronic intermittent hypoxia (CIH) yields persistent elevations in sympathetic nerve activity (SNA) and mean arterial pressure(MAP)with exaggerated sympathetic chemoreflexes. We examined the impact of CIH upon other sympathoexcitatory reflexes and a potential central mechanism underlying the altered regulation of SNA.Male Sprague-Dawley rats were exposed to CIH for 2 weeks (40 s at 6% O2 every 9 min, 8 h day⁻¹). After exposure to CIH, urethane-anaesthetized, vagotomized, ventilated, paralysed rats had significantly elevated MAP, splanchnic SNA, and rate of phrenic nerve discharge (PND; P<0.05). Elimination of SNA by ganglionic blockade produced a larger fall in MAP in rats exposed to CIH (P<0.05). Like acute hypoxia, stimulation of the sciatic nerve or the nasal mucosa evoked greater increases in SNA after exposure to CIH (P<0.05). In addition, acute hypoxia promoted exaggerated increases in PND amplitude after CIH (P<0.05). In contrast, the nasopharyngeal reflex evoked exaggerated increases in SNA during apnoea. These sympathoexcitatory reflexes are mediated by glutamatergic activation of the rostral ventrolateral medulla (RVLM), and accordingly, microinjections of glutamate into RVLM evoked larger increases in SNA after CIH (P<0.05). Paradoxically, none of these exaggerated acute rises in SNA was accompanied by enhanced pressor responses. Reduced adrenergic vascular reactivity may contribute to the blunted sympathetically mediated pressor responses, because bolus doses of phenylephrine evoked attenuated pressor responses after CIH (P<0.01).These data suggest exposure to CIH facilitates activation of SNA, potentially by changes within the RVLM. However, the exaggerated rises in SNA are not dependent upon stimulation of inspiratory drive. Although elevated SNA may contribute to CIH-induced hypertension, reduced adrenergic vascular reactivity buffers the cardiovascular impact of exaggerated acute rises in SNA.

Activation of MEK1/ERK1/2/iNOS/sGC/PKG pathway associated with peroxynitrite formation contributes to hypotension and vascular hyporeactivity in endotoxemic rats

Increased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO contributes to fall in blood pressure and vascular reactivity during endotoxemia. We investigated whether an increase in protein expression and activity of the enzymes involved in mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2)/iNOS/soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway associated with peroxynitrite production would contribute to endotoxin-induced decrease in mean blood pressure (MAP) and vascular reactivity in rats. A selective iNOS inhibitor, 1,3-PBIT (10 mg/kg, i.p.), or a selective inhibitor ERK1/2 phosphorylation by MEK1, U0126 (5 mg/kg, i.p.), prevented endotoxin (10 mg/kg, i.p.)-induced decrease in MAP and vascular reactivity to norepinephrine (0.001–100 μM) in endothelium-intact and -denuded arteries associated with increased levels of nitrite (an index for NO production), cyclic GMP (an index for sGC activity), phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), and nitrotyrosine (an index for peroxynitrite production). Endotoxin-induced increase in the phosphorylated MEK1 protein levels were not changed by 1,3-PBIT or U0126. U0126 prevented the endotoxin-induced increase in phosphorylated ERK1/2 and iNOS expressions. A selective sGC inhibitor, ODQ (3 μM), prevented the endotoxin-induced decrease in the E max values and increase in the EC 50 values of norepinephrine in endothelium-intact aortic rings isolated from endotoxemic rats in vitro. ODQ also reversed the effect of endotoxin on the increase in the EC 50 values of norepinephrine in endothelium-denuded rings. A selective PKG inhibitor, KT5823 (1 μM), only prevented the endotoxin-induced decrease in the E max values of norepinephrine in arteries with endothelium. These results suggest that activation of MEK1/ERK1/2 pathway leading to an increase in iNOS protein expression and NO production associated with an increase in sGC and PKG activity and peroxynitrite formation results in hypotension and vascular hyporeactivity in endotoxemic rats. However, further study is needed to confirm the involvement of PKG to the fall in vascular reactivity in the rat model of endotoxemia.