Fatima Z.G.A. Cyrino

Effects of oral administration of different doses of purified micronized flavonoid fraction on microvascular reactivity after ischaemia/reperfusion in the hamster cheek pouch

1 The effects of a purified micronized flavonoid fraction (S5682) on mean internal diameter and blood flow of arterioles and venules, as well as the functional capillary density (FCD) were evaluated in the hamster cheek pouch microvasculature before and after 90 min of total ischaemia. 2 Male hamsters were treated for ten days, twice a day, with oral doses of S5682 (5, 20, 80 and 160 mg kg−1 day−1) or placebo (10% lactose solution). The cheek pouch preparation was placed under an intravital microscope coupled to a closed circuit TV system. Local ischaemia was obtained by a cuff mounted around the neck of the everted pouch where it leaves the mouth of the hamster. 3 Measurements were performed before ischaemia, at the onset of reperfusion and 10, 20, 30, 45 and 60 min thereafter. Diameters were measured by means of an image shearing device. Red blood cell (RBC) velocity was analysed by use of the dual‐slit photometric technique. Blood flow was calculated from diameters and RBC velocities. FCD, defined as the number of capillaries with flowing blood per field of observation, was also assessed. 4 During reperfusion, placebo‐treated animals showed a significant vasodilatation, a decrease in blood flow and FCD and S5682‐treated animals showed a clear trend, dose‐dependent, towards maintaining these parameters closer to the value found before ischaemia. 5 In conclusion, our results indicate that S5682 improves the microvascular reactivity and FCD after ischaemia/reperfusion. These data suggest that S5682 could function as an antioxidant, which may explain its beneficial therapeutic effect in chronic venous insufficiency where oxidative stress is involved in the pathological mechanism.

Leukocyte Adhesion after Oxidant Challenge in the Hamster Cheek Pouch Microcirculation

Oral administration of S-5682 (Daflon 500 mg, 90% diosmin, 10% hesperidin) inhibits oxidant-induced increase in macromolecular permeability in the postcapillary venules of the hamster cheek pouch microcirculation. In this study, the effect of S-5682 on leukocyte-endothelium interaction was evaluated using the same experimental model. Hamsters kept on a standard diet were divided into 5 groups (n = 6) and treated orally, twice a day, with placebo (10% lactose solution), S-5682, 5, 20 or 80 mg/kg/day (suspended in 10% lactose solution) or α-tocopherol, 1 mg/kg/day, for 10 days prior to the oxidant challenge with tert-butylhydroperoxide (TBOOH). Topical application of TBOOH (10–4 M for 5 min) to hamsters given acridine orange prior to TBOOH resulted in increases in the number of rolling and sticking (no movement for at least 30 s) leukocytes in postcapillary venules. No changes in the number of rolling leukocytes could be observed in the treated groups compared with the placebo group (p > 0.05). On the contrary, leukocyte adhesion was inhibited in groups treated with S-5682 (5, 20 and 80 mg/kg/day) or α-tocopherol: placebo 105 ± 3/6 mm2 (mean ± SEM); S-5682, 5 mg/kg/day 68 ± 3/6 mm2 (p < 0.01), 20 mg/kg/day 55 ± 3/6 mm2 (p < 0.001) and 80 mg/ kg/day 39 ± 2/6 mm2 (p < 0.001) and α-tocopherol 36 ± 1/6 mm2 (p < 0.001). The inhibition of oxidant-induced leukocyte adhesion by S-5682 was similar to that seen for ischemia-reperfusion and the higher dose of S-5682 was as effective as α-tocopherol in inhibiting it.

Possible mechanisms for the inhibitory effect of Ruscus extract on increased microvascular permeability induced by histamine in hamster cheek pouch.

Extract of Ruscus aculeatus is used in treatment of venous insufficiency. In the present study, we used the hamster cheek pouch preparation and investigated in vivo the effects of an alpha 1 and alpha 2 adrenoceptor antagonists, a calcium blocker, Ruscus extract, and their combination on increased microvascular permeability induced by histamine. Experiments were performed on male hamsters; 30 min after completion of the cheek pouch preparation, fluorescein-labeled dextran (molecular weight 150,000) was given intravenously (i.v.). Histamine, applied topically, increased the number of fluorescent vascular leakage sites from postcapillary venules, evidence of an increase in macromolecular permeability, which was quantified by ultraviolet light microscopy as the number of leaky sites in the prepared area. Prazosin (alpha 1-adrenoceptor antagonist), diltiazem (calcium blocker), and Ruscus extract applied topically dose-dependently inhibited the macromolecular permeability-increasing effect of histamine. Rauwolscine (alpha 2-adrenoceptor antagonist), also applied topically, had no effect on histamine-induced permeability increase. Inhibition of the histamine-induced permeability increase evoked by Ruscus extract could be blocked by prazosin and by diltiazem but not by rauwolscine. These results indicate that any variation in the transmembrane flux of calcium impairs formation of microvascular leaky sites by histamine. Our results show that Ruscus extract has a protective effect against the leakage of FITC-dextran in hamster cheek pouch after administration of histamine that is modulated by calcium and selectively by alpha 1-adrenoceptors.

Effects of Ruscus extract on the internal diameter of arterioles and venules of the hamster cheek pouch microcirculation

Summary: In the present study, we investigated (a) the effects of the extract of Ruscus aculeatus, which is used to increase peripheral venous tone, on the diameter of arterioles (ID range 10–70 u.m) and venules (ID range 20–135 (μm) of hamster cheek pouch microvasculature in vivo and (b) the influence of temperature on the observed effects. For microcirculatory measurements, the preparations were placed under an intravital microscope and coupled to a closed-circuit TV (ccTV) system. The TV monitor display was used to obtain arteriolar and venular ID recordings (always at the same site) by an image shearing device. For systemic intravenous (i.v.) administration, the measurements were performed every 10 min, before (control) and after injection of the extract (5 mg/kg). During topical application, the extract was tested, in different concentrations, at 25°, 36.5°, and 40°C. Systemic i.v. administration of Ruscus extract evoked venular constriction and did not affect the arteriolar diameter or mean arterial pressure (MAP). Topical application of Ruscus extract elicited concentration- and temperature-dependent responses in the vessels. At 25°C, arterioles and venules dilated; at 36.5°C, the arterioles remained unchanged while the venules constricted, and at 40°C, the arterioles remained unchanged or constricted depending on the concentration used while the venules further constricted. The effects of Ruscus extract observed in vivo at the microcirculatory level further support the data previously reported on larger vessels and on patients with venous insufficiency.

High fat diet induces central obesity, insulin resistance and microvascular dysfunction in hamsters

Microvascular dysfunction is an early finding in obesity possibly related to co-morbidities like diabetes and hypertension. Therefore we have investigated changes on microvascular function, body composition, glucose and insulin tolerance tests (GTT and ITT) on male hamsters fed either with high fat (HFD, n=20) or standard (Control, n=21) diet during 16 weeks. Total body fat and protein content were determined by carcass analysis, aorta eNOS and iNOS expression by immunoblotting assay and mean blood pressure (MAP) and heart rate (HR) by an arterial catheter. Microvascular reactivity in response to acetylcholine and sodium nitroprusside, functional capillary density (FCD), capillary recruitment induced by a hyperinsulinemic status and macromolecular permeability after 30 min ischemia was assessed on either cheek pouch or cremaster muscle preparations. Compared to Control, HFD animals have shown increased visceral fat (6.0 ± 0.8 vs. 13.8 ± 0.6g/100g BW), impaired endothelial dependent vasodilatation, decreased FCD (11.3 ± 1.3 vs. 6.8 ± 1.2/field) and capillary recruitment during hyperinsulinemia and increased macromolecular permeability after ischemia/reperfusion (86.4 ± 5.2 vs.105.2 ± 5.1 leaks/cm(2)), iNOS expression and insulin resistance. MAP, HR, endothelial independent vasodilatation and eNOS expression were not different between groups. Our results have shown that HFD elicits an increase on visceral fat deposition, microvascular dysfunction and insulin resistance in hamsters.

Inhibitory effect of the Ruscus extract and of the flavonoid hesperidine methylchalcone on increased microvascular permeability induced by various agents in the hamster cheek pouch.

Summary: The Ruscus extract and the flavonoid hesperidine methylchalcone (HMC) are used in treatment of venous insufficiency. In the present study, we used the hamster cheek pouch preparation and investigated the effects of these substances on increased microvascular permeability induced by bradykinin, histamine, and leukotriene B4 (LTB4) applied topically. Experiments were performed on male hamsters; 30 min after completion of the cheek pouch preparation, fluorescein-labeled dextran [molecular weight (mol wt) 150,000] was given intravenously (i.v.). Bradykinin, histamine, and LTB4 increased the number of fluorescent vascular leakage sites from postcapillary venules, evidence for an increase in macro-molecular permeability, which was quantified in ultraviolet (UV)-light microscope as the number of leaky sites in the prepared area. Ruscus extract and HMC, given i.v., significantly inhibited the macromolecular permeability-increasing effect of bradykinin, LTB4, and histamine. Ruscus extract, applied topically, dose dependently inhibited the macromolecular permeability-increasing effect of histamine. Our results show that Ruscus extract and HMC have a protective effect against leakage of FITC-dextran in the cheek pouch after administration of various permeability-increasing substances, which further supports data previously reported on patients with venous insufficiency

Effects of Insulin and the Combination of Insulin Plus Metformin (Glucophage) on Microvascular Reactivity in Control and Diabetic Hamsters

The purpose of this study was to determine the in vivo microvascular reactivity of arte rioles (mean internal diameter range: 16.0 to 106.4 μm) and venules (mean internal diameter range: 24.0 to 117.3 μm) in the hamster cheek pouch to insulin and to the mixture insulin + metformin. Experiments were performed using an intravital micro scope coupled to a closed-circuit TV system and a videotape. The TV monitor display was used to obtain arteriolar and venular internal diameter measurements by an image- shearing device. The studied drugs were applied topically, added to the superfusion solution, to avoid systemic effects that would complicate the analysis of the results. In control animals (glycemia 7.7 ±0.4 mmol/L), application of insulin (10 to 500 μU/mL/min) evoked vasodilatation in a dose-dependent fashion in arterioles (4.9 ±3.2% to 50.9 ±6.5%, smallest and largest concentration, respectively, values expressed in percent of the initial diameter as mean ±SE) and venules (-2.1 ±3.1% to 14.3 ±5.1%), decreased and finally abolished the spontaneous vasomotion frequency (from 9.5 ± 0.3 cycles per minute [cpm] to 0.0 ±0.0 cpm) and amplitude (from 8.6 ±0.3 to 0.0 ±0.0 μm). Addition of metformin, 0.2 mg/mL/min, did not significantly change either the observed vasodilatation in arterioles and venules or the vasomotion frequency and amplitude curves. Two types of diabetic hamsters were studied: severely diabetic, induced with three intraperitoneal injections of streptozotocin, diluted in physiological saline, 50 mg/kg/dose, given in three consecutive days, and mildly diabetic, induced by a single dose of streptozotocin. All diabetic animals were studied four weeks after the onset of diabetes and no specific treatment for diabetes was given. In severely diabetic hamsters (glycemia 18.0 ±2.2 mmol/L), application of insulin, in the same concentration range, evoked a significantly reduced vasodilatation in arterioles as compared with control animals (5.9 ±1.3% to 18.9 ±3.5%) and did not change the vasodilatation observed in the venules (5.9 ±1.4% to 21.3 ±2.5%). In these preparations no spontaneous arteriolar vasomotion could be detected. Addition of metformin did not significantly improve the impaired vasodilatation. In mildly diabetic hamsters (glycemia 12.1 ±0.8 mmol/L), application of insulin, in the same concentration range, evoked vasodilatation, in a dose- dependent fashion, equivalent to the one observed in control animals, in arterioles (3.1 ±2.5% to 53.4 ±10.0%) and venules (7.1 ±3.0% to 29.9 ±4.8%) and also reduced the vasomotion frequency (from 10.1 ±0.3 to 0.1 ±0.1 cpm) and amplitude (from 9.2 ±0.6 to 0.2 ± 0.2 μm). Addition of metformin tended to increase the observed arteriolar dilata tion (6.6 ±3.0% to 67.8 ±5.5%), did not change the venular dilatation (6.7 ±4.8% to 28.0 ±3.3%), and tended to preserve vasomotion frequency and amplitude. These experiments show that (1) insulin has a direct dilatatory effect on arterioles and venules; (2) the vasodilatation evoked by insulin is impaired in severe diabetes, and (3) no significant abnormality could be detected on microvascular reactivity in mild diabetes. Further addition of metformin helped to maintain the spontaneous arteriolar vasomotion even during moderate vasodilatation and tended to augment the arteriolar dilatation evoked by insulin in mildly diabetic animals.

Possible mechanisms for the venular constriction elicited by ruscus extract on hamster cheek pouch

We investigated the influence of alpha-adrenoceptors blockers and calcium blockers on the effects of the venotonic agent Ruscus extract on the diameter of arterioles (ID 10-70 microns) and venules (ID 20-135 microns) of hamster cheek pouch microvasculature in vivo. For microcirculatory measurements, the preparations were placed under an intravital microscope coupled to a closed-circuit TV system. The TV monitor display was used to obtain arteriolar and venular internal diameter recordings (always at the same site) by an image shearing device. All drugs were applied topically. Ruscus extract was tested in different concentrations and in combination with prazosin (alpha 1-adrenoceptor antagonist), rauwolscine (alpha 2-adrenoceptor antagonist), or diltiazem (calcium blocker). Topical application of Ruscus extract elicited concentration-dependent responses in the studied vessels: arterioles remained unchanged in the concentration range tested, whereas venules remained unchanged or constricted depending on the concentration used. The observed venular constriction could be blocked by low concentrations (10(-9) M) of prazosin or diltiazem and by high concentrations (> 10(-6) M) of rauwolscine. Our results suggest that the venular constriction elicited by Ruscus extract in vivo, at the microcirculatory level, is mediated by calcium and by alpha-adrenoceptors and further support data previously reported on larger vessels and on patients with venous insufficiency.

Waist circumference leads to prolonged microvascular reactive hyperemia response in young overweight/obese women.

OBJECTIVES Previous data in our laboratory have shown microvascular dysfunction in normoglycaemic subjects with metabolic syndrome (MS). In a step further, we have investigated which clinical parameters related or not to MS would elicit microvascular dysfunction and the need of diagnosing MS for the establishment of microcirculatory impairment in overweight/obese women. METHODS Nineteen lean [23.6±3.1years, body mass index (BMI) 21.9±1.8kg/m(2)] and 59 overweight/obese [24.6±3.7years; BMI 34.4±5.9kg/m(2)] sedentary non-smoking women, divided in overweight/obese without (MS negative, n=36) and obese with MS (MS positive, n=23) were evaluated. Blood biochemistry, HOMA-IR index and anthropometric variables were determined. Morphological (capillary diameters) and functional [functional capillary density, red blood cell velocity (RBCV) at baseline and peak and time (TRBCV(max)) to reach it during post-occlusive reactive hyperemia after 1min ischemia] microcirculatory variables were examined by nailfold videocapillaroscopy. RESULTS Compared to controls, overweight/obese MS negative and obese MS positive presented longer TRBCV(max); the presence of two MS components was sufficient to prolong it and the MS diagnosis did not add any significant impairment to the microcirculation. Among clinical parameters investigated, a direct relationship between TRBCV(max) and waist circumference and insulin concentrations was found. CONCLUSION Our results have shown that microvascular dysfunction is independent of metabolic syndrome diagnosis and could be predicted by the waist circumference on young overweight/obese women, reinforcing the relationship between obesity-related microvascular/metabolic disturbances.

Oxidant-lnduced Increase in Vascular Permeability Is Inhibited by Oral Administration of S-5682 (Daflon® 500 mg) and Alpha-Tocopherol

The aim was to study the effect of oral administration of three different doses of S-5682 and alpha-tocopherol on an oxidant-induced injury by tert-butylhydroperoxide (TBOOH) resulting in increased plasma leakage from postcapillary venules in the hamster cheek pouch microcirculation. Hamsters were on a standard laboratory animal diet with normal vitamin E and C content. Five groups of hamsters (n = 6) were treated orally with placebo (10% lactose solution), S-5682 (5, 20 or 80 mg/kg/day) suspended in 10% lactose solution, or alpha-tocopherol (1 mg/kg/day) for 10 days prior to oxidant challenge with TBOOH. Topical application of 10(-4) M TBOOH for 5 min to hamsters given FITC-dextran 30 min prior to TBOOH resulted in reversible increases in the number (mean +/- SE) of leaks in postcapillary venules: placebo, 117+/-7 leaks/cm2; S-5682, 5 mg/kg/day, 68+/-3 leaks/cm2 (p < 0.01); S-5682, 20 mg/kg/day, 41+/-3 leaks/cm2 (p < 0.01); S-5682, 80 mg/kg/day, 25+/-2 leaks/cm2 (p < 0.001), and alpha-tocopherol, 1 mg/kg/day, 18+/-1 leaks/cm2 (p < 0.001). The efficacy of inhibition of oxidant-induced leakage by S-5682 was similar to that seen with the same dose (20 mg/kg/day) of histamine, bradykinin, leukotriene B4 or ischemia/reperfusion-induced leakage, suggesting a common pathway for the induction of plasma leakage by these mediators. The maximal dose of S-5682 (80 mg/kg/day) was as effective as alpha-tocopherol (1 mg/kg/day) in inhibiting plasma leakage.