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Reproducibility of Laser Doppler Imaging of Skin Blood Flow as a Tool to Assess Endothelial Function

Kubli, Sandrine; Waeber, Bernard; Dalle-Ave, Anne; Feihl, François

Journal of Cardiovascular Pharmacology: November 2000 - Volume 36 - Issue 5 - p 640-648
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Endothelial dysfunction might be an important and early event in the pathogenesis of major cardiovascular diseases. Therefore, the evaluation of endothelial function in humans may be of great clinical relevance. Usual methods for that purpose are either invasive and/or technically demanding. In the dermal microcirculation, endothelial function may be assessed noninvasively from the laser Doppler measurement of increases in blood flow after either the transdermal application of acetylcholine by iontophoresis, or the release of transient arterial occlusion (reactive hyperemia). An endothelium-independent response may be provided by the iontophoresis of sodium nitroprusside. This approach is notable for technical simplicity, but of uncertain reproducibility. Sixteen young, healthy, nonsmoking males were examined in the fasting state. Changes in skin blood flow were measured with a laser Doppler imager during the iontophoresis of acetylcholine and sodium nitroprusside, as well as during reactive hyperemia, on two different days, at each of two different sites on the volar face of the forearm. Nonspecific effects related to the stimulation of terminal nerve fibers by the iontophoretic current were suppressed by prior surface anesthesia. The iontophoresis of acetylcholine and sodium nitroprusside induced a seven- to eightfold increase in dermal blood flow. The corresponding figure for peak reactive hyperemia was approximately fourfold. The mean coefficients of variation of responses recorded on different days, on the same site, in the same individual were <10% for iontophoresis of acetylcholine and for peak reactive hyperemia, and between 10 and 20% for iontophoresis of sodium nitroprusside. This day-to-day variation was significantly smaller than the site-to-site variation (p < 0.01 for all three responses). Endothelium-dependent and -independent responses of dermal blood flow evaluated with laser Doppler imaging are highly reproducible from day to day, at least in healthy nonsmoking young male subjects, and provided some simple precautions are observed, foremost among which is the strict standardization of the recording site. These observations may have implications for the testing of endothelial function in clinical studies.

Division of Clinical Pathophysiology and Medical Teaching, Department of Internal Medicine, Lausanne University Hospital, Lausanne, Switzerland

Received May 9, 2000; revision accepted August 18, 2000.

Address correspondence and reprint requests to Dr. F. Feihl at Division of Clinical Pathophysiology and Medical Teaching, BH19-640, CHUV, CH-1011 Lausanne, Switzerland. E-mail: Francois.Feihl@chuv.hospvd.ch

The vascular endothelium plays an essential role in the biology of blood vessels. An altered behavior of vascular endothelial cells (endothelial dysfunction) seems to occur early in the course of major cardiovascular diseases, such as atherosclerosis, coronary heart disease, essential hypertension, and diabetes (1-5). It is likely that endothelial dysfunction has a crucial importance in the pathogenesis of functional and structural blood vessel abnormalities characteristic of these conditions. It might therefore be hoped that the evaluation of endothelial function could serve several useful purposes in the clinical setting, for instance, in the early detection of vascular abnormalities in subjects with known risk factors, or risk stratification and the provision of surrogate end points in therapeutic trials (5,6). Thus, methods for the evaluation of endothelial function in humans have attracted considerable interest in the last decade.

To be used beyond a restricted experimental setting, such methods must be noninvasive and should not require high technical skills. Additional requirements are the possibility of repeated measurements in an individual patient and adequate reproducibility. Endothelial function can be evaluated noninvasively in the radial artery, a conductance vessel, from the ultrasonic measurement of arterial diameter changes after a reactive hyperemia (flow-mediated vasodilation) (7). This method has excellent reproducibility (8), but requires a highly trained operator for reliability (9). Alternatively, endothelium-dependent vascular responses can be observed in the dermal microcirculation with laser Doppler flowmetry, the relevant stimuli being either reactive hyperemia (10,11) or the noninvasive local application of acetylcholine with iontophoresis (12-14). These methods are simple to implement, but may suffer from limited reproducibility, at least when the measurement of blood flow is restricted to the minute area in contact with an optical fiber, as is the case with standard laser Doppler flowmetry.

Recent technological developments raise the hope of removing the latter limitation. It has become feasible to measure dermal blood flow in predefined areas of arbitrary size with a laser Doppler imager. Rather than an optical fiber, this device uses a moving mirror to direct the laser beam onto the skin. A skin region can thus be scanned for blood flow within seconds or minutes, depending on the desired spatial resolution and surface area to be explored (15). Limited data indicate that scanning a surface with laser Doppler flowmetry rather than resorting to single-point laser Doppler flowmetry may significantly reduce the variability of dermal blood flow responses to the iontophoresis of either acetylcholine (16,17) or the endothelium-independent vasodilator sodium nitroprusside (17).

In the present study, we assessed the reproducibility of dermal blood flow responses with a recently developed laser Doppler imaging system, to either locally applied acetylcholine and sodium nitroprusside, or to reactive hyperemia. Although presently restricted to laboratory conditions, our data make this method a promising candidate for the clinical evaluation of endothelial function in the dermal microcirculation.

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METHODS

Subjects

Sixteen male healthy subjects, aged 20-28 years, were included. They were all nonsmokers and had no personal history of hypertension, diabetes, or hypercholesterolemia. None of the subjects had taken any vasoactive medication or antiinflammatory drugs during 10 days before the beginning of the study. The volunteers were fully informed about the protocol of the study and gave their written consent. The study conformed with the principles outlined in the Declaration of Helsinki, and was approved by the Ethical Committee of the Medical Faculty of Lausanne, Switzerland.

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Laser Doppler imaging

We used a recently developed laser Doppler Imager (Moor Instruments, software version 3.01, Axminster, U.K.) (18), whose principle of operation is presented schematically in Fig. 1A. In essence, a moving mirror directs a beam of coherent red light generated by a 633-nm helium-neon laser on the skin. Computer-controlled rotations of the mirror around two perpendicular axes allow the scanning of a square region. The surface of the scanned area can be chosen in a range from 1 mm2 to a complete body part such as the hand or thorax, depending on angular amplitudes and skin-mirror distance. From the analysis of the backscattered Doppler-shifted light, microvascular blood flow in each of up to 256 × 256 adjacent spots ("pixels") is calculated, with a computation time of 4 ms/pixel. Thus, at full spatial resolution, a complete scan is obtained in ∼5 min. Scan time can be shortened by reducing resolution and/or the size of the scanned area. For example, a 64 × 64 scan would take only ∼25 s. The final result is a computer-generated, color-coded image of the spatial distribution of microvascular blood flow. Total flow, expressed in perfusion units (PU) according to the principle of laser Doppler flowmetry, can be calculated later by summing the pixel values in an arbitrarily shaped region of interest within the scanned area, using the software provided by the laser Doppler imaging system manufacturer. In comparison with usual fiber-optic laser Doppler probes, this system allows the measurement of microvascular blood flow of a much larger area, with no skin contact.

FIG. 1

FIG. 1

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Iontophoresis of vasoactive agents

Iontophoresis is a noninvasive method for transdermal transfer of charged molecules locally on the skin by means of an externally applied electrical current (16).

To follow with the laser Doppler imaging system the response of skin blood flow to iontophoretically applied vasoactive agents, we constructed a ring-shaped chamber in black neoprene fitted on the inside with an annular cooper electrode connected to a iontophoresis controller (MIC1-e; Moor Instruments) (Fig. 1B). The chamber was affixed to the skin with double-sided tape, and filled to the rim with a solution of either 1% acetylcholine (endothelium-dependent stimulus) or 0.1% sodium nitroprusside (endothelium-independent stimulus) in distilled water. The internal diameter of the chamber was 10 mm, so that exposed skin area was 0.78 mm2. To avoid optical artefacts generated at the air-liquid interface, the chamber was covered with a thin clear glass lid, with care taken to avoid the trapping of any bubble underneath. The iontophoresis controller was also connected to an indifferent ECG electrode placed on the wrist. Polarity was adapted to the electric charge of the vasoactive molecule (chamber positive for acetylcholine, and negative for sodium nitroprusside). The pulsed iontophoretic protocols shown in Fig. 1C were used. They were adapted from those described by Morris et al. (13) and Morris and Shore (16), to obtain a maximal response to each agent (preliminary data, not shown). The total current charges were 22 and 38 mC for the iontophoresis of acetylcholine and sodium nitroprusside, respectively. Electrical current alone may induce a vasodilatation due to stimulation of local sensory nerves, a response which may be inhibited by local anesthesia (15,16). In the present study, acetylcholine and sodium nitroprusside were therefore applied on skin pretreated for 1 h with a local anesthetic cream (Emla cream 5%; Astra Pharmaceutica AG, Dietikon, Switzerland), under an occlusive dressing (Tegaderm; 3M Health Care Ltd., U.K.). The effective inhibition of current-induced vasodilatation was systematically controlled by applying the same amount of current to a control chamber filled with isotonic saline. The current was simultaneously applied to both the control and the active-compound chambers by two different iontophoresis controllers operating in synchrony. Isotonic saline was chosen as control iontophoresis rather than the deionized water used as drug solvent, because iontophoresis of deionized water alone occasionally induced small (<1 mm2) lesions typical of current burns in the exposed skin, possibly explained by an inhomogeneous distribution of current density in these particular conditions. Such lesions were never observed with the iontophoresis of saline, acetylcholine, and sodium nitroprusside.

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Reactive hyperemia

Reactive hyperemia is an temporary increase in blood flow after the release of temporary occlusion of arterial inflow. In the present study, postischemic vasodilatation was assessed with the laser Doppler imaging system in the anterior forearm skin, at sites not exposed to the anesthetic cream. The brachial artery occlusion was achieved by a pressure cuff placed on the arm and inflated at a suprasystolic pressure (200 mm Hg) for 3 min.

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Protocol

The study was carried out in the early morning, in a quiet room with air conditioning. Ambient temperature was system-atically measured and ranged from 21 to 23.5°C. The subjects were asked to come in the fasting state on two different days 48-72 h apart. On arrival at 8 a.m., the anesthetic cream was applied on the sites elected for iontophoresis on the anterior face of the forearms. One arm was randomly chosen for the application of acetylcholine, and the other for sodium nitroprusside. Each agent was applied within 7 cm of the wrist (distal site) and of the antecubital fossa (proximal site). These sites were selected so as to exclude visible veins, and were marked with permanent ink, so as to be used identically on day 1 and day 2. Reactive hyperemia was recorded in two sites not exposed to anesthetic cream, only one on each arm, one distal and the other proximal in random allocation. Subjects were examined in the supine position with the arm supported by a cushion. The distance traveled by the incident laser beam from the laser aperture to the skin was set at 40-42 cm.

During the hour necessary for the surface anesthesia cream to be effective, the reactive hyperemia was performed. The laser Doppler imaging device was set for repetitive scanning of a 1.16 × 0.16-cm skin surface, corresponding to 116 pixels. Two scans were used to assess the baseline, and two others for the determination of the biologic zero during arterial occlusion. Simultaneous with cuff deflation, repetitive scanning was launched at a frequency of one image each 5 s. This time interval was chosen to follow the rapid change in skin blood that followed.

After exposure to the anesthetic cream for 1 h, the cream was taken off on one site, which was gently wiped with alcohol 70% followed by deionized water and then allowed to dry. Two chambers were stuck in place. One was then filled with the appropriate drug solution, and the other with the control isotonic saline. The thin glass lid was placed in position without bubbles. The laser beam was positioned to scan a 4.9 × 3.1-cm rectangular area including both chambers in slightly less than 1 min. Sixteen such scans were taken at a frequency of one per minute, with the appropriate iontophoretic protocol started just before the beginning of the third scan and terminated slightly after the onset of the ninth one. The same procedure was then repeated sequentially at each of the remaining sites. The background setting of the Moor laser Doppler imaging system was chosen to ensure zero flow reading on the black neoprene that made up the chambers. Their rims were thus clearly seen on each scanned image as annular areas of zero flow, allowing the precise determination of mean flow in the exposed skin (∼1,500 pixels per chamber).

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Drugs

The drug solutions were prepared freshly on each day. All drugs were dissolved in deionized water. For acetylcholine 1%, a powder of acetylcholine chloride (Sigma Chemie, Buchs, Switzerland)-stored at −20°C to avoid hyperhydration-was directly prepared at a concentration of 1%. For sodium nitroprusside 0.1% (Nipruss; Schwarz Pharma, Monheim, Germany), the dried powder was first dissolved to form a 1% solution, and then deep frozen at −80°C in aliquots of 1 ml. On each day, one aliquot was thawed and diluted to a 0.1% solution. The light-sensitive sodium nitroprusside was kept in the dark (aluminum foil) until immediately before use. The deionized water was obtained from the facilities of our hospital, isotonic saline from Braun Medical (Switzerland), and the anesthetic cream (Emla 5%) from Astra Pharmaceutics (Dietikon, Switzerland).

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Data analysis

Data are presented as the mean ± SD. For statistical analysis, each response was first reduced to a single number (i.e., the maximal change in perfusion from baseline, in perfusion units). In each subject, coefficients of variation were computed for pairs of responses obtained either at the same site on the two different days (day-to-day variation) or on the same day at the two different sites (site-to-site variation). These individual coefficients of variation were then averaged across subjects to provide overall estimates of intraindividual variability. In addition, the interindividual coefficients of variation of each response were computed for each day and site. Finally, a mixed-model analysis of variance was applied, in which Day and Site of measurement were specified as fixed factors, whereas Subject and Subject by Site interaction were random. These effects were examined for significance only if the overall F value exceeded the critical point. The alpha level of all tests was set at 0.05. All computations were performed with the JMP software, version 3.2.2 (SAS Institute, Cary, NJ, U.S.A.).

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RESULTS

Iontophoresis of vasoactive agents

In the conditions used (surface anesthesia with a cream), the iontophoresis of isotonic saline had no apparent vasoactive effect in the forearm skin (Fig. 2). In contrast, marked vasodilatation was induced by the iontophoresis of both acetylcholine and sodium nitroprus-side, with a seven- to eightfold increase of flow above baseline (Fig. 3, and Table 1). The time course of response was somewhat more rapid for acetylcholine than for sodium nitroprusside. With both agents, a plateau was reached at the end of iontophoresis or shortly there-after, and lasted at least until the end of repeated laser Doppler scanning (i.e., 7 min after the last current pulse). At any given site (i.e., proximal or distal on the forearm skin), the average time courses and maximal responses were quasi identical on the two days of study (Fig. 3 and Table 1). The maximal response to acetylcholine, but not to sodium nitroprusside, was slightly lower at the distal, compared with the proximal site (p < 0.05).

FIG. 2

FIG. 2

FIG. 3

FIG. 3

TABLE 1

TABLE 1

The first two lines of Table 2 show estimates of the intraindividual coefficients of variation of iontophoretic data, computed as described in Methods. Day-to-day variability of maximal responses to acetylcholine measured at a constant site was remarkably low (i.e., <10%), whereas the site-to-site variation appeared larger (∼20%). This was borne out by plots of individual data (Fig. 4, top), as well as by the mixed-model analysis of variance, which disclosed a very significant Site × Subject interaction (p < 0.001).

TABLE 2

TABLE 2

FIG. 4

FIG. 4

Compared with acetylcholine responses, sodium nitroprusside responses were more variable from day to day, but not from site to site. With this agent, a similar, although less marked pattern of greater site-to-site compared with day-to-day variability was noted in the plots of individual data (Fig. 4, middle), as well as in the results of mixed-model analysis of variance (Site × Subject interaction: p = 0.006).

Estimates of the interindividual coefficients of variation of iontophoretic data, computed as described in Methods, were mostly between 30% and 40% (Table 3). The variance between was significantly larger than the variance within subjects (p values of the subject factor: 0.012 and 0.021 for maximal responses to acetylcholine and sodium nitroprusside, respectively).

TABLE 3

TABLE 3

The spatial heterogeneity of microvascular skin blood flow response to one challenge with acetylcholine administered by iontophoresis is illustrated in Fig. 5. The heterogeneity was assess by comparing the responses calculated from the pixels located in five small zones (each <2 mm2) within the 0.78-cm2 area encompassed by the iontophoresis chamber and that calculated as the average of all pixels within the chamber area.

FIG. 5

FIG. 5

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Reactive hyperemia

The time resolution offered by the laser Doppler imaging system-one measurement every 5 s-was largely sufficient to capture the typical flow profiles of reactive hyperemia in the skin microcirculation (Fig. 6). There was no statistically significant effect of either time or site on the average responses (last line of Table 1). The day-to-day coefficients of variation of the peak increase in flow were intermediate between the values for responses to acetylcholine and sodium nitroprusside (Table 2, last line). Once again, there was a pattern of greater site-to-site compared with day-to-day variability (Fig. 4, bottom; Site × Subject interaction: p < 0.001).

FIG. 6

FIG. 6

Estimates of the interindividual coefficients of variation of peak changes in blood flow clustered around 30% (Table 3, last line). The variance between was significantly larger than the variance within subjects (p = 0.003).

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DISCUSSION

Reproducibility

Exploration of the skin microcirculation with laser Doppler technology has often been considered poorly reproducible (19). In the present study, we systematically investigated the variability of several endothelium-dependent and -independent responses assessed noninvasively in the skin microcirculation, using our laser Doppler imaging system. Our major finding is that such responses are highly reproducible from day to day, at least in healthy nonsmoking young men, and provided some simple precautions are observed, foremost among which is the strict standardization of the recording site. These observations may have implications for the testing of endothelial function in clinical studies.

Laser Doppler technology has been used relatively often to monitor the response of skin blood flow to the transdermal iontophoretic application of acetylcholine (11,13,14,16,17,20-23) or sodium nitroprusside (11,13,14,16,17,21-23). These various authors reported day-to-day coefficients of variation ranging from 13.7% (20) to 34% (14), and from 14% (22) to 34% (14), for responses to acetylcholine and sodium nitroprusside, respectively. Interstudy comparisons are made difficult by differences in study populations, laser Doppler and iontophoresis techniques, and by general lack of detailed information on the exact conditions under which reproducibility was tested.

The laser Doppler imager used in the present study allows the quasi-simultaneous measurement of skin blood flow at a very large number of points, thus allowing the "averaging out" of spatial variations encountered within the explored area. In addition, any mechanical contact with the latter is avoided. In contrast, single-point laser Doppler samples only ∼1 mm2 of skin, and is thus very sensitive to the variations in dermal perfusion that characteristically occur with a spatial frequency of one to two per millimeter (24). Furthermore, mechanical contact made by the sensor has the potential to disturb the underlying microcirculation (25). On the basis of these features, it is rational to expect a general enhancement in the reproducibility of any data obtained with this device, in comparison with the more traditional single-point technique. An example of the variability introduced in an acetylcholine response by millimetric displacements of the measurement point is shown in Fig. 5. Morris et al. (16) have provided a formal demonstration of the superior site-to-site reproducibility provided by scanning a surface, in comparison with single-point laser Doppler, for the measurement of acetylcholine responses.

Although a likely contributor, the use of our laser Doppler imaging system is not sufficient to account for the excellent day-to-day reproducibility of our data. This is suggested by the day-to-day coefficients of variation clearly higher than those shown in Table 2 reported by some studies that used laser Doppler scanning technique for the measurement of acetylcholine [22% (13); 23% (16); 14% (20) and sodium nitroprusside responses (31%) (16)]. It is not clear in these reports what efforts were made to ensure an identical location of tests repeated in the same subject on different occasions. In the present study, all three responses investigated were more variable when measured on the same day at two different sites distant from 10 to 15 cm on the forearm skin, compared with measurements carried out on strictly the same site on different days (Fig. 4). The reasons for this regional variation remain to be determined. Heterogeneity in microvessel density and function, in the depth sampled by the laser beam (26), in the relationship of the Doppler signal to actual blood flow (25), and finally in the efficiency of iontophoretic transfer could be all be involved. Whatever the explanation, our results clearly indicate the importance of using the same site on different occasions if one wishes to optimize the day-to-day reproducibility of the skin microvascular reactivity tests examined in the present study.

A further factor that may bear on the reproducibility of acetylcholine and sodium nitroprusside responses is the possible vasodilatory reaction to iontophoretic current alone, which is mediated by the local activation of terminal nociceptive nerve endings (axon reflex) (15,16). Several of the previously cited studies ignored this problem entirely (17,20,21,23,27). Others determined a control response to the iontophoresis of solvent alone, which was subtracted from the response to the vasoactive agent (11,13,16). Spatial variations of the former might then introduce an error. Thus, because the control and test responses cannot be recorded simultaneously on the same site, this procedure is subject to errors introduced by spatial variations. Furthermore, strict additivity between drug and current effects is assumed without proof. Thus, selective suppression of the latter would seem preferable. Noon et al. (14) reported a complete absence of current-induced vasodilation when using a 2% methylcellulose gel in ultrapure water as a support for the vasoactive agent. Measurement of blood flow with a laser Doppler device might be difficult in such condition, because of poor transmission of the laser beam through the gel. In agreement with the work of others (15,16), we successfully suppressed current-induced vasodilatation with surface anesthesia (Fig. 2), which was systematically applied to all sites tested with iontophoretic drug application. In these conditions, we were able to choose iontophoretic protocols that provided the maximal effect of acetylcholine agent (preliminary data, not shown). We used the pulsed protocols described in the work of Morris et al. (16), adapted essentially by increasing the total current charge (acetylcholine: 22 vs. 18 mC; sodium nitroprusside: 38 vs. 8 mC). It is noteworthy that the acetylcholine and sodium nitroprusside responses appeared respectively slightly (sixfold increase of flow above baseline) and largely (threefold increase of flow) smaller in this previous work than in the present study (seven- to eightfold increase for both responses). One might reasonably expect that suppressing current-induced vasodilatation and working on the flat part of the dose-effect relationship should contribute to the reproducibility of dermal blood flow responses to the iontophoresis of vasoactive agents.

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Limitations

The present study was carried out in young, healthy, nonsmoking adult men. In addition, examinations occurred in the early morning and in the fasting state. How departures from these rather restrictive conditions could affect reproducibility remains largely to be determined. The investigation of vascular reactivity in women is complicated by the known influence of estrogen levels on the endothelial function of both peripheral conduit arteries (28) and skin microvessels (21). The effects of postprandial state on endothelium-dependent flowmediated vasodilatation of the brachial artery are well established (29), but remain unknown in the case of the skin microcirculatory responses examined in the present work.

A different concern, also not addressed by our study, is the actual information content of these responses on endothelial function in the skin microcirculation. In this vascular bed, the acetylcholine response has been variously reported to be partially blocked by the inhibition of either nitric oxide synthase (12) or cyclooxygenase (14), suggesting a mediation by endothelium-derived vasodilatory products. There would be little dispute that the vasodilatation induced by sodium nitroprusside, an NO donor, could serve as an endothelium-independent control. Reactive hyperemia is partly mediated by the endothelium in skeletal muscle, both in the rat (10) and in humans (30), but its mechanisms have not been specifically studied in dermal microvessels.

Finally, assessing endothelial function in a specific territory only, the results may or may not be similar to those from other parts of the circulation. This concern is in fact inherent to all methods invasive and noninvasive, proposed for that purpose. It may be partly alleviated by correlative studies conducted in different vascular beds. For instance, the amplitude of flow-mediated vasodilatation of the brachial artery was found related to the change in coronary vascular tone induced by the intracoronary infusion of acetylcholine (31). Along a similar line, the responses to iontophoresis of acetylcholine correlated well with peak oxygen consumption during exercise in heart transplant recipients, suggesting that microvascular responses of the skin reflected those of the resistance vessels in working muscles in these conditions (22).

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CONCLUSIONS

The present study defines basic requirements under which laser Doppler monitoring of skin blood flow responses to the iontophoresis of acetylcholine and sodium nitroprusside, as well as reactive hyperemia, may be carried out with acceptable reproducibility. Our data demonstrate the importance of removing sources of variation related to spatial heterogeneity of skin blood flow. The new technology of laser Doppler imaging appears ideally suited to that effect. In addition, we suggest the usefulness of surface anesthesia for the suppression of current-induced vasodilatation during the iontophoresis of acetylcholine and sodium nitroprusside. Utility of these methods for the testing of endothelial function in clinical studies remains to be demonstrated.

Acknowledgment: We thank Camille Anglada for excellent technical assistance. Financial support was provided by the Tossiza Foundation.

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Keywords:

Acetylcholine; Endothelial function; Nitric oxide; Vasoactive agents

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