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A total of 16 female Korean volunteers with melasma were recruited. The mean age was 44.2±5.36 years old (31-55 years old), and the average of disease duration was 12.9±7.2 years. All subjects have Fitzpatrick skin type III or IV. All subjects had a washout period of at least one month from a bleaching agent or steroid containing triple agents and a washout period of at least one year from lasers, dermabrasion, and chemical peeling. No skin care products had been applied to the measured sites 24 hours prior to the measurements, and the measured sites had not been washed with soaps or surfactants for at least two hours prior to the study. A two-millimeter punch biopsy from lesional and perilesional skin of melasma was performed in 11 of 16 volunteers. Informed written consent was obtained. This study was approved by the institutional review board of Ajou University Hospital (IRB number:

AJIRB-MED-DEO-10-152).

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B. Measurements of biophysical characteristics

The melanin/erythema index, SC hydration capacitance, TEWL and amount of sebum were measured on lesional and perilesional skin by respective probes (Mexameter:

MX16®; Corneometer: CM825®; Tewameter: TM210®; Sebumeter: SM810®) connected to MPA-5® (Courage+Khazaka electronic GmbH, Köln, Germany). SC capacitance and melanin/erythema index were measured on the right side of the face, and the final value was expressed as the mean of three readings. TEWL was assessed on the left side of the face.

After baseline measurement of TEWL, barrier perturbation was performed by 5 times repeated tape stripping (D-squame®) for barrier recovery rate measurement. TEWL was measured immediately and 5 hours after tape stripping. Sebum amount was measured at 30 minutes after facial washing and at 5 hours after degreasing to evaluate sebum excretion rate.

All measurements were performed in an environmentally controlled research facility that maintained an ambient temperature at 22-24 and relative humidity of 50℃ -55%. All volunteers were allowed to acclimatize for 30 minutes prior to the test in order to adapt to the room conditions.

C. Measurement of stratum corneum thickness and immunohistochemistry

Hematoxylin and eosin staining was performed using standard protocols. Under H&E stain, digital images were captured (SPOT Flex®, SPOT Imaging Solutions, Sterling Heights, MI, USA) for estimating the area of SC and length of SC baseline. SC thickness was measured by the ratio of area and length of SC baseline.

In immunohistochemial analyses, formalin-fixed and paraffin-embedded tissue was

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-serial sectioned at 4.5μm and deparaffinized and rehydrated by sequential immersion in xylene, graded concentrations of ethanol. The deparaffinized tissue sections were incubated for 30 minutes at room temperature in a solution of 0.5% hydrogen peroxidase in methanol to quench endogenous peroxidase activity, followed by heated for 20 minutes with citrate buffer at pH 6.0 in a microwave or pretreated with trypsine for 10 minutes in 42 for ℃ antigen retrieval. After washing by Tris-buffered saline (TBS, 0.1mol/L, pH7.4, Dako, Carpinteria, CA), tissue sections were incubated with primary antibodies for 30 minutes at 38 . ℃ A monoclonal antibody to peroxisome proliferator-activated receptor-α (PPAR-α;

Abcam, Cambridge, U.K.) and arachidonate 15-lipoxygenase, type B (ALOX15B, Lifespan Biosciences, Seattle, WA, USA) were used with 1:200 and 1:100 dilution respectively. After incubation with secondary antibody for 10 minutes, horseradish peroxidase (HRP) complex and AEC chromogen were added for 15 minutes and 20 minutes respectively, followed by, counterstaing was performed with hematoxylin. The image was analyzed using Image-Pro Plus Version 4.5 (Media Cybernetics co., Silver Spring, MD, USA).

D. Statistical analysis

The SPSS 14.0 statistics program (SPSS, Inc., Chicago, IL, USA) and the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for all statistical analysis. If the data showed normality distribution, the independent t-test was used to analyze the statistically significant difference between the melasma-involved and perilesional normal skin; if it does not, Mann-Whitney test was used. The relationship between each biophysical property was tested with Spearson correlation. A value of less than

0.05 was considered to be statistically significant. Data are expressed as mean ± standard deviation (SD).

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-III. RESULTS

A. Melanin index and erythema index

The melanin index was significantly higher in lesional skin than in perilesional normal skin (221.4±54.3 vs. 150.4±29.9, p<0.001; Fig 1A). The erythema index was significantly increased in lesional skin than perilesional normal skin (323.2±60.3 vs.

272.3±34.8, p<0.012; Fig 1B). The melanin and erythema index showed a positive correlation (Correlation coefficient: 0.703, p<0.01; Fig 1C).

A.

B.

C.

Fig. 1. Melanin/Erythema index; Comparison of melanin/erythema index between lesional skin and perilesional normal skin; A. Melanin index, B. Erythema index, C. Correlation between melanin/erythema index; Melanin index and erythema index showed positive correlation. (*p< 0.05)

- 10 - B. Basal stratum corneum properties

The SC hydration was significantly increased in lesional skin compared to perilesional normal skin (66.9±9.9 vs. 58.4±10.1; p<0.029; Fig.2). However, the basal TEWL showed no significant difference between lesional and perilesional normal skin (15.6±4.6 vs. 13.5±4.0, p=0.210; Fig 3).

Fig. 2. Stratum corneum capacitance; Stratum corneum capacitance was significantly higher in melasma-involved skin than perilesional normal skin. (*p< 0.05)

Fig. 3. Basal TEWL: There was no significant difference between lesional skin and perilesional normal skin. (*p< 0.05)

C. Epidermal permeability barrier function

Immediately after tape stripping, the increased rate of TEWL in lesional skin was significantly higher than that of perilesional normal skin (%, 143.2±116.4 vs. 79.5±40.9, p=0.014; Fig 4). Furthermore, 5 hours after 5 times of tape stripping, a significant delayed barrier recovery rate was demonstrated in lesional skin in comparison to perilesional normal skin (%, 62.5±22.8 vs. 76.3±12.3, p=0.043; Fig 4). The melanin level was found to be inversely correlated with TEWL levels after tape stripping (Correlation coefficient: -0.503, p<0.05; Fig 5).

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Fig. 4. Barrier recovery rate: Immediately after tape-stripping 5 times, the rate of TEWL in lesional skin was significantly higher in comparison to perilesional normal skin. In addition, 5 hours after the tape stripping, the barrier recovery rate was significantly delayed in lesional skin in comparison to perilesional normal skin. (*p< 0.05)

Fig. 5. Correlation between melanin index and epidermal barrier recovery rate: There was negative correlation between the melanin index and barrier recovery rate.

D. Sebum content

Sebum amount measurement showed no significant difference between lesional skin and perilesional normal skin at 30 minutes after facial washing (μg/cm2, 7.9±5.8 vs.

10.6±14.5, p=0.897, Fig 6). After 5 hours for acclimatization, the amount of sebum and the sebum excretion rate during 5 hours showed no significant difference (μg/cm2, 5 hours after degreasing: 20.5±15.4 vs. 22.4±24.1, p=0.752, Fig 6).

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Fig. 6. Sebum contents: There were no significant differences in sebum amount at both 30 minutes and 5 hours after facial degreasing.

E. Expressions of PPAR-α and ALOX15B and SC thickness

Because lipid metabolism related genes, such as PPARA and ALOX15B were found to be down-regulated in melasma, we examined their protein levels.(Kang et al., 2011) There was no significant difference in immunoreactivity of PPAR-α and ALOX15B between lesional and perilesional normal skin (Fig 7). However, we found that the SC thickness was reduced in the lesional skin of melasma in comparison with perilesional normal skin (µm, 10.4±2.9 vs. 14.5±5.5, p=0.052, Fig 7, 8A). The SC thickness showed positive correlation with epidermal barrier recovery rate (Correlation coefficient: 0.721, p=0.02; Fig 8B)

Fig. 7. Expressions of PPAR-α and ALOX15B and stratum corneum thickness: A.

Stratum corneum thickness was lower in lesional skin compared to perilesional normal skin.

B, C. There was no significant difference of PPAR-α and ALOX15B expression between lesional and perilesional normal skin. (N: perilesional normal skin, L: Lesional skin. original magnification x200)

- 16 - A.

B.

Fig. 8. Stratum corneum thickness and correlation with barrier recovery rate; A. SC of lesional skin showed reduced tendency in comparison with perilesional normal skin. B. SC thickness showed positive correlation between epidermal barrier recovery rate.

IV. DISCUSSION

The present study demonstrated that melasma skin showed a normal hydration state

and sebaceous gland activity. However, the SC integrity and barrier function were impaired

in the lesional skin of melasma. The SC thickness was reduced in lesional skin, and it

correlated with the barrier recovery rate. The melanin index was also significantly related

with delayed barrier recovery.

The mechanisms underlying abnormal barrier function in melasma are unclear, but

there are possible explanations. Recently, we found the lipid metabolism-associated genes

such as peroxisome proliferator-activated receptor alpha (PPARA), arachidonate

15-lipoxygenase, and type B (ALOX15B) were down-regulated in the lesional skin of melasma

(Kang et al., 2011). It is well known that the lipids of SC play an important role in

maintaining cutaneous barrier homeostasis. PPAR-α is an important regulator of lipid

catabolism, mediating fatty acid oxidation, fatty acid uptake, and lipoprotein assembly and

transport.(Mao-Qiang et al., 2004) Therefore, down-regulated lipid metabolism-associated

genes may be a causal factor of the impairment of epidermal barrier function in melasma,

although we could not confirm the down-regulation of these proteins by

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immnohistochemical staining. The reduced SC thickness may play another role in barrier

impairment in lesional skin. In our study, reduced SC thickness was significantly related with

delayed barrier recovery. SC thickness is correlated linearly to the 1/TEWL value

(Weigmann et al., 2005). Furthermore, it has been reported that SC thickness is significantly

correlated with the objective score of atopic dermatitis (Nemoto-Hasebe et al., 2009). Also,

SC thickness is significantly decreased in acute eczematous atopic skin compared to

non-lesional and control healthy skin (Voegeli et al., 2009). That means that SC thickness may

influence the epidermal barrier homeostasis. Taken together, it is speculated that reduced SC

thickness and down-regulation of lipid metabolism related genes in melasma affect the

barrier function in melasma.

Chronic UV exposure may be another possible explanation for impaired barrier

function in melasma. UV exposure is a major triggering or aggravating factor for melasma

development. Indeed, previous studies have indicated that melasma lesions show a higher

degree of UV-induced damage. Increased solar elastosis in lesional skin has been shown. It

has also been shown that melasma is characterized by increased vasculature in the lesional

skin both clinically and histologically (Voegeli et al., 2009). Expression of VEGF, a major

angiogenic factor of UV irradiated skin, is upregulated in melasma lesions compared to

perilesional normal skin. In literature, chronic UV exposure influences cutaneous fatty acid

metabolism and barrier function (Merle et al., 2010). Also, chronic UV irradiation reduces

the epidermal free fatty acid and triglyceride synthesis that has an important role in

epidermal barrier homeostasis (Kim et al., 2010). In addition, UVB exposure is detrimental

to the epidermal permeability barrier in a dose- and time- dependent manner (Haratake et al.,

1997). In photo-aged skin, barrier recovery is known to be significantly delayed (Reed et al.,

1997). Therefore, an altered barrier function in melasma might be a result of the chronic UV

exposure and accompanying epidermal hyperpigmentation. Interestingly, we found a

negative correlation between melanin index and barrier recovery rate. It means that patients

with severe melasma may have poor SC.

Very recently, it has been suggested that barrier function is influenced by

pigmentation in the SC (Elias et al., 2009). Gunathilake et al. (Gunathilake et al., 2009)

reported that skin type IV-V subjects have more acidic SC due to more melanosomes than

pale-skinned subjects, and these acidic conditions were attributed to enhanced SC integrity

and accelerated barrier recovery in darker skins (Bhatnagar et al., 1993; Puri et al., 2000).

- 20 -

Unfortunately, we did not measure the skin surface pH but the present study does not reveal

a positive relationship between melanin index and barrier recovery rate. Rather, there was a

negative correlation. These difference might be explained by the fact that molecules of

melanin in melasma are different from normal melanin (Moncada et al., 2009). Especially,

Raman skin spectroscopy measurements showed that melasma patients have degraded

melanin in the SC of lesional skin. Of course, there are inter-individual differences in

subjects’ race and geographic location.

The present study also demonstrated a significant increase of both the melanin and

erythema index in melasma-involved skin. Also, a positive correlation appeared between the

melanin index and the erythema index. The increased melanin index was reflected as the

hallmark of melasma in histological studies such as epidermal hyperpigmentation (Kang et

al., 2002). Furthermore, the increased erythema index in melasma-involved skin

corresponded with the results of earlier studies, which reported that melasma patients

showed higher erythema intensity and increased vascularity in the melasma lesions than that

of perilesional normal skin (Kim et al., 2007). In a histopathologic study, the number of

dermal vessels had a positive relationship with pigmentation in lesional skin (Kim et al.,

2007). These results have suggested that the connection between vessels and cutaneous

pigmentation. It should be further studied on a large scale in the future.

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V. CONCLUSION

In conclusion, in the present study, I have demonstrated that the melanin index, erythema index and SC hydration were significantly higher in lesional skin compared to perilesional normal skin. However, the basal TEWL and sebum excretion rate showed no significant difference. Interestingly, the epidermal barrier recovery in lesional skin is delayed, and SC integrity is decreased in lesional skin compared to perilesional normal skin. The melanin index showed to be inversely correlated with the barrier recovery rate. In histopathologically, SC thickness in lesional skin decreased than perilesional normal skin and correlated with the barrier recovery rate. These findings suggested that melasma skin have a normal hydration state and sebaceous gland activity, whereas the SC integrity and barrier function were impaired in melasma skin. Decreased SC integrity is one of major biophysical characteristic of melasma.

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proliferator-activated receptor alpha (PPAR-α), and arachidonate 15-lipoxygenase, type B

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