Pilot-study of photodamaged skin and melasma using reflectance confocal microscopy

Received on: 30/01/2018

Approved on: 29/05/2018

This study was performed at the Faculdade de Ciências Farmacêuticas de Ribeirão Preto da University of São Paulo - Ribeirão Preto (SP), Brazil

Financial support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) e Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp) and Foundation for Research Support of the State of São Paulo (Fapesp)

Conflict of interests: None

Abstract

Introduction: Photoaging and melasma are frequent dermatological complaints. Confocal reflectance microscopy (CRM) is a recent technique that can be used for diagnostic evaluation of these dermatoses.
Objectives: To evaluate the characteristics of the epidermis and dermis containing pigmentary alterations caused by photodamage and melasma with the assistance of CRM, and compare the findings linked to these changes with the perilesional region.
Methods: A pilot study was conducted with eight female individuals (aged 38 to 50 years, Fitzpatrick phototypes from II to IV) with clinical diagnoses of photodamage (n = 4) and melasma (n = 4) in the facial malar region. The perilesional and lesional regions were compared regarding the thickness of the stratum corneum and viable epidermis, the depth of the interpapillary crests, and the presence of hyper-refractive structures.
Results: The pigmentary alterations in the photodamaged skin revealed a morphological pattern — such as an increase in the depth of the interpapillary ridges in the lesion region — typical of solar lentigo. In the lesional region of volunteers bearing melasma, it was possible to observe the presence of dendritic cells in the epidermis and melanophages in the dermis. All volunteers had hyper-refractive keratinocytes in the lesional epidermis region.
Conclusions: Considering the number of patients evaluated, it was possible to characterize and compare cutaneous pigmentary alterations caused by photodamage to those cause by melasma.

Keywords: Aging; Microscopy, confocal; Diagnosis

INTRODUCTION

Photoaging and melasma are frequent complaints in dermatological practices.^1-3 From a clinical point of view, photodamaged skin has wrinkles, changes in texture and pigmentation, and loss of elasticity and firmness.^3-5 Patients with melasma typically have brown spots with irregular borders and clear delimitation, located in areas exposed to the sun, especially in the face and in women. Both dermatoses can have impacts on the patients’ quality of life.^5-9 In Brazil, roughly 8.4% of the population have some pigmentation disorder, while 10% of the Latino population living in the United States have melasma.^7,10

Solar lentigo, also known as senile lentigo or aging spot, is a benign pigmentary disorder that arises with the process of photoaging. This hyperchromia occurs in areas exposed to the sun, especially in the dorsum of the hands, forearms and face.^11-14 It affects more than 90% of the Caucasian population over 50 years of age.¹⁵ Factors linked to its onset would be related to exposure of the skin to external factors, such as ultraviolet (UV) radiation, polycyclic aromatic hydrocarbons (pollution), and the expression of growth and inflammatory factors.^12,16,17

Melasma is a pigmentary disorder characterized by the presence of macules located mainly in the central, malar and / or mandibular regions of the face.^1,7,16-18 Ultraviolet (UV) radiation, female hormones (pregnancy, endocrine dysfunction, use of oral contraceptives ) and inflammatory processes are involved in its pathogenesis as triggering factors, associated with genetic predisposition.^1,7,19,20 This disorder’s pathophysiology has not yet been fully elucidated, however some theories suggest that its emergence would be linked to the increase in the expression of melanogenic factors and specific receptors such as estrogen.^1,19 The increase in the number and caliber of blood vessels in the affected region, as well as the increase in the expression of vascular endothelial growth factor, is also involved.^16,20

The diagnosis of pigmentary changes of the face is predominantly performed by clinical examination and / or histological examination, when there is suspicion of malignancy. A new noninvasive technique, such as confocal reflectance microscopy (CRM), can assist in the clinical diagnosis and also contribute to increase therapeutic efficacy.^21-23 In addition, CRM can be applied in the quantification of epidermal pigmentation, ²⁴ since this methodology’s principle consists of the emission of infrared light on the skin and its subsequent selective capture when reflected by cutaneous structures that have different refractive indices, yielding black and white images. Keratin, melanin, and collagen fibers of the dermis are hyperrefractive structures, being visualized in a lighter color. ^25-28

Methods for instrumental evaluation of photodamaged skin and melasma

There are currently a variety of techniques for instrumentally evaluating the skin, assisting clinical diagnosis.²⁹ Among the methods available for evaluating melasma and photodamage, reflectance spectroscopy and high-resolution image analysis stand out in the investigation of skin color and melanin distribution.^30,31 The Cutometer^® device (Courage-Khazaka, Germany) evaluates the mechanical properties (changes in the elasticity and firmness) of the photodamaged skin. In addition, the analysis of epidermis’ and dermis’ thickness using high frequency ultrasonography substantially contributes to the evaluation of the therapeutic efficacy.^32,33

Confocal reflectance microscopy (CRM) is an advanced technique that allows examination of the epidermis and papillary dermis with a resolution close to that of histological examination, with the ability of identifying structures and cells with high resolution.^2,34 This technique’s basic principle involves beaming infrared light on the skin and the selective capture of this light, after being reflected by cutaneous structures such as keratin, melanin and collagen fibers, whose refractive indexes are diverse.²⁶ Confocal reflectance microscopy is considered a tool for diagnostic confirmation of pigmentary changes in the photodamaged skin and melasma, in this manner avoiding cutaneous biopsy in the face.²

In light of this, the objective of the present study was to evaluate the characteristics of the epidermis and papillary dermis caused by pigmentary alterations linked to melasma and cutaneous photodamage using CRM, and to compare the findings related to these alterations with the characteristics of the perilesional region.

METHODS

Recruitment

This pilot study was performed after approval by the Research Ethics Committee of the Faculdade de Ciências Farmacêuticas de Ribeirão Preto (CEP / FCFRP, Protocol No. 1,418,673 / 2015). The study sample corresponded to 8 female participants aged 38-50 years, Fitzpatrick phototypes II to IV, with alterations of hyperpigmentation in the malar region, diagnosed with pigmentary skin alterations (n = 4) and melasma (n = 4).

Instrumental evaluation

Images of the malar regions were obtained in triplicate (lesional and perilesional) using a confocal reflectance laser microscope VivaScope 1500 (Lucid, USA), having been standardized using the coupled software Vivastack (Lucid, USA). The images were obtained at every 1.5 µm, starting from the stratum corneum up until the depth of 37.5µam, and at every 3µam up until the depth of 132.5µm. Based on the obtained images, the thickness of the stratum corneum, the viable epidermis (granulosum, spinosum and basal layers), and the depth of the interpapillary ridges were evaluated quantitatively and objectively. Likewise, the presence and absence of hyperrefractive structures in the perilesional and lesional region were evaluated qualitatively and subjectively. These evaluations were performed all volunteers.

Statistical analysis

The data had normal distribution, meaning that the t-test was used to compare the morphological alterations between the lesional and perilesional regions. Results were expressed as mean values and standard deviations. A significance level of p <0.05 was used. The Origin8Pro^® software (OriginLab, USA) was employed to evaluate the distribution of the data, while GraphPad Prism 5^® software (GraphPad Software, USA) assisted in the statistical analysis.

RESULTS

The quantitative and objective analysis of the data obtained from the volunteers diagnosed with photodamage showed a non-significant increase in the values of the thickness of the stratum corneum and viable epidermis in the lesional region as compared to the perilesional region. Also, a significant increase (p <0.05) in the depth of the interpapillary ridges of the lesional region was observed as compared with the perilesional region. These findings were not observed among volunteers bearers of melasma (Table 1).

Based on the qualitative and subjective analysis of the images, it was possible to observe the presence of hyperrefractive structures in the lesional region of all volunteers (Table 2 and Figures 1L.e, 2L.e and 3L.e).

In all volunteers diagnosed with photodamage, it was possible to observe a disorganized pattern for the interpapillary ridges and accumulation of hyperrefractive keratinocytes in the lesional region when compared to the perilesional region (Table 2 and Figure 1 L. JDE).

Two volunteers diagnosed with melasma presented hyperrefractive keratinocytes in the perilesional region (Table 2). In addition, it was possible to observe the presence of dendritic cells in the lesional region of a volunteer diagnosed with melasma (Table 2 and Figure 2L.e) and of melanophages in the lesional region’s dermis of another volunteer who was also diagnosed with melasma (Table 2 and Figure 3 L.d).

In a third volunteer bearer of melasma, it was possible to observe a disorganized pattern for the interpapillary ridges in the perilesional region (Table 2).

DISCUSSION

The images obtained by CRM evidenced the presence of hyperrefractive keratinocytes in the epidermis of the regions with benign pigmentation disorders. This outcome indicates the accumulation of melanin in keratinocytes, which is one of the morphological consequences of photoaging.³⁶ Irregular deposition of melanin in the skin has been reported in CRM studies and is characterized by the observation of bright structures due to the high refractive index of melanin.^{21, 36,37}

The disorganized pattern of the interpapillary ridges – represented by a modification in the shape of the papillae, which become polygonal – associated with irregular alignment, was observed at the dermoepidermal junction in the lesional region of the volunteers with pigmentary alterations due to photodamage. These changes are characteristic of solar lentigo, as described in the literature.^21,37,38 A significant increase (p <0.05) in the depth of the interpapillary ridges of the lesional region and a non-significant increase in the thickness of the viable epidermis were observed in the volunteers who presented photodamaged skin with solar lentigo. To date, according to histological studies, the increase in the keratinocytes’ thickness in solar lentigo might be related to hypertrophy or increased cell proliferation.^39,40

According to the literature, the presence of dendritic cells, commonly observed in melasma, might correspond to active melanocytes.¹⁹ In another volunteer diagnosed with melasma, the presence of ovoid contrast cells located in the dermis was observed, which, according to reports in the literature, could be melanophages.^41,42 The outcomes obtained are aligned to those observed in previous investigations, suggesting a possible morphological difference between these pigmentary disorders, that are detectable by CRM.^19,21,36-24

CONCLUSION

Considering the number of patients evaluated, it was possible to characterize the pigmentary alterations in the photodamaged skin and in the melasma. Based on the analyses performed with assistance of CRM, it was possible to identify differences between pigmentary alterations and the perilesional areas in the malar region, both in melasma and in photodamaged skin.

DECLARATION OF PARTICIPATION:

Francine César | ORCID 0000-0002-3748-1341
Instrumental evaluation, image acquisition and manuscript write up

Ana Paula Martini | ORCID 0000-0003-3705-4339
Image interpretation, manuscript development and write up

Maria Cláudia Issa | ORCID 0000- 0003- 1031- 7370
Structural organization of the manuscript and final review

Patrícia Maia Campos | ORCID 0000-0001-6678-1207
Study coordination, manuscript development and final review

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