Endothelial dysfunction, assessed at the macrocirculation, has been proven as an early marker of vascular complications in several diseases, including diabetes, dyslipi-demia, and hypertension. The development of techniques capable to measure the skin blood flow has also enabled the study of the vascular reactivity at the microcirculation level. More specifically, the noninvasive measurement of cutaneous blood perfusion can be performed by the laser Doppler.
Currently, laser Doppler flowmetry is the most widely accepted technique for evaluating blood flow in the skin microcirculation. Basically, it measures the capillary flux, which is a combination of the velocity and the number of moving blood cells. This is achieved by using red laser light, which is transmitted to the skin through a fiberoptic cable. The frequency shift of light back-scattered from the moving blood cells beneath the probe tip is computed to give a measure of the superficial microvascular perfusion.
There are mainly two different types of instruments available: the laser Doppler perfusion imager (LDPI) and the laser Doppler blood flow monitor (LDM). The LDPI, or laser scanner, enables the quantification of superficial skin blood perfusion in a multiple number of adjacent sites on the skin and calculates the mean blood perfusion in a particular region (Fig. 6). The LDM, which is characterized for having two single-point laser probes is capable to measure the blood flow changes only in a small skin area (about 2-3 mm diameter)—that corresponds to the area where the probes are placed— and records the blood flow changes in response to the vasodilatory stimulus in a continuous way (Fig. 7).
The LDPI is best-suited for studying the relative changes in flow induced by a variety of physiological manoeuvres or pharmaceutical intervention procedures. The singlepoint laser probe is used mainly for evaluating the hyperemic response to heat stimulus or for evaluating the nerve-axon related hyperemic response. Both these two laser Doppler instruments have been extensively used to evaluate the skin microcirculatory flow of patients with diabetes in response to the delivery of two vasodilatory substances
by iontophoresis: a 1% acetylcholine chloride solution (endothelium-dependent vasodilation) and a 1% sodium nitroprusside solution (endothelium-independent vasodilation).
To use these methods for longitudinal analysis, a certain degree of confidence is needed to ensure that the results are not skewed for instrumental inaccuracies or other experimental factors. The main limitation of both techniques is, in fact, the variability, which is higher for the single-point laser Doppler than for the LDPI. The single-point technique has been validated against direct measurements of the capillary blood flow velocity (37). The day-to-day reproducibility of the technique was evaluated in healthy subjects who were repeatedly tested at their foot and arm for 10 consecutive days in our lab. The coefficient of variation for the maximal response to heat was 27.9%, whereas for the maximal hyperemic response after Ach and/or SNP-iontophoresis was 35.2% (18). The variability of this technique is mostly a spatial one, i.e., it is mainly because of the high heterogeneity of the skin microcirculation and not to the technique itself. In fact, the technique reproducibility can be significantly enhanced if one pays attention to place the laser probe approximately at the same skin area for repeated measurements (38).
The laser scanner has a significantly better reproducibility (which is mainly because of the minor spatial variation of blood flow assessment) with the coefficient of variation at the foot and forearms level being between 14 and 19%, and can therefore be used for
Indirect response ft 2
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