Dorsal skin in a young mouse showing epidermis with a simple organization (E) and dermal collagen fibers (D, pink or red) (Verhoeff–van Gieson stain, original magnification ×400).
Dorsal skin in an old mouse showing atrophy of the epidermis and an abundance of elastic fibers (arrow) (Verhoeff–van Gieson stain, original magnification ×400).
Ventral skin in a young mouse (Verhoeff–van Gieson stain, original magnification ×400).
Ventral skin in a young adult mouse showing thin, folded epidermis and sebaceous follicles with smaller, pyknotic nuclei (Verhoeff–van Gieson stain, original magnification ×400).
Scatter diagram (with regression line) of the number of epidermal cells from the dorsal skin in the 3 age groups of CBA mice.
Percentage of elastic fibers in the footpad relative to age. Vertical lines and bars are means ± SDs.
Bhattacharyya TK, Thomas JR. Histomorphologic Changes in Aging SkinObservations in the CBA Mouse Model. Arch Facial Plast Surg. 2004;6(1):21-25. doi:10.1001/archfaci.6.1.21
From the Department of Otolaryngology–Head and Neck Surgery, University of Illinois at Chicago.
Background Aging of human skin is a phenomenon resulting from a combination of chronological aging and environmental stressors such as sunlight.
Objectives To study the effects of intrinsic aging on the skin in laboratory-raised CBA mice in 3 age groups, and to assess histological alterations as a function of age in this model.
Methods Skin samples from CBA mice in 3 age groups (young, young adult, and old) were obtained from the dorsal and ventral areas, pinna, and hind foot to study the following variables using light microscopic manual morphometric methods: the depth of the epidermis and number of epidermal cells, depth of the dermis, and percentage area of dermal collagen, elastic fibers, pilosebaceous units, blood vessels, and tissue space. The obtained values were analyzed using 1-way analysis of variance to detect any significant effects of age.
Results There was a notable attrition of the epidermal thickness and number of cells that could be correlated with age. A reduced number of pilosebaceous units was noted in skin samples from the dorsal region and the footpad. No conspicuous change was noted in the depth of the dermis or percentage area of collagen in aging animals. A proliferation of stainable elastic fibers was demonstrated in the dorsal skin and footpad of older mice.
Conclusions CBA mice show unique age-related histological modifications of the skin that are different from other rodent species. These baseline data will be helpful in further studies of regenerative effects of pharmaceutical agents on the histological structure of skin and in photoaging studies.
Aging of facial skin is a topic of current interest in view of ongoing searches for noninvasive methods to maintain a youthful appearance and regeneration of skin in older people. The phenomenon of aging in human skin comprises 2 elements: intrinsic, or the chronological aging process, and extrinsic aging from environmental stressors, often called photoaging. Cumulative sun exposure is the cause of most structural and physiologic changes in aging skin in humans.1-2 The morphometric distinction of the 2 kinds of aging changes in human skin has been the subject of research during the last few decades.
To rejuvenate photoaged skin or the skin of postmenopausal women, topical agents such as retinoids and vitamin C or oral hormone administration has been used in clinical trials with the hope that these can stimulate the skin or reverse the aging process.3-5 These trials need to be supported by in vivo experimental data gathered from colony-raised animals with well-delineated aging records. Such animals can be useful models to test the cutaneous response of the aging skin to chemotherapeutic agents and to distinguish histomorphologic effects of chronological aging. In the present study, CBA mice of 3 age groups (young, young adult, and old) were chosen to investigate the histological and morphometric condition of skin as a function of age, as a prelude to ultrastructural studies subsequent to experimental and pharmaceutical manipulations.
CBA mice were purchased from the National Institute on Aging, Bethesda, Md, and 3 age groups of mice were used in this study: 1 month (young), 6 months (young adult), and 27 months (old) of age. Each age group comprised 6 animals. Institutional guidelines regarding humane use and handling for animal experiments were followed, and the animals were housed in the animal care facilities of the university's Biological Resources Laboratory for 1 week before they were humanely killed. At the time of death, skin samples were excised from the dorsal and ventral areas, pinna, and hind footpad and were immersed in Bouin-Hollande fixative for 48 hours before further processing. Samples were paraffin-embedded, and 5-µm sections were stained using hematoxylin-eosin-phloxine sequence and Verhoeff–van Gieson staining technique to distinguish collagen and elastic fibers.
The method of morphometric measurement was similar to that used for a skin expansion study.6 All observations were made with a ×45 objective lens and a ×100 oil immersion lens. The thickness of the epidermis (excluding the stratum corneum) and dermis and the number of epidermal nuclei per millimeter of interfollicular epidermis were measured using a calibrated ocular micrometer scale. A point-counting method was used to assess change in the percentage of dermal structures in the skin samples. A 10 × 10 square grid (105 × 105 µm) with 121 intersecting points was placed over the section at magnifications of ×1000. The number of intersection points falling on a given dermal element (collagen, elastic fibers, blood vessels, pilosebaceous units, tissue space containing cellular elements, muscle, etc) was recorded. The number of points overlying the element of interest divided by the total number of points yielded the relative percentage or area fraction of that particular dermal element. Individual means ± SDs for each variable in all age groups were generated and analyzed for correlation with age. For all variables, the mean values of the age groups were compared using 1-way analysis of variance to detect significant effects of age. Differences were considered to be significant at P<.05, and all data were analyzed using SYSTAT version 10.2 (Systat Software Inc, Richmond, Calif) and SPSS version 10.0 (SPSS Inc, Chicago, Ill) software.
Table 1 gives the mean ± SD measurements obtained for various skin variables in the 3 age groups of mice. Epidermal thickness (depth) in the young age group ranged from 11.5 ± 1.0 µm in the ventral skin to 41.4 ± 3.0 µm in the footpad epidermis. With increasing age, a trend of diminution was noted in the thickness of the epidermal nucleated cell layers in all 4 skin samples (Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5). The overall analysis of variance indicated significant differences among the 3 age groups in all of the representative skin areas (P≤.001) (Table 2). Correlation analysis showed a negative linear effect of age on the epidermis depth variable (range, P = .04 to P<.001). A significant reduction in the epidermal cell count was noted in all 4 types of skin (ventral: F = 4.66, P = .02; pinna: F = 6.30, P = .01; dorsal: F = 16.90, P<.001; and footpad: F = 16.99, P<.001). The number of cells was also negatively correlated with age (range, r = −0.61 [P = .004] to r = −0.83 [P<.001]). Therefore, an aging effect on both of these epidermal variables was observed, indicating a gradual attrition of the superficial layers of the skin. Qualitative changes, such as the formation of crypts due to collapse of the cell layers, and flattening of the epidermis were also noted.
A reduced number of pilosebaceous unit profiles was observed in the dorsal skin samples (F = 9.84, P = .002) and in the footpad (F = 7.88, P = .01). Correlation with age in these 2 areas was also significant (P = .01 and P = .002, respectively). The sebaceous gland appeared to be atrophied, with pyknotic nuclei in some areas, although no quantitative evaluation was done on this aspect. The profiles of capillaries showed a significant reduction only in the skin sections of the ear (F = 4.86, P = .02; r = −0.63, P = .003).
In a comparison across the 3 age groups of mice, a similarity in the depth of the dermis was observed. Only in pinna skin was a thicker dermis noted in the young adult stage compared with the youngest animals, with a decrease in thickness in the 27-month-old (old) animals (F = 3.76, P = .05). Results were not significant for the area fraction of collagen. A significant age effect on dermal elastic fibers as shown by Verhoeff–van Gieson staining was seen in skin from the footpad (F = 7.09, P = .007) and in the dorsal skin (F = 6.54, P = .009). Higher percentage fraction values of elastic fiber profiles were demonstrated in young adult and old mice in these 2 body regions (Table 1 and Figure 6).
Data on intrinsic aging in humans are difficult to acquire because of problems with reliable sampling, confounding effects of disease, and environmental effects on the skin. The mouse, apart from being genetically similar to humans, lends other conveniences, such as affordability for experimental study, accuracy of the chronological aging record, and husbandry in disease-free colonies protected from environmental hazards. Experimental findings from aging mice may reveal innate age-induced cutaneous modifications, which may be compared with human data and help to identify and separate intrinsic aging factors vs secondary effects. Murine basic scientific data may also have clinical applications in studies related to wound healing or diseases of the integumentary system. In fact, inbred laboratory mice are increasingly used to study the biology and pathological systems of skin in an effort to understand human diseases of the skin and hair.7
CBA mice used in this study showed distinct alterations in epidermal variables, indicating thinning or atrophy that may be related to intrinsic aging. The present observation of epidermal attrition in skin samples from different body regions in this strain does not concur with observations in other rodent species. In CBA agouti mice, triphasic mitotic changes were recorded in the pinna skin, demonstrating a high degree of mitosis in the immature age, followed by a lowering of the mitotic rate at maturity and a final increase during senility.8 These results were not statistically analyzed. In C57BL/6N mice, the number of epidermal cell layers and the epidermal thickness remained constant from 1 to 22 months of age,9 whereas in C57B1/6NNia mice, epidermis from the ear and footpad showed a significant increase in thickness.10 In female Balb/c mice, observed between 2 and 20 months of age, the number of epidermal nuclei per millimeter of interfollicular epidermis was slightly reduced, with no significant change in DNA content of the epidermis, but a decrease in RNA and proteins.11 In Wistar rats studied from 2 days to 34 months of age, the epidermal thickness decreased up until the fourth week and remained constant thereafter.12 Therefore, among rodent species studied, it appears that the CBA mouse is unique in showing a uniformity in age-induced epidermal atrophy in all of the tested body regions, possibly because of inhibited cell proliferation. Differences in inherent genetic constitution, and the ensuing balance between DNA damage and its restitution capacity, might explain the variety of epidermal responses in different strains of colony-raised rats and mice. Aging in humans is believed to be a consequence of genetic programming and cumulative environmental stressors, and the aged skin may reflect changes resulting from the inhibited proliferative capacity of epidermal keratinocytes.13
There are conflicting data regarding epidermal thinning with aging in humans.14 In one study, an age-dependent decrease in epidermal thickness was statistically significant only among men in the 20 to 30 and 30 to 40 age groups.15 No difference in epidermal thickness between younger and older volunteers was reported in a study16 of wound healing. Qualitative changes such as a thinner, flattened epidermis and less organized basal and spinous layers were described by Smith.17 In humans, such morphologic changes, along with impaired synthesis of cytokines or cell-to-cell signals in response to environmental stress, may result in impaired wound healing and the formation of surgical scars in aged individuals. Contet-Audonneau et al18 noted decreased markers of epidermal differentiation (ie, filaggrin, keratohyalin, and transglutaminase) at the bottom of wrinkles in older human skin, along with increased thinning atrophy. Recently, an increase in thickness of the epidermis was observed in younger and older volunteers, as shown by confocal laser scanning microscopic methods using histometric measurements.19 In contrast, in another study,20 in vivo confocal microscopy and ultrasound imaging revealed a slight but significant decrease in epidermal thickness in aging volunteers.
The absence of dermal change, ie, width of the dermis and percentage area of collagen, in CBA mice is comparable to observations reported in other strains of aging rats. In C57BL/6N mice, the dermal thickness decreased from age 3 to 22 months, whereas in the same study9 the thickness of the dermis remained constant in Fisher rats from age 2 to 22 months. In Wistar rats, after initial fluctuations for up to 4 weeks, the dermis width increased until 1 year of age and thereafter remained constant. However, in the same animals, the surface density of collagen bundles did not change from age 1 to 34 months, as seen using morphometric image analysis.12 Admittedly, light microscopic evaluation of collagen may not be sensitive enough to detect more subtle age-induced alterations in the dermis, which can be detected only with morphometric electron microscopy of collagen fibrils or with biochemical analysis. In C57BL/6αα Swiss mice studied between 1 and 2 years of age, skin hyaluronic acid and chondroitin sulfate were decreased.21
A correlation between the structural and functional properties of connective tissue in aging human skin has been made in several studies, and the morphologic picture did not always corroborate the biochemical alterations. In one human study,22 the collagen fiber density per unit of dermal surface, as studied microscopically, did not change with age, despite a decrease in the collagen content of the skin. In addition, no significant difference in the dermis thickness between younger and older women was found when measured using in vivo confocal microscopy.20 However, a stereological analysis of collagen fibers in normal human dermis and its variability with age, as demonstrated by the point-counting method in sections, showed that the collagen fiber density continued to increase with age for up to 30 to 40 years, after which it started decreasing.23 It has been postulated that a dysfunction of the cell-interstitial matrix unit and metabolic changes in the dermal extracellular matrix play a role in the loss of elasticity or formation of wrinkles in sagging skin.24
No morphologic evaluation of dermal elastic fibers has been reported in previous studies of aging, colony-raised rodents. The abundance of elastic fibers in the older age groups of mice may signify proliferation or an accelerated continuing synthesis of these elements concomitant with chronological aging. It is interesting to note that elastic fiber hyperplasia without histological evidence of collagen damage was observed in a UV-A–irradiated hairless mouse model.25 An age-dependent increase in elastic fiber volume in men has also been described.23 Recently, immunocytochemical tests and confocal microscopy showed increased areas of elastin and fibrillin fibers in the reticular dermis, as well as fragmentation of randomly oriented fibers in the subepidermal areas in aged human subjects.26 In CBA mice, the increase in percentage area of elastic fibers appears to be a compensatory mechanism to provide mechanical support to the skin undergoing thinning of the superficial layers.
Because of the complexity of skin organization at a molecular level, a morphologic account of the aging process of the skin gives at best a partial picture of the biology of cutaneous senescence. The aging skin of CBA mice studied in the present investigation using simple histomorphologic methods shows some noteworthy alterations and quantifiable structural changes that distinguish this species from other available rodents and mimic some age-related alterations reported in human skin. These morphologic changes can be presumed to be related to intrinsic aging, and the current availability of this species from the colonies of the National Institute on Aging makes the CBA mouse an interesting model suitable for studying the aging process of mammalian skin. The process of human cutaneous aging is a multifactorial phenomenon, resulting from a combination of environmental factors, disease, trauma, traction forces, nutrition, and metabolic and genetic factors. Because many of these factors can be eliminated in laboratory-raised rodents, this model is suitable to test the effects of pharmacological tools to prevent or retard skin aging. The baseline data from the present investigation will be helpful in contemplated studies of (1) the effects of UV radiation on skin for comparing intrinsic aging vs photoaging and (2) noninvasive methods to rejuvenate the aged skin by topical application of cosmetic preparations or by chemotherapeutic agents.
Corresponding author: Tapan K. Bhattacharyya, PhD, DSc, Department of Otolaryngology–Head and Neck Surgery, University of Illinois at Chicago, 1855 W Taylor St, Chicago, IL 60612 (e-mail: email@example.com).
Accepted for publication February 25, 2003.