Increase In Macromolecular Permeability Research Articles

Effects of Selenium in the Microcirculation of Fructose-Fed Hamsters

Background and Aims: Fructose intake is directly related to vascular dysfunction and it is a risk factor for the development of metabolic and cardiovascular diseases, such as obesity, diabetes, and hypertension. Selenium, a component of antioxidant enzymes, improves hyperglycemia and vascular function in diabetic animals. The aim of this study was to evaluate the effects of dietary selenium supplementation on microcirculatory and metabolic parameters of fructose-fed hamsters. Methods and Results: Male hamsters (Mesocricetus auratus) had their drinking water substituted or not by 10% fructose solution for 60 days, during which their microcirculatory function was evaluated in the cheek pouch preparation. Blood glucose and serum insulin levels were also tested. Microcirculatory responses to acetylcholine (an endothelium-dependent vasodilator) and to sodium nitroprusside (SNP, an endothelium-independent vasodilator), and macromolecular permeability increase induced by a 30-min ischemia/reperfusion (I/R) procedure, showed that endothelium-dependent and independent vasodilatation was significantly increased in animals that had high selenium supplementation, in both the control and fructose-fed groups. Selenium supplementation protected against plasma leakage induced by I/R in all control and fructose-fed groups. Conclusion: Our results indicate that dietary selenium supplementation reduces microvascular dysfunction by increasing endothelial-dependent and independent dilatation and reducing macromolecular permeability increase in fructose-fed animals.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract.Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking.Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50–100μg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction.Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

Microcirculatory effects of zinc on fructose-fed hamsters

Background and aimsFructose is a major dietary component directly related to vascular dysfunction and diseases such as obesity, diabetes, and hypertension. Zinc is considered a non-pharmacological alternative for treating diabetes due to its antioxidant and hyperglycemia-lowering effects in diabetic animals. Therefore, the aim of this study was to evaluate the effects of dietary zinc supplementation on the microcirculatory parameters of fructose-fed hamsters. Methods and ResultsMale hamsters (Mesocricetus auratus) were fed drinking water substituted by 10% fructose solution for 60 days, whereas control animals were fed drinking water alone. Their microcirculatory function was evaluated using cheek pouch preparation, as well as their blood glucose and serum insulin levels. Their microcirculatory responses to acetylcholine (ACh, an endothelium-dependent vasodilator) and to sodium nitroprusside (SNP, an endothelium-independent vasodilator) as well as the increase in macromolecular permeability induced by 30 min of ischemia/reperfusion (I/R) were noted. Endothelium-dependent vasodilation was significantly increased in control animals with high zinc supplementation compared to the groups without zinc supplementation. Zinc was able to protect against plasma leakage induced by I/R in all control and fructose-fed groups, although the microvascular permeability was higher in animals fed drinking water substituted by 10% fructose solution compared to those fed filtered drinking water alone. ConclusionOur results indicate that dietary zinc supplementation can improve microvascular dysfunction by increasing endothelial-dependent dilatation and reducing the increase in macromolecular permeability induced by I/R in fructose-fed animals.

α-Tocopherol Improves Microcirculatory Dysfunction on Fructose Fed Hamsters.

Fructose, an everyday component of western diet associated to chronic hyperglycemia and enhanced free radical production, impairs endothelial function and supplementation with antioxidants might improve it. In this study we investigated if vitamin E could reverse the microvascular damage elicited by fructose. Male Syrian golden hamsters drank either 10% fructose solution (F) or filtered water (C), combined with three concentrations of vitamin E in their chows [zero, normal (VE) or 5X (5XVE)] during 60 days. Microvascular reactivity in response to topical application of acetylcholine (Ach; endothelium-dependent vasodilator) or sodium nitroprusside (SNP; endothelium-independent vasodilator) and macromolecular permeability increase induced by either 30 min ischemia followed by reperfusion (I/R) or topical application of histamine (5 μM) were assessed using the cheek pouch preparation. Compared to controls (drinking filtered water), fructose-drinking animals showed decreased vasodilatation to acetylcholine in all concentrations tested (-56.2% for 10-9M, -53.9% for 10-7M and -43.7% for 10-5M). On the other hand, vitamin E supplementation resulted in increased responses for both water and fructose drinking groups (177.4% for F vs. F/5XVE and 241.6% for C vs. C/5XVE for 10-5M Ach). Endothelial-independent vasodilatation explored by topical application of SNP was restored and even enhanced with the supplementation of 5X vitamin E in both groups (80.1% for F vs. F/5XVE; 144.2% for C vs. C/5XVE; 3.4% of difference for C/5XVE vs. F/5XVE on 10-5M SNP). The number of leaky sites after I/R and histamine stimuli in vitamin E supplemented animals decreased (-25.1% and -15.3% for F vs. F/5XVE; and -21.7% and -16% of leaky sites comparing C vs. C/5XVE, respectively for I/R and histamine stimuli) pointing to tightening of the endothelial barrier for macromolecular permeability. Our results strongly suggest that vitamin E could improve the endothelial function and permeability barrier and also reverse impairments elicited by sugar overload.

N-3 PUFA induce microvascular protective changes during ischemia/reperfusion.

Ischemia/reperfusion (I/R) injury can occur in consequence of myocardial infarction, stroke and multiple organ failure, the most prevalent cause of death in critically ill patients. I/R injury encompass impairment of endothelial dependent relaxation, increase in macromolecular permeability and leukocyte-endothelium interactions. Polyunsaturated fatty acids (n-3 PUFA), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) found in fish oil have several anti-inflammatory properties and their potential benefits against I/R injury were investigated using the hamster cheek pouch preparation before and after ischemia. Before the experiments, hamsters were treated orally with saline, olive oil, fish oil and triacylglycerol (TAG) and ethyl ester (EE) forms of EPA and DHA at different daily doses for 14 days. Fish oil restored the arteriolar diameter to pre ischemic values during reperfusion. At onset and during reperfusion, Fish oil and DHA TAG significantly reduced the number of rolling leukocytes compared to saline and olive oil treatments. Fish oil, EPA TAG and DHA TAG significantly prevented the rise on leukocyte adhesion compared to saline. Fish oil (44.83 ± 3.02 leaks/cm2), EPA TAG (31.67 ± 2.65 leaks/cm2), DHA TAG (41.14 ± 3.63 leaks/cm2), and EPA EE (30.63 ± 2.25 leaks/cm2), but not DHA EE (73.17 ± 2.82 leaks/cm2) prevented the increase in macromolecular permeability compared to saline and olive oil (134.80 ± 1.49 and 121.00 ± 4.93 leaks/cm2, respectively). On the basis of our findings, we may conclude that consumption of n-3 polyunsaturated fatty acids, especially in the triacylglycerol form, could be a promising therapy to prevent microvascular damage induced by ischemia/reperfusion and its consequent clinical sequelae.

Gender differences in microcirculation: Observation using the hamster cheek pouch

OBJECTIVES:Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation.METHODS:Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of post-capillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (α1-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent).RESULTS:The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17–22) leaks/cm2 vs. 124 (123–128) leaks/cm2, respectively, p<0.001), but the number of rolling or adherent leukocytes was not different between the groups. Phenylephrine-induced arteriolar constriction was significantly lower in females compared with males [77 (73–102)% vs. 64 (55–69)%, p<0.04], but there were no detectable differences in endothelin-1-dependent vasoreactivity. Additionally, arteriolar vasodilatation elicited by acetylcholine or sodium nitroprusside did not differ between the groups.CONCLUSION:The female gender could have a direct protective role in microvascular reactivity and the increase in macromolecular permeability induced by ischemia/reperfusion.

Effects of babassu nut oil on ischemia/reperfusion-induced leukocyte adhesion and macromolecular leakage in the microcirculation: Observation in the hamster cheek pouch

BackgroundThe babassu palm tree is native to Brazil and is most densely distributed in the Cocais region of the state of Maranhão, in northeastern Brazil. In addition to the industrial use of refined babassu oil, the milk, the unrefined oil and the nuts in natura are used by families from several communities of African descendants as one of the principal sources of food energy. The objective of this study was to evaluate the effects of babassu oil on microvascular permeability and leukocyte-endothelial interactions induced by ischemia/reperfusion using the hamster cheek pouch microcirculation as experimental model.MethodsTwice a day for 14 days, male hamsters received unrefined babassu oil (0.02 ml/dose [BO-2 group], 0.06 ml/dose [BO-6 group], 0.18 ml/dose [BO-18 group]) or mineral oil (0.18 ml/dose [MO group]). Observations were made in the cheek pouch and macromolecular permeability increase induced by ischemia/reperfusion (I/R) or topical application of histamine, as well as leukocyte-endothelial interaction after I/R were evaluated.ResultsThe mean value of I/R-induced microvascular leakage, determined during reperfusion, was significantly lower in the BO-6 and BO-18 groups than in the MO one (P < 0.001). In addition, histamine-induced increase of microvascular permeability was significantly less pronounced in BO groups compared to MO one. No significant differences among groups in terms of leukocyte adhesion, concentrations of tumor necrosis factor alpha, interleukin 1, and interleukin 6 were found.ConclusionsOur findings suggest that unrefined babassu oil reduced microvascular leakage and protected against histamine-induced effects in postcapillary venules and highlights that these almost unexploited nut and its oil might be secure sources of food energy.

Hyaluronan impairs vascular function and drug delivery in a mouse model of pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton...

N- tert-Butyl and N-methyl nitrones derived from aromatic aldehydes inhibit macromolecular permeability increase induced by ischemia/reperfusion in hamsters

N-Alquil nitrones 1c and 3– 6 were prepared from aromatic aldehydes and N-tert-butylhydroxylamine or N-methylhydroxylamine in good yields and soft conditions. Their protective effect against microvascular damages caused by ischemia/reperfusion in ‘hamster cheek pouch’ assay was investigated and compare with that observed for nitrones 1a, b and 2, previously studied. Nitrones 3b, 4b and 4c were the most active ones in inhibiting macromolecular permeability increase induced by ischemia/reperfusion when administered by gavage and intravenous, while 3a and 4a were active only after intravenous administration. N- tert-butylhydroxylamine and Nt-methylhydroxylamine, products of the hydrolysis of these nitrones, were weakly active when administered by gavage or intravenous. Nitrone ( 4a) was the most potent in inhibiting macromolecular permeability increase induced by histamine. In this case, N-tert-butylhydroxylamine was as active as 4a. The lypophylicity in nitrones, specially in N-methyl nitrones, play an important role on the protective action when compounds were administered by gavage.

Micronization enhances the protective effect of purified flavonoid fraction against postischaemic microvascular injury in the hamster cheek pouch.

1. The present study was designed to evaluate the effect of micronization on the protective effect of the purified flavonoid fraction (MPFF) on increases in macromolecular permeability induced by ischaemia-reperfusion in the hamster cheek pouch microcirculation. 2. Male hamsters (Mesocricetus auratus) were treated orally, twice a day, with vehicle (lactose), MPFF and non-micronized purified flavonoid fraction (PFF) at 5, 20, 80 and 320 mg/kg per day for 10 consecutive days. On the 11th day, cheek pouches of anaesthetized animals were prepared for intravital microscopy. 3. Local ischaemia was obtained by clamping the neck of the everted pouch and the increase in microvascular permeability was quantified as leakage (leaks) of intravenously injected fluorescein isothiocyanate-labelled dextran (FITC-dextran 150; MW = 150 000). 4. Reperfusion, after 30 min ischaemia, resulted in an immediate but reversible increase in post-capillary leakage. The MPFF induced a significant dose-related reduction in the increased permeability, with 83.4% inhibition compared with control at 320 mg/kg per day (19.2 +/- 1.9 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001). Non-micronized PFF was significantly less effective: only 47.9% inhibition compared with control was observed at 320 mg/kg per day (60.3 +/- 1.0 vs 115.7 +/- 4.1 leaks/cm2; P < 0.0001) and there was no dose-effect relationship. 5. In conclusion, micronization significantly enhances the protective effects of the purified flavonoid fraction on reperfusion injury in the hamster cheek pouch. This improvement is likely to be related to the better absorption of the micronized formulation, which could explain the superior clinical efficacy shown in previous studies.

Bradykinin and alpha-thrombin increase human umbilical vein endothelial macromolecular permeability by different mechanisms.

Bradykinin and alpha-thrombin both increase endothelial macromolecular permeability, however the mechanism for this effect is unclear. Human umbilical vein endothelial cell (HUVEC) permeability to human serum albumin was increased by 1 microM alpha-thrombin (AT) or bradykinin (BK), but the kinetics of the permeability response were different. Intracellular calcium mobilization of HUVEC by AT was increased, yet BK had no effect on intracellular calcium. Distribution of F-actin and content was increased by AT as early as 10 minutes after administration, yet BK had no affect on F-actin when compared to control. We hypothesized that BK may increase HUVEC permeability by producing matrix metalloproteinase-2 (MMP-2). The AT-treated HUVEC produced an intermediate 64 kDa MMP-2, whereas BK-treated HUVEC increased the intermediate 64 kDa MMP-2 and also an active 62 kDa MMP-2. Pre-treatment of the HUVEC with tissue inhibitor of matrix metalloproteinase-2 slightly decreased the AT-induced increase in macromolecular permeability and completely inhibited the BK-induced increase in macromolecular permeability.

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.

Microvascular permeability with sulfonylureas in normal and diabetic hamsters

The hamster cheek pouch is an experimental model in which quantitative studies of macromolecular permeability can be made by direct observation of extravasated fluorescein isothiocyanate (FITC)-dextran (leaks). The advantage of this model is that simultaneous light and fluorescent-light microscopy observations can be performed with instantaneous correlations between the site of FITC-dextran extravasation and the vessel morphology. The aims of our study were to compare, using the cheek pouch preparation, the effects of two sulfonylureas, gliclazide and glibenclamide, on the macromolecular permeability increase induced by histamine using control (normoglycemic) hamsters. In these studies, FITC-labeled dextran 150,000 daltons was administered intravenously and quantified by UV-light microscopy, and the drugs used were applied topically at therapeutic concentrations. Gliclazide and glibenclamide dose-dependently decreased the macromolecular permeability increase induced by histamine. This effect of gliclazide could be blocked by nifedipine (Ca 2+ channel blocker) and not by diazoxide K + channel opener), whereas for glibenclamide it could be blocked by diazoxide and not by nifedipine. To better characterize the antioxidant capacity of gliclazide and glibenclamide, their effect on the macromolecular permeability increase induced by ischemia/reperfusion was also compared with the effect of vitamin C in diabetic hamsters (glycemia > 240 mg/dL). Total ischemia of the preparation was obtained with a cuff placed around the neck of the everted pouch. Diabetes was induced by three intraperitoneal injections of streptozotocin 50 mg/kg/d in 3 days. In diabetic hamsters during ischemia/reperfusion, gliclazide was more effective in inhibiting the macromolecular permeability increase than glibenclamide (136.0 ± 5.8 leaks/cm 2 for placebo; 68.0 ± 2.9 for 1.2 × 10 −6 mol/L gliclazide; 55.3 ± 3.5 for 1.2 × 10 −5 mol/L gliclazide; 89.2 ± 5.7 for 8 × 10 −8 mol/L glibenclamide; 107.0 ± 3.8 for 8 × 10 −7 mol/L glibenclamide; 56.7 ± 3.4 for 10 −6 mol/L vitamin C; and 20.5 ± 0.6 for 10 −5 mol/L vitamin C). Our results suggest that (1) the inhibition of the permeability increase induced by histamine elicited by gliclazide may be mediated by Ca 2+ channels, while that of glibenclamide may be mediated by K + channels, and (2) gliclazide appears to have an antioxidant capacity in ischemia/reperfusion injury similar to that of 10 −6 mol/L vitamin C. Improvement in the microcirculation was independent of the hypoglycemic properties of the drug.

Effects of oral administration of purified micronized flavonoid fraction on increased microvascular permeability induced by various agents and on ischemia/reperfusion in the hamster cheek pouch.

The effects of a clinically used purified micronized flavonoid fraction (S 5682) containing 90% diosmin and 10% hesperidin on increased microvascular permeability induced by histamine, bradykinin, and leukotriene B4 (LTB4) were investigated by intravital microscopy in the hamster cheek pouch preparation. The authors also investigated the effects of S 5682 on macromolecular permeability increase and leukocyte adhesion during ischemia-reperfusion by using the same preparation. S 5682, suspended in 10% lactose solution, or vehicle (10% lactose) was administered orally to male hamsters for ten days at 20 mg/kg/day (10 mg/kg twice a day). Fluorescein isothiocyanate (FITC)-labeled dextran (mol wt 150,000) was given intravenously, thirty minutes after completion of the cheek pouch preparation. The leukocytes were stained by continuous IV infusion of acridine orange (0.5 mg/kg/minute). Histamine (2 microM), bradykinin (1 microM), and LTB4 (0.01 microM), applied topically for five minutes, increased the number of fluorescent vascular leakage sites in postcapillary venules. A temporary ischemia with total circulatory arrest of the cheek pouch was obtained by clamping the neck of the everted pouch. The maximum number of leaky sites (per cm2 in the prepared area) that occurred either at five minutes after the beginning of each topical application or ten minutes after the onset of reperfusion was quantified in ultraviolet light microscopy. The results from 60 animals divided into 10 groups of 6 animals each are presented as means +/- SEM. In comparison with vehicle, S 5682 significantly inhibited the macromolecular permeability increasing effect of histamine (343.5 +/- 22.3 versus 207.5 +/- 32.0 leaks/cm2; P < 0.01), bradykinin (345.2 +/- 19.0 versus 206.2 +/- 21.6 leaks/cm2; P < 0.01), and LTB4 (353.3 +/- 27.5 versus 242.7 +/- 33.6 leaks/cm2; P < 0.05). At reperfusion, after thirty minutes of ischemia, S 5682 significantly decreased the observed macromolecular permeability (103.6 +/- 15.4 versus 42.6 +/- 9.3 leaks/cm2; P < 0.01). Flavonoid-treated animals also displayed a statistically significant lower number of adhering leukocytes to the venular endothelium (83.5 +/- 9.5 versus 48.4 +/- 12.3 per 6 mm2; P < 0.05). These results demonstrate that oral administration of S 5682 for ten days at 20 mg/kg body weight/day had a protective effect against leakage of macromolecules after application of permeability-increasing substances and during ischemia-reperfusion in the cheek pouch microvasculature. Since firm leukocyte attachment to the endothelial wall and subsequent emigration of leukocytes into the interstitium is a mechanism for tissue damage during inflammation, attenuation of this phenomenon during conditions of ischemia-reperfusion can in part explain previous observations that this purified micronized flavonoid fraction decreases edema formation. The present data illustrating the inhibitory effect of a clinically relevant dose of S 5682 on the inflammatory processes induced in this in vivo model of microcirculation may serve as a rational basis to explain its clinical efficacy.

L-arginine does not reverse impaired agonist-induced increases in macromolecular efflux during diabetes mellitus.

The first goal of this study was to determine the effect of diabetes mellitus on agonist-induced increases in venular macromolecular permeability of the hamster cheek pouch. The second goal was examine the role for an alteration in the availability of L-arginine to nitric oxide synthase in impaired agonist-induced increases in macromolecular permeability during diabetes. We used intravital fluorescent microscopy and fluorescein isothiocyanate dextran (FITC-dextran; mw = 70 K) to examine macromolecular extravasation from post-capillary venules in non-diabetic and diabetic (2 weeks after injection of streptozotocin) hamsters in response to histamine and substance P. Increases in extravasation of macromolecules were quantitated by counting the number of venular leaky sites and by calculating the clearance (ml/s x 10(-6)) of FITC-dextran-70 K. In non-diabetic hamsters, histamine (1.0 and 5.0 microM) and substance P (50 and 100 nM) increased permeability of the cheek pouch microcirculation to FITC-dextran-70 K. In contrast, histamine- and substance P-induced increases in macromolecular extravasation were markedly reduced in diabetic hamsters. Next, we investigated whether alterations in histamine- and substance P-induced changes in macromolecular extravasation in diabetic hamsters may be related to an alteration in the availability of L-arginine. We examined whether exogenous application of L-arginine (100 microM) could restore impaired histamine- and substance-P-induced increases in macromolecular extravasation in diabetic hamsters. We found that L-arginine potentiated agonist-induced increases in macromolecular extravasation in non-diabetic hamsters, but did not alter responses in diabetic hamsters. These findings suggest that short-term diabetes mellitus alters agonist-induced alterations in microvascular permeability. The mechanism of altered microvascular permeability during diabetes mellitus does not appear to be related to an impaired availability of L-arginine.

Effects of iloprost, a stable prostacyclin analog, and its combination with NW-nitro-L-arginine on early events following lipopolysaccharide injection: observations in the hamster cheek pouch microcirculation.

The effects of iloprost, a stable prostacyclin analog, and of its combination with NW-nitro-L-arginine (L-NAG) on the microcirculatory changes observed in early stages of endotoxemia were investigated in male hamsters treated with Escherichia coli lipopolysaccharide (LPS). The cheek pouch was studied in vivo by means of intravital microscopy and mean arterial and venous pressures, mean arteriolar internal diameter, spontaneous arteriolar vasomotion, microvascular blood flow, macromolecular permeability, leukocyte adhesion and mean survival time were evaluated in animals treated with LPS alone or with the combination of LPS+iloprost and LPS+iloprost+L-NAG. Intravenous injection of LPS (100 mg/kg) per se elicited a significant reduction in mean arterial blood pressure (MABP) and arteriolar blood flow. The observed arterioles dilated and spontaneous vasomotion ceased. Iloprost (40 ng/kg/min) prevented LPS-induced reduction of MABP, ameliorated the decrease in arteriolar blood flow and reduced the vasodilation. Arteriolar vasomotion was reduced but it did not cease. The combination of L-NAG (0.5 mg/kg) + iloprost (40 ng/kg/min) with LPS (100 mg/kg) did not improve the results obtained with iloprost alone, except that it prevented the vasodilation and the vasomotion ceased 1 h after the bolus injection of LPS+L-NAG. The mean survival time compared to LPS alone (57 +/- 7 h) was significantly increased by the combination of LPS+iloprost (102 +/- 6 h) and it did not change significantly with the combination L-NAG+iloprost (64 +/- 9 h). Topical addition of iloprost (10 ng/kg/min) evoked an early increase in macromolecular permeability and a significant decrease in leukocyte adhesion compared with animals treated with topical LPS (0.7 microgram/ml/min) alone. The combination of L-NAG+iloprost (1.3 + 10 ng/ml/min) enhanced the early increase in macromolecular permeability even further, tended to increase the macromolecular permeability and evoked a smaller decrease in leukocyte adhesion than the one observed with iloprost combined with LPS. Our results, in the hamster cheek pouch microcirculation, suggest that the use of prostacyclin could be beneficial in the treatment of early changes in endotoxic shock, but L-NAG showed no benefit in this preparation.

Possible Mechanisms for the Inhibitory Effect of Ruscus Extract on Increased Microvascular Permeability Induced by Histamine in Hamster Cheek Pouch

Summary: 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 α1 and α2adrenoceptor 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 (α1-adrenoceptor antagonist), diltiazem (calcium blocker), and Ruscus extract applied topically dosedependently inhibited the macromolecular permeability-increasing effect of histamine. Rauwolscine (α2-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 α1-adrenoceptors.

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.

Water and protein permeability is regulated by the glomerular epithelial slit diaphragm.

The glomerular barriers to water and macromolecular movement were examined 2 and 24 h after the administration of a monoclonal antibody (mAb) specific to an antigen located on the epithelial slit diaphragm and the external aspect of the glomerular basement membrane. By micropuncture techniques 2 h after mAb administration, single-nephron GFR (SNGFR) and plasma flow were unchanged but the glomerular capillary hydrostatic pressure gradient and glomerular capillary hydrostatic pressure were increased and the glomerular ultrafiltration coefficient (LpA) decreased to values that were 50% of the normal control. There was no increase in urinary protein excretion at 2 h. However, at 24 h after mAb, nephron plasma flow (SNPF) and SNGFR increased and the glomerular ultrafiltration coefficient returned to values indistinguishable from the normal control. At 24 h, there was a marked increase in protein excretion. The administration of meclofenamate decreased values for SNGFR and SNPF to normal. Immunoglobulin G was exclusively bound to glomerular capillary walls in a linear or continuous fashion at 2 h, but in a discontinuous, granular pattern at 24 h. These studies suggest that after mAb, important limiting glomerular barriers for hydraulic conductivity and protein excretion reside on the epithelial aspect of the glomerular capillary basement membrane, specifically at the level of the slit diaphragm. Studies also suggest that alterations in glomerular capillary hydraulic conductivity can be effectively separated from increases in macromolecular permeability.

Cryptosporidium parvum infection of intestinal epithelium: morphologic and functional studies in an in vitro model.

A monolayer of mature polarized colonic epithelial cells (T84) able to generate and maintain a barrier to macromolecular flow was used to study pathophysiologic events that occur on microvillus cell exposure to Cryptosporidium parvum. By 24-48 h, several life cycle forms were seen in parasitophorous vacuoles near the apical cell surface, along with a time- and oocyst dose-dependent reduction in epithelial barrier function. As few as 10(5) organisms constituted a successful infecting dose, and heat inactivation of organisms markedly reduced the monolayer barrier alteration. Horseradish peroxidase flux studies demonstrated a substantial increase in macromolecular permeability of the monolayer, and lactate dehydrogenase determinations indicated modest injury of the T84 epithelial cells on exposure to oocysts. Thus, disruption of the epithelial cell barrier, not just opening of transcellular channels for ion flow as reported previously, is responsible for the effects of C. parvum oocysts on intestinal epithelium.