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School of Medical and Surgical Sciences, Centre for Integrated Systems Biology and Medicine, Derby, UKDepartment of Vascular Surgery, Derby Hospitals NHS Foundation Trust, Derby, UK
Corresponding author. Prof Richard Donnelly, MD, PhD, FRCP, University of Nottingham Medical School, Derby City General Hospital, Uttoxeter Rd, Derby DE22 3DT, UK.
To evaluate the effects of varicose vein surgery on cutaneous microvascular perfusion and vasodilator responses to acetylcholine (Ach) and sodium nitroprusside (SNP) in the gaiter area of patients with great saphenous vein insufficiency.
Methods
Twenty-nine patients with isolated great saphenous vein incompetence attended three study mornings (before surgery, and 6–8 weeks and 6 months after sapheno-femoral ligation+partial stripping) during which cutaneous microvascular responses were measured in the supine and standing positions using laser Doppler fluximetry (LDF) combined with incremental-dose iontophoretic administration of endothelial-dependent (Ach) and -independent (SNP) vasodilators.
Results
Varicose vein surgery had no significant effect on baseline cutaneous perfusion or the microvascular response to Ach: e.g. peak vasodilator responses to the 1000 μC stimulus were mean 58 SEM 7, 64 SEM 6 and 65 SEM 7 PU on the pre-operative, 6–8 weeks and 6 months assessments. In contrast, the corresponding responses to SNP were significantly increased following surgery: e.g. at 2000 μC, mean 63 SEM 9, 142 SEM 4 and 157 SEM 9 PU (p<0.0001) in the upright position.
Conclusions
Sapheno-femoral ligation and partial stripping in patients with great saphenous vein insufficiency improves endothelial-independent cutaneous vasodilator function at the gaiter area, which may at least partly explain the benefits of surgery in reducing the risk of venous ulceration.
Chronic venous insufficiency is the most common cause of gravitational ulceration around the gaiter area (above the medial or lateral malleolus). Dilatation and incompetence of the great saphenous vein leads to increased ambulatory venous pressure, microvascular ischaemia and cutaneous tissue breakdown.
Several factors affect tissue viability and susceptibility to ulceration, but the structural and functional integrity of the microcirculation to maintain blood flow, tissue oxygenation and nutrient delivery despite changes in systemic and local haemodynamics may be especially important.
For example, we have previously shown that cutaneous microvascular vasodilator responses in the perimalleolar region are impaired in the upright position in patients with isolated great saphenous vein incompetence compared with healthy controls.
Thus, venous disease seems to be associated with a reduction in the compensatory vasodilator mechanisms which preserve cutaneous blood flow in the standing position, while similar microvascular abnormalities have been linked to the increased risk of foot ulceration in patients with sickle cell disease.
Varicose vein surgery for patients with isolated great saphenous vein incompetence, i.e. sapheno-femoral ligation or subfascial ligation and stripping, reduces the risk of ulceration and improves ulcer healing and recurrence rates,
but the effects of surgery on cutaneous microvascular perfusion and reactivity in the perimalleolar region have not been previously reported. Recent technological improvements in laser Doppler fluximetry (LDF) and the associated analytical software have provided more reliable diagnostic and prognostic information in various clinical settings, especially when LDF is combined with incremental iontophoretic administration of vasoactive drugs.
Studies with iontophoretic administration of drugs to human dermal vessels in vivo: cholinergic vasodilation is mediated by dilator prostanoids rather than nitric oxide.
Thus, the purpose of this study was to evaluate the effects of varicose vein surgery on cutaneous endothelial-dependent and endothelial-independent vasodilator responses in the perimalleolar region in both the supine and standing positions.
2. Methods
Thirty patients awaiting varicose vein surgery (unilateral sapheno-femoral ligation and partial stripping) agreed to participate in this study which was approved by the Southern Derbyshire Local Research Ethics Committee and the Derby Hospitals NHS Foundation Trust. All patients had isolated great saphenous vein reflux defined by duplex scanning. Patients with present or past venous ulceration, lower limb arterial disease, peripheral oedema or cardiac failure, and those with major skin changes in the gaiter area were excluded. All patients gave written informed consent. Following a baseline assessment, which included measurements of height, weight, BP, routine serum biochemistry and full blood count, each patient attended the Clinical Research Unit (CRU) for three study mornings during which microvascular perfusion and vasodilator responses in the gaiter area were measured under temperature-controlled conditions using a standard clinical protocol. Each patient attended on three occasions: (1) 2 weeks prior to varicose vein surgery; (2) 6–8 weeks following surgery; and (3) 6 months after surgery.
2.1 Clinical protocol for each study day
On each of three study mornings, patients attended the CRU at 8 a.m. and acclimatised to the room during a 30 min period of supine rest with the leg to be studied supported at a 30° angle. Ambient room temperature was maintained at 24 °C. The gaiter area was cleaned and dried thoroughly before applying two perspex iontophoresis chambers (Moor Instruments Ltd, Axminster, UK) to the surface of the leg 4–8 cm proximal to the medial malleolus. The chambers were positioned over healthy looking skin, approximately 2–5 cm apart, avoiding any area of lipodermatosclerosis or superficial veins. The anodal and cathodal chambers were filled with 0.25 ml of 1% acetylcholine (Ach) (Sigma Chemicals, UK) and 1% sodium nitroprusside (SNP) (Nipride, Roche Pharmaceuticals Ltd), respectively, and a laser Doppler probe positioned through the centre of each chamber.
After establishing a stable recording of baseline cutaneous flux, LDF responses to transcutaneous administration of the endothelial-dependent vasodilator Ach and the endothelial-independent vasodilator SNP were measured using an incremental-dose iontophoresis protocol.
Thereafter, following 10 min recovery and stabilization of baseline flux recordings in the standing position, repeat measurements of vasodilator responses were performed with the patient upright.
The DRT4 LDF system (Moor Instruments, Axminster, UK) was used throughout, together with the Moor Instruments iontophoresis controller and the DRT4 software which provides automated data analysis of skin temperature, cutaneous microvascular flux and dose-response curves for each iontophoretic challenge.
2.2 Iontophoretic protocol for measuring endothelial-dependent and -independent cutaneous microvascular responses
Transcutaneous iontophoresis of vasodilator drugs involves creating an electrical potential difference which actively causes ions in solution (Ach and SNP) to migrate according to the size of the electrical charge. The magnitude of the iontophoretic charge (Q) is, therefore, calculated from the duration (t) and size of the applied current (I), i.e Q (C) =I (A)×t (s). Thus, the dose of agonist administered in a given iontophoretic challenge is proportional to the charge applied.
The automated iontophoretic protocol is designed to provide short, incremental doses of Ach and SNP with time-dependent monitoring of the subsequent vasodilator responses, as described previously.
Each burst of current is followed by a 4 min period of laser Doppler recording. Thus, dose-response curves for Ach and SNP induced vasodilation were characterised using the following procedure to apply incremental charge-stimuli: 25 μA applied for 10 s (i.e. 250 μC), 50 μA for 10 s (500 μC), 100 μA for 10 s (1000 μC), and 100 μA for 20 s (2000 μC), with a 4 min recording period between each electrical charge.
2.3 Statistical analysis
The LDF commercial software system (Moorsoft V1, Moor Instruments, UK) was used to provide automated analysis of the microvascular Doppler flow responses to incremental doses of each agonist. Measurements of maximum perfusion (in arbitrary perfusion units, PU) for Ach and SNP in individual patients on each of three study days was compared using ANOVA for repeated measures. Data are presented as mean and SEM.
3. Results
One patient withdrew from the study due to cancellation of surgery. The remaining 29 patients (18 female, mean age 60 years) completed the study. There were no significant differences in body weight or BP between the pre-operative and post-operative study days.
3.1 Microvascular vasodilator responses to Ach
Incremental administration of the endothelial-dependent vasodilator Ach produced a dose-dependent increase in microvascular perfusion in both the supine (Fig. 1) and standing (Fig. 2) positions, but there were no significant differences in the response to Ach post-operatively compared with the pre-operative assessment. For example, peak vasodilator responses to the 1000 μC stimulus were mean 58 SEM 7, 64 SEM 6 and 65 SEM 7 PU on the pre-operative, 6–8 weeks and 6 months post-operative assessments, respectively. Maximum vasodilator responses to Ach at 2000 μC were similarly unchanged 6 months after surgery (mean 98 SEM 11 vs 82 SEM 10 PU).
Fig. 1Cutaneous microvascular vasodilator responses to incremental doses (proportional to charge) of acetylcholine (Ach) (upper panel) and sodium nitroprusside (SNP) (lower panel), administered by transcutaneous iontophoresis, in patients with isolated great saphenous vein incompetence (n=29). Data are represented by mean and SEM. These measurements were undertaken in the supine position before surgery and at 6–8 weeks and 6 months following surgery. There were no differences between the three study days in the endothelial-dependent vasodilator dose-response relationship to Ach, but responses to SNP were significantly increased. *p<0.041, **p<0.003, ***p<0.0001 for 6 months vs baseline (ANOVA).
Fig. 2Cutaneous microvascular vasodilator responses to incremental doses (proportional to charge) of acetylcholine (Ach) (upper panel) and sodium nitroprusside (SNP) (lower panel), administered by transcutaneous iontophoresis, in patients with isolated great saphenous vein incompetence (n=29). Data are represented by mean and SEM. These measurements were undertaken in the standing position before surgery and at 6–8 weeks and 6 months following surgery. There were no differences between the three study days in the endothelial-dependent vasodilator dose-response relationship to Ach, but responses to SNP were significantly increased. **p<0.01, ***p<0.001 for 6 months vs baseline (ANOVA).
In contrast to Ach, cutaneous endothelial-independent vasodilator responses to SNP were significantly increased following varicose vein surgery, both in the supine (Fig. 1) and standing (Fig. 2) positions. The increased responsiveness to SNP tended to be greater at 6 months compared with 6 weeks following surgery, and was especially evident in the upright position. For example, in response to SNP 2000 μC in the supine position peak vasodilator responses were mean 100 SEM 5, 158 SEM 9 and 190 SEM 8 PU at baseline, 6 weeks and 6 months (Fig. 1, p<0.0001), and correspondingly mean 63 SEM 9, 142 SEM 4, and 157 SEM 9 in the upright position (Fig. 2, p<0.001).
4. Discussion
We have previously shown that patients with uncomplicated great saphenous vein incompetence have impaired cutaneous vasodilator responses in the standing position.
Compared with healthy control subjects, upright posture had an adverse effect on microvascular perfusion in the gaiter area and the response to locally administered vasodilators. Absolute skin flux, even in healthy subjects, is much lower in the perimalleolar region compared with the shin or the dorsum of the foot,
which may in part explain why this area is highly susceptible to tissue breakdown. Factors that affect the inter-play between venous pressure, posture and endothelial function may have clinically important effects on tissue perfusion and the risk of ulceration. There is some information about the effects of herbal products on microvascular perfusion in the gaiter area of patients with venous incompetence,
Improvement of cutaneous microcirculation and oxygen supply in patients with chronic venous insufficiency by orally administered extract of red vine leaves AS195: a randomised, double-blind, placebo-controlled, crossover trial.
but nothing has yet been published about the impact of surgical interventions on microvascular haemodynamics.
The purpose of the present study was to assess whether venous haemodynamic changes that occur as a result of surgery (sapheno-femoral ligation) have immediate and/or longer term effects on cutaneous perfusion, microvascular reactivity and the compensatory mechanisms (e.g. in response to upright posture) which affect cutaneous blood flow in the perimalleolar region. The main finding was that varicose vein surgery has contrasting effects on endothelial-dependent and -independent vasodilator responses. Whereas baseline perfusion and cutaneous reactivity to Ach were unchanged, microvascular responses to SNP were markedly increased both at 6–8 weeks and at 6 months following surgery, especially in the upright position.
The interpretation and significance of these results can be discussed at three levels. Firstly, cutaneous vasoconstriction in response to standing is often referred to as the venoarteriolar reflex (or ‘oedema protection reflex’).
Local neurovascular mechanisms, including the sympathetic nervous system, cause vasoconstriction in response to venous hypertension and thus tend to buffer the rise in capillary pressure on standing. That vasodilator responses to exogenous SNP were enhanced post-operatively in the standing position would be consistent with a reduction in the venoarteriolar reflex following surgery, i.e. less activation of local neurovascular pathways, which cause vascular smooth muscle (VSM) contraction and oppose the effects of SNP. A reduction in the venoarteriolar reflex might be explained by differences in venous pressure as a result of surgery, or by changes in the sensory or effector arms of the reflex pathway, including down-regulation of cutaneous vasoconstrictor mechanisms. Extracellular matrix remodelling is one of several mechanisms which might affect the balance of vasodilator and vasoconstrictor responses of the blood vessel wall.
Secondly, an important observation in this study is that varicose vein surgery had no significant effect on endothelial-dependent microvascular vasodilation. Ach-induced relaxation of large conduit arteries is mediated by endothelial release of nitric oxide, but in the microcirculation the underlying mechanisms also include Ach-induced release of vasodilator prostanoids and/or endothelium-derived hyperpolarizing factor (EDHF).
Studies with iontophoretic administration of drugs to human dermal vessels in vivo: cholinergic vasodilation is mediated by dilator prostanoids rather than nitric oxide.
However, in the present study it is clear that surgical intervention, even though it improves venous pressure and clinical outcome, had no significant effect on microvascular endothelial dysfunction either in the supine or standing position.
The third dimension of this study compared microvascular responses at 6–8 weeks and 6 months after surgery. In the supine position, vasodilator responses to SNP were significantly increased at the later timepoint, which suggests that longer term adaptive changes may be occurring in response to surgery. A number of structural and histological changes in the vein wall and the microcirculation have been reported in patients with varicose veins,
and it seems plausible that improvements in microvascular vasodilator function following surgery will occur gradually over several months. For example, changes in matrix metalloproteinases,
may occur more slowly as part of vascular remodelling and adaptation following surgery.
In conclusion, surgical treatment of great saphenous vein incompetence has contrasting effects on endothelial-dependent and -independent cutaneous microvascular responses at the perimalleolar region. In particular, there were significant increases in SNP-induced vasodilation post-operatively, but surgery had no effect on microvascular endothelial function.
Studies with iontophoretic administration of drugs to human dermal vessels in vivo: cholinergic vasodilation is mediated by dilator prostanoids rather than nitric oxide.
Improvement of cutaneous microcirculation and oxygen supply in patients with chronic venous insufficiency by orally administered extract of red vine leaves AS195: a randomised, double-blind, placebo-controlled, crossover trial.
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