Beneficial effects of the micronized purified flavonoid fraction (MPFF *,Daflon® 500 mg) on microvascular damage elicited by sclerotherapy

Abstract

Objectives

To evaluate if the micronized purified flavonoid fraction (MPFF) treatment could reduce the side effects of sclerotherapy (a procedure frequently used to treat venous disease manifestations) by minimizing the inflammatory response within the surrounding tissues.

Method

Twenty-two male New Zealand rabbits were treated by gavage with micronized purified flavonoid fraction (MPFF; 300 mg/kg/day) or vehicle (10% lactose solution) during 21 consecutive days, starting 7 days before sclerotherapy. The sclerotherapy consisted of an injection containing 5% ethanolamine oleate solution in the rabbit’s dorsal ear vein. Before and after sclerotherapy, venular and arteriolar diameters, microvascular permeability, functional capillary density (FCD), number of rolling and sticking leukocytes were evaluated on ear microcirculation. Images of the sclerotherapy site were taken before and after the procedure.

Results

Compared to placebo, MPFF treatment prevented the increase in venular diameter, preserved FCD (P < 0.001) and reduced the number of leaky sites (P < 0.001) and sticking leukocytes (P < 0.001). Imaging confirmed these effects on thrombosis and perivascular edema of the sclerosed vein, 14 days after procedure.

Conclusion

MPFF treatment limited the postsclerotherapy inflammation in surrounding microvascular network, suggesting that MPFF may prevent undesirable secondary effects of the procedure in this animal model. This study warrants further investigation for its use in clinical conditions.

Keywords

Inflammation leukocyte-endothelial interaction macromolecular permeability MPFF rabbit

Introduction

Telangiectasia and varicose veins are the visible surface manifestations of venous insufficiency syndrome, arising primarily in venules consequent to persistent passive venous dilatation as well as distensibility of the vessel wall.^1–2 Treatment of these vessels is a common dermatologic request, as women in particular are concerned about the unpleasant appearance.

Sclerotherapy (endovenous chemical ablation) represents the treatment of choice for telangiectasia and is also indicated in the treatment of reticular and short saphenous varicose veins.^2,3 However, this procedure could induce several possible side effects such as postsclerosis hyperpigmentation, matting, or ulceration,^4–7 which should be avoided for both clinical and esthetic reasons.^7–9 So far no strategy has aimed to limit the inflammatory response induced by the sclerotherapeutic process.

Several clinical and experimental studies have illustrated the beneficial effects of micronized purified flavonoid fraction (MPFF) for treatment of chronic venous disease (CVD) and its complications.^10–13 According to its vascular anti-inflammatory properties^11–12 demonstrated in experimental microcirculation models,^14–16 we postulated that MPFF treatment of patients selected for sclerotherapy might help prevent its secondary effects and lead to an improvement in the outcome of the procedure. Hence, this study was aimed to evaluate possible beneficial effects of MPFF treatment on capillary network in surrounding tissues, in a rabbit dorsal ear vein model of sclerotherapy.^6,17,18

Methods

Rabbits and procedures

Twenty-two male New Zealand white rabbits, 13–16 weeks old, weighing between 1.1 and 2.9 kg, housed in individual cages with free access to food and water were used. All procedures were performed in accordance with the protocol approved by the Ethical Committee of the State University of Rio de Janeiro (CEA/215/2007)

Treatment

Treatment (gavage 2 ml/kg), either with drug MPFF or vehicle was performed during 21 consecutive days, starting 7 days before sclerotherapy.

MPFF consists of 90% micronized diosmin and 10% flavonoids (hesperidin, diosmetin, linarin, and isorhoifolin) obtained from Servier Laboratories (Suresnes, France). The dose of MPFF (300 mg/kg/day), was chosen based on previous experiments¹⁹; the drug was suspended in 10% lactose solution, which was used as vehicle.

Sclerotherapy

Liquid sclerotherapy was performed at D0 (after 7 days of treatment) in both treatment groups including 11 rabbits (22 ears) each. Both dorsal lateral ears were shaved and received local anesthesia with prilocaine cream (Emla® 5% cream, Astra Zeneca, Brazil).

The method used was similar to that described by Goldman et al.^6,17 Injections (0.25 ml) with 5% ethanolamine oleate solution (Ethamolin®, Zest Farmacêutica, Brazil) were performed 4 cm distal to the tip of the auricular artery-dorsal marginal ear vein anastomosis. Immediately following the injection, pressure was applied for 20 seconds proximal to and 5–6 cm distal to the injection site to allow an increased contact time between the agent and the vein wall.

Microcirculatory parameters

Anesthesia was induced by an intraperitoneal injection of 70 mg/kg of Dopalen (Ketamine hydrochloride, Vetbrands, Brazil, 116 mg/ml) plus 7 mg/kg of Anasedan (Xylazine hydrochloride, Vetbrands, Brazil, 20 mg/ml) and maintained with intraperitoneal injection containing one-third of this solution. Anesthetized rabbits were placed under the intravital microscope (Leitz, Wetzlar, Germany), coupled to a closed circuit TV system. The microcirculation of the rabbit’s ear was observed with optical magnification of 50 ×for microvascular permeability experiments and 100 ×for evaluation of the venular and arteriolar diameters, functional capillary density (FCD), and number of rolling and sticking leukocytes.

Venular and arteriolar diameters

In five rabbits of each group (10 ears), venular and arteriolar diameters were measured at: (i) baseline, (ii) 24 h postsclerotherapy, and (iii) 8 days postsclerotherapy as previously described.¹⁴ Two to three arterioles and venules were selected for analysis in each preparation.

Functional capillary density

FCD, defined as the number of capillaries with flowing red blood cells per unit of tissue area, was assessed at baseline, 24 h and 8 days postsclerotherapy in five rabbits of each group (10 ears) in sites of 1 mm² within the preparation.¹⁴

Macromolecular permeability

Microvascular permeability for large molecules was quantified as the number of leaky sites (= leaks, extravascular spots with diameter >40 µm) of fluorescein labeled dextran in the observed area (n = 6 ears from three rabbits): 2 h, 24 h and 8 days after sclerotherapy.¹⁵

Leukocyte rolling and sticking

Leukocytes were stained by an intravenous infusion of rhodamine-6 G (Sigma-Aldrich Co, MO, USA, 0.5 mg/kg/min) and their interaction to the endothelium (per 6 mm²) was observed and quantified at 2 h and 8 days after sclerotherapy.^15,16

Images

Images of the ear vein injection site were taken before, 2 h, and 14 days post sclerotherapy.

Statistical analysis

Statistical analysis was performed using two-way analysis of variance (ANOVA) with repeated measures on time. When the (group vs time) interaction was significant, a test of group effect was done at a fixed level of time using the Dunnett test (Easystat SAS). Values are expressed as mean ± SEM and P ≤ 0.05 was considered significant. Graphs were drawn using the Graph Pad Prism version 5.0 software (Graph Pad Software, San Diego, CA, USA).

Results

Arteriolar and venular diameters

Statistical analysis of mean venular diameter showed a treatment effect (P < 0.05) when all-confounding time points were considered. An increase in venular diameter was observed after 8 days which was partially prevented by MPFF treatment (27% vs 42% increase in venular diameter in MPFF- versus the lactose-treated group, respectively; Figure 1(a)).

Figure 1.

Comparison between MPFF- (300 mg/kg/day) and lactose-treated groups with respect to venular (a) and arteriolar (b) diameters at baseline, 24 h, and 8 days of dorsal marginal rabbit ear vein sclerotherapy. Data are expressed as mean ± SEM. *Significantly different from the lactose-treated group at all-confounding time points (P < 0.05). No statistically significant difference in the interaction treatment time.

The internal diameter of arterioles was significantly lower in MPFF-treated group at all-confounding time points (P < 0.05). As arteriolar diameter was not measured before treatment, it is not possible to determine if this difference in arteriolar diameter could be attributed to MPFF treatment. Nevertheless, arteriolar diameter expressed as percentage of variation from baseline values, showed that slight and similar changes occurred in both groups, suggesting that MPFF treatment did not influence arteriolar diameter variation after sclerotherapy (Figure 1b).

Functional capillary density

Sclerotherapy induced a decrease in the number of functional capillaries in both groups at 24 hours and up to 8 days. However, this reduction was significantly prevented by MPFF treatment when compared with the control group (6.3 ± 0.5 versus 2.7 ± 0.4 capillaries/mm² P < 0.001 after 24 h; and 6.7 ± 0.5 versus 2.8 ± 0.5 capillaries/mm²; P < 0.001 after eight days) (Figure 2).

Figure 2.

Effects of MPFF treatment (300 mg/kg/day) on FCD at baseline, 24 h, and 8 days of dorsal marginal rabbit ear vein sclerotherapy. Data are expressed as mean ± SEM. ***Significantly different from the lactose-treated group (P < 0.001).

Microvascular permeability

In the studied ear area of control rabbits (n = 10), the number of leaky sites averaged 58.6 ± 2.7 leaks/6 mm² at 2 h and 60.3 ± 1.4 leaks/6 mm² at 24 h post-sclerotherapy. Significantly lower leaks were noted at 2 and 24 hours after sclerotherapy in MPFF-treated animals, (36.0 ± 1.8 leaks/6 mm² at 2 h; P < 0.001) and 25.6 ± 1.4 leaks/6 mm² at 24 h (P < 0.001). Eight days postsclerotherapy, microvascular permeability was dramatically decreased in both treated groups, but remained lower in the MPFF-treated group (7.3 ± 0.5 versus 12.6 ± 0.6 leaks/6 mm² in MPFF- and lactose-treated animals, respectively; P < 0.05) (Figure 3).

Figure 3.

Effects of MPFF treatment (300 mg/kg/day) on microvascular permeability 2 h, 24 h, and 8 days after dorsal marginal rabbit ear vein sclerotherapy. ***Significantly different from the lactose-treated group (P < 0.001).

Leukocyte-endothelium interaction

A huge reduction in the number of rolling and sticking leukocytes was observed in MPFF-treated animals 2 h postsclerotherapy when compared with control rabbits (1.3 ± 0.5 vs. 2.9 ± 0.8 rolling leukocytes/mm², respectively (P < 0.001); and 8.3 ± 0.60 vs. 12.1 ± 0.7 sticking leukocytes/mm², respectively). Due to excessive edema observed 24 h postsclerotherapy, it was not possible to assess the number of rolling and sticking leukocytes in the control group. At eight days postsclerotherapy, the number of rolling and sticking leukocytes in the control group decreased to a value comparable with the MPPF-treated group (Figure 4(a) and (b)).

Figure 4.

Effects of MPFF treatment (300 mg/kg/day) on leukocyte rolling and sticking. Number of rolling (a) and sticking (b) leukocytes 2 h and 8 days after dorsal marginal rabbit ear vein sclerotherapy. Data are expressed as mean ± SEM. ***Significantly different from the lactose-treated group (P < 0.001).

Images

Imaging confirmed the beneficial effect of MPFF shown by decreased perivascular inflammation without affecting the sclerosant effect (Figures 5 to 7).

Figure 5.

Aspect of the rabbit dorsal ear vein before the procedure. Animal treated with lactose (left) and MPFF (right).

Figure 6.

Aspect of the rabbit dorsal ear vein 2 h after the sclerotherapy procedure on the dorsal ear vein. Note that vasospasm of treated vessels are similar in lactose-treated (left) and MPFF-treated (right) rabbits.

Figure 7.

Fourteen days after the sclerotherapy procedure. In the rabbit treated with lactose (left), there is a persistency of inflammatory signals around the sclerotic ear vein. In the rabbit treated with MPFF (right), a sclerosant effect is observed with partial disappearance of the dorsal ear vein, but there is no persistency of perivenous inflammatory process.

Discussion

The lead principle in sclerotherapy is to cause irreversible endothelial injury in the desired vessels while avoiding damage to normal collateral vessels and surrounding tissues. Consequently, the inflammatory phase must be controlled because excessive intravascular and perivascular inflammation induce vessel damage leading to extravasation of red blood cells and sclerosing agent into the subcutaneous tissue resulting in hyperpigmentation and necrosis. All currently available sclerosants cause side effects and we have used ethanolamin oleate, popular in Brazil. Clinical trials already support indication of Daflon® 500 mg in the peri-stripping period (DEFANS trial),²⁰ endovenous procedures (DECISION study),²¹ or combination with microsclerosing treatment (SYNERGY and SATISFY).²²

These experiments were designed, using a well-described animal model of sclerotherapy, the rabbit dorsal ear vein model,^6,8,17,18 to analyze possible beneficial actions of MPFF on microcirculatory parameters, close to the site of sclerotherapy. The dorsal marginal rabbit ear vein is similar in size to telangiectasias in humans and previous studies demonstrated that this model provides a relevant method to compare different sclerosing solutions and concentrations.^6,17,18

Dose selection for pharmacological studies depends on species (absorption and metabolism vary between species) as well as experimental model and purposes.

Most of the experimental studies demonstrating phlebotonic activity and anti-inflammatory properties of MPFF were performed in hamster and rat at doses between 5 and 320 mg/kg.

In the present work, the dose of 300 mg/kg p.o. was chosen based on the results of a previous study performed in a model of skin irradiation in the rabbit, leading to an increase in the permeability of capillaries of the skin. In this experiment, the maximal effect on capillaries density and colorant extravasation was observed after treatment with Daflon at the dose of 300 mg/kg.¹⁹

Analysis of MPFF-treatment effects on hemodynamic microcirculatory parameters demonstrated that it did not affect arteriolar tone and prevented the increase in venular diameter observed in the control group after sclerotherapy. This effect may be due to MPFF’s property to increase venous tone by prolonging noradrenergic activity.²³ Reducing the diameter of the vein may improve the efficacy of the sclerosing agent, and may also reduce complications following it, such as necrosis or matting, which represent, with hyperpigmentation, the most frequent side effect (occurring in up to 30% of patients). FCD reduced under inflammatory processes, was also investigated. During inflammation, endothelial cell edema reduces capillaries’ lumen leading to leukocyte plugging and obstruction of blood flow.^24,25 A remarkable finding of the present study is the preservation of the number of functional capillaries noted in animals treated with MPFF. This beneficial effect could be explained by vascular anti-inflammatory effects of MPFF as demonstrated by the observed huge decrease in the number of rolling and adherent leukocytes. MPFF probably acts by inhibiting endothelial cell swelling, preventing capillary obstruction, and preserving the number of functional capillaries. Further evidences for the anti-inflammatory effects of MPFF were demonstrated by the significant decrease in the number of leaky sites at postcapillary venules level, suggesting that MPFF is effective in preventing edema formation after sclerotherapy. Previous experiments performed in different species and models (ischemia-reperfusion, edema-inducing substances, oxidant challenge) confirmed that MPFF administration had protective effects on the venous wall by inhibiting the inflammatory process, decreasing the expression of adhesion molecules, and protecting against oxygen-derived free radicals.^{10–16,19,23} These results suggest that MPFF is capable of maintaining better oxygenation of tissues surrounding the sclerosed vein, without altering the efficacy of sclerotherapy on targeted varicose veins or telangiectases. In addition, MPFF by decreasing vascular inflammation may prevent rupture of fragile vessels and secondary extravasation of red blood cells into the dermis resulting in hemosiderin deposition and hyperpigmentation. Macroscopic observation confirmed perivascular inflammation disappearance in the MPFF-treated group. However, a clinical assay is necessary to confirm these promising results, as the action of a sclerosing solution was studied on a normal vessel in the rabbit ear model.

Conclusion

In conclusion, this experimental study points to the therapeutic efficacy of MPFF in limiting excessive postsclerotherapy microvascular disorders such as inflammation, suggesting that MPFF treatment could reduce secondary adverse effects of sclerotherapy and provide a better aesthetic aspect in clinical conditions without reducing efficacy of local sclerosing agents.

Footnotes

Conflict of interest

None declared.

Funding

This work was supported by Institut de Recherche International Servier, Suresnes, France.

Ethical approval

The ethics committee from State University of Rio de Janeiro approved this study (CEA/215/2007).

Guarantor

Contributorship

EB conceived the study, gaining ethical approval, revised the manuscript; EB and CEVM developed the experimental protocol; MGCS and TJV performed data interpretation and wrote the manuscript; FZGAC, MRM, and JJC performed the experiments and data acquisition; FLS performed the statistical analysis. All authors read and approved the final version of the manuscript.

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