Eduardo Tibiriçá

Murine models for pharmacological studies of the metabolic syndrome

Metabolic syndrome has been described as the association of insulin resistance, hypertension, hyperlipidemia and obesity. Its prevalence increased dramatically, mainly in developed countries. Animal models are essential to understand the pathophysiology of this syndrome. This review presents the murine models of metabolic syndrome the most often used in pharmacological studies. The most common metabolic syndrome models exhibit a non-functional leptin pathway, or metabolic disorders induced by high fat diets. In a first part, and after a short introduction on leptin, its receptor and mechanism of action, we provide a detailed description of each model: SHROB, SHHF, JCR:LA-cp, Zucker, ZDF, Wistar Ottawa Karlsburg W, and Otsuka Long-Evans Tokushima Fatty rats, ob/ob, db/db, agouti yellow and Mc4R KO mice. The second part of this review is dedicated to metabolic syndrome models obtained by high fat feeding.

Statins decrease neuroinflammation and prevent cognitive impairment after cerebral malaria

Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.

Evaluation of systemic microvascular endothelial function using laser speckle contrast imaging

OBJECTIVE The aim of this study was to compare cutaneous microvascular function in young healthy subjects (n=50) with that of cardiometabolic diseased patients (n=50) using laser speckle contrast imaging (LSCI) coupled with transdermal iontophoretic delivery of acetylcholine (ACh) and post-occlusive reactive hyperemia (PORH). METHODS Cutaneous blood flow was assessed in the forearm using LSCI at rest, during PORH and during iontophoresis of ACh with increasing anodal currents of 30, 60, 90, 120, 150 and 180 μA during 10-second intervals spaced 1 min apart. RESULTS Endothelium-dependent skin microvascular vasodilator responses induced by both ACh and PORH were significantly reduced in cardiometabolic diseased patients compared to healthy subjects. Vasodilator responses induced by ACh were significantly higher in young women than in young men. Iontophoresis charges up to 1.5 mC do not induce nonspecific effects on skin microvascular flux. CONCLUSION LSCI appears to be a promising noninvasive technique for evaluating systemic microvascular endothelial function.

Skin capillary density and microvascular reactivity in obese subjects with and without metabolic syndrome.

Obesity is associated with increased cardiovascular morbidity and mortality. We hypothesized that microvascular function may be impaired in obese subjects with metabolic syndrome (OB-MetSnd) compared to obese subjects without MetSnd (OB) and healthy subjects (HS). In this cross-sectional study, we evaluated skin capillary density (SCD) in OB-MetSnd (n=20, 12 women, BMI=36.5±1.1kg/m(2)), OB (n=25, 16 women, BMI=34.5±0.7kg/m(2)), and HS (n=30, 22 women, BMI=22.8±0.3kg/m(2)) groups. SCD was evaluated by intravital video-microscopy at rest and after post-occlusive reactive hyperemia (PORH) and venous congestion (VC). OB-MetSnd subjects exhibited significant differences in the values of MetSnd components and in leptin and HOMA-IR levels compared to OB and HS individuals. There were no differences in SCD among groups in resting conditions. The OB-MetSnd group failed to show a significant increase in the number of recruited capillaries during PORH and VC compared to the SCD evaluated at rest. A negative correlation of SCD with waist circumference, BMI, blood pressure, and HOMA-IR was observed after PORH and VC. When obese subjects were analyzed according to their HOMA-IR quartiles, a significant decrease in SCD was observed during POHR (P=0.02). Our findings showed that obese subjects have structural and functional alterations in skin microcirculation that are proportional to the increase in the degree of global and central obesity. In addition, in OB-MetSnd subjects, the cutaneous capillaries at rest are already maximally recruited, indicating an absence of functional capillary reserve. This may be related to the insulin resistance observed in OB-MetSnd individuals.

Glucose levels observed in daily clinical practice induce endothelial dysfunction in the rabbit macro‐ and microcirculation

We investigated whether different concentrations of elevated glucose – corresponding to levels observed in patients with type 2 diabetes under routine care (post‐prandial mean and maximum values) and those used for diagnosing diabetes – induce impairment of vascular reactivity of the macro‐ and microcirculation in non‐diabetic rabbits. Aortic rings and isolated perfused kidneys from normal rabbits were acutely exposed (3 h) to normal (5.5 mm) or high (7–25 mm) d‐glucose concentrations. Vascular reactivity was evaluated with endothelium‐dependent [acetylcholine (ACh) and bradykinin (BK)] and independent [sodium nitroprusside (SNP)] vasodilating agents. Endothelium‐dependent relaxation of the thoracic aorta induced by ACh or BK was significantly attenuated after a 3‐h exposure to high d‐glucose (15–25 mm) but not after corresponding increased osmolarity with mannitol solutions. Relaxation induced by SNP (endothelium‐independent) was not affected by high d‐glucose concentrations. Moreover, endothelium‐dependent but not independent vasodilation of the isolated rabbit kidney was also impaired after 3‐h perfusion with high d‐glucose (11.1–25 mm). Perfusion with mannitol solutions (15–25 mm) partially blunted endothelium‐dependent renal vasodilation. It is concluded that acute hyperglycemia corresponding to post‐prandial levels in patients with type 2 diabetes induces endothelial dysfunction of conduit vessels as well as of the renal circulation of non‐diabetic rabbits.

Effects of Antihypertensive Drugs on Capillary Rarefaction in Spontaneously Hypertensive Rats : Intravital Microscopy and Histologic Analysis

We investigated the effects of chronic oral antihypertensive treatment on functional and structural capillary rarefaction in spontaneously hypertensive rats (SHR). Wistar Kyoto rats (WKY) were used as a normotensive control group. In untreated rats, intravital videomicroscopy showed that functional capillary density was lower in SHR skeletal muscle (WKY 395 ± 17 and SHR 258 ± 13 capillaries/mm2, P < 0.01) and ear skin (WKY 391 ± 18 and SHR 210 ± 15 capillaries/mm2, P < 0.01). A linear relationship was seen between skeletal muscle and skin capillary densities (r = 0.654, P < 0.0001). Histologic analysis showed that SHR had a lower capillary-to-fiber ratio in the skeletal muscle (WKY 1.74 ± 0.08 and SHR 1.40 ± 0.06, P < 0.01). Capillary volume density-to-fiber volume density ratio in the left ventricle of SHR was also reduced (WKY 0.55 ± 0.09 and SHR 0.42 ± 0.09, P < 0.01). The animals were treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril, the angiotensin II type I receptor (AT1) receptor antagonist losartan, the β-blocker atenolol, or the calcium channel blocker nifedipine, resulting in similar reductions in systolic blood pressure (19.8%, 19.1%, 17.4%, and 18.2%, respectively, P > 0.05). Atenolol did not induce any change in functional capillary density of SHR. Losartan and nifedipine completely reversed functional capillary rarefaction in both muscle and cutaneous tissues, whereas enalapril significantly increased functional capillary density only in the skin. The skeletal muscle capillary-to-fiber ratio was normalized by enalapril, losartan, and nifedipine. Treatments with enalapril or losartan normalized the cardiac structural capillary rarefaction of SHRs, whereas atenolol and nifedipine had no effect. Our results suggest that different pharmacologic classes of antihypertensive drugs with similar effect on blood pressure differ in terms of their effect on the microcirculation.

Structural and functional microvascular alterations in a rat model of metabolic syndrome induced by a high‐fat diet

To investigate microvascular alterations in an experimental model of metabolic syndrome induced by a high‐fat diet (HFD) associated with salt supplementation (0.5% NaCl).

Antihypertensive Treatment Improves Microvascular Rarefaction and Reactivity in Low-Risk Hypertensive Individuals

To test whether long‐term antihypertensive treatment with metoprolol succinate (a β1‐adrenoceptor blocker) or olmesartan medoxomil (an angiotensin II AT1‐receptor blocker) reverses microvascular dysfunction in hypertensive patients.

PPAR gamma activation protects the brain against microvascular dysfunction in sepsis

Sepsis is a severe disorder characterized by systemic inflammatory responses in the presence of an infection and may progress to multiple organ dysfunction and death. Alterations in cerebral microcirculation fulfill a crucial role in the pathogenesis of severe sepsis, and include a decrease in capillary density and disturbances in leukocyte movement along capillaries. Nevertheless, the mechanisms involved in sepsis-associated cerebral microcirculatory alterations have so far not been defined. We investigated the effect of the peroxisome proliferator-activated receptor gamma (PPARγ) selective agonist rosiglitazone on leukocyte/endothelial cell interaction and functional capillary density in the brain in the cecal ligation and puncture (CLP) model of sepsis. Anti-inflammatory effects of rosiglitazone on the cerebral microcirculation were marked. Functional capillary density increased and leukocyte rolling and adhesion were decreased in animals submitted to CLP and treated with rosiglitazone. Our data provide evidence for involvement of PPARγ activation in leukocyte-endothelium interactions and alterations in capillary density. Improved cerebral perfusion in animals treated with rosiglitazone, suggests that PPARγ activation is protective against cerebral microvascular dysfunction in sepsis.

Antihypertensive effects of crude extracts from leaves of Echinodorus grandiflorus

The antihypertensive action of a crude ethanolic extract (EEEG) from leaves of Echinodorus grandiflorus (Alismataceae) was investigated in spontaneously hypertensive rats. The intraperitoneal injection of increasing doses of EEEG (300–1000 mg/kg) elicited dose‐dependent reductions in mean arterial pressure (MAP) that were paralleled by reductions of cardiac output and systemic vascular resistance, reaching the maximum of 23 ± 5%, 13 ± 3% and 18 ± 4%, respectively (n = 5, P < 0.05). Comparable reductions of MAP were obtained upon i.v. administration of EEEG (3–100 mg/kg), reaching the maximum decrease of 51 ± 6% (n = 7; P < 0.001). The blockade of nitric oxide synthesis significantly reduced the hypotension induced by i.v. administration of EEEG. Moreover, the pre‐treatment of the animals with a selective antagonist of cholinergic muscarinic receptors or of platelet‐activating factor (PAF) receptors partially blunted the cardiovascular effects of EEEG. The i.v. pre‐treatment with the selective B2 bradykinin receptor antagonist HOE 140 or with indomethacin, an inhibitor of the enzyme cyclooxygenase, did not prevent the hypotensive effects induced by EEEG. Finally, the chronic oral treatment with EEEG presented a significant antihypertensive effect that was comparable to that of reference antihypertensive drugs currently used to treat arterial hypertension. It is concluded that EEEG elicits significant acute antihypertensive effects through the release of nitric oxide and the stimulation of cholinergic muscarinic and PAF receptors. Moreover, our results suggest that EEEG may be appropriate to chronic oral treatment of arterial hypertension.