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. 2005 May;59(5):511-9.
doi: 10.1111/j.1365-2125.2004.02344.x.

Laser Doppler imager (LDI) scanner and intradermal injection for in vivo pharmacology in human skin microcirculation: responses to acetylcholine, endothelin-1 and their repeatability

Anabelle M Opazo Saez  1 , Frank Mosel, Jens Nürnberger, U Rushentsova, Mario Gössl, Anna Mitchell, Rafael F Schäfers, Thomas Philipp, René R Wenzel
Affiliations

Laser Doppler imager (LDI) scanner and intradermal injection for in vivo pharmacology in human skin microcirculation: responses to acetylcholine, endothelin-1 and their repeatability

Anabelle M Opazo Saez et al. Br J Clin Pharmacol. 2005 May.

Abstract

Aims: The purpose of this study was to evaluate the repeatability of forearm skin blood flow responses to intradermal injections of acetylcholine (ACh) and endothelin-1 (ET-1) using a double injection technique (DIT) and a laser Doppler imager (LDI) scanner in the human skin microcirculation.

Methods: We used a laser Doppler imager (Moor LDI V3.01) to continuously monitor the change in skin blood flow during intradermal administration of physiological saline (0.9% NaCl), acetylcholine (ACh 10(-7), 10(-8), 10(-9) M) and endothelin-1 (ET-1 10(-14), 10(-16), 10(-18) M) in 10 healthy male subjects. Subjects were examined on 3 different days for assessment of interday and interobserver repeatability. Injections of either drug were randomly placed on different sites of the forearm. Laser Doppler images were collected before and after injection at 2.5 min intervals for 30 min. Data were analysed after the completion of each experiment using Moor Software V.3.01. Results are expressed as changes from baseline in arbitrary perfusion units (PU).

Results: ACh caused a significant vasodilation (P < 0.0001 anova, mean +/- SE: 766 +/- 152 PU, ACh 10(-9) M; 1868 +/- 360 PU, ACh 10(-8) M; 4188 +/- 848 PU, ACh 10(-7) M; mean of days 1 and 2, n = 10), and ET-1 induced a significant vasoconstrictive response (P < 0.0001 anova, -421 +/- 83 PU, ET-1 10(-18) M; -553 +/- 66 PU, ET-1 10(-16) M; -936 +/- 90 PU, ET-1 10(-14) M; mean of days 1 and 2, n = 10). There was no difference on the response to either drug on repeated days. Bland-Altman analyses showed a close agreement of responses between days with repeatability coefficients of 1625.4 PU for ACh, and 386.0 PU for ET-1 (95% CI: ACh, -1438 to 1747 PU, ET-1, -399 to 358 PU) and between observers with repeatability coefficients of 1057.2 PU for ACh and 255.8 PU for ET-1 (95% CI: ACh, -1024 to 1048 PU, ET-1, -252 to 249 PU). The variability between these responses was independent of average flux values for both ACh and ET-1. There was a significant correlation between responses measured in the same site, in the same individual on two different days by the same observer (ACh, r = 0.94, P < 0.0001; ET-1, r = 0.90, P < 0.0006), and between responses measured by two different observers (ACh, r = 0.94, P < 0.0001; ET-1, r = 0.91, P < 0.0003).

Conclusion: We have shown that interday and intraobserver responses to intradermal injections of ET-1 and ACh, assessed using the DIT in combination with an LDI scanner, exhibited good reproducibility and may be a useful tool for studying the skin microcirculation in vivo.

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Figures

Figure 1
Figure 1
Laser Doppler images recorded 2 min after intradermal injection of ACh (10−9, 10−8, 10−7 M) and ET-1 (10−18, 10−16, 10−14 M) on the forearm of a young healthy male volunteer are shown. The colour scale illustrates increasing flux in arbitrary perfusion units. At the injection site, ACh markedly increased blood flow for all three concentrations. ACh did not induce a flare reaction. In contrast, ET-1 induced vasoconstriction. A distinct weal and flare response was observed for ET-1 10−14 M
Figure 2
Figure 2
Changes in skin blood flow in arbitrary perfusion units (PU) are shown as a function of time for 12 time intervals assessed with the LDI scanner during a 30-min period. (A) Changes in skin blood flow in response to 0.9% NaCl. Responses to saline were measured on the forearm of left and right arms on two different days to assess interday variability. Measurements of blood flow were obtained after injection of NaCl, and subsequently at 2.5 min intervals for a period of 30 min. The responses shown correspond to those measured in the left arm, and were nearly identical to those of the right arm (data not shown). There was no difference between values obtained in day 1 (filled circles) or day 2 (empty circles). Day 1 (•), day 2 (○). (B) Changes in skin blood flow (PU) in response to ACh (10−9, 10−8, 10−7 M). ACh-10−7 (•), ACh-10−8 (▵), ACh-10−9 (▿). (C) Changes in skin blood flow (PU) in response to ET-1 (10−18, 10−16, 10−14 M). ET-1 10−14 (•), ET-1 10−16 (▵), ET-1 10−18 (▿). For ACh and ET-1 responses correspond to images collected in day 1 and were similar to those obtained in day 2 (data not shown). Values are expressed as mean ± SEM.
Figure 3
Figure 3
Changes in the area under the curve (AUC), after injection with ACh (A) or ET-1 (B). Injections of ACh caused marked vasodilation, while ET-1 resulted in vasoconstriction, in a dose-dependent manner. There was no difference between measurements performed on two different days. Day 1 (•), day 2 (△)
Figure 4
Figure 4
Bland–Altman plots for assessment of interday repeatability. The difference in responses measured on the same site on two different days is plotted vs. the average of the responses for ACh (A) and ET-1 (B). Each response is the change from baseline in skin blood flow, expressed in arbitrary perfusion units. The solid line corresponds to the mean of the differences. The dotted lines indicate the limits of agreement defined as the mean difference ± SD
Figure 5
Figure 5
Correlations between measurements of skin blood flow obtained in the same site of the arm, on two different days in 10 subjects are shown for ACh (A) and ET-1 (B). There was a significant relationship between day 1 and day 2 in response to ACh (r= 0.94, P < 0.0001) and ET-1 (r= 0.90, P < 0.0006). Dotted lines show 95% confidence intervals (CI)
Figure 6
Figure 6
Bland–Altman plots for interobserver variability. The difference in responses measured on the same site, by two different observers, is plotted vs. the average of the responses for ACh (A) and ET-1 (B). The solid line is the mean of the differences, and the dotted lines show the limits of agreement
Figure 7
Figure 7
Correlations between measurements of skin blood flow performed on the same site, by two different observers are shown. There was a significant correlation between measurements performed by two different observers for both ACh (A, r = 0.94, P < 0.0001) and ET-1 (B, r = 0.91, P < 0.0003). Dotted lines show 95% confidence intervals (CI)
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