assessments were chosen for further analysis.
Corneocyte Cornified Envelope (CE) Maturation
Corneocyte CE maturation technique is based on the doublestaining of CE-bound lipids with Nile red and CE structural protein with involucrin. CE maturation was evaluated from the fourth D-Squame of the six mentioned subjects. Briefly, half of the tapes were extracted following Sylnevia laboratory (Labège, France) isolation protocol. Isolated CEs in suspension were placed onto microscope slides and incubated with involucrin primary and respective secondary antibodies before being washed and mounted with Nile red. Images of both Nile redstained and involucrin immunostained corneocytes were taken separately with a fluorescence microscope (ZEISS, ApoTome). IMAGEJ image analysis software was used to analyze the red pixels obtained from the Nile red stained mature cells and the green pixels from the involucrin immunostained immature cells. The ratio of red â„green pixels corresponds to the CE maturation.
Skin Surface Isotropy Assessment by Scanning Electron Microscopy (SEM)
The other half of the fourth D-Squame of the six mentioned subjects were prepared for visualization with SEM (Quanta 250 FEG FEI; ThermoFisher Scientific, USA) by Sylnevia laboratory. Briefly, after being coated with a thin layer of gold, the samples were placed in the microscope, where 36 images per group were taken (6 subjects; 2 timepoints; 3 magnifications: x50, x250, and x500), for a total of 180 images. High-resolution pictures were taken and evaluated in a blinded fashion by one scientist. Adapting the semiquantitative scoring system of Fluhr et al, for SC surface isotropy (ie, micromorphology organizational patterns), three parameters were assessed: cellular clusters at x50, dispersion at x250, and differentiated single cells appearance at x500.16 Scoring for each parameter according to defined criteria was translated into a quantitative scale from 0 to 3. The sum of individual scores obtained after evaluation of the three parameters gives a skin surface isotropy score. Lower score corresponds to a more disorganized SC surface morphology (low isotropy).
Statistical Analysis
For pigmentation and erythema clinical scores, TEWL and hydration index, linear mixed models were used to analyze longitudinal data with change from baseline as response vector; baseline, time, treatment and treatment-time interaction as fixed effect; and subject as random effect. P values were adjusted with Benjamini-Hochberg approach for TEWL and Hydration Index, and a signed-rank Wilcoxon test for pigmentation and erythema scores.
For tape-stripping analysis endpoints, data were analyzed to determine mean, and standard error with normality not assumed according to the number of samples per group. Bonferroni's multiple comparison test was first performed, followed by a Wilcoxon matched-pairs signed rank test to compare each condition at each time points. P values <0.05 were considered statistically significant.
Corneocyte Cornified Envelope (CE) Maturation
Corneocyte CE maturation technique is based on the doublestaining of CE-bound lipids with Nile red and CE structural protein with involucrin. CE maturation was evaluated from the fourth D-Squame of the six mentioned subjects. Briefly, half of the tapes were extracted following Sylnevia laboratory (Labège, France) isolation protocol. Isolated CEs in suspension were placed onto microscope slides and incubated with involucrin primary and respective secondary antibodies before being washed and mounted with Nile red. Images of both Nile redstained and involucrin immunostained corneocytes were taken separately with a fluorescence microscope (ZEISS, ApoTome). IMAGEJ image analysis software was used to analyze the red pixels obtained from the Nile red stained mature cells and the green pixels from the involucrin immunostained immature cells. The ratio of red â„green pixels corresponds to the CE maturation.
Skin Surface Isotropy Assessment by Scanning Electron Microscopy (SEM)
The other half of the fourth D-Squame of the six mentioned subjects were prepared for visualization with SEM (Quanta 250 FEG FEI; ThermoFisher Scientific, USA) by Sylnevia laboratory. Briefly, after being coated with a thin layer of gold, the samples were placed in the microscope, where 36 images per group were taken (6 subjects; 2 timepoints; 3 magnifications: x50, x250, and x500), for a total of 180 images. High-resolution pictures were taken and evaluated in a blinded fashion by one scientist. Adapting the semiquantitative scoring system of Fluhr et al, for SC surface isotropy (ie, micromorphology organizational patterns), three parameters were assessed: cellular clusters at x50, dispersion at x250, and differentiated single cells appearance at x500.16 Scoring for each parameter according to defined criteria was translated into a quantitative scale from 0 to 3. The sum of individual scores obtained after evaluation of the three parameters gives a skin surface isotropy score. Lower score corresponds to a more disorganized SC surface morphology (low isotropy).
Statistical Analysis
For pigmentation and erythema clinical scores, TEWL and hydration index, linear mixed models were used to analyze longitudinal data with change from baseline as response vector; baseline, time, treatment and treatment-time interaction as fixed effect; and subject as random effect. P values were adjusted with Benjamini-Hochberg approach for TEWL and Hydration Index, and a signed-rank Wilcoxon test for pigmentation and erythema scores.
For tape-stripping analysis endpoints, data were analyzed to determine mean, and standard error with normality not assumed according to the number of samples per group. Bonferroni's multiple comparison test was first performed, followed by a Wilcoxon matched-pairs signed rank test to compare each condition at each time points. P values <0.05 were considered statistically significant.
RESULTS
Skin Color Change after UVR
Clinical assessment for erythema and skin pigmentation are illustrated in Figure 1A and 1B, respectively. UVR elicited a perceivable and statistically significant increase in erythema, peaking at day 1 and recovering to baseline by day 7. Treatment with SPF or SPF+Moisturizer routine presented with a significantly less-marked increase in erythema; while treatment with Moisturizer showed no significant effect and was similar to UV only (Figure 1A). For skin pigmentation, UV induced a noticeable and statistically significant skin darkening response, which persisted up to day 14. Treatment with SPF or SPF+Moisturizer routine presented a statistically significant, but less-pronounced increase in pigmentation, which was maintained at minimal level following irradiation until day 14; whereas treatment with Moisturizer showed no significant effect (Figure 1B and 1C). Pairwise comparisons reveal no statistical difference between UV only and Moisturizer for erythema and pigmentation. Treatment with SPF or SPF+Moisturizer routine showed similar performance and were most effective in reducing both erythema and hyper-pigmentation after UVR at all timepoints (Table 1).
Skin Barrier Properties (Hydration and TEWL) after UVR
Next, we investigated the impact of UV on skin barrier by assessing skin hydration and TEWL. There was no statistical difference in skin hydration between control and UV only zones (Figure 2A). Compared to UV only, SPF+Moisturizer routine showed an increasing statistical trend in skin hydration at day 1, and demonstrated significant higher hydration levels by day 3, day 7 and day 14. Treatment with Moisturizer alone showed an increasing trend in skin hydration at day 3 and significant improvement by day 14 compared to UV only, while treatment with SPF showed improved skin hydration only at day 14 (Figure 2A and Table 1). These results suggest that SPF+Moisturizer routine and Moisturizer alone, to a lesser extent, were both effective in promoting skin hydration following UVR.
TEWL showed smaller variations over time following UV, inducing no significant change in all conditions (Figure 2B). Table 1 illustrates no statistical differences in performance between treatments, except at day 3, where SPF+Moisturizer routine showed significant reduced TEWL compared to UV only.
Corneocyte Visualization and Maturation after UVR
To further elucidate the impact of UV on skin barrier integrity, we determined whether UVR affects the superficial SC surface
Clinical assessment for erythema and skin pigmentation are illustrated in Figure 1A and 1B, respectively. UVR elicited a perceivable and statistically significant increase in erythema, peaking at day 1 and recovering to baseline by day 7. Treatment with SPF or SPF+Moisturizer routine presented with a significantly less-marked increase in erythema; while treatment with Moisturizer showed no significant effect and was similar to UV only (Figure 1A). For skin pigmentation, UV induced a noticeable and statistically significant skin darkening response, which persisted up to day 14. Treatment with SPF or SPF+Moisturizer routine presented a statistically significant, but less-pronounced increase in pigmentation, which was maintained at minimal level following irradiation until day 14; whereas treatment with Moisturizer showed no significant effect (Figure 1B and 1C). Pairwise comparisons reveal no statistical difference between UV only and Moisturizer for erythema and pigmentation. Treatment with SPF or SPF+Moisturizer routine showed similar performance and were most effective in reducing both erythema and hyper-pigmentation after UVR at all timepoints (Table 1).
Skin Barrier Properties (Hydration and TEWL) after UVR
Next, we investigated the impact of UV on skin barrier by assessing skin hydration and TEWL. There was no statistical difference in skin hydration between control and UV only zones (Figure 2A). Compared to UV only, SPF+Moisturizer routine showed an increasing statistical trend in skin hydration at day 1, and demonstrated significant higher hydration levels by day 3, day 7 and day 14. Treatment with Moisturizer alone showed an increasing trend in skin hydration at day 3 and significant improvement by day 14 compared to UV only, while treatment with SPF showed improved skin hydration only at day 14 (Figure 2A and Table 1). These results suggest that SPF+Moisturizer routine and Moisturizer alone, to a lesser extent, were both effective in promoting skin hydration following UVR.
TEWL showed smaller variations over time following UV, inducing no significant change in all conditions (Figure 2B). Table 1 illustrates no statistical differences in performance between treatments, except at day 3, where SPF+Moisturizer routine showed significant reduced TEWL compared to UV only.
Corneocyte Visualization and Maturation after UVR
To further elucidate the impact of UV on skin barrier integrity, we determined whether UVR affects the superficial SC surface
