Epidermal dermal junction and spots in human skin
Authors: Nizard C. , Cario‐André M. , Lepreux S. , Pain C. , Noblesse E. , Schnebert S. , Mahé C. , Taïeb A. 
Published: September 2010

IntroductionSenile lentigo (SL) (lentigo senilis, aging spot, liver spot, solar lentigine) is a common component of photoaged skin and women could be affected by these non‐aesthetic spots. SL is characterized by hyperpigmented macules which affects mostly face (forehead and cheeks) or scalp (in bald patients), forearms and dorsa of hands after age 50. Hodgson [1] in a comprehensive study among residents of a home for the aged has shown that the number of spots is clearly related to age and associated features of skin senility. Macules vary in color from yellow‐light brown to black, the medium hue being dark brown, depending probably on underlying skin (photo)type. The coloration is usually homogeneous but the darker lesions have frequently a mottled appearance. Size and shape are much variable, the commonest lesions being round/oval with smooth outlines, but larger lesions are irregular in shape and measure up to several centimeters in diameter, and small patches may coalesce in larger ones especially on the dorsum of hands. The surface of SL is usually smooth in skin showing moderate senile changes but shows roughness and scaling in older subjects like surrounding skin in the context of diffuse actinic keratoses. Miescher et al. [2] have proposed a classification in three categories of lentigo senilis, namely the small macular type (kleinfleckige Typ, ‘senile ephelides’), the large macular type (grossfleckige Typ) and the leucomelanodermic type (Leukomelanoderm), which however overlaps and may correspond to chronological stages of the disease [3].MethodsA systematic comparison between lesional versus perilesional skin using immunohistochemistry and electron microscopy was done to detect precursor lesions of SL and to determine whether melanocytes or keratinocytes were first affected in the evolution of lesions.PatientsThe study was approved by the University Bordeaux Hospitals internal review board. Twelve patients recruited in the out‐ and inpatient clinics volunteered to participate, after giving informed consent. Seven females and five males aged 58–92 years were included in the study.Biopsy procedure and sample preparationAfter local anesthesia with lidocaine, a 4 mm punch biopsy was done at the center of a typical lesion on the dorsum of one hand and at a control site situated immediately at the border from the lesion in clinically non‐pigmented skin. Half of the biopsy was fixed in 4% formaldehyde and the rest was fixed in a 6.25% glutaraldehyde, 0.1 M cacodylate pH 7.4 solution.Histopathologic studiesBiopsies were embedded in paraffin, cut in 5 μm sections and stained using hematoxylin–eosin and Fontana‐Masson techniques to assess the general morphology of the epidermis and melanin distribution. An anti‐MelanA antibody (Dako, Trappes, France) was used to stain melanocytes. As secondary antibody, we used the Envision Kit HRP+ (Dako, Trappes, France), which was revealed with aminoethylcarbamide (AEC). AEC was chosen because red staining facilitates the count of melanocytes in the pigmented basal layer. To analyze the dermis, we stained sections with the orcein stain to visualize elastic fiber, and with Masson's trichrome to visualize collagen fibers. An anti alpha smooth muscle actin monoclonal antibody (clone 1A4, Sigma, St Louis, MO, U.S.A.) was used to detect myofibroblasts. The secondary horseradish peroxidase (HRP) antibody was revealed with diaminobenzidine (DAB).Electron microscopyBiopsies were embedded in Epon and cut in 0.6 μm sections. Sections were examined after post fixation in osmium tetroxide with a CM 10 (Philips, FEI Company, Eindhoven, The Netherlands) electron microscope.ResultsEpidermal morphology of lesional versus perilesional skinConventional microscopyAn accumulation of melanin was noted in epidermal basal layers together with an elongation of rete ridges. This phenomenon increased with the presumed age of the lesion, determined by the age of the patient, and SL could be classified histopathologically in three grades of severity (Fig. 1). Observation of perilesional skin revealed the presence of cluster of cells accumulating pigment (Fig. 1). When comparing lesional skin with perilesional skin, a significant increase was detected in the number of melanocytes by mm of stratum corneum with anti‐melanA antibody. Melanocytes did not accumulate in the ridges, a finding which was confirmed by electron microscopy.Histologic evolution of senile lentigo. Fontanna‐Masson staining (A, B, D–F) and anti‐melan‐A staining (C). (A–C) Perilesional skin. Note in (B) and (C), clusters of keratinocytes retaining pigment capping the nucleus (arrow) and in C melanocyte (arrow head). (D–F) Lesional skin, (D) stage 1, (E) stage 2, and (F) stage 3 corresponding to a progressive accumulation of pigment at the tip of epidermal rete ridges of lesser thickness and increased length (scale bar: 10 μm).imageElectron microscopyIn melanocytes, we observed melanosomes of normal size at all maturation stages. In lesional skin many melanocytes showed an activated phenotype, i.e. numerous mitochondria in the cytoplasm and melanosomes in migration within dendrites. In two perilesional skin samples we observed an increase number of mitochondria in melanocytes, whereas most other melanocytes looked poorly active. Melanocytes ‘hanging down’ in the dermis were occasionally noted in lesional and perilesional skin. A massive capping formed by melanosomial complexes, some of which of unusual large size, was found in basal keratinocytes (Fig. 2).Observation of lesional skin by electron microcopy. Melanosomes are present in dendrites. Some are unusually large and suggest complexation of single melanosomes (arrows), whereas in perinuclear cytoplasm, melenosomes are normal size. In Keratinocytes, melanosomial complexes form a massive capping of nuclei (D). Numerous mitochondria (arrows heads) can be seen in melanocytes. M, melanocyte; K, keratinocyte; and D, dendrite (scale bar: 1 μm).imageDermal morphology of lesional versus perilesional skinConventional microscopyAfter staining with Orcein, Masson's trichrome or AML, there was no difference detected between lesional and perilesional skin, which showed age‐related elastotic changes (data not shown). In lesional and perilesional skin, beneath a superficial dermis showing fibrotic changes, a thick band of elastotic deposits was noted in the reticular dermis corresponding to closely packed and randomly orientated elastic fibers on hematoxylin–eosin and orcein stains.Immunohistochemistry using the anti alpha smooth muscle actin antibody showed in the dermis a low number of interstitial stained cell corresponding to myofibroblasts, without quantitative differences between lesional and perilesional skin.Electron microscopyElectron microscopic examination of areas of solar elastosis in both cases showed aggregates of thick elastotic fibers. These elastotic fibers were composed in their thin periphery by electron dense tubular microfibrils and mainly in the center by elastin characterized by a fine granular matrix of low/medium electron density containing electron dense inclusions.DiscussionSL responds to destructive local treatments such as cryotherapy, carbon dioxide laser, and peelings. Based on the reasoning that retinoic acid inhibits melanogenesis [4] and improves cutaneous photoaging[5], Rafal et al. [6] performed a double blind vehicle controlled study showing that this drug was efficacious, but the exact reason of efficacy remains unknown, since retinoids modify the proliferation and differentiation of a variety of cells. A keratinocyte driven dysregulation of endothelin‐1 production leading to melanocyte dysfunction and excessive production of pigment has been investigated [7]. ET‐1 is mitogenic and stimulates pigment formation in melanocytes following UVB irradiation, via its receptor (ETbR) which is present on melanocytes. These authors have shown an increased number of melanocytes in lesional skin, but the sites studied were not typical [7]. They found ET‐1 and TNF‐α to be more expressed in lesional basal epidermal layers and an increased expression of ET‐1 mARN and EtbR as well.In conclusion, SL is possibly related to an impaired homeostasis of the epidermal melanin unit, triggered by chronic sun exposure, in an irradiation spectrum which seems different from UVA. The nomenclature in this field is confused by the number of supposed synonyms, and a consensus clinico‐pathologic terminology would be useful. If the cosmetic prejudice is clearly related to hyperpigmentation, the elementary histological events suggest that keratinocytes may play an initiating role because of pigment retention. This early event may trigger further epidermal remodeling and hyperplasia. The demonstrated role of retinoids may influence first keratinocyte targets, rather than influencing primarily melanogenesis.Our results on understanding Senile Lentigo, could let us foresee new investigations and future development of cosmetic products new generation.References1. Hodgson, C. Senile lentigo. Arch. Dermatol. 87, 197 (1963).2. Miescher, G., Häberlin, L. and Guggenheim L. Über fleckförmige Alterspigmentierungen: Ihre Beziehungen zur melanotischen präcancerose und zur senilen Warze. Arch. Derm. Syph. (Berl.)174, 105 (1936).3. Braun‐Falco, O. and Schoefinius, H.H. Lentigo senilis. Übersicht und eigene Untersuchungen. Hautarzt. 22, 277 (1971).4. Orlow, S.J., Chakraborty, A.K. and Pawelek, J.M. Retinoic acid is a potent inhibitor of inducible pigmentation in murine and hamster melanoma cell lines. J. Invest. Dermatol. 94, 461 (1990).5. Weiss, J.S., Ellis, C.N., Headington, J.T. and Voorhees, J.J. Topical tretinoin in the treatment of aging skin. J. Am. Acad. Dermatol. 19(1 Pt 2), 169 (1988).6. Rafal, E.S., Griffiths, C.E., Ditre, C.M., Finkel, L.J., Hamilton, T.A., Ellis, C.N. and VoorheesJJ. Topical tretinoin (retinoic acid) treatment for liver spots associated with photodamage. N. Engl. J. Med. 326, 368 (1992).7. Kadono, S., Manaka, I., Kawashima, M., Kobayashi, T. and Imokawa G. The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J. Invest. Dermatol. 116, 571 (2001).