Histological variation of Japanese skin with aging
Authors: Dumas M. , Langle S. , Noblesse E. , Bonnet‐Duquennoy M. , Pelle de Queral D. , Tadokoro T. , Bonté F. 
Published: September 2010

The extent of changes with aging depends largely on how much the skin is exposed to sunlight and also on the genetic disposition of the individual. There are thus two main processes, extrinsic aging because of environmental stress, and intrinsic genetically programmed aging [1]. Hence, the processes of aging depends on a person's ethnic origin and the part of the world in which they live.

Methods

The present study investigates the influence of age and exposure to sunlight on the facial skin of 31 healthy Japanese women, aged 20–78 years old, living in Osaka city. Skin samples were obtained during plastic surgery from the face, from areas exposed to varying intensities of sunlight (forehead, cheek, nose, upper eyelid). Samples were fixed in formalin and embedded in paraffin. Sections (5–7 μm) were cut and stained with hematoxylin–phloxyn–safran, orcein or Masson's trichrome. Others were immunostained for p63, β1‐integrin, type IV collagen CD1a and AQP3. Statistical analysis of quantitative and qualitative parameters were performed by analysis of variance (anova) with linear regressions, and the chi‐squared test.

Results and discussion

Epidermis and dermal‐epidermal junction: histological organization

We confirmed that the whole living epidermis becomes thinner with increasing age, with an average decrease of about 5 μm per decade. This thinning is mainly because of a significant reduction in the number of keratinocyte layers. The thinning of the epidermis and the reduction of the keratinocytes layers in Japanese skin do not seem to be reflected in the thickness of the stratum corneum, which appears to remain constant whatever the age in the sample studied.

The epidermal papillary structures also became flatter with age, associated with an increase in the thickness of the dermal–epidermal junction (DEJ). Thus, in addition to the loss of epidermal cells, the epidermis and dermis become less overlapped, so reducing the surface area for exchange between the two compartments. The DEJ also become thicker as Japanese ages and the expression of type IV collagen, the main constituent of the lamina densa and anchoring plaques is reduced in the most photoexposed skin areas. This accounts for the major changes in the function and molecular structure of DEJ components, as in aged Caucasian skin [2].

Keratinocyte growth and differentiation –β1‐integrin and p63

There is no doubt that the rate of cell turnover decreases in the flat aged epidermis, as indicated by the smaller number of proliferative cells [3, 4]. This study focused on two key regulatory proteins. One was p63, that is involved in maintaining the proliferative potential of basal keratinocytes and blocking their calcium‐induced differentiation [5]. The other was β1‐integrin, an adhesion protein present in basal keratinocytes and linked to their clonogenic potential [6].

We found p63‐positive cells in the basal layer of the epidermis and in the suprabasal layers (Fig. 1a), in agreement with others [7]. The used pan anti‐p63 antibody suggest that other isoforms of the protein in addition to the major ΔNp63α mainly expressed in the basal cells, could take part to other functions like differentiation in the suprabasal layers [5]. The great interindividual variation in the staining intensity for p63 in the samples studied made it impossible to detect significant changes in the number of p63‐positive cells with age. Only an increase of p63 was observed in photoexposed areas compared to others within this case, a possible relation to the epidermal thickening.

Immunostaining of Japanese facial skin showing AQP3 expression at the plasma membrane of keratinocytes throughout the living epidermis (a) and the age‐related change in AQP3 (b).

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β1‐integrin was detected only in the basal keratinocytes, and the staining intensity varied from one segment of the basal layer to another. We evaluated the length of immune‐labeled epidermis and the intensity of labeling in each fragment, using an arbitrary colorimetric scale (0–5). We observed a significant decrease in the intermediate intensities (equal to 2 only) with age in the zone least exposed to sunlight. This is consistent with the loss of adhesive properties of freshly isolated epidermal cells in aged skin [2] and the existence of different pools of basal keratinocytes [6]. It suggests that the effect of aging could affect particularly on the transit amplifying cells containing intermediate quantities of β1‐integrin.

Osmotic and water homeostasis – aquaporin‐3

In addition to the stratum corneum (SC) that limits transepidermal water loss, the osmotic equilibrium inside the epidermis and hydration is controlled by the aquaglyceroporins 3 (AQP3) [8, 9]. Immunostaining for AQP‐3 confirmed the presence of the protein in the plasma membranes of keratinocytes throughout the epidermis, together with AQP‐3 cytoplasmic expression of the basal layer cells (Fig. 1a). There was no immunostaining in the SC (Fig. 1a) to retain water in the epidermis via the tight junction proteins [10], so maintaining the water‐lipid barrier within the SC. The immunostaining for AQP3 decreased significantly with the skin age (Fig. 1b), but there was no significant difference between areas exposed to sunlight and those not exposed. This suggests that there is an overall reduction in the natural hydration potential as Japanese facial epidermis ages.

The immune system – epidermal CD1a‐positive cells

Epidermal dendritic cells, mainly Langerhans cells, control the skin immune system. These cells are CD1a positive (CD1a+). Immunostaining showed a major population of highly dendritic cells throughout the epidermis of all the Japanese skin samples. It also showed that the areas most exposed to sunlight (cheeks, forehead and nose) had significantly more CD1a+ cells than less exposed areas (upper eyelid) (Fig. 2a). This confirms previous findings on the wrinkling of area of Caucasian facial skin after chronic exposure to UV light [11]. The number of CD1a+ cells in less exposed areas of skin increased significantly with donor age (Fig. 2b). This shows that chronological aging and exposure to sunlight give rise to an epidermis which cellular immune homeostasis is perturbed.

Immunostaining for CD1a in Japanese facial skin: numbers of CD1a‐positive (CD1a+) epidermal cells per mm of dermal–epidermal junction (DEJ) in skin exposed to sunlight and skin from protected areas (a), and sun‐protected area of facial skin from subjects of different ages (b).

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The pigmentation system – melanocytes and melanin deposits

Pigmentation is part of the protective system developed by the skin epidermis, but unwanted pigmentation is one of the first signs of aging and has a major social impact. The pigmentation of the facial skin of Japanese women increases with age, and spots develop, particularly on the cheeks [12]. We found a significantly greater density of melanocytes in the epidermis of older donors in both sun‐exposed and protected areas. But there were significantly fewer melanocytes in the areas exposed to sunlight than in protected areas. As expected, there were greater deposits of melanin in keratinocytes in the regions exposed to sunlight. This suggests that older donors had a pool of highly active melanocytes in areas of skin exposed to sunlight, with a high rate of melanin transfer to keratinocytes and/or reduced keratinocyte turnover.

The dermal constituents – the elastic fibrils

The dermis is also a crucial contributor to skin aging through the loss of skin biomechanical properties that result in sagging [13]. Elastosis appears in cases of severe photoaging, and was observed only in those areas of the face exposed to sunlight, never in the upper eyelids. This confirms, together with the melanin content, that this area is more protected from sunlight. The density of vertically oriented, thin, elastic oxytalan fibers in the papillary dermis of Japanese facial skin decreased during aging, supporting a previous report that these microfibrils tend to decrease in this ethnic group [14]. But we observed no difference between the areas exposed to sunlight (cheek, nose forehead) and protected areas (upper eyelid) for the same age range and the more severe stores (equal to 4) are only observed in the sun‐exposed areas. In addition, elastosis frequently increased significantly with photoexposure. The fragmentation of elastin fibers in the dermis increased significantly with age. This could be related to the reduction in fibrillin‐1, a major component of the elastic fibers that has been seen in areas of aged wrinkled Caucasian facial skin exposed to sunlight [15].

We conclude that aging affects those areas of Japanese facial skin that are exposed to sunlight and those that are protected, to alter the skin histology and cellular and molecular homeostasis, as indicated by the changes in β1‐integrins, p63 aquaporine‐3 and epidermal immune CD1a+ cells. These parameter may thus be new key targets in the prevention and treatment of Japanese skin aging.

Acknowledgements

We thank P. Gasser (Bio‐EC, Clamart, France) for technical assistance and J.B. Grieu (LVMH Recherche) for support.

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