2.1.

Subjects

A total of 29 healthy subjects ($M=14$, $F=15$), ages 18 to 35 (average age: 22.62 years), were enrolled in the study. All subjects graduated or were enrolled in a higher education program, and they participated in the study on a voluntary basis. All subjects signed a written consent after having been fully informed about the study, the confidentiality criteria, the rights, and the ethics criteria.

Those with cardiovascular or respiratory diseases, autoimmune diseases, neoplasia, organ transplant, psychiatric disorders, infectious diseases during the previous month, or allergic reactions to the substances used in the study or their derivatives were excluded from the study, as well as those taking medication that might influence the physiological parameters under study. Pregnant or breast feeding women were also excluded from the study.

Study participants were asked to avoid psychoactive substances, alcohol, coffee, tea, energy drinks, beverages containing caffeine, smoking, taking drugs, and strenuous physical effort 24 h before being tested.

The subjects included in the study were randomly divided into two groups. A test group with 15 subjects ($M=8$, $F=7$) was administered locally capsaicin dissolved in a vehicle solution (capsaicin treated group—CAP), and a control group with 14 subjects ($M=6$, $F=8$) was administered locally only the vehicle (noncapsaicin treated group – nonCAP).

2.2.

Performance of the Experiment

The study was conducted in the Dermato-oncology Research Laboratory of Carol Davila Medicine and Pharmacy University, Bucharest, after the approval of the local Ethics Committee.

The experiments were performed in the afternoon (12 p.m. to 6 p.m.) at a room temperature of $22\pm 1°\mathrm{C}$ and a humidity of $50\pm 5\%$. They lasted approximately 2 h for each subject. Following their arrival at the laboratory, the subjects were seated comfortably and given a period of 45 min to adjust to the local conditions. During that time, they filled in the forms of inclusion in the study, and they watched a slide show on the monitor.

At the end of the adjustment period, the region to be investigated was delimited on the dorsal side of the nondominant hand. Any previous injuries were avoided, as were any maneuvers that might cause an inflammatory reaction at the investigated region. Cutaneous microvascularization was evaluated using in vivo RCM. Two sessions of measurements were performed for every subject. Initial evaluation allowed the identification of a baseline level for the parameters under study. The second measurement session was performed after the topical administration of the capsaicin solution (for subjects in the CAP group) or the vehicle administration (for subjects in nonCAP group) on the investigated area. Cutaneous microvascularization changes in the investigated area were quantified at 0, 10, 25, and 40 min after the beginning of administration.

2.3.

Administration of Substances on the Investigated Region

Capsaicin (M-2028; Sigma Chemical Co, St Louis, MO) was dissolved at a concentration of 1% in the immersion oil (Crodamol STS oil; Croda Inc., Edison, NJ) used in a standard manner for image acquisition with in vivo RCM. A volume of 7.5 µL capsaicin solution in immersion oil was administered with a pipette (Biohit Proline Single-Channel Pipettor, Variable Volume, 0.5 to 10 µL) on the investigated area in the CAP group. The investigated region was isolated from the adjacent skin with a plastic adhesive disc attached to a metallic ring, which was used to fix the image acquisition device. A similar method of administration was used for subjects in the nonCAP group, but the immersion oil did not contain capsaicin.

2.4.

Image Acquisition by RCM

We used the VivaScope® 1500 (Lucid Inc, Rochester, NY) to acquire the images taken by RCM. The wavelength of the laser source was 830 nm, which enabled the visualization of structural details in cutaneous microvascularization at the dermoepidermal junction. The magnetic objective of the microscope was placed in contact with the metallic ring fixed to the skin, without applying any pressure.

We acquired sets of images from the center of the investigated region with an area of $4\times 4\mathrm{mm}$, situated in the dermoepidermal junction, for every evaluation stage. Although previous studies did not show an influence of image acquisition depth on morphological parameters of papillary capillaries,³⁵ in our study, images were acquired at a similar depth for every subject throughout the experiment.

2.5.

Cutaneous Microvascularization Evaluation

Images taken by RCM were analyzed using the Java-based image processing and analysis program ImageJ 1.45 ( http://rsbweb.nih.gov/ij/), which can be downloaded and used free of charge for scientific research.

For every image, we identified the papillae with visible capillary lumina. After marking the capillary lumina margins, the program allowed automatic calculation of micromorfological parameters. The capillary section area, perimeter, and Feret’s diameter (maximum caliper) of capillaries from the dermal papillae were evaluated (see Fig. 1) for a minimum of 14 dermal papillae per subject.

Fig. 1

With in vivo RCM images, dermal papillae apear as dark round-oval areas corresponding to dermal tissue, surrounded by bright rings representing cells from the basal layer of the epidermis. Capillary ansae in the structure of dermal papillae appear in transversal section as very dark discs, in which some bright elements can sometimes be noticed representing blood cells. High resolution of the images acquired with RCM enables the micromorphological evaluation of papillary vascular ansae using as parameters the area of section, perimeter, and Feret’s diameter of the capillaries.

The confocal microscopy images were evaluated in a blinded manner.

2.6.

Statistical Analysis

Values of the investigated parameters were calculated for every subject and for every evaluation stage. Values obtained during the time intervals of 0, 10, 25, and 40 min from administration were expressed as a percentage against the baseline value.

The program SPSS 12.0 (SPSS, Chicago, IL) was used for statistical analysis of data.

Differences between the sexes, and testing of equivalence between the two groups regarding the mean baseline values for each of investigated parameters, were analyzed with one-way between groups analysis of variance (ANOVA). One-way repeated measures ANOVA, followed by Tukey post hoc tests, were used to quantify differences within the two groups for values calculated at every time interval against the baseline for each investigated parameter. When statistically significant differences were revealed, evaluation of correlations existing between these changes was performed, and correlation with the mean baseline values was tested using the Pearson Correlation test.

One-way between groups ANOVA was used for comparative analysis between the two groups for the changes calculated against the baseline value for every time interval.

The results were presented as an average $\pm \mathrm{SD}$. A $P$ value $<0.05$ was considered significant.

3.1.

Mean Baseline Values

We performed an overall analysis of mean baseline values for each investigated parameter to evaluate possible differences between female and male subjects, and no statistically significant differences were found (all $P$ $\text{values}>0.81$, ANOVA), as shown in Table 1.

Table 1

Comparative analysis between female and male subjects of the mean baseline values of the investigated parameters.

Likewise, possible differences in mean baseline values for the investigated parameters between the two groups were analyzed, and no statistically significant differences were revealed (all $P$ $\text{values}>0.13$, ANOVA), as shown in Table 2.

Table 2

Comparative analysis between the two groups of the mean baseline values of the investigated parameters.

3.2.

Changes of Investigated Parameters After Capsaicin or Vehicle Solution Application

We analyzed the values of investigated parameters at 0, 10, 25, and 40 min from administration, searching possible differences between sexes, and similar values were obtained, without revealing statistically significant differences for any parameter.

Subsequently, we evaluated within each group the changes of parameters associated to every time interval, and we drew an analysis of correlations between them.

The nonCAP group had relatively constant values for capillary section area, perimeter, and Feret’s diameter at 0, 10, 25, and 40 min, without revealing any statistically significant differences (see Fig. 2).

Fig. 2

Analysis performed within each group regarding the changes of parameters at 0, 10, 25, and 40 min. (a) area of the section, (b) perimeter, and (c) Feret’s diameter of capillaries did not change significantly in the nonCAP group, whereas in the CAP group, a significant elevation was registered 10 min after the application of capsaicin, followed by a slower increasing trend after 25 and 40 min. Error bars represent the standard deviation. *$P<0.05$, **$P<0.01$, Tukey post hoc test.

By contrast, the CAP group had a significant increase of these parameters 10 min after capsaicin application, followed by a slower increase trend after 25 min and 40 min, as shown in Figs. 2(a) to 2(c) and 3. This increase was not influenced by baseline values of the above mentioned parameters, since no correlations were revealed between baseline values and changes found at the investigated time intervals (Pearson coefficient between $-0.09560$ and 0.25327; all $P$ $\text{values}>0.36241$). However, in this group, strong positive correlations were found between values calculated at time intervals of 10, 25, and 40 min after capsaicin application for all parameters (Pearson coefficient between 0.70501 and 0.83267; all $P$ $\text{values}<0.00334$). Subjects with a more intense reaction to capsaicin administration at 10 min maintained a similar response trend at 25 and 40 min after active substance application. These changes in dermal vascular circulation can be easily monitored in real-time examination using RCM (see Videos 1Fig. 2Fig. 3 to 4).

Fig. 3

Images of RCM of the same dermal papilla acquired (a) 0 min, (b) 10 min, (c) 25 min, and (d) 40 min after the application of capsaicin. Dilation of the lumina of papillary capillaries can be noticed after 10 min. This effect becomes stronger 25 and 40 min after the application of the active substance.

Video 1

Papillary dermis vascular circulation at 0 min from capsaicin application (MPEG, 4.75 MB) [URI: http://dx.doi.org/10.1117/1.JBO.17.8.085003.1].

Video 2

Papillary dermis vascular circulation 10 min after capsaicin application (MPEG, 5.37 MB) [URI: http://dx.doi.org/10.1117/1.JBO.17.8.085003.2].

Video 3

Papillary dermis vascular circulation 25 min after capsaicin application (MPEG, 5.64 MB) [URI: http://dx.doi.org/10.1117/1.JBO.17.8.085003.3].

Video 4

Papillary dermis vascular circulation 40 min after capsaicin application (MPEG, 5.23 MB) [URI: http://dx.doi.org/10.1117/1.JBO.17.8.085003.4].

For every time interval, a comparative analysis of values calculated for capillary section area, perimeter, and Feret’s diameter was conducted between the two groups (see Table 3 and Fig. 4). Micromorphological evaluation showed a rapid vasodilation induced by capsaicin. As early as 10 min after active substance application, the capillary section area, perimeter, and Feret’s diameter became significantly larger in the CAP group, and this difference was maintained until the experiment’s conclusion.

Table 3

Comparative analysis between the two groups of the values at every moment of time of the investigated parameters expressed as a percentage against their baseline value.

Fig. 4

Comparative analysis between the two groups of values calculated for the investigated parameters for every time interval. (a) Area of section, (b) perimeter, and (c) Feret’s diameter of capillaries were significantly higher in the CAP group as against the the nonCAP group as early as after 10 min, these differences were mantained after 25 and 40 min. Error bars represent the standard deviation. **$P<0.01$, and ***$P<0.001$, one way ANOVA test.