Effects of venotonic drugs on the microcirculation: Comparison between Ruscus extract and micronized diosmine 1

Abstract

BACKGROUND:

Chronic venous disease of the lower limbs is a common public health problem worldwide with negative impact on quality of life and results with drugs used to treat it are sparse, probably due to lack of good experimental models.

OBJECTIVE:

In this investigation we have tested the effects of two commonly used venotonic substances, Ruscus extract and micronized diosmine, on the microcirculation in vivo.

METHODS:

These substances were given orally, by gavage, during two weeks, twice daily and observations were made using the hamster cheek pouch preparation.

RESULTS:

The drugs elicited a dose-dependent inhibition of (1) macromolecular permeability increase induced by histamine or ischemia followed by reperfusion, being the Ruscus extract more active on both and (2) leukocyte-endothelium interaction, again being the Ruscus extract more effective in the inhibition of the number of adherent and rolling leukocytes. About the duration of the effect after the end of the treatment, both drugs had similar effects but Ruscus extract showed greater permanence of its effect on all observed parameters.

CONCLUSIONS:

Our results suggest that both drugs have antioxidant and anti-inflammatory properties being Ruscus extract more active. It should be added that only Ruscus extract showed a significant venular constriction.

Keywords

Microvascular reactivity macromolecular permeability ischemia/reperfusion leukocyte-endothelium interaction duration of action

1 Introduction

Chronic venous disease (CVD) of the lower limbs is a common public health problem worldwide with negative impact on quality of life and experimental results with agents (venotonic drugs) used to treat it are sparse, probably owing to lack of good experimental models for the disease. It should be mentioned that compression stockings are an alternative therapy in CVD [1].

The exact role of the microcirculation in the physiopathology of CVD is still not completely defined [2] but it is well-accepted that venous hemodynamic alterations leading to venous hypertension play an important role in the development of the observed microangiopathy [3 –5]. Elevated ambulatory pressure damages not only the macrocirculation with the development of varicose veins but also the microcirculation that becomes dysfunctional. It is possible to observe using the orthogonal polarized image system the morphology of cutaneous capillaries that become enlarged, tortuous and with the progression of the disease look like “glomerulus-like” capillaries [6]. The endothelial cells also change and become enlarged, with larger interendothelial pores responsible for the increase in microvascular permeability, with extravasation of plasma, blood cells and macromolecules. The persistence of venous stasis and hypertension results in chronic inflammation and edema, normally found in CVD patients.

Hemodynamic forces, such as venous hypertension, stasis and alterations in shear stress play an important role in the activation of the inflammatory cascade. As consequence of venous hypertension, transmitted backwards to the microcirculation, there is an increase in the extravasation of fluid from the vessels, mainly from post capillary venules, with edema formation and an increase in lymphatic content as well as in blood viscosity. Alterations in shear stress in consequence of abnormal blood flow lead to changes in morphology, function and gene expression in endothelial cells [7].

CVD is also accompanied by an increase in leukocyte infiltration in the affected leg [8] and its adhesion to post capillary venules and bigger veins could be facilitated by the expression of selectins, integrins and members of the immunoglobulin superfamily [9].

In this investigation we have tested the effects of two commonly used venotonic substances, namely Ruscus extract and micronized diosmine, on hamsters and observed their effects on the microcirculation in vivo using the cheek pouch preparation.

2 Material and methods

Experiments were performed on the cheek pouch of male Syrian golden hamsters (Mesocricetus auratus), aged 7 to 10 weeks. We did not work with female hamsters because the venous system is particularly sensitive to estrogen and progesterone levels [10]. The experimental protocol was approved by the Ethical Committee of the State University of Rio de Janeiro (CEUA 058/2012). For oral treatment (gavage), Ruscus extract (50, 150 and 450 mg/kg/day), its placebo (filtered water) micronized diosmine (50, 150 and 450 mg/kg/day) or its placebo (10% lactose solution) was given twice a day, at 8 : 00 AM and 5 : 00 PM, for 2 weeks. Each group had at least 6 animals. The dry extract of Ruscus titrated in sterolic heterosides was obtained from Ruscus aculeatus L rhizomes and roots by hydroalcoholic extraction (Pierre Fabre Laboratories) and micronized diosmine from Euromedex (Strasbourg, France). We are aware about the effects of endotoxins to the microcirculation [11 –14] and they were not detectable in any of the animals used in the present investigation.

In a separate set of experiments, in order to test the permanence of the effects, animals were treated by gavage during 2 weeks with either Ruscus extract or micronized diosmine, 450 mg/kg/day, twice a day, at 8 : 00 AM and 5 : 00 PM and the same parameters were observed 5, 10, 15, 20, 25, 30, and 35 days after the end of treatment.

Anesthesia was induced by an intraperitoneal injection of 0.1–0.2 ml of sodium pentobarbital (Pentobarbital sodique, Sanofi, Paris, France, 60 mg/ml) and maintained with α-chloralose (Sigma Chemicals, St. Louis, MO, USA, 100 mg/kg) administered through the femoral vein. The femoral artery was also cannulated for pressure measurements to check the viability of the in vivo experiment. Throughout operation and the subsequent experiment, the temperature of the animals was maintained at 37.5°C with a heating pad controlled by a rectal thermistor. A tracheal tube was inserted to facilitate spontaneous breathing.

The hamster was placed on a microscope stage similar to that described by Duling [15] with minor modifications [16]. The cheek pouch was gently everted and pinned with four to five needles into a circular well filled with silicone rubber to provide a plane bottom layer, thus avoiding stretching of the tissue but preventing shrinkage. In this position, the pouch is submerged in a superfusion solution that continuously flushed the pool of the microscope stage. Before the pouch is pinned, larger arterioles and venules were located with the aid of a Zeiss binocular stereomicroscope.

Fashioning of a single-layer preparation starts with incision of the upper layer to swing a triangular flap to one side. The exposed area is dissected at X10– 16 under the stereomicroscope, and the fibrous, almost avascular, connective tissue covering the vessels is removed with ophthalmic surgical instruments. The dissected part of the pouch is 125–150 μm thick. Dissected pouches with petechial formations or those without blood flow in all vessels are discarded.

The superfusion solution was a HEPES-supported HCO₃-buffered saline solution (composition in mM: NaCl 110.0, KCl 4.7, CaCl₂ 2.0, MgSO₄ 1.2, NaHCO₃ 18.0, HEPES 15.39 and HEPES Na⁺ salt 14.61); temperature of the solution was maintained at 36.5°C, and superfusion rate varied from 4 ml/min (arteriolar and venular diameters and leukocyte-endothelium interaction) to 6 ml/min (macromolecular permeability). pH was set to 7.40 by bubbling the solutions continuously with 5% CO₂ in 95% N₂.

For measurements of arteriolar and venular diameters, the preparations were placed under an intravital microscope where they were allowed to rest for 30 min. If after this time there was (a) an indication of good vascular tone; (b) brisk blood flow in all parts of the vascular bed, including the larger veins (where individual erythrocytes should not be discernible in the image of the blood stream) and (c) no tendency for leukocytes to adhere to the vessel wall, the experiments were performed by taking DVD recordings of each of one to four selected arterioles and venules per preparation [16]. The TV monitor display was used to obtain arteriolar and venular internal diameter measurements by an image shearing monitor (IPM model 907, San Diego, CA, USA), final magnification x720. In practice, we have used DVD replay for final determination of vessel diameters, since greater attention could be given to this measurement than is possible during the conduct of the experiment.

For macromolecular permeability measurements, 30 min after completion of the preparative procedure, FITC-dextran (molecular weight 150,000 Dalton; Bioflor HB, Uppsala, Sweden), with a degree of substitution of FITC molecules 2/1,000 glucose molecules in the polysaccharide chain, was given in a dose of 25 mg/100 g body weight as an i.v. injection of a 5% solution in 0.9% saline [17]. Observations were made with an intravital microscope equipped with a 100 W Hg DC lamp and specific filters.

The total observed area of the pouch, 1 cm², is roughly circular and observations of the number of leakage sites (= leaks) are made by scanning manually the total area at selected time intervals, i.e., at 2, 5, 7, 10, 15, 20 and 30 min after the initiation of each topical application of histamine (5 μM, Sigma-Aldrich, St. Louis, MO, USA) that lasted 5 min.

Local ischemia of the cheek pouch was obtained by a cuff, made of thin latex tubing, mounted around the neck of the everted pouch where it leaves the mouth of the hamster [18]. The cuff can be placed without any visible interference with local blood flow and the intratubular pressure can be rapidly increased by air compression with a syringe and also as rapidly decreased at evacuation. An intratubular pressure of 200–220 mmHg in the cuff results in a complete arrest of the microvascular blood flow within a few seconds. Throughout the 30 min occlusion period minor adjustments of blood movements could be seen in the larger vessels. At evacuation there was an immediate increase in blood flow, which eventually returned to a level similar to that observed before the occlusion [18].

For leukocyte-endothelium interaction, prior to ischemia and 30 min after the onset of reperfusion, leukocytes were stained with an i.v. infusion of rhodamine G (Sigma Chemicals, St. Louis, WO, USA) and the fluorescent leukocytes adhering to the endothelium or rolling closer to the venular wall were quantified in UV-light microscope [19].

Results were presented as means±S.D., unless otherwise noted. Statistical analyses were divided in two approaches: intra- and inter-groups. The aim of the intra-group analyses was the comparison between microvascular parameters of groups with different concentrations of Ruscus extract or micronized diosmine and their respective placebos. Inter-group analyses were performed between groups of Ruscus extract and micronized diosmine, according to the respective comparable concentration; or, in a second approach of the study, the comparison between groups with the same concentration (450 mg/kg/day) of above mentioned substances along 5, 10, 15, 20, 25, 30, and 35 days after the end of treatment.

Each group (with different concentrations of each substance or days after the end of treatment) was assessed concerning their distribution of frequencies (kurtosis, skewness, and Shapiro-Wilk’s W test for normality), as well as their medians and quartile ranges. Comparisons between groups (intra- and inter-group analyses) were made through pairwise Mann-Whitney’s U test. In order to keep a conservative look upon the results, the significance level adopted was alpha = 0.01 (Bonferroni correction).

3 Results

3.1 Comparison between Ruscus extract and micronized diosmine

3.1.1 Effects on mean arteriolar and venular diameters

Experiments were performed on 57 hamsters, 120.4±6.1 g and 95.0±0.3 mmHg. Compared to its respective placebo, the oral treatment with Ruscus extract elicited venular constriction and no significant effect on mean arteriolar diameters (data not shown) while the oral treatment with micronized diosmine did not significantly change either arteriolar or venular mean internal diameters (Fig. 1).

Fig.1

Effects of different oral doses of either Ruscus extract or micronized diosmine and their respective placebos (given twice a day for 2 weeks) on mean venular diameters. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^# #p < 0.001.

3.1.2 Effects on histamine induced macromolecular permeability

Experiments were performed on 49 hamsters, 122.8±5.5 g and 95.1±0.3 mmHg. Compared to its respective placebo, the oral treatment with Ruscus extract elicited more pronounced dose-dependent inhibition of histamine induced macromolecular permeability increase than micronized diosmine, evaluated at 5 min after the beginning of the topical application of histamine (Fig. 2).

Fig.2

Effects of different oral doses of either Ruscus extract or micronized diosmine and their respective placebos (given twice a day for 2 weeks) on macromolecular permeability increase induced by topical application of histamine. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

3.1.3 Effects on ischemia/reperfusion induced macromolecular permeability

Experiments were performed on 48 hamsters, 123.7±5.7 g and 95.2±0.3 mmHg. Compared to its respective placebo, the oral treatment with Ruscus extract or micronized diosmine elicited a dose-dependent inhibition of ischemia/reperfusion induced macromolecular permeability, evaluated 10 min after the onset of reperfusion (Fig. 3). Again, the Ruscus extract elicited a more pronounced inhibition of the macromolecular permeability.

Fig.3

Effects of different oral doses of either Ruscus extract or micronized diosmine and their respective placebos (given twice a day for 2 weeks) on macromolecular permeability increase induced by ischemia/reperfusion. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

3.1.4 Effects on leukocyte rolling and sticking induced by ischemia/reperfusion

Experiments were performed on 48 hamsters, 136.4±18.0 g and 95.3±0.6 mmHg. Compared to its respective placebo, the oral treatment with Ruscus extract elicited more pronounced dose-dependent inhibition of the leukocyte-endothelium interaction than micronized diosmine (Figs. 4 and 5). Leukocyte-endothelium interaction was induced by 30 min ischemia followed by reperfusion and evaluated 15 min after the onset of reperfusion.

Fig.4

Effects of different oral doses of either Ruscus extract or micronized diosmine and their respective placebo (given twice a day for 2 weeks) on the number of rolling leukocytes induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

Fig.5

Effects of different oral doses of either Ruscus extract or micronized diosmine and their respective placebo (given twice a day for 2 weeks) on the number of sticking leukocytes induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

3.1.5 Comparison between Ruscus extract and micronized diosmine for the duration of the effect

We have tested for duration of the effect all parameters evaluated for the comparison between the two drugs in 336 hamsters, 125.4±3.8 g, 95.2±04 mmHg, 5, 10, 15, 20, 25, 30, and 35 days after the end of treatment and found greater permanence of the effect of Ruscus extract compared with diosmine for macromolecular permeability increase induced by either topical application of histamine (Fig. 6) or ischemia followed by reperfusion (Fig. 7) and leukocyte-endothelium interaction, for both number of rolling (Fig. 8) and adherent (Fig. 9) leukocytes.

Fig.6

Duration of the effect of an oral dose of 450 mg/kg/day given twice a day during 2 weeks of either Ruscus extract or micronized diosmine and their respective placebos on macromolecular permeability increase induced by topical application of histamine. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

Fig.7

Duration of the effect of an oral dose of 450 mg/kg/day given twice a day during 2 weeks of either Ruscus extract or micronized diosmine and their respective placebos on macromolecular permeability increase induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

Fig.8

Duration of the effect of an oral dose of 450 mg/kg/day given twice a day during 2 weeks of either Ruscus extract or micronized diosmine and their respective placebos on the number of rolling leukocytes induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

Fig.9

Duration of the effect of an oral dose of 450 mg/kg/day given twice a day during 2 weeks of either Ruscus extract or micronized diosmine and their respective placebos on the number of sticking leukocytes induced by 30 min ischemia followed by reperfusion. Observation in the hamster cheek pouch microcirculation. Intragroup differences ^*p < 0.01; ^**p < 0.001. Intergroup differences ^#p < 0.01; ^# #p < 0.001.

4 Discussion

The aim of this investigation was to compare the effects on the microcirculation of two commonly used venoactive drugs, namely Ruscus extract and micronized diosmine testing also the duration of their effect after the end of the treatment, up to 35 days using the hamster cheek pouch preparation.

The main findings of our study are that oral administration of Ruscus extract or micronized diosmine elicited a dose-dependent inhibition of (1) macromolecular permeability increase induced by histamine or ischemia followed by reperfusion, being the Ruscus extract more active on histamine and ischemia/reperfusion induced macromolecular permeability increase and (2) leukocyte-endothelium interaction, again being the Ruscus extract more effective in the inhibition of the number of rolling and adherent leukocytes. About the duration of the effect after the end of the treatment, both drugs had similar effects but Ruscus extract showed greater permanence of its effect on macromolecular permeability increase induced by either topical application of histamine or ischemia followed by reperfusion and on leukocyte-endothelium interaction, for both number of rolling and sticking leukocytes. It should be added that only Ruscus extract showed a significant venular constriction.

The use of the hamster cheek pouch as a site of observation of microcirculation was described in the late 1940s and early 1950s by Fulton, Jackson and Lutz [20 –22] and has since been used in a wide range of studies on microcirculatory physiology and microvascular responses. This microvascular preparation has intact nervous and blood supplies and all classes of microcirculatory vessels can usually be seen in the microscopic field. Arterioles could be defined as any of the small subdivisions of an artery that form thin-walled vessels ending in capillaries while venules are small blood vessels in the microcirculation that allow blood to return from the capillary beds to drain into the larger blood vessels, the veins. The preparation is thus suitable for observing the effects of different drugs on the microvasculature, which are either given orally (by gavage or added to the drinking water) or added to the superfusion solution (topical application). According to Handler and Shepro [23], the histology of the pouch is similar to the cutaneous tissue and it could be considered as “skin minus hair follicles and glands”. The effects of different drugs could be observed after oral, intravenous or topical application, making it a rather versatile microvascular preparation obtained from a rodent mammal.

The two studied drugs are clinically used for the treatment of chronic venous disease and hemorrhoid attacks [24, 25]. The venular constriction observed in animals treated with Ruscus extract could result from activation of both post-junctional α₁- and α₂-adrenergic receptors, release of endogenous norepinephrine from adrenergic nerve endings and/or a direct action on venous smooth muscle [10 , 26–28]. Using the hamster cheek pouch preparation, we have shown that the observed venular constriction could be blocked by topical applications of low concentrations (10^–9 M) of prazocin (α₁-adrenoceptor antagonist) or diltiazem (calcium blocker) and high concentrations (>10^–6 M) of rauwolscine (α₂-adrenoceptor antagonist) [29]. The difference between the responses of arterioles and venules could be due to increased liberation of endothelium-derived relaxing factor on the arteriolar side [30].

Histamine stimulates endothelial cells directly through specific receptors and the extravasation of macromolecules occurs between adjacent cells through the formation of a wider gap [31]. The inhibitory effect of Ruscus extract on histamine induced macromolecular permeability increase could be blocked by prazocin and diltiazem, but not by rauwolscine [32]. Previous experiments suggest that micronized diosmine could interfere in the mechanism of gap formation between endothelial cells, in which calcium flux plays an important role [33, 34].

Ischemia and reoxygenation of the tissue during reperfusion initiate a sequence of oxygen-dependent biochemical reactions that cause endothelial cell damage. The tissue damage due to ischemia is augmented during reperfusion because oxygen transported into a previously ischemic tissue promotes the generation of oxygen-derived free radicals [24, 35]. Our results have shown that both Ruscus extract and micronized diosmine inhibited in a dose-dependent fashion the macromolecular permeability increase induced by ischemia/reperfusion suggesting that they could function as antioxidants.

The increase in the number of leukocytes rolling and sticking to the venular wall after ischemia followed by reperfusion also depends on endothelial cell damage [5, 9] and the fact that such effect could be inhibited by both drugs in a dose-dependent fashion further reinforces their antioxidant properties.

The main limitation of this study is the fact that it was not performed in an animal model of chronic venous disease or at least in a model where the microcirculation was investigated in a situation of high venous pressure and low blood flow.

In conclusion, our results suggest that both drugs have antioxidant and anti-inflammatory properties being Ruscus extract more active in the inhibition of leukocyte-endothelium interaction and macromolecular permeability increase induced by histamine or ischemia/reperfusion. It should be added that only Ruscus extract showed a significant venular constriction.

Financial support

The study was supported by grants from Rio de Janeiro State Foundation for Research Support (FAPERJ), project number E-26/010.001248/2015.

Footnotes

Acknowledgments

We would like to thank Mr. Paulo José Ferreira Lopes and Mr. Claudio Natalino Ribeiro for animal care.

References

Konschake

, Riebe

, Pediaditi

, Haase

, Jünger

, Lutze

. Compression in the treatment of chronic venous insufficiency: Efficacy depending on the length of the stocking. Clin Hemorheol Microcirc. 2016;64(3):425–34.

Schmid-Shöenbein

, Takase

, Bergan

. New advances in the understanding of the pathophysiology of chronic venous insufficiency. Angiology. 2001;52(Suppl 1):S27–8.

Cesarone

, De Sanctis

, Incandela

, Belcaro

, Griffin

. Microvascular changes in venous hypertension due to varicose veins after standardized application of Essaven Gel. Angiology. 2001;52(Suppl. 3):S11–6.

Porter

, Moneta

. Reporting standards in venous disease: An update. J Vasc Surg. 1995;21:635–45.

Takase

, Schmid-Shöenbein

, Bergan

. Leucocyte activation in patients with venous insufficiency. J Vasc Surg. 1999;30:148–56.

Virgini-Magalhães

, Lascasas-Porto

, Fernandes

, Dorigo

, Bottino

, Bouskela

. Use of microcirculatory parameters to evaluate chronic venous insufficiency. J Vasc Surg. 2006;43:1037–44.

Ando

, Yamamoto

. Vascular mechanobiology: Endothelial cell responses to fluid shear stress. Circ J. 2009;73(11):1983–92.

Cheatle

, Scott

, Scurr

, Coleridge Smith

. White cells, skin blood flow and venous ulcers. Br J Dermatol. 1991;125(3):288–90.

Pascarella

, Penn

, Schmid-Schöenbein

. Venous hypertension and the inflammatory cascade: Major manifestations and trigger mechanisms. Angiology. 2005;(Suppl 1):S3–10.

10.

Harker

, Marcelon

, Vanhoutte

. Temperature, oestrogens and contractions of venous smooth muscle of the rabbit. Phlebology. 1988;3(suppl. 1):77–82.

11.

Santos

, Furtado

, Villela

, Bouskela

. Microcirculatory effects of fluid therapy and dopamine, associated or not to fluid therapy, in endotoxemic hamsters. Clin Hemorheol Microcirc. 2011;47(1):1–13.

12.

Miranda

, Balarini

, Bouskela

. Dexmedetomidine attenuates the microcirculatory derangements evoked by experimental sepsis. Anesthesiology. 2015;122(3):619–30.

13.

Miranda

, Pereira

, Santos

, Villela

, Kraemer-Aguiar

, Bouskela

. Milrinone attenuates arteriolar vasoconstriction and capillary perfusion deficits on endotoxemic hamsters. PLoS One. 2015;10(2):e0117004.

14.

Miranda

, Balarini

, Caixeta

, Bouskela

. Microcirculatory dysfunction in sepsis: Pathophysiology, clinical monitoring and potential therapies. Am J Physiol Hear Circ Physiol. 2016;311(1):H24–35.

15.

Duling

. The preparation and use of the hamster cheek pouch for studies of the microcirculation. Microvasc Res. 1973;5:423–9.

16.

Bouskela

, Grampp

. Spontaneous vasomotion in the hamster cheek pouch arterioles in varying experimental conditions. Am J Physiol. 1992;262:H478–85.

17.

Svensjö

, Arfors

K-E

, Arturson

, Rutili

. The hamster cheek pouch as a model for studies of macromolecular permeability of the microvasculature. Uppsala J Med Sci. 1978;83:71–9.

18.

Persson

, Erlansson

, Svensjö

, Takolander

, Berqvist

. The hamster cheek pouch – an experimental model to study postischemic macromolecular permeability. Int J Microcirc Clin Exp. 1985;4:257–63.

19.

Bouskela

, Cyrino

FZGA

, Lerond

. Leukocyte adhesion after oxidant challenge in the hamster cheek pouch microcirculation. J Vasc Res. 1999;36(suppl 1):11–14.

20.

Lutz

, Fulton

. The use of the hamster cheek pouch for the study of vascular changes at the microscopic level. Anat Rec. 1954;120(1):293–307.

21.

Fulton

, Jackson

, Lutz

. Cinephotomicroscopy of Normal Blood Circulation in the Cheek Pouch of the Hamster. Science. 1947;105(2727):361–2.

22.

Fulton

, Jackson

, Lutz

. Cinephotomicroscopy of normal blood circulation in the cheek pouch of the hamster. Anat Rec. 1946;96(4):537.

23.

Handler

, Shepro

. Cheek pouch technology: Uses and applications. In: Hoffman

, Robinson

, Magalhães

, eds. The golden hamster. AMES, IA: Iowa State University Press; 1968;195–202.

24.

Bouskela

, Cyrino

FZGA

, Lerond

. Effects of oral administration of different doses of purified micronized flavonoid fraction on microvascular reactivity after ischaemia/reperfusion in the hamster cheek pouch. Br J Pharmacol. 1997;122:1611–16.

25.

Bouskela

, Cyrino

FZGA

, Marcelon

. Effects of Ruscus extract on the internal diameter of arterioles and venules of the hamster cheek pouch microcirculation. J Cardiovasc Pharmacol. 1993;22:221–24.

26.

Marcelon

, Vanhoutte

. Venotonic effect of Ruscus extract under variable temperature conditions in vitro. Phlebology. 3(suppl 1); 1988)51–4.

27.

Marcelon

, Verbeuren

, Lauressergues

, Vanhotte

. Effect of Ruscus aculeatus on isolated canine cutaneous veins. Gen Pharmacol. 1983;14:103–6.

28.

Ruabanyi

, Marcelon

, Vanhoutte

. Effect of temperature on the responsiveness of cutaneous veins to the extract of Ruscus aculeatus. Gen Pharmacol. 1984;15:431–4.

29.

Bouskela

, Cyrino

FZGA

, Marcelon

. Possible mechanisms for the venular constriction elicited by Ruscus extract on the hamster cheek pouch. J Cardiovasc Pharmacol. 1994;24:165–70.

30.

Miller

, Marcelon

, Vanhoutte

. Ruscus extract releases endothelial-derived relaxing factor in arteries and veins. In: Vanhoutte

, ed. Return circulation and norepinephrine: An update. Paris: John Libbey Eurotext; 1991)31–42.

31.

Hultström

, Svensjö

. Intravital and electron microscopic study of bradykinin-induced vascular permeability changes using FITC-dextran as a tracer. J Pathol. 1979;129:125.

32.

Bouskela

, Cyrino

FZGA

, Marcelon

. Possible mechanisms for the inhibitory effect of Ruscus extract on increased microvascular permeability induced by histamine in the hamster cheek pouch. J Cardiovasc Pharmacol. 1994;24:281–85.

33.

, Curry

. Depolarization modulates endothelial cell calcium influx and microvessel permeability. Am J Physiol. 1991;261:H1246–54.

34.

Mayhan

, Joyner

. The effect of altering the external calciumconcentration and a calcium channel blocker, verapamil, onmicrovascular leaky sites and dextran clearance in the hamstercheek pouch. Microvasc Res. 1984;28:159–79.

35.

Ley

, Arfors

K-E

. Changes in macromolecular permeability by intravascular generation of oxygen-derived free radicals. Microvasc Res. 1982;24:25–33.