INTRODUCTION

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TENLAND ET AL. (9) REPORTED large variations in Doppler flux recordings from skin sites spaced only a few millimeters apart. This large spatial variation in cutaneous blood flow raised some questions about the validity of a single laser Doppler flux recording being a representative index of skin blood flow (SkBF). Anatomic studies (1) show that the spacing of ascending arterioles on the ventral surface of the human forearm averages 1.7 mm. These data support the possibility that a laser Doppler flow probe placed randomly on the forearm skin has a good chance of being located at a skin site that may not contain an ascending arteriole, a blood vessel important in the regulation of regional blood flow. Another interesting observation was that ~20% of the 1-cm² area of skin examined could be considered avascular, containing few microvessels within the volume of tissue (1 mm³) thought to be sampled by laser Doppler velocimetry (1). Several alternative approaches have been used to overcome the limitations of a single-site recording, including use of multiple single-site recordings, use of integrating probes, and construction of topographical perfusion maps. Topographical perfusion maps of human skin have been created by using manual methods (2, 3) and more recently by using laser Doppler scanning instruments (12, 13).

The use of laser Doppler scanning techniques should enable researchers to address many early concerns associated with the large spatial heterogeneity of SkBF. However, rapid laser Doppler scanning may have its own inherent limitations. For example, single-site laser Doppler recordings provide a continuous view of the cutaneous circulation and provide information related to the temporal pattern of the cutaneous blood flow and its dynamic response to specific perturbations. Laser Doppler scanning procedures produce a blood flow map that represents a "slice" in time of the blood flow pattern and may neglect temporal variations in the data. An earlier paper has provided preliminary data outlining some of the possible limitations of laser Doppler imaging (12). However, at present it is not entirely clear how one accurately evaluates changes in the pattern of blood flow when using laser Doppler scanning or the reproducibility of this measurement. The purpose of this communication is to describe preliminary work with laser Doppler imaging, its reproducibility, and specific methodological procedures that aid in data analysis. These preliminary studies should provide insight into the usefulness of laser Doppler imaging in understanding reflex control of SkBF.

	METHODS

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Topographical perfusion maps of the skin were generated by using two types of systems: a semiautomated mapping system or a commercially available laser Doppler scanner. The semiautomated system consisted of a laser Doppler velocimetry unit with a needle-type probe (Perimed PF2), which was mounted in the penholder of a flatbed X-Y plotter with the pen movements controlled by stepping motors (resolution of 0.025 mm; model 7000-C, United Innovations). The laser Doppler flux data were acquired by using a National Instruments analog-to-digital data-acquisition board (LabView II software), and pen movements were controlled via an RS-232 interface with the computer (Macintosh II). A special plastic probe holder for the needle probe was designed to fit into the plotter's existing penholder with a counterbalance of <4 g when in contact with the skin. The plotter was programmed to lift and move the probe at a velocity of 17 cm/s (<0.5-s movement time) and move in a rectilinear fashion over a 10 × 10 grid (1-cm² area). The digitizing function of the plotter was used to establish reference points outside the desired sample area and allowed identification of sample sites on successive days. Topographical perfusion maps were constructed from data collected at each site (10 samples/s for 7 s). A 1-cm² area of skin was scanned in ~15 min with a resolution of 1 mm²/pixel. After creation of the topographical perfusion map, six skin sites were identified within the 1-cm² area and were programmed for monitoring via the digitizing function of the plotter. Blood flow measurements at each site were made once per minute for a 10-s period.

Two laser Doppler imaging systems were used in these studies: a Moor laser Doppler imager (Moor Instruments, Axminster, UK) and LISCA laser Doppler perfusion imager 1.0 (LISCA Development). Both systems used laser radiation generated by a visible red helium-neon laser with a wavelength of 632.8 nm and a nominal power of <2.0 mW. The Moor system was used for evaluating scanning speeds and reproducibility studies, whereas the LISCA system was used to evaluate the SkBF responses to whole body heating and lower body negative pressure (LBNP).

In all studies, we examined the ventral forearm skin. This site was chosen because it represented a common site examined by earlier studies with the use of single-site laser Doppler flow probes (4, 5). The scanning rate of the LISCA system was fixed at 10 ms/pixel, and a 2.5 × 2.5-cm area of skin was sampled at a resolution of 28 × 28 pixels, producing a spatial resolution of ~0.8 mm²/pixel. The Moor system allowed more flexibility with adjustable scanning speeds of 4, 10, and 50 ms/pixel. An additional slow-scan mode was incorporated into the standard programming (courtesy of Moor Instruments) to allow for a 1,000 ms/pixel scan rate. The 1,000-ms scan rate was considered the standard for comparison on the basis of the work of Wårdell et al. (12). With the Moor system, a 25 × 25-pixel map was generated over a 2.5 × 2.5-cm area of skin, producing a spatial resolution of 1.0 mm²/pixel. These resolutions were chosen to allow the scanner to sample at a density similar to that possible when using the manual procedures described above and in the literature (2, 3).

The ability of topographical perfusion maps to characterize the spatial pattern of blood flow was evaluated as a function of scanning rate. The Moor scanning system was used for these studies, which involved comparison of perfusion maps created at four speeds: 4, 10, 50, and 1,000 ms/pixel. Ten sequential scans were performed at 4, 10, and 50 ms/pixel, whereas only one scan was collected at the 1,000 ms/pixel rate (25 × 25-pixel map, requiring ~11 min). Perfusion maps were produced at the four different speeds in random order. The ten sequential scans at 4, 10, and 50 ms/pixel were averaged to create a "collapsed" perfusion map, which was compared with the single perfusion map produced at 1,000 ms/pixel.

The day-to-day variability in the spatial pattern of resting SkBF was examined on 3 nonconsecutive days over a 7-day period. Measurements were made after the subjects rested in the supine position for 60 min at an ambient temperature of 27.0 ± 0.1°C. Subjects wore a liquid-perfusion garment that covered the entire skin surface except for the head, hands, feet, and one forearm that was perfused at 34°C during control measurements. One scan per minute for 10 min was performed at a 50 ms/pixel scan rate. After control measurements were made, the suit was perfused with 1°C water for 6 min, and scanning continued on a minute-by-minute basis. Analysis was performed on the first nine control scans and the last four cold scans. The middle 2 × 2-cm area of skin was cropped from the perfusion map for data analysis. The skin just outside the analysis area but within the scanned area was marked with indelible ink to allow for boundary identification on successive days and for cropping purposes during analysis. On subsequent days, the location of the scanner was adjusted to ensure that the 2.5 × 2.5-cm area of skin was identical to that on the first day. Our ability to align scanned areas was evaluated with the use of a program that determined, via an iterative process, the positioning of two perfusion maps that produced the least significant difference between corresponding pixels. Arterial blood pressure was measured once per minute with a noninvasive arm cuff on the opposite arm (Colin STPD monitor). Calculated mean arterial blood pressure {MAP = [(2 × diastolic blood pressure + systolic blood pressure)/3]} was used to estimate cutaneous vascular conductance (CVC).

The SkBF response to increased body temperature and application of LBNP was evaluated in four subjects. Subjects were tested in the supine position, with the lower portion of their body enclosed in a LBNP box (sealed at the iliac crest) and the lower legs immersed in a water bath controlled at 34°C water while the ambient temperature of the room was controlled at 28°C. Laser imaging consisted of four control scans in each of the following conditions: control, normothermia; 40-mmHg LBNP, normothermia; heat stress; and 40-mmHg LBNP plus heat stress. Heat stress involved an increase in the lower leg water bath from 34 to 43°C for 30 min, which raised esophageal temperature by ~0.5°C and elicited significant sweating. Great care was taken to maintain the area of interest during the experiment, which proved difficult during LBNP and which tended to shift subjects slightly toward the box. Ink marks on the forearm skin within the scan area were used to select a region of interest during postprocessing if the limb moved. Arterial blood pressure was measured once per minute on the opposite arm with a standard clinical blood pressure monitor (Colin STBP-780 monitor). MAP was used to calculate CVC. In addition, zero flux was determined during 3 min of brachial artery occlusion, and maximal conductance was determined after 30 min of local skin heating to 43°C by using a servo-controlled infrared lamp. The infrared lamp was turned off during laser Doppler scanning measurements. Data from this protocol are presented as a percentage of maximal CVC.

Data analysis. In the perfusion maps, color gradation was used to illustrate the degree of SkBF (flux) or CVC: dark blue represents low values and red represents high values, respectively. The range of colors chosen to represent the gradation of flux or conductance was maintained across logical comparisons.

	RESULTS

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The resolution of topographical perfusion maps of human skin by using laser Doppler scanning can depend on the rate at which the laser beam travels across the tissue and the reflected light is sampled. Figure 1 is a representative comparison of perfusion maps created from the same skin site (6.25 cm² on forearm) at four different scan speeds (4, 10, 50, and 1,000 ms/pixel). At scan speeds of <1,000 ms/pixel, each image represents the average of 10 consecutive scans. Figure 1 shows that the ability to discriminate variations in SkBF is improved as scan speed decreases from 4 to 1,000 ms/pixel. At the fastest scan rate (4 ms/pixel), the high-flux areas appear as a band or "peninsula" of higher perfusion. As scan rate decreased, this band of flux began to break off into discrete "islands." This transition from a homogeneous band to more discrete islands of high flux is subtle, primarily because the maps at the faster scan speeds represent the average of 10 consecutive scans. The correlation coefficient (r²) for pixel-by-pixel comparisons between images was 0.47 for the comparison of 4 and 1,000 ms/pixel scan speeds. The correlation increased to 0.59 when scan speed was slowed to 10 ms/pixel and increased further to 0.67 at a scan speed of 50 ms/pixel. In subsequent experiments that used the Moor Instruments laser scanner, a 50 ms/pixel setting was selected as the optimal scanning speed, as it combined good resolution with a relatively short scan period (<60 s) for a 6.25-cm² area of skin.

View larger version (122K): [in this window] [in a new window]   Fig. 1.   Comparison of topographical perfusion maps generated from scanning 6.25-cm2 area of skin from ventral forearm at rates of 4, 10, 50, and 1,000 ms/pixel. Perfusion maps scanned at rates <1,000 ms/pixel represent collapsed mean of 10 consecutive scans. Color gradation of laser Doppler voltage output at each pixel from highest (black) to lowest (blue) is shown.

After 60 min of supine rest in a thermoneutral environment, consecutive CVC perfusion maps showed a coefficient of variation of 11 ± 1% (range 7-13%). The variability in the data represents the combined effect of temporal and spatial variations. Consecutive resting scans were collapsed pixel by pixel to provide a basis for detection of significant changes in the spatial distribution pattern during various treatments. Under strictly controlled thermal conditions, the spatial pattern of cutaneous blood flow in the forearm at rest appeared highly reproducible. The coefficient of variation comparing the collapsed image on 3 separate days averaged 6 ± 3% (range 2-9%). When the consecutive images are collapsed, the contribution of temporal variability to the calculated coefficient of variation is reduced. Figure 2 illustrates a representative collapsed perfusion map from one subject on 3 separate days. During the cold stimulus, MAP rose from 86 ± 5 to 98 ± 5 mmHg, and mean CVC decreased by 37 ± 2% (Fig. 2). Compared with the collapsed resting scan, 89 ± 2% of the pixels decreased in response to cold, 9 ± 2% of the pixels were unchanged (changed <1 SE), and 2 ± 1% of the pixels showed an increase in conductance. Figure 2 illustrates that the pattern of blood flow response to a uniform cold stimulus was quite reproducible.

View larger version (149K): [in this window] [in a new window]   Fig. 2.   Representative data from 1 subject comparing topographical perfusion maps of 6.25-cm2 area of skin from ventral forearm scanned at 50 ms/pixel on 3 nonconsecutive days within a 7-day period. Each perfusion map represents mean of 9 consecutive scans under control conditions and 4 scans during cold stress.

The usefulness of topographical perfusion mapping in examining both the spatial pattern of blood flow in the skin and its control is shown in Fig. 3. The perfusion map in Fig. 3 was generated by movement of a standard single-site laser Doppler flow probe, over a 1-cm² area of skin, in a rectilinear fashion, by using a semiautomated mapping system. After creation of the perfusion map, six skin sites were selected (4 high-flux and 2 low-flux sites). Each site showed a stable baseline flux before application of LBNP, indicating that the changes in SkBF during LBNP were not due to an artifact associated with data collection (i.e., movement artifact). Three of the four high-flux sites demonstrated a reduction in SkBF at the onset of 40-mmHg LBNP. The reduction in SkBF was sustained for the entire 10-min LBNP period in two sites but tended to recover at one site. One high-flux skin site increased SkBF during LBNP, although this increase was not sustained. In the low-flux skin sites, SkBF was unchanged or increased during application of LBNP. The increase in SkBF at these sites was not sustained and returned toward control levels toward the end of 10 min of 40-mmHg LBNP.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Topographical perfusion map created by computer-controlled stereotaxic placement of needle-type laser Doppler probe over 10 × 10 mm grid on ventral surface of forearm. Six skin sites were selected from map and sampled 1/min (10-s sample time) during 5 min of rest and 10 min of 40-mmHg lower body negative pressure (LBNP). Note variability in response of skin vasculature to baroreceptor unloading induced by LBNP. LDV, laser Doppler velocimetry.

In a separate series of experiments, we used laser Doppler scanning to evaluate reflex control of SkBF during LBNP. In normothermic conditions, at an ambient temperature of 28°C, resting CVC averaged 8.2 ± 2.9% of maximal CVC. In response to 40-mmHg LBNP, the mean CVC was unchanged at 8.0 ± 3.7% of maximal CVC. The pattern of response was not uniform, and frequency analysis indicated that 47 ± 5% of the pixels showed a significant reduction in CVC (changed >1 SE of the resting value), 28 ± 2% of the skin area was unaffected, and the remaining 26 ± 5% of the pixels showed some increase (>1 SE) in CVC. In response to 30 min of lower leg immersion, 94 ± 5% of the pixels showed an increase in CVC (to 22.0 ± 6.2% of maximal), whereas 5 ± 4% were unchanged. During the increase in body core temperature, application of LBNP produced a reduction in the mean perfusion map CVC to 14.2 ± 1.5% of maximal. Within the perfusion map, 54 ± 19% of the pixels showed a decrease in CVC, 21 ± 8% of the pixels were unresponsive, and 26 ± 11% of the pixels showed an increase in CVC during application of 40-mmHg LBNP. Figure 4 illustrates the complex pattern of response of SkBF during baroreceptor unloading and passive body heating.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Representative data from 1 subject showing spatial pattern of skin blood flow response to baroreceptor unloading with application of 40-mmHg LBNP and passive body heating. Data are presented as %maximal cutaneous vascular conductance (CVC) determined after 30 min of local heating to skin temperature of 43°C. Arrows represent 2 original topographical perfusion maps used to produce subtraction map. Subtraction maps illustrate area and magnitude of response in 2.5 × 2.5-cm area of forearm skin. Color gradation of calculated CVC presented as %maximal value obtained after 30 min of local heating at each pixel from highest (black) to lowest (blue) is shown. CVC, change in CVC.

	DISCUSSION

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Topographical perfusion mapping provides a unique opportunity to evaluate reflex control of SkBF. When posture and the ambient temperature are strictly controlled, the spatial pattern of blood flow in the skin is highly reproducible from day to day. By the production of serial topographical perfusion maps under control conditions and collapse of these maps into a single perfusion map, strict criteria can be established from which to judge subsequent changes in the cutaneous blood flow. Thus it is possible to quantify both the magnitude and direction of changes in SkBF in a given area of skin. With the use of these techniques, the changes in CVC during thermal stimuli (cooling skin or increasing body core temperature) were judged consistent (>95% of the pixels responded in an expected manner) and reproducible. An important finding of these preliminary studies was that the changes in SkBF during baroreceptor unloading were not consistent, and the range of responses varied widely within an imaged skin site and between individuals. During baroreceptor unloading, the SkBF response was characterized by areas of vasoconstriction and vasodilation. These data were supported by observations from more conventional laser Doppler single-spot recording techniques (Fig. 3).

The purpose of this report was to characterize the usefulness of topographical perfusion mapping in evaluating reflex control of SkBF. Some insight into methodological considerations that were helpful in acquiring reproducible data is provided. Under the present experimental conditions, the spatial pattern of SkBF could be identified by using a wide range of scanning speeds (4, 10, 50, and 1,000 ms/pixel). Yet the scanning speed of 50 ms/pixel provided an optimal combination of speed and resolution for imaging relatively small areas of skin. This conclusion is based on the improvement of the correlation coefficient (pixel by pixel) between the slow (1,000 ms/pixel) scan speed and the mean (collapsed) perfusion maps as the scan speed moved from 4 to 50 ms/pixel. In addition, under less-ideal conditions (i.e., lower ambient temperature), when resting SkBF is reduced, slower scan speeds should help to improve spatial resolution by increasing the signal-to-noise ratio. The technique of collapsing consecutive scans to create a mean resting perfusion map and a pixel-by-pixel error term provides an objective method to analyze subsequent perfusion maps in a quantitative manner. With the use of surface marks (natural or ink), it is possible to precisely align and crop areas of skin for subsequent analysis. The ability to scan the same area of skin on different days was judged sufficiently accurate based on the results of our computer program, which aligned perfusion maps after they were cropped. The program aligned perfusion maps such that it produced the least significant difference between the corresponding pixels. When collapsed perfusion maps from different days were compared, the maximal shift of any two perfusion maps was never greater than one or two pixels, indicating that our visual cropping technique resulted in perfusion maps with very similar profiles of skin perfusion. Many periodic events (i.e., vasomotion and respiration) can influence blood flow and produce "false" variations in the spatial pattern. However, the fact that the areas of high and low flux persisted throughout 10 consecutive scans suggests that the variations are not artifacts of some periodic event. In addition, the collapsing of 10 consecutive scans helps to smooth some of these false values from the data. Also, during scanning, artifacts generally appear as stripes along the sample axis and not as discrete areas of low or high perfusion.

During the response to thermal stimuli, the spatial pattern of SkBF response was consistent with the reported literature and was highly reproducible. The cutaneous vascular response to thermal stimuli consists of both veno- and vasomotor adjustments (8). As such, cold stress will result in a reduction in skin blood volume due to a reduction in arteriolar inflow and increased venoconstriction. In this respect, we speculate that changes in the laser Doppler flow output during vasoconstriction induced by cooling represent changes in the scattering volume under the probe and possibly the scattering properties of the tissue (i.e., path length). It is likely that this feature of the vascular response to thermal stimuli biases the output in favor of detection of consistent changes in SkBF by laser Doppler velocimetry.

Baroreceptor unloading during application of LBNP is thought to result in cutaneous vasoconstriction, yet recent data have revived some of the earlier controversy on this topic (10, 11). In addition, LBNP does not produce a consistent effect on the venous circulation (7). Using topographical perfusion mapping, we noted that only 47% of the skin area showed a significant decrease in blood flow during LBNP. A small area (~28%) of skin did not respond to LBNP with any significant change in blood flow, yet 22% of the skin area showed some degree of vasodilation (increased CVC >1 SE of control). These observations were consistent with measurements made with the use of a more conventional single-spot recording laser Doppler velocimetry unit (Fig. 3). In addition, preliminary data indicate that laser Doppler velocimetry data are inconsistent with simultaneous measurements of venous occlusion plethysmography measurements of forearm SkBF during LBNP (6). These observations provide a puzzling dichotomy of the role of baroreceptors in reflex control of SkBF. It is possible that the shallow sample depth of the laser Doppler system, in combination with the underlying microvascular architecture of the skin, contributes to the observed responses to LBNP. In addition, the impact of increased cutaneous sympathetic vasoconstrictor activity may be more upstream (deeper) in the tissue. On the basis of current dogma, the cutaneous vasculature is thought to be innervated by sympathetic vasoconstrictor and vasodilator systems, and the level of SkBF is set by the competition of these two systems. During cold stress, the large fraction of skin area responded with a decrease in flow; thus it is likely that most cutaneous vessels are innervated with sympathetic vasoconstrictor nerves. The large increase in flow during passive heating indicates a uniform sympathetic vasodilator system. The inability of baroreceptor unloading to produce a uniform reduction in blood flow leaves room for several alternative hypotheses. One such hypothesis, first proposed by Braverman et al. (2), is that two populations of microvessels exist in the skin: one primarily responsible for tissue perfusion, and one primarily responsible for temperature regulation. The topographical perfusion mapping technique should provide a useful tool for evaluating the controversy surrounding baroreflex control of SkBF.

In summary, laser Doppler imaging can provide quantitative and reproducible information about the spatial distribution of cutaneous blood flow. Reflex control of SkBF during thermal stimuli is uniform across most of the measured area, whereas responses to baroreceptor unloading vary from site to site within a given area of skin. Under thermoneutral conditions and with appropriate control for posture, topographical perfusion mapping will provide valuable insight into the function of the cutaneous vasculature; however, caution must still be used in evaluating baroreflex responses.