Review
Methodological issues in the assessment of skin microvascular endothelial function in humans

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The study of microvascular function can be performed in humans using laser Doppler flowmetry of the skin. This technology lends itself to a wide range of applications for studying the endothelial function of skin blood vessels. We review the advantages and limitations of postocclusive hyperemia, local thermal hyperemia, acetylcholine iontophoresis, flowmotion and association with microdialysis as tools with which to investigate skin microvascular endothelial function in humans. Postocclusive hyperemia, thermal hyperemia and acetylcholine iontophoresis provide integrated indexes of microvascular function rather than specific endothelial markers. However, they are valuable tools and can be used as surrogate endpoints in clinical trials in which the assessment of microvascular function in humans is required.

Introduction

The functions of the vascular endothelium include the control of vascular tone, the inhibition of platelet aggregation, the modulation of leukocyte tethering, rolling and transmigration, the regulation of smooth muscle cell proliferation and the modulation of vascular wall permeability. However, the term ‘endothelial function’ is, in most cases, used to refer to the ability of the endothelium to release compounds that can induce a direct relaxation of smooth muscle cells within the vascular wall, namely nitric oxide (NO), prostacyclin and endothelium-derived hyperpolarizing factors (EDHFs).

Section snippets

Endothelial and microvascular function as a biomarker in vascular diseases

Great emphasis has recently been placed on the role of endothelial dysfunction in the pathogenesis of a wide spectrum of cardiovascular diseases such as atherosclerosis and hypertension. Most studies aimed at studying endothelial dysfunction have focused on conductance arteries as a surrogate endpoint of coronary artery disease. By contrast, connective tissue diseases such as systemic sclerosis involve mostly microvascular dysfunction, which is a crucial step in complications of other diseases

Assessment of endothelial function in the microvasculature

Unlike flow-mediated dilatation of the brachial artery that is assessed using brachial artery ultrasonography, the assessment of endothelial function in the microvasculature is not standardized. Isolated subcutaneous microvessels can be mounted on isometric myographs, but such an approach remains invasive and exhibits the numerous limitations of in vitro studies. Another approach is to study the forearm blood flow using strain-gauge venous plethysmography 1, 2. Although reproducible, this

Using laser Doppler to investigate microvascular function in human skin

Laser Doppler is based on the reflection of a beam of laser light. Light undergoes changes in wavelength (Doppler shift) when it hits moving blood cells. The magnitude and frequency distribution of these changes in wavelength are related to the number and velocity of blood cells. Several different signals can be recorded but the red blood cell flux (i.e. the product of the velocity and concentration of moving blood cells within the measuring volume) is used most often.

Laser Doppler flowmetry

The measurement of cutaneous blood flux rather than blood flow

One major limitation of laser Doppler flowmetry is that it is not possible to measure absolute perfusion values (i.e. cutaneous blood flow in ml/min relative to the volume or weight of tissue). Although some researchers have tried to convert millivolts to conventional blood flow units using theoretical calculations, this is not widely accepted. Measurements in most studies are expressed as arbitrary perfusion units (PU) or millivolts (1 PU = 10 mV) and are often referred to as flux rather than

Postocclusive hyperemia

Postocclusive skin reactive hyperemia refers to the increase in skin blood flow above baseline levels following the release of a brief arterial occlusion. It is also called postischemic or reactive hyperemia. It can be characterized by an initial peak in flux that occurs within a few seconds of removal of the occlusion, and a sustained hyperemia. This test is performed by placing a cuff on the distal part of the upper arm and increasing the pressure to 50 mm Hg above the systolic blood pressure.

Local thermal hyperemia

Local thermal hyperemia leads to a temperature-dependent sustained increase in skin cutaneous flow [18] and achieves a maximal vasodilatation between 42 °C and 44 °C [3]. This maximal thermal vasodilatation corresponds to the maximal vasodilator capacity of the vessels [3]. Local-heating-evoked vasodilatation is mediated by at least two independent mechanisms: an initial peak in cutaneous blood flow during the first 10 min, followed by a plateau after 20–30 min of warming. The initial rapid phase

Acetylcholine iontophoresis

Iontophoresis is based on the principle that a charged drug in solution will migrate across the skin under the influence of a direct low-intensity electric current [30]. The quantity of drug delivered depends on the magnitude and duration of the current applied and on the skin barrier. When combined with laser Doppler flowmetry or perfusion imaging, this method enables the detection of alterations in cutaneous blood flow in response to the time-controlled delivery of the vasoactive drug to a

Laser Doppler flowmetry coupled with microdialysis

Microdialysis is a technique used to introduce or remove ions, molecules and drugs of interest to or from the interstitial space in skin and/or muscle. It can be used to deliver pharmacological agents to a small area of tissue so that no confounding systemic effects occur while simultaneously sampling the effluent fluid. In addition, the concentration of substances released in response to the pharmacological action can be measured in the dialysate (effluent fluid) [37].

Microdialysis fibers can

Flowmotion

Periodic oscillations of cutaneous blood flow, also called flowmotion, can be quantified by spectral analysis of the signal. Spectral analysis can be performed by the classical Fourier transformation but, because signal characteristics change continuously, it is better analyzed using the wavelet transform, which takes into account the time component. Five characteristic frequencies are reported on the available frequency spectrum [39]. These oscillations represent the influence of the heartbeat

Concluding remarks

The endothelial function of human skin can be assessed by different applications of laser Doppler. Postocclusive hyperemia, thermal hyperemia and acetylcholine iontophoresis provide integrated indexes of microvascular function rather than specific endothelial markers. However, these tests are of clinical use for the assessment of global microvascular function in humans.

Acknowledgements

J-L.C. was supported by grants from AGIRàDom and ANTADIR. C.M. was supported by HL-070928.

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