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Reflection Spectroscopy of Analgesized Skin
Microvascular Research 62, 392– 400 (2001) doi:10.1006/mvre.2001.2358, available online at http://www.idealibrary.com on
Reflection Spectroscopy of Analgesized Skin Erik Ha¨ggblad, Marcus Larsson, Mikael Arildsson, Tomas Stro¨mberg, and E. Go¨ran Salerud Department of Biomedical Engineering, University Hospital, Linko¨pings Universitet, SE-581 85 Linko¨ping, Sweden Received January 18, 2001; published online September 12, 2001
Analgesized skin, when subjected to heat stimuli, responds by increasing skin perfusion. This response does not originate from increased perfusion in superficial capillaries, but rather in the deeper lying vessels. The aim of this study was to assess changes in blood chromophore content, measured by reflection spectroscopy, in relation to the perfusion increase, especially regarding the chromophores oxyhemoglobin and deoxyhemoglobin. Eleven normal subjects were treated with analgesic cream (EMLA) and placebo for 20, 40, 60, 120, and 180 min. Individual reactions to local heating were classified as responses if the change in reflection data or the change in perfusion, as measured by laser Doppler blood flowmetry, exceeded 2 standard deviations of normal variation. The increase in blood perfusion or in blood content gave rise to an increased absorption, interpreted as an increase due mainly to the chromophore oxyhemoglobin. The number of responses increased with increased treatment time for EMLA-treated areas. In general, there was a good agreement between both methods; 44 of 55 classifications coincided for the two methods used. In conclusion, analgesized forearm skin, which had been exposed to local heating, responded with an elevated perfusion consisting of oxygenated blood. This strengthens the hypothesis that the flow increase occurs through dilatation of larger deeper lying skin vessels and not in the capillaries. © 2001 Academic Press
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Key Words: spectroscopy; laser Doppler flowmetry; EMLA; hemoglobin; analgesia; heat stimuli; skin microcirculation.
INTRODUCTION The eye, together with the perceptual system, is perhaps the most sensitive biological sensor, able to perform discrimination analysis even at low differences, in both color intensity and saturation. Despite its excellent sensitivity, visual classification shows a large intra- and interindividual variability and is, therefore, subjective in its nature (Andersen et al., 1991a; Bjerring and Andersen, 1987; Takiwaki and Serup, 1995). In irritancy tests and other dermatological applications in which graded information is necessary, a more objective comparison has to be performed. Reflectance spectroscopy (Andersen and Bjerring, 1990a; Bjerring and Andersen, 1987) is, therefore, used in many areas such as skin patch testing (Andersen et al., 1991b) and evaluation of UV-induced erythemas (Andersen et al., 1991a; Kollias and Baqer, 1988). In superficial skin, melanin and hemoglobin are the two dominant chromophores, having considerable impact on the reflection spectra (Andersen and Bjerring, 1990b). In human blood, hemoglobin appears in dif-
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Reflection Spectroscopy of Analgesized Skin
ferent forms, of which oxyhemoglobin (OH), i.e., hemoglobin containing bound oxygen, and deoxyhemoglobin (DOH), i.e., not containing any bound oxygen, are the most common (Dorland, 1988). The different hemoglobin forms exhibit characteristic absorption spectra that are distinguishable (Anderson and Parrish, 1982). Therefore, any changes in hemoglobin concentration, or the composition of the different forms, will affect the captured spectra according to the change. In a previous investigation, skin perfusion together with the physiological consequences of local skin analgesia and, in particular, the perfusion response to different stimuli in the microcirculation were investigated (Arildsson et al., 2000b). It was postulated that the observed increase in perfusion occurs in deeper lying vessels, since the number of physiologically active capillaries decreased (Arildsson et al., 2000a). By applying reflection spectroscopy in a similar set-up, the relative change of oxygenation in these vessels can be determined. The aim was to assess changes in reflection spectra associated with local heating of analgesized skin in relation to skin chromophores and skin perfusion.
MATERIAL AND METHODS Subjects This study comprised 12 Caucasian subjects (6 male and 6 female, ages 21–32 years), who gave their informed consent. All subjects were healthy, with no prescription medication and no visible skin disorders and did not use any form of nicotine. Ingestion of food or caffeine, less than 1 h prior to the start of the examination, was not allowed. All measurements were made with the subject sitting comfortably in a chair in an upright position.
The Dermal Sensitivity Tester The dermal sensitivity tester (Desensor; Cenova AB, Sweden), a computerized robotic system, delivers
standardized preset skin stimuli to a well-defined area of the skin (Arildsson et al., 2000b). In this study, the Desensor was used to deliver a local heat stimulus to a predefined and analgesized skin site on the lower forearm. The stimulus was delivered using a copper probe ( ⫽ 12 mm, temperature 45°C) in contact with the skin surface for 9 s.
Reflection Spectroscopy Each chromophore affects the reflected spectra differently, leaving a unique and characteristic fingerprint. Reflection spectroscopy (RS) is therefore a suitable method for analyzing the chromophore content in superficial tissue (Buckley and Grum, 1961; Dawson et al., 1980). Blood flow redistribution, between skin plexa, or changes in blood composition alter the amount of chromophores and hence the recorded reflectance spectra of the tissue. The spectrometer (S-1000; Ocean Optics, U.S.A.) consists of a grating and an optical system that projects an image of the light spectrum onto a CCD array. The data stored in the CCD are then transferred as an analog signal and sampled by a computer. The illuminating light source (LS-1; Ocean Optics) was a stabilized tungsten halogen lamp (99.7% stabilization within 5 min and 100% in 40 min). To ensure stable light conditions, a warm-up period of at least 40 min was allowed prior to each experiment. A fiber-optic probe (FCR-7IR200-2; Ocean Optics) guides the light to the area under study, through six fibers. The reflected light is collected and guided back to the spectrometer through a single fiber encircled by the illuminating fibers. The spectrometer, in combination with the light source, used in this study operates in the spectral range from 500 to 1000 nm. During each measurement session three different, consecutive, spectroscopic measurements were carried out to minimize the effects of any spatial differences in the skin color and microcirculation. These spectroscopic measurements were made by applying the probe, firmly but with mild pressure, perpendicular to the skin surface so that contact was
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FIG. 1. Typical reflectance spectra from: (a) a piece of polyacetal plastic (reference spectrum), (b) EMLA-treated tissue without heat provocation, and (c) EMLA-treated tissue after heat provocation; (b) and (c) are normalized against the maximum intensity. (d) Relative absorption spectrum (c– b; see Material and Methods).
made with it. The time taken for each single measurement was not more than a couple of seconds, with a total time of less than a minute for all three measurements. To control the inherent characteristics of the spectrometer and light source, a reference spectrum was collected (Fig. 1a). The reference spectrum of the equipment was obtained using a piece of reflective polyacetal plastic.
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Laser Doppler Perfusion Imaging Coherent laser light, interacting with moving red blood cells in tissue according to the Doppler principle, can be used to estimate the tissue perfusion. This technique is generally referred to as laser Doppler blood flowmetry (LDF) (Stern, 1975). Laser Doppler perfusion imaging (LDPI) (Essex and Byrne, 1991; Wårdell et al., 1993, 1994) uses the LDF technique in
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order to generate a tissue perfusion map by moving a freely impinging laser beam across the surface of interest. The investigations in the present study were made using a LDPI system (PIM1.0; Lisca Development AB, Sweden). All recorded images were of size 10 ⫻ 10 measurement points, covering approximately 20 ⫻ 20 mm.
EMLA and Placebo Cream Astra Pain Control, Sweden, provided the placebo cream and analgesic cream (EMLA) used in this study. The EMLA (eutectic mixture of local analgesics) (Juhlin and Evers, 1990; Nielsen et al., 1992) cream contains the local analgesic substances lidocaine (2.5%) and prilocaine (2.5%), while in the placebo cream these substances have been substituted by coconut oil. The pH of the two creams is the same. Approximately 2.5 g of cream was applied to the test area and was fixated using a transparent plastic dressing (Tegaderm; 3M, Canada).
Experimental Set-up This study was approved by the Ethics Committee for Human Research at Linko¨ping University Hospital, Sweden. The subject was acclimatized for 30 min prior to each experiment. The volar sides of the lower forearms were randomly assigned a treatment, either EMLA or placebo cream. Each arm was subdivided into five test areas (10 cm 2) and randomly assigned treatment times of 20, 40, 60, 120, and 180 min (Fig. 2a). Corresponding test areas, on each arm, were assigned the same treatment time. For every test area, three measurement sessions were performed: before treatment, after treatment, and 6 min after delivered heat stimulus (Fig. 2b). The effect of heat stimulation on placebo-treated skin has previously been reported and it has been shown that the placebo treatment did not affect the LDPI recordings (Arildsson et al., 2000b). LDPI images were, therefore, only captured on the EMLA-treated arm.
FIG. 2. (a) Schematic illustration of treatment areas on the volar sides of the lower forearms. (b) Example of the spectroscopic measurement within the test area. x denotes initial measurements, y denotes measurements after EMLA treatment, z denotes measurements outside of the heat-stimulated area, and r denotes measurements inside of the heat-stimulated area.
Initially, spectroscopic measurements (x) and LDPI images were performed on untreated skin, after which the creams were applied to the test areas. All applications, except for the 40-min treatment, were carried out in one session, with EMLA and placebo separated by a period of 12 min, allowing a controlled and comparable environment for all measurements. The application of EMLA and placebo for the 40-min treatment was made 15 min prior to the 120-min measurement session. After the predetermined treatment time, the plastic dressing was removed and the cream gently wiped off with a cloth, and a second set of RS ( y) and LDPI data was collected. The heat stimulus was then delivered by the Desensor to the center of the treated test area. A final session with collection of RS and LDPI data was performed 6 min after the heat stimulus. During this session, two measurements were made with the spectrometer, one in the unstimulated area (z) and one in
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the stimulated area (r), to conclude the effect of heat stimulus and EMLA treatment on the microcirculation. During all LDPI measurements, the subject’s arm was fixed, using a vacuum pillow, in order to prevent artifacts due to arm movement.
Data Analysis In order to minimize the illuminating intensity variances in the RS measurements, all spectra were normalized against the maximum intensity at a wavelength outside the area of interest (635– 642 nm). To minimize the spatial variations depending on skin site, the average of three normalized spectra were calculated for each treatment time and measurement (r and z, Fig. 2b). As proposed by Kollias and Baqer (1988), the change in absorption (calculated from differential measurements) reflects the change in chromophore content. Thus, to assess the change in light absorption in the skin due to heat stimuli, the difference between the logarithm of inverse reflectance for the average spectra was calculated. This ensures that chromophores that do not change as a result of the heat stimulus are subtracted and do not interfere with the calculations. The plotted change contains the characteristic absorption pattern of OH (Fig. 1d). By fitting tabulated OH and DOH absorption spectra to the change in absorption spectra, using a linear regression model, the difference in chromophore content was analyzed (Fig. 3a). The absorption data for OH and DOH used in this study have been acquired from tabulated data for the molar extinction coefficients from Prahl (1999) (Fig. 3b). To compare the results from the RS with visual assessment and LDPI, the sum of the OH and DOH coefficients was used. In order to classify areas as responding or not, the normal variations in the spectroscopic measurement data of untreated skin were studied by evaluating the three central test areas. The sum of the OH and DOH coefficients for adjacent skin areas (2–3 and 3– 4, Fig. 2a) was compared according to the same protocol as for the heat-stimulated areas, resulting in 44 differences. Two standard deviations (SD) were arbitrarily chosen as the classification threshold. The average LDPI skin perfusion before treatment,
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FIG. 3. (a) The absorption spectrum for 180 min EMLA treatment of a typical subject. Also shown is the sum of deoxy- and oxyhemoglobin with the respective coefficients of 3.56 ⫻ 10 ⫺2 and 1.10 ⫻ 10 ⫺2 derived from curvefitting. The normalized absorption spectra for deoxy- and oxyhemoglobin are shown in (b), based on data for the extinction coefficient from Prahl (1999).
after treatment, and after provocation was estimated by calculating the mean value of the corresponding 10 ⫻ 10 measurement points LDPI images. To classify an area as responding or nonresponding, the normal perfusion variation was evaluated in the same way as
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TABLE 1 Relative Amounts of Oxyhemoglobin (OH) and Deoxyhemoglobin (DOH) in the Heat-Stimulated Area Compared to the Unstimulated Adjacent Skin Site (see Material and Methods) Placebo—180 min
EMLA—180 min
Subject
OH
DOH
Sum
OH
DOH
Sum
⌬Perf
1 2 3 4 5 6 7 8 9 10 11 Average SD
0.0016 ⫺0.0012 0.0032 0.0009 0.0048 0.0095 0.0028 0.0058 0.0034 0.0007 ⫺0.0008 0.0028 0.0031
⫺0.0038 ⫺0.0018 ⫺0.0010 0.0013 0.0000 0.0030 ⫺0.0030 0.0073 0.0006 ⫺0.0048 ⫺0.0023 ⫺0.0004 0.0034
⫺0.0022 ⫺0.0030 0.0022 0.0022 0.0049 0.0125 ⫺0.0002 0.0131 0.0040 ⫺0.0040 ⫺0.0031 0.0024 0.0059
0.0303 0.0356 0.0358 0.0124 0.0318 0.0143 0.0305 0.0386 0.0081 0.0261 0.0180 0.0256 0.0106
0.0051 0.0110 0.0065 0.0014 0.0031 0.0026 0.0007 0.0097 ⫺0.0046 0.0056 0.0006 0.0038 0.0044
0.0353 0.0466 0.0423 0.0138 0.0350 0.0168 0.0312 0.0483 0.0035 0.0317 0.0186 0.0294 0.0144
75 103 124 0 158 ⫺21 56 27 ⫺14 51 ⫺4 50.5 59.7
Note. For a comparison the change in perfusion (in arbitrary units), as measured by laser Doppler perfusion imaging, can be seen in the rightmost column.
for RS, i.e., 2 SD were chosen as a classification threshold.
RESULTS One female subject was excluded due to equipment failure. Among the remaining 11 subjects, a visually perceived distinguishable red spot appeared after the heat stimulus was applied. The red spot was similar in shape and size to the probe contact area used to deliver the heat provocation. In EMLA-treated areas, this red mark lasted for a period of at least 8 min. The red spot was also seen in some placebo-treated areas, but generally disappeared within a minute after cessation of provocation. The reflectance spectra of the red spot showed a decreased reflection for wavelengths shorter than 600 nm (Fig. 1c). This decrease in reflection was not found in areas treated with EMLA but without heat stimuli (Fig. 1b). The increase in absorption, calculated as the difference between areas without and with heat stimulation, showed characteristic wavelength peaks at 542 and 577 nm (Fig. 1d). This increased absorption
was identified as being due to an increase in hemoglobin and, in particular, oxygenated hemoglobin. The relative change of OH and DOH, estimated by curve fitting, is presented for the 3-h treatment time in Table 1. Among the nine RS responses in EMLAtreated skin, OH was the predominant chromophore. The goodness of fit for these responses ranged from 0.95 to 0.99. In general for all responses in EMLAtreated skin, the increased absorption consisted of OH (Table 1). This could not be shown for the placebo responses. The RS difference (M ⫾ SD) between test areas 2 and 3 was 0.0054 ⫾ 0.0109, with a calculated 95% confidence interval of (⫺0.0012; 0.012), and between test areas 3 and 4 the difference was 0.0061 ⫾ 0.0067, giving a 95% confidence interval of (0.0021; 0.010). The threshold for a significant response in RS was 0.015 (2 SD of normal skin variation). The LDPI difference (M ⫾ SD) between test areas 2 and 3 was ⫺4.9 ⫾ 4.5, with a 95% confidence interval of (⫺14; 4.0), and between test areas 3 and 4 the difference was 2.0 ⫾ 4.3 with a 95% confidence interval of (⫺6.6; 11). The threshold for a significant LDPI response was 29 arbitrary units (2 SD of normal variation). With the threshold chosen, the absorption results
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only twice (Fig. 4a). For a comparison between number of responses classified by RS and LDPI, respectively, see Fig. 4b; for individual responses see Table 2. There were 19 LDPI and 20 RS responses. The methods agreed for 14 responses.
DISCUSSION In summary, this study has examined the use of reflectance spectroscopy, in conjunction with laser Doppler perfusion imaging, in a provocation model consisting of a heat load delivered to an analgesized skin site on the lower forearm. Skin sites exhibiting an elevated perfusion were associated with an increase of the blood chromophore content, of which the oxyhemoglobin change was sixfold greater than that of deoxyhemoglobin. The observed reflection spectra were found to have good short-term stability. Thus, only reflection spectra with a short-term lag between measurement points were analyzed, i.e., those of the treated and heated area were compared to those of the treated but not heated area. The spectra obtained before and after treatment, inspected only by eye, confirmed that no major changes in blood content had occurred during TABLE 2 Distribution of Responding Areas Classified Using Reflectance Spectroscopy/Laser Doppler Perfusion Imaging FIG. 4. (a) The number of responses, classified by reflection spectroscopy, for the different treatment times in EMLA- and placebo-treated areas, respectively. A response was defined as an increase in the relative amount of oxy- and deoxyhemoglobin exceeding 2 standard deviations of the variability of normal skin. (b) The number of responses classified by reflectance spectroscopy and LDPI, where LDPI response was defined as a change in perfusion exceeding 2 standard deviations of the variability found in normal skin.
demonstrated an increasing number of RS responses with increasing treatment time for EMLA-treated areas (Fig. 4a). The placebo-treated areas responded
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EMLA treatment time Subject
20 min
40 min
60 min
120 min
180 min
1 2 3 4 5 6 7 8 9 10 11 Total
0/0 0/0 0/0 0/0 0/1 0/0 1/0 0/0 0/0 0/0 0/0 1/1
0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/1 0/0 1/1 0/0 2/2
0/0 1/1 0/0 0/0 1/1 0/0 0/1 0/0 0/1 0/0 0/0 2/4
0/0 1/0 1/0 0/0 1/1 1/1 1/1 0/1 0/0 1/1 0/1 6/6
1/1 1/1 1/1 0/0 1/1 1/0 1/1 1/0 0/0 1/1 1/0 9/6
Note. Responses are denoted by a “1”.
Reflection Spectroscopy of Analgesized Skin
the treatment procedure. However, the results obtained cannot exclude the possibility that the blood content and the inherent chromophores of the nonheated adjacent area were changed due to the heat stimuli. This is, however, not plausible, since the reaction to the heat stimulus, observed visually, consisted of a clearly defined red spot. Moreover, it has been shown previously (Arildsson et al., 2000b) that elevated perfusion occurs in relation to this red spot. Curve fitting of recorded absorption spectra was performed only on the hemoglobin chromophores. The reason for not including melanin, a dominant chromophore in superficial skin, is that our results are calculations of difference spectra and melanin remains constant during the short recording time (even during UVB stimulation (Andersen et al., 1991a), melanin changes are slow). Tabulated values were used (Prahl, 1999) for the oxy- and deoxyhemoglobin reference spectra. It is possible that measurement with our equipment would have given slightly different reference spectra. Nevertheless, the goodness of the fit ranged from 0.95 to 0.99. Classification of an area as being a response or not was not performed using a graded scale. The threshold to pass, in order to be classified as a response, was estimated as 2 SD of the natural variability between three central sites located on the forearm. Although both the RS and the LDPI limits were obtained using the same methodology, some differences exist. In the LDPI data, no systematic differences were found between untreated skin sites, although the site closest to the wrist showed, in general, a higher perfusion. The sum of OH and DOH showed a similar pattern, with higher values closer to the wrist (in declining order: sites 2, 3, and 4). By subtracting values from the same site and using a randomized set-up, systematic baseline differences were avoided. In previous studies (Arildsson et al., 2000a,b), using the EMLA and heat provocation model, a large and time-dependent perfusion increase was documented. RS has been used with the same model and compared to LDPI in order to strengthen the perfusion regulation hypothesis. Comparing RS and LDPI measurements from the same skin sites, an 80% agreement was found (44/55). Of the LDPI responses, 74% also exhib-
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ited an RS response (14/19). However, it cannot be concluded that RS measures the same vascular bed as LDPI from these data alone, since the number of active capillaries also increases after heat provocation. The number of active capillaries almost halves due to the EMLA treatment (Arildsson et al., 2000a). However, the absorption spectra before and after treatment, as well as LDPI measurements (Arildsson et al., 2000b), showed that there was no major change in blood content due to EMLA treatment. Hence, the RS and LDPI data track each other and we can assume that they see essentially the same vascular bed. EMLA treatment does not inherently alter the blood volume, but a small, induced heat load results in skin reddening in the area in contact with the heat probe. In a few subjects, a red spot was seen in the placebotreated skin area. In contrast to the long-lasting response in EMLA-treated skin (also found in two placebo areas), this response vanished quickly. A possible explanation is that the placebo response involves primary thermoregulatory responses, while in EMLAtreated skin tissue, the autoregulatory control is affected and brings about a sustained inflow to the tissue being investigated. The main purpose of capillary perfusion is to support the living tissue according to its metabolic demands (Guyton and Hall, 1996). In the present study, the change in DOH was not statistically significant, while the change in OH was obvious, indicating that the metabolic rate is unchanged. This implies that the observed OH is a secondary effect emanating from an increase in deeper lying perfusion plexas. When examining the LDPI response versus capillary appearance, in the same provocation model, it was concluded that the vascular bed involved in the perfusion response was not that of the superficial capillaries, but rather vessels deeper into the skin (Arildsson et al., 2000a). Data from the previous studies are, however, still compatible with the hypothesis that a substantial amount of the blood flow was due to flow in deeper lying capillary vessels. However, it seems unlikely that the deeper lying capillaries should respond differently. The RS signal from such blood is likely to be a mixture of oxygenated precapillary blood and deoxygenated postcap-
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illary blood. However, the main finding of this study is that the increased blood flow is almost exclusively composed of oxygenated blood. This strengthens the hypothesis from Arildsson et al. (2000a) that superficial capillaries are not involved and, in addition, shows that neither are deeper lying capillaries involved. The blood flow can thus be characterized as a shunt flow consisting of oxygenated blood. Similar results demonstrating the reddening of the skin and increase in OH have been reported previously by other authors (Andersen et al., 1991a,b; Kollias and Baqer, 1988), though it should be noted that they used different provocation models. In conclusion, local heating of analgesized forearm skin was associated with increased blood flow consisting of oxygenated blood. This supports the hypothesis that the flow increase occurs through dilatation of larger deeper lying skin vessels and not in the capillaries.
ACKNOWLEDGMENTS The Swedish National Center of Excellence for Non-invasive Medical Measurements (NIMED) supported this study. We also acknowledge ASTRA Pain Control, Sweden, for their valuable cooperation.
REFERENCES Andersen, P. H., Abrams, K., Bjerring, P., and Maibach, H. (1991a). A time-correlation study of ultraviolet B-induced erythema measured by reflectance spectroscopy and laser Doppler flowmetry. Photodermatol. Photoimmunol. Photomed. 8, 123–128. Andersen, P. H., and Bjerring, P. (1990a). Noninvasive computerized analysis of skin chromophores in vivo by reflectance spectroscopy. Photodermatol. Photoimmunol. Photomed. 7, 249 –257. Andersen, P. H., and Bjerring, P. (1990b). Spectral reflectance of human skin in vivo. Photodermatol. Photoimmunol. Photomed. 7, 5–12. Andersen, P. H., Nangia, A., Bjerring, P., and Maibach, H. I. (1991b). Chemical and pharmacologic skin irritation in man. A reflectance spectroscopic study. Contact Dermatitis 25, 283–289.
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Anderson, R. R., and Parrish, J. A. (1982). Optical properties of human skin. In “The Science of Photomedicine” (J. D. Regan and J. A. Parrish, Eds.), pp. 147–194. Plenum, New York. Arildsson, M., Asker, C. L., Salerud, E. G., and Stro¨mberg, T. (2000a). Skin capillary appearance and skin microvascular perfusion due to topical application of analgesia cream. Microvasc. Res. 59, 14 –23. Arildsson, M., Nilsson, G. E., and Stro¨mberg, T. (2000b). Effects on skin blood flow by provocation during local analgesia. Microvasc. Res. 59, 122–130. Bjerring, P., and Andersen, P. H. (1987). Skin reflectance spectrophotometry. Photodermatology 4, 167–171. Buckley, W. R., and Grum, F. (1961). Reflection spectrophotometry. Arch. Dermatol. 83, 249 –261. Dawson, J. B., Barker, D. J., Ellis, D. J., Grassam, E., Cotterill, J. A., Fisher, G. W., and Feather, J. W. (1980). A theoretical and experimental study of light absorption and scattering by in vivo skin. Phys. Med. Biol. 25, 695–709. Dorland, W. A. N. (1988). “Dorland’s Illustrated Medical Dictionary.” Saunders, Philadelphia. Essex, T. J., and Byrne, P. O. (1991). A laser Doppler scanner for imaging blood flow in skin. J. Biomed. Eng. 13, 189 –194. Guyton, A. C., and Hall, J. E. (1996). “Textbook of Medical Physiology.” Saunders, Philadelphia. Juhlin, L., and Evers, H. (1990). Emla: A new topical anesthetic. Adv. Dermatol. 5, 75–91. Kollias, N., and Baqer, A. H. (1988). Quantitative assessment of uv-induced pigmentation and erythema. Photodermatology 5, 53– 60. Nielsen, J. C., Arendt-Nielsen, L., Bjerring, P., and Svensson, P. (1992). The analgesic effect of Emla cream on facial skin. Quantitative evaluation using argon laser stimulation. Acta Dermatol. Venereol. 72, 281–284. Prahl, S. (1999). Optical absorption of hemoglobin. Oregon Medical Laser Center, http://omlc.ogi.edu/spectra/hemoglobin/. Stern, M. D. (1975). In vivo evaluation of microcirculation by coherent light scattering. Nature 254, 56 –58. Takiwaki, H., and Serup, J. (1995). Measurement of erythema and melanin indices. In “Handbook of Non-invasive Methods and the Skin” (J. Serup and G. B. E. Jemec, Eds.), pp. 377–384. CRC Press, Boca Raton, FL. Wårdell, K., Braverman, I. M., Silverman, D. G., and Nilsson, G. E. (1994). Spatial heterogeneity in normal skin perfusion recorded with laser Doppler imaging and flowmetry. Microvasc. Res. 48, 26 –38. Wårdell, K., Jakobsson, A., and Nilsson, G. E. (1993). Laser Doppler perfusion imaging by dynamic light scattering. IEEE Trans. Biomed. Eng. 40, 309 –316.
Reflection Spectroscopy of Analgesized Skin Erik Ha¨ggblad, Marcus Larsson, Mikael Arildsson, Tomas Stro¨mberg, and E. Go¨ran Salerud Department of Biomedical Engineering, University Hospital, Linko¨pings Universitet, SE-581 85 Linko¨ping, Sweden Received January 18, 2001; published online September 12, 2001
Analgesized skin, when subjected to heat stimuli, responds by increasing skin perfusion. This response does not originate from increased perfusion in superficial capillaries, but rather in the deeper lying vessels. The aim of this study was to assess changes in blood chromophore content, measured by reflection spectroscopy, in relation to the perfusion increase, especially regarding the chromophores oxyhemoglobin and deoxyhemoglobin. Eleven normal subjects were treated with analgesic cream (EMLA) and placebo for 20, 40, 60, 120, and 180 min. Individual reactions to local heating were classified as responses if the change in reflection data or the change in perfusion, as measured by laser Doppler blood flowmetry, exceeded 2 standard deviations of normal variation. The increase in blood perfusion or in blood content gave rise to an increased absorption, interpreted as an increase due mainly to the chromophore oxyhemoglobin. The number of responses increased with increased treatment time for EMLA-treated areas. In general, there was a good agreement between both methods; 44 of 55 classifications coincided for the two methods used. In conclusion, analgesized forearm skin, which had been exposed to local heating, responded with an elevated perfusion consisting of oxygenated blood. This strengthens the hypothesis that the flow increase occurs through dilatation of larger deeper lying skin vessels and not in the capillaries. © 2001 Academic Press
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Key Words: spectroscopy; laser Doppler flowmetry; EMLA; hemoglobin; analgesia; heat stimuli; skin microcirculation.
INTRODUCTION The eye, together with the perceptual system, is perhaps the most sensitive biological sensor, able to perform discrimination analysis even at low differences, in both color intensity and saturation. Despite its excellent sensitivity, visual classification shows a large intra- and interindividual variability and is, therefore, subjective in its nature (Andersen et al., 1991a; Bjerring and Andersen, 1987; Takiwaki and Serup, 1995). In irritancy tests and other dermatological applications in which graded information is necessary, a more objective comparison has to be performed. Reflectance spectroscopy (Andersen and Bjerring, 1990a; Bjerring and Andersen, 1987) is, therefore, used in many areas such as skin patch testing (Andersen et al., 1991b) and evaluation of UV-induced erythemas (Andersen et al., 1991a; Kollias and Baqer, 1988). In superficial skin, melanin and hemoglobin are the two dominant chromophores, having considerable impact on the reflection spectra (Andersen and Bjerring, 1990b). In human blood, hemoglobin appears in dif-
0026-2862/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
393
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ferent forms, of which oxyhemoglobin (OH), i.e., hemoglobin containing bound oxygen, and deoxyhemoglobin (DOH), i.e., not containing any bound oxygen, are the most common (Dorland, 1988). The different hemoglobin forms exhibit characteristic absorption spectra that are distinguishable (Anderson and Parrish, 1982). Therefore, any changes in hemoglobin concentration, or the composition of the different forms, will affect the captured spectra according to the change. In a previous investigation, skin perfusion together with the physiological consequences of local skin analgesia and, in particular, the perfusion response to different stimuli in the microcirculation were investigated (Arildsson et al., 2000b). It was postulated that the observed increase in perfusion occurs in deeper lying vessels, since the number of physiologically active capillaries decreased (Arildsson et al., 2000a). By applying reflection spectroscopy in a similar set-up, the relative change of oxygenation in these vessels can be determined. The aim was to assess changes in reflection spectra associated with local heating of analgesized skin in relation to skin chromophores and skin perfusion.
MATERIAL AND METHODS Subjects This study comprised 12 Caucasian subjects (6 male and 6 female, ages 21–32 years), who gave their informed consent. All subjects were healthy, with no prescription medication and no visible skin disorders and did not use any form of nicotine. Ingestion of food or caffeine, less than 1 h prior to the start of the examination, was not allowed. All measurements were made with the subject sitting comfortably in a chair in an upright position.
The Dermal Sensitivity Tester The dermal sensitivity tester (Desensor; Cenova AB, Sweden), a computerized robotic system, delivers
standardized preset skin stimuli to a well-defined area of the skin (Arildsson et al., 2000b). In this study, the Desensor was used to deliver a local heat stimulus to a predefined and analgesized skin site on the lower forearm. The stimulus was delivered using a copper probe ( ⫽ 12 mm, temperature 45°C) in contact with the skin surface for 9 s.
Reflection Spectroscopy Each chromophore affects the reflected spectra differently, leaving a unique and characteristic fingerprint. Reflection spectroscopy (RS) is therefore a suitable method for analyzing the chromophore content in superficial tissue (Buckley and Grum, 1961; Dawson et al., 1980). Blood flow redistribution, between skin plexa, or changes in blood composition alter the amount of chromophores and hence the recorded reflectance spectra of the tissue. The spectrometer (S-1000; Ocean Optics, U.S.A.) consists of a grating and an optical system that projects an image of the light spectrum onto a CCD array. The data stored in the CCD are then transferred as an analog signal and sampled by a computer. The illuminating light source (LS-1; Ocean Optics) was a stabilized tungsten halogen lamp (99.7% stabilization within 5 min and 100% in 40 min). To ensure stable light conditions, a warm-up period of at least 40 min was allowed prior to each experiment. A fiber-optic probe (FCR-7IR200-2; Ocean Optics) guides the light to the area under study, through six fibers. The reflected light is collected and guided back to the spectrometer through a single fiber encircled by the illuminating fibers. The spectrometer, in combination with the light source, used in this study operates in the spectral range from 500 to 1000 nm. During each measurement session three different, consecutive, spectroscopic measurements were carried out to minimize the effects of any spatial differences in the skin color and microcirculation. These spectroscopic measurements were made by applying the probe, firmly but with mild pressure, perpendicular to the skin surface so that contact was
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FIG. 1. Typical reflectance spectra from: (a) a piece of polyacetal plastic (reference spectrum), (b) EMLA-treated tissue without heat provocation, and (c) EMLA-treated tissue after heat provocation; (b) and (c) are normalized against the maximum intensity. (d) Relative absorption spectrum (c– b; see Material and Methods).
made with it. The time taken for each single measurement was not more than a couple of seconds, with a total time of less than a minute for all three measurements. To control the inherent characteristics of the spectrometer and light source, a reference spectrum was collected (Fig. 1a). The reference spectrum of the equipment was obtained using a piece of reflective polyacetal plastic.
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Laser Doppler Perfusion Imaging Coherent laser light, interacting with moving red blood cells in tissue according to the Doppler principle, can be used to estimate the tissue perfusion. This technique is generally referred to as laser Doppler blood flowmetry (LDF) (Stern, 1975). Laser Doppler perfusion imaging (LDPI) (Essex and Byrne, 1991; Wårdell et al., 1993, 1994) uses the LDF technique in
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order to generate a tissue perfusion map by moving a freely impinging laser beam across the surface of interest. The investigations in the present study were made using a LDPI system (PIM1.0; Lisca Development AB, Sweden). All recorded images were of size 10 ⫻ 10 measurement points, covering approximately 20 ⫻ 20 mm.
EMLA and Placebo Cream Astra Pain Control, Sweden, provided the placebo cream and analgesic cream (EMLA) used in this study. The EMLA (eutectic mixture of local analgesics) (Juhlin and Evers, 1990; Nielsen et al., 1992) cream contains the local analgesic substances lidocaine (2.5%) and prilocaine (2.5%), while in the placebo cream these substances have been substituted by coconut oil. The pH of the two creams is the same. Approximately 2.5 g of cream was applied to the test area and was fixated using a transparent plastic dressing (Tegaderm; 3M, Canada).
Experimental Set-up This study was approved by the Ethics Committee for Human Research at Linko¨ping University Hospital, Sweden. The subject was acclimatized for 30 min prior to each experiment. The volar sides of the lower forearms were randomly assigned a treatment, either EMLA or placebo cream. Each arm was subdivided into five test areas (10 cm 2) and randomly assigned treatment times of 20, 40, 60, 120, and 180 min (Fig. 2a). Corresponding test areas, on each arm, were assigned the same treatment time. For every test area, three measurement sessions were performed: before treatment, after treatment, and 6 min after delivered heat stimulus (Fig. 2b). The effect of heat stimulation on placebo-treated skin has previously been reported and it has been shown that the placebo treatment did not affect the LDPI recordings (Arildsson et al., 2000b). LDPI images were, therefore, only captured on the EMLA-treated arm.
FIG. 2. (a) Schematic illustration of treatment areas on the volar sides of the lower forearms. (b) Example of the spectroscopic measurement within the test area. x denotes initial measurements, y denotes measurements after EMLA treatment, z denotes measurements outside of the heat-stimulated area, and r denotes measurements inside of the heat-stimulated area.
Initially, spectroscopic measurements (x) and LDPI images were performed on untreated skin, after which the creams were applied to the test areas. All applications, except for the 40-min treatment, were carried out in one session, with EMLA and placebo separated by a period of 12 min, allowing a controlled and comparable environment for all measurements. The application of EMLA and placebo for the 40-min treatment was made 15 min prior to the 120-min measurement session. After the predetermined treatment time, the plastic dressing was removed and the cream gently wiped off with a cloth, and a second set of RS ( y) and LDPI data was collected. The heat stimulus was then delivered by the Desensor to the center of the treated test area. A final session with collection of RS and LDPI data was performed 6 min after the heat stimulus. During this session, two measurements were made with the spectrometer, one in the unstimulated area (z) and one in
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the stimulated area (r), to conclude the effect of heat stimulus and EMLA treatment on the microcirculation. During all LDPI measurements, the subject’s arm was fixed, using a vacuum pillow, in order to prevent artifacts due to arm movement.
Data Analysis In order to minimize the illuminating intensity variances in the RS measurements, all spectra were normalized against the maximum intensity at a wavelength outside the area of interest (635– 642 nm). To minimize the spatial variations depending on skin site, the average of three normalized spectra were calculated for each treatment time and measurement (r and z, Fig. 2b). As proposed by Kollias and Baqer (1988), the change in absorption (calculated from differential measurements) reflects the change in chromophore content. Thus, to assess the change in light absorption in the skin due to heat stimuli, the difference between the logarithm of inverse reflectance for the average spectra was calculated. This ensures that chromophores that do not change as a result of the heat stimulus are subtracted and do not interfere with the calculations. The plotted change contains the characteristic absorption pattern of OH (Fig. 1d). By fitting tabulated OH and DOH absorption spectra to the change in absorption spectra, using a linear regression model, the difference in chromophore content was analyzed (Fig. 3a). The absorption data for OH and DOH used in this study have been acquired from tabulated data for the molar extinction coefficients from Prahl (1999) (Fig. 3b). To compare the results from the RS with visual assessment and LDPI, the sum of the OH and DOH coefficients was used. In order to classify areas as responding or not, the normal variations in the spectroscopic measurement data of untreated skin were studied by evaluating the three central test areas. The sum of the OH and DOH coefficients for adjacent skin areas (2–3 and 3– 4, Fig. 2a) was compared according to the same protocol as for the heat-stimulated areas, resulting in 44 differences. Two standard deviations (SD) were arbitrarily chosen as the classification threshold. The average LDPI skin perfusion before treatment,
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FIG. 3. (a) The absorption spectrum for 180 min EMLA treatment of a typical subject. Also shown is the sum of deoxy- and oxyhemoglobin with the respective coefficients of 3.56 ⫻ 10 ⫺2 and 1.10 ⫻ 10 ⫺2 derived from curvefitting. The normalized absorption spectra for deoxy- and oxyhemoglobin are shown in (b), based on data for the extinction coefficient from Prahl (1999).
after treatment, and after provocation was estimated by calculating the mean value of the corresponding 10 ⫻ 10 measurement points LDPI images. To classify an area as responding or nonresponding, the normal perfusion variation was evaluated in the same way as
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TABLE 1 Relative Amounts of Oxyhemoglobin (OH) and Deoxyhemoglobin (DOH) in the Heat-Stimulated Area Compared to the Unstimulated Adjacent Skin Site (see Material and Methods) Placebo—180 min
EMLA—180 min
Subject
OH
DOH
Sum
OH
DOH
Sum
⌬Perf
1 2 3 4 5 6 7 8 9 10 11 Average SD
0.0016 ⫺0.0012 0.0032 0.0009 0.0048 0.0095 0.0028 0.0058 0.0034 0.0007 ⫺0.0008 0.0028 0.0031
⫺0.0038 ⫺0.0018 ⫺0.0010 0.0013 0.0000 0.0030 ⫺0.0030 0.0073 0.0006 ⫺0.0048 ⫺0.0023 ⫺0.0004 0.0034
⫺0.0022 ⫺0.0030 0.0022 0.0022 0.0049 0.0125 ⫺0.0002 0.0131 0.0040 ⫺0.0040 ⫺0.0031 0.0024 0.0059
0.0303 0.0356 0.0358 0.0124 0.0318 0.0143 0.0305 0.0386 0.0081 0.0261 0.0180 0.0256 0.0106
0.0051 0.0110 0.0065 0.0014 0.0031 0.0026 0.0007 0.0097 ⫺0.0046 0.0056 0.0006 0.0038 0.0044
0.0353 0.0466 0.0423 0.0138 0.0350 0.0168 0.0312 0.0483 0.0035 0.0317 0.0186 0.0294 0.0144
75 103 124 0 158 ⫺21 56 27 ⫺14 51 ⫺4 50.5 59.7
Note. For a comparison the change in perfusion (in arbitrary units), as measured by laser Doppler perfusion imaging, can be seen in the rightmost column.
for RS, i.e., 2 SD were chosen as a classification threshold.
RESULTS One female subject was excluded due to equipment failure. Among the remaining 11 subjects, a visually perceived distinguishable red spot appeared after the heat stimulus was applied. The red spot was similar in shape and size to the probe contact area used to deliver the heat provocation. In EMLA-treated areas, this red mark lasted for a period of at least 8 min. The red spot was also seen in some placebo-treated areas, but generally disappeared within a minute after cessation of provocation. The reflectance spectra of the red spot showed a decreased reflection for wavelengths shorter than 600 nm (Fig. 1c). This decrease in reflection was not found in areas treated with EMLA but without heat stimuli (Fig. 1b). The increase in absorption, calculated as the difference between areas without and with heat stimulation, showed characteristic wavelength peaks at 542 and 577 nm (Fig. 1d). This increased absorption
was identified as being due to an increase in hemoglobin and, in particular, oxygenated hemoglobin. The relative change of OH and DOH, estimated by curve fitting, is presented for the 3-h treatment time in Table 1. Among the nine RS responses in EMLAtreated skin, OH was the predominant chromophore. The goodness of fit for these responses ranged from 0.95 to 0.99. In general for all responses in EMLAtreated skin, the increased absorption consisted of OH (Table 1). This could not be shown for the placebo responses. The RS difference (M ⫾ SD) between test areas 2 and 3 was 0.0054 ⫾ 0.0109, with a calculated 95% confidence interval of (⫺0.0012; 0.012), and between test areas 3 and 4 the difference was 0.0061 ⫾ 0.0067, giving a 95% confidence interval of (0.0021; 0.010). The threshold for a significant response in RS was 0.015 (2 SD of normal skin variation). The LDPI difference (M ⫾ SD) between test areas 2 and 3 was ⫺4.9 ⫾ 4.5, with a 95% confidence interval of (⫺14; 4.0), and between test areas 3 and 4 the difference was 2.0 ⫾ 4.3 with a 95% confidence interval of (⫺6.6; 11). The threshold for a significant LDPI response was 29 arbitrary units (2 SD of normal variation). With the threshold chosen, the absorption results
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only twice (Fig. 4a). For a comparison between number of responses classified by RS and LDPI, respectively, see Fig. 4b; for individual responses see Table 2. There were 19 LDPI and 20 RS responses. The methods agreed for 14 responses.
DISCUSSION In summary, this study has examined the use of reflectance spectroscopy, in conjunction with laser Doppler perfusion imaging, in a provocation model consisting of a heat load delivered to an analgesized skin site on the lower forearm. Skin sites exhibiting an elevated perfusion were associated with an increase of the blood chromophore content, of which the oxyhemoglobin change was sixfold greater than that of deoxyhemoglobin. The observed reflection spectra were found to have good short-term stability. Thus, only reflection spectra with a short-term lag between measurement points were analyzed, i.e., those of the treated and heated area were compared to those of the treated but not heated area. The spectra obtained before and after treatment, inspected only by eye, confirmed that no major changes in blood content had occurred during TABLE 2 Distribution of Responding Areas Classified Using Reflectance Spectroscopy/Laser Doppler Perfusion Imaging FIG. 4. (a) The number of responses, classified by reflection spectroscopy, for the different treatment times in EMLA- and placebo-treated areas, respectively. A response was defined as an increase in the relative amount of oxy- and deoxyhemoglobin exceeding 2 standard deviations of the variability of normal skin. (b) The number of responses classified by reflectance spectroscopy and LDPI, where LDPI response was defined as a change in perfusion exceeding 2 standard deviations of the variability found in normal skin.
demonstrated an increasing number of RS responses with increasing treatment time for EMLA-treated areas (Fig. 4a). The placebo-treated areas responded
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EMLA treatment time Subject
20 min
40 min
60 min
120 min
180 min
1 2 3 4 5 6 7 8 9 10 11 Total
0/0 0/0 0/0 0/0 0/1 0/0 1/0 0/0 0/0 0/0 0/0 1/1
0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/1 0/0 1/1 0/0 2/2
0/0 1/1 0/0 0/0 1/1 0/0 0/1 0/0 0/1 0/0 0/0 2/4
0/0 1/0 1/0 0/0 1/1 1/1 1/1 0/1 0/0 1/1 0/1 6/6
1/1 1/1 1/1 0/0 1/1 1/0 1/1 1/0 0/0 1/1 1/0 9/6
Note. Responses are denoted by a “1”.
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the treatment procedure. However, the results obtained cannot exclude the possibility that the blood content and the inherent chromophores of the nonheated adjacent area were changed due to the heat stimuli. This is, however, not plausible, since the reaction to the heat stimulus, observed visually, consisted of a clearly defined red spot. Moreover, it has been shown previously (Arildsson et al., 2000b) that elevated perfusion occurs in relation to this red spot. Curve fitting of recorded absorption spectra was performed only on the hemoglobin chromophores. The reason for not including melanin, a dominant chromophore in superficial skin, is that our results are calculations of difference spectra and melanin remains constant during the short recording time (even during UVB stimulation (Andersen et al., 1991a), melanin changes are slow). Tabulated values were used (Prahl, 1999) for the oxy- and deoxyhemoglobin reference spectra. It is possible that measurement with our equipment would have given slightly different reference spectra. Nevertheless, the goodness of the fit ranged from 0.95 to 0.99. Classification of an area as being a response or not was not performed using a graded scale. The threshold to pass, in order to be classified as a response, was estimated as 2 SD of the natural variability between three central sites located on the forearm. Although both the RS and the LDPI limits were obtained using the same methodology, some differences exist. In the LDPI data, no systematic differences were found between untreated skin sites, although the site closest to the wrist showed, in general, a higher perfusion. The sum of OH and DOH showed a similar pattern, with higher values closer to the wrist (in declining order: sites 2, 3, and 4). By subtracting values from the same site and using a randomized set-up, systematic baseline differences were avoided. In previous studies (Arildsson et al., 2000a,b), using the EMLA and heat provocation model, a large and time-dependent perfusion increase was documented. RS has been used with the same model and compared to LDPI in order to strengthen the perfusion regulation hypothesis. Comparing RS and LDPI measurements from the same skin sites, an 80% agreement was found (44/55). Of the LDPI responses, 74% also exhib-
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ited an RS response (14/19). However, it cannot be concluded that RS measures the same vascular bed as LDPI from these data alone, since the number of active capillaries also increases after heat provocation. The number of active capillaries almost halves due to the EMLA treatment (Arildsson et al., 2000a). However, the absorption spectra before and after treatment, as well as LDPI measurements (Arildsson et al., 2000b), showed that there was no major change in blood content due to EMLA treatment. Hence, the RS and LDPI data track each other and we can assume that they see essentially the same vascular bed. EMLA treatment does not inherently alter the blood volume, but a small, induced heat load results in skin reddening in the area in contact with the heat probe. In a few subjects, a red spot was seen in the placebotreated skin area. In contrast to the long-lasting response in EMLA-treated skin (also found in two placebo areas), this response vanished quickly. A possible explanation is that the placebo response involves primary thermoregulatory responses, while in EMLAtreated skin tissue, the autoregulatory control is affected and brings about a sustained inflow to the tissue being investigated. The main purpose of capillary perfusion is to support the living tissue according to its metabolic demands (Guyton and Hall, 1996). In the present study, the change in DOH was not statistically significant, while the change in OH was obvious, indicating that the metabolic rate is unchanged. This implies that the observed OH is a secondary effect emanating from an increase in deeper lying perfusion plexas. When examining the LDPI response versus capillary appearance, in the same provocation model, it was concluded that the vascular bed involved in the perfusion response was not that of the superficial capillaries, but rather vessels deeper into the skin (Arildsson et al., 2000a). Data from the previous studies are, however, still compatible with the hypothesis that a substantial amount of the blood flow was due to flow in deeper lying capillary vessels. However, it seems unlikely that the deeper lying capillaries should respond differently. The RS signal from such blood is likely to be a mixture of oxygenated precapillary blood and deoxygenated postcap-
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illary blood. However, the main finding of this study is that the increased blood flow is almost exclusively composed of oxygenated blood. This strengthens the hypothesis from Arildsson et al. (2000a) that superficial capillaries are not involved and, in addition, shows that neither are deeper lying capillaries involved. The blood flow can thus be characterized as a shunt flow consisting of oxygenated blood. Similar results demonstrating the reddening of the skin and increase in OH have been reported previously by other authors (Andersen et al., 1991a,b; Kollias and Baqer, 1988), though it should be noted that they used different provocation models. In conclusion, local heating of analgesized forearm skin was associated with increased blood flow consisting of oxygenated blood. This supports the hypothesis that the flow increase occurs through dilatation of larger deeper lying skin vessels and not in the capillaries.
ACKNOWLEDGMENTS The Swedish National Center of Excellence for Non-invasive Medical Measurements (NIMED) supported this study. We also acknowledge ASTRA Pain Control, Sweden, for their valuable cooperation.
REFERENCES Andersen, P. H., Abrams, K., Bjerring, P., and Maibach, H. (1991a). A time-correlation study of ultraviolet B-induced erythema measured by reflectance spectroscopy and laser Doppler flowmetry. Photodermatol. Photoimmunol. Photomed. 8, 123–128. Andersen, P. H., and Bjerring, P. (1990a). Noninvasive computerized analysis of skin chromophores in vivo by reflectance spectroscopy. Photodermatol. Photoimmunol. Photomed. 7, 249 –257. Andersen, P. H., and Bjerring, P. (1990b). Spectral reflectance of human skin in vivo. Photodermatol. Photoimmunol. Photomed. 7, 5–12. Andersen, P. H., Nangia, A., Bjerring, P., and Maibach, H. I. (1991b). Chemical and pharmacologic skin irritation in man. A reflectance spectroscopic study. Contact Dermatitis 25, 283–289.
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Anderson, R. R., and Parrish, J. A. (1982). Optical properties of human skin. In “The Science of Photomedicine” (J. D. Regan and J. A. Parrish, Eds.), pp. 147–194. Plenum, New York. Arildsson, M., Asker, C. L., Salerud, E. G., and Stro¨mberg, T. (2000a). Skin capillary appearance and skin microvascular perfusion due to topical application of analgesia cream. Microvasc. Res. 59, 14 –23. Arildsson, M., Nilsson, G. E., and Stro¨mberg, T. (2000b). Effects on skin blood flow by provocation during local analgesia. Microvasc. Res. 59, 122–130. Bjerring, P., and Andersen, P. H. (1987). Skin reflectance spectrophotometry. Photodermatology 4, 167–171. Buckley, W. R., and Grum, F. (1961). Reflection spectrophotometry. Arch. Dermatol. 83, 249 –261. Dawson, J. B., Barker, D. J., Ellis, D. J., Grassam, E., Cotterill, J. A., Fisher, G. W., and Feather, J. W. (1980). A theoretical and experimental study of light absorption and scattering by in vivo skin. Phys. Med. Biol. 25, 695–709. Dorland, W. A. N. (1988). “Dorland’s Illustrated Medical Dictionary.” Saunders, Philadelphia. Essex, T. J., and Byrne, P. O. (1991). A laser Doppler scanner for imaging blood flow in skin. J. Biomed. Eng. 13, 189 –194. Guyton, A. C., and Hall, J. E. (1996). “Textbook of Medical Physiology.” Saunders, Philadelphia. Juhlin, L., and Evers, H. (1990). Emla: A new topical anesthetic. Adv. Dermatol. 5, 75–91. Kollias, N., and Baqer, A. H. (1988). Quantitative assessment of uv-induced pigmentation and erythema. Photodermatology 5, 53– 60. Nielsen, J. C., Arendt-Nielsen, L., Bjerring, P., and Svensson, P. (1992). The analgesic effect of Emla cream on facial skin. Quantitative evaluation using argon laser stimulation. Acta Dermatol. Venereol. 72, 281–284. Prahl, S. (1999). Optical absorption of hemoglobin. Oregon Medical Laser Center, http://omlc.ogi.edu/spectra/hemoglobin/. Stern, M. D. (1975). In vivo evaluation of microcirculation by coherent light scattering. Nature 254, 56 –58. Takiwaki, H., and Serup, J. (1995). Measurement of erythema and melanin indices. In “Handbook of Non-invasive Methods and the Skin” (J. Serup and G. B. E. Jemec, Eds.), pp. 377–384. CRC Press, Boca Raton, FL. Wårdell, K., Braverman, I. M., Silverman, D. G., and Nilsson, G. E. (1994). Spatial heterogeneity in normal skin perfusion recorded with laser Doppler imaging and flowmetry. Microvasc. Res. 48, 26 –38. Wårdell, K., Jakobsson, A., and Nilsson, G. E. (1993). Laser Doppler perfusion imaging by dynamic light scattering. IEEE Trans. Biomed. Eng. 40, 309 –316.