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Effect of Age and Anatomical Site on Density of Sensory Innervation in Human Epidermis
Isabelle Besné, PhD;
Caroline Descombes;
Lionel Breton, PhD
Arch Dermatol. 2002;138:1445-1450.
ABSTRACT
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Background Aging leads to decline of multiple cutaneous physiological functions including decreased sweating, immune responsiveness, thermoregulation, DNA repair, and sensory and tactile perception. Interestingly, sensory perception, like that for pain or spatial acuity, varies in different body parts.
Objective To evaluate epidermal innervation according to age and anatomical site.
Methods Eighty-two biopsy samples from surgical procedures involving 82 patients of different ages (20-93 years) were analyzed. Four anatomical sites were examined: 2 from facial areas (upper eyelid and preauricular area) and 2 from truncal areas (abdomen and mammary area). Epidermal innervation was detected using a marker of neural cells, the protein gene product 9.5. The basement membrane was stained with type IV collagen antibodies. The epidermal area occupied by nerve endings was then calculated using image analysis.
Results A trend displaying age-associated decreased epidermal innervation of facial skin was found. Epidermal innervation of abdominal skin did not change with age, and an age-associated increased innervation was observed in mammary skin. Also, the number of epidermal nerves in facial areas tested (palpebral and preauricular areas) was significantly higher than their number in the abdomen and mammary area. Eyelid epidermis showed the highest ratio of nerve fiber surface to epidermal surface.
Conclusions Epidermal nerve density variations could explain the different sensitivity threshold in different parts of the body. Decreased spatial discrimination with aging may be associated with decreased epidermal nerve density.
INTRODUCTION
SENSORY INNERVATION consists of free nerve endings and endings associated with corpuscular structures. Free nerve endings, which instantly transfer stimuli from the external environment, belong to the classes of A and C fibers.1 They are located in the dermis and epidermis, reaching the granular layer, the uppermost viable layer.2-3 Cutaneous innervation is closely associated with major skin functions such as immune function, inflammation, wound healing, thermoregulation, and hair growth, and it contributes to skin homeostasis.
Aging of the skin involves 2 major processes: chronological aging and photoaging. They are both associated with changes in neuronal and nonneuronal structures of the skin.4-6 Inflammatory and repair processes decrease with age and are probably associated with a decline in neurogenic inflammation.7 A decrease in sweat function associated with reduced pseudomotor nerve density and atrophy of sweat glands has been reported in subjects older than 80 years.8 The sense of touch was shown to be altered in older age as measured by Stevens and Choo,9 Stevens and Cruz,10 and Gescheider et al.11 In parallel, the perception of thermal stimuli decreases with age. Surprisingly, however, painful sensation is relatively conserved.12 The loss of tactile sensation can be related to the decrease in skin innervation13 or to different factors also affected by aging, such as skin hydration14 or a decline in peripheral microcirculation.15 Some authors reported no variations in epidermal innervation with aging,16 while more recent publications reported age-associated decreases in sensory nerve function and epidermal innervation.17-18 In addition to the influence of aging, variations in cutaneous innervation and sensory perception as a function of anatomical sites were reported.9, 19 However, the relation between sensory epidermal innervation density, anatomical sites, and aging is still unclear.
Different methods have been used to evaluate the density as well as function of sensory innervation.20-21 Among those, computer analysis appears to be the most reliable and easiest to use.22-23 In the present study, we used protein gene product 9.5 (PGP 9.5) immunostaining to identify nerve fibers24-25 and computer-assisted morphometric analysis to recognize them and calculate epidermal nerve density in different body parts and as a function of aging.
METHODS
BIOPSY SAMPLES
Human skin samples from the upper eyelid, the preauricular and mammary areas, and abdomen were obtained from elective plastic surgery performed on white women aged 20 to 93 years. Each biopsy sample was cut into small pieces and maintained in Zamboni fixative containing paraformaldehyde and picric acid for 3 hours at 4°C and then rinsed in 15% sucrose and 0.15mM sodium azide phosphate-buffered saline (PBS) overnight at 4°C. The tissue pieces were embedded in ornithine carbamyl transferase compound (Tissue-Tek OCT Compound, Sakura Fine Technical Co Ltd, Tokyo, Japan) and stored at -80°C until immunostained.
IMMUNOHISTOCHEMICAL STAINING
Transverse cryostat sections 10-µm thick were processed for immunofluorescent indirect double staining. Nerve fibers were labeled with a rabbit primary antibody directed to the pan-neuronal marker PGP 9.5 (1:2000 dilution; Ultraclone, Cambridge, England) and a cyanine 3-conjugated donkey anti-rabbit secondary antibody (1:400 dilution; Jackson Immunoresearch Laboratories, West Grove, Pa). Basal membrane of the dermoepidermal junction as well as basal membrane of blood vessels was visualized with a monoclonal antibody directed to collagen type IV (1:25 dilution; Chemicon International Inc, Temecula, Calif) and a rhodol green–conjugated goat anti-mouse secondary antibody (1:400 dilution; Molecular Probes Inc, Eugene, Ore). Immunolabeling was performed in PBS containing 0.1% bovine serum albumin and 0.3% Triton X-100 for 1 hour. Sections were then rinsed, incubated with secondary antibodies, and mounted in aqueous antifading medium (Fluorescent mounting medium; Dako Corporation, Carpinteria, Calif). Immunostained sections were used to evaluate epidermal nerve density.
QUANTITATIVE ANALYSIS OF EPIDERMAL NERVE FIBER DENSITY
Immunolabeled sections were observed with a Leica DM RB microscope (Leica Camera AG, Solms, Germany) equipped for epifluorescence. Double immunostaining was used to localize the dermoepidermal junction and nerve free endings surface in the same preparation. Two digital images were captured by a video camera (Sony DXC 930P; Sony, Tokyo, Japan), one corresponding to the basal membrane labeling by collagen type IV antibody and the other corresponding to the free-endings labeling by the PGP 9.5 antibody. Images were digitized, and the areas occupied by the PGP 9.5 stain were automatically delineated by the computer and analyzed using a Leica Quantimet 600 S analyzer (Leica Camera AG). Collagen type IV immunostaining was used to identify the dermoepidermal junction and to measure the epidermal surface. To determine nerve density, the ratio between epidermal area occupied by nerve endings and epidermal surfaces was calculated and expressed as a percentage. This ratio was compared between different anatomical sites and with aging. All samples were blindly evaluated by 2 different examiners. Fifty fields of each sample, corresponding to 4 sections, were processed for quantification, which corresponds to a 2-cm length of epidermis.
SAMPLE PREPARATION FOR CONFOCAL LASER SCANNING MICROSCOPY
Frozen 50-µm-thick sections were cut with a cryostat microtome. Floating sections were incubated in normal serum (5% goat serum and 5% donkey serum in PBS) for 1 hour at room temperature, rinsed in PBS, and incubated in the primary antibodies (rabbit antibody to PGP 9.5, 1:5000; mouse antibody to collagen type IV, 1:600) for 72 hours at 4°C. Samples were then rinsed in PBS for 2 hours at room temperature and incubated in the secondary antibodies (cyanine 3–conjugated donkey anti-rabbit antibody, 1:800; rhodol green–conjugated goat anti-mouse antibody, 1:1000) for 1 hour in the dark at room temperature and then rinsed and mounted in aqueous antifading medium. All antibodies were diluted in PBS containing 0.1% bovine serum albumin and 0.3% Triton X-100.
Confocal microscopy was carried out using a Zeiss LSM 510 laser scanning microscope, equipped with x40 Plan-Neofluar apochromat objective and appropriate filters (Carl Zeiss, Jena, Germany). Digitalized images were collected in successive frames of 1-µm serial optical sections (z series) and projected into a single image of 50-µm section.
STATISTICAL ANALYSIS
The ratio of the different samples were compared. Nonparametric statistical analysis was performed using the Mann-Whitney test, with the SPSS packages (Statistical Product and Service Solutions; SPSS Inc, Chicago, Ill). A P value lower than .05 was considered significant.
RESULTS
CHARACTERISTICS OF SKIN SAMPLES ANALYZED AND EVALUATION OF EPIDERMAL NERVE FIBERS
As shown in Table 1, samples from 82 patients (1 sample per patient) were analyzed, and the 4 most common plastic surgery sites were evaluated. Twenty samples were taken from the mammary area, 11 from the abdomen, 29 from upper eyelids, and 22 from the preauricular area. Ages of patients ranged from 20 to 70 years for mammary samples, 22 to 56 years for the abdomen samples, 34 to 76 years for the eyelid samples, and 35 to 93 years for the preauricular samples.
Confocal laser microscopy of the entire 50-µM section from the eyelid (Figure 1) shows the general organization of cutaneous sensory innervation labeled by PGP 9.5 antibody. The different steps of morphometric analysis were carried out on 10-µM sections and are shown in Figure 2.
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Figure 1. Confocal microscopy micrograph showing innervation of human eyelid skin. Nerve fibers (NF) were immunolabeled with a protein gene product 9.5 antibody with Cy-3 fluorophore. Basal membranes (BM) of the dermoepidermal junction and blood vessels (BV) were immunostained using collagen type IV antibody with rhodol green fluorophore. Bundles of nerve fibers (labeled in red) are located close to the microvasculature in the dermis (D) and branch out into the superficial part and penetrate the basal membrane. In the superficial part of the dermis, the microcirculation (labeled in green) and nerve fibers establish fine contacts together (labeled in yellow) and terminate as free nerve endings in the epidermis (E) just beneath the stratum corneum (SC). Bar = 50 µm. Inset shows details of the epidermal innervation. Bar = 30 µm.
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Figure 2. Illustration of the computer-assisted structure recognition and the different steps of the morphometric analysis for intraepidermal nerve fiber quantification. A, Step 1: detection of the lower part of the epidermis using collagen type IV immunostaining. B, Step 2: computer-generated image assigning a green color to all areas not stained with collagen type IV antibodies. C, Step 3: computer-generated image assigning yellow color to the epidermis used to calculate total epidermal area. D, Step 4: detection of the intraepidermal nerve fibers, using protein gene product 9.5 immunostaining. Measurement of nerve fiber surface was performed by automatic delineation of the stained areas. E, Step 5: the stratum corneum was delineated manually and epidermal length was measured. Bar = 30 µm.
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TOPOGRAPHIC VARIATIONS OF EPIDERMAL NERVE FIBER DENSITY
The epidermal innervation of the 4 anatomical sites was determined in all patients (Figure 3). The results were expressed as mean ± SD density. The density of the intraepidermal nerves varied in the different body areas. The epidermis of the face was more densely innervated compared with the 2 other body areas. Eyelid epidermis was the most densely innervated with a 0.420 ± 0.13 ratio, then the preauricular area with a 0.313 ± 0.12 ratio. The ratio for abdominal epidermis was 0.231 ± 0.14, while mammary epidermis was the least densely innervated (ratio, 0.183 ± 0.12). The differences of epidermal innervation between the 4 areas were statistically significant (P = .005) except for the difference between the abdomen and mammary skin or the abdomen and the preauricular skin. Epidermal innervation was then plotted as a function of patient age (Figure 4).
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Figure 3. Histograms showing the mean epidermal innervation density of 4 anatomical sites measured by morphometric analysis and expressed as a ratio between the nerve fiber surface and epidermal surfaces. Error bars show SD.
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Figure 4. Intraepidermal nerve fiber density: comparison between 2 truncal (A) and 2 facial (B) body areas. In both facial areas, regression lines display similar decrease with age. The preauricular region shows significantly lower innervation density compared with the eyelid area. Conversely, both sun-protected areas demonstrated a slight increase in epidermal innervation with aging reflected by similar regression line slopes. Also, innervation density was comparable between abdominal and mammary skin.
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As shown in Figure 3, there were no statistically significant differences between the innervation of abdomen and mammary epidermis. Conversely, the eyelid area was significantly more densely innervated compared with the preauricular area (P<.05).
EFFECT OF AGING ON EPIDERMAL NERVE FIBER DENSITY
To further analyze age-related modulations, epidermal nerve fiber density was displayed separately for each decade (Figure 5). A trend displaying decreased epidermal innervation of facial skin was observed, although it was not statistically significant (Figure 5A and B). Epidermal innervation of abdominal skin did not change with age (Figure 5C), and epidermal innervation of mammary skin (Figure 5D) displayed a tendency to increase, although the increase was not statistically significant. These results are consistent with those displayed in Figure 4 in which skin sites are compared 2 by 2 at comparable ages.
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Figure 5. Variations of epidermal nerve density according to age groups in the 4 anatomical sites analyzed: eyelid (A), preauricular area (B), abdomen (C), and mammary area (D). Samples are distributed by 10-year range. Results are expressed as ratio between nerve fibers and epidermal surfaces.
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SENSORY INNERVATION OF SKIN AS A FUNCTION OF AGE AND ANATOMICAL SITE
Figure 6 illustrates epidermal innervation of representative samples. Density of nerve fibers varied between different body areas and with aging. Consistent with Figure 1, small nerve bundles in the upper dermis give rise to nerve fibers that penetrate the basal membrane and ramify in the epidermis. This is particularly obvious for the eyelids (Figure 6A and B), which are the most densely innervated areas. Dense innervation is also observed in the preauricular area of younger women (Figure 6C), whereas less epidermal innervation is visualized in samples from the abdomen and mammary area (Figure 6E and G). In samples from older volunteers, the density of thin nerve fibers is decreased in the epidermis of the eyelid and preauricular area, as shown in Figure 6B and D. In nonfacial areas, the epidermal sensory fibers remain sparse irrespective of age (Figure 6E-H).
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Figure 6. Micrographs showing representative immunofluorescence labeling of different anatomical sites evaluated at 2 ages. Eyelid at ages 37 (A) and 76 (B) years; preauricular area at ages 48 (C) and 75 (D) years; mammary area at ages 27 (E) and 49 (F) years; and abdomen at ages 22 (G) and 56 (H) years. Bar = 25 µm.
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COMMENT
In the present study, the sensory epidermal nerve fiber density was characterized and quantified by anatomical site and patient age in biopsy specimens of normal skin. Thin nerve fibers were immunohistologically visualized using PGP 9.5, a cytoplasmic protein of peripheral or central neurons that belongs to the family of ubiquitin C-terminal hydrolases, which is routinely used to investigate cutaneous innervation.26-27 Different methods such as silver staining, methylene blue, or acetylcholine esterase detection28-30 have also been used to characterize the cutaneous innervation. However, comparative studies24, 31 have demonstrated that PGP 9.5 labeling is easier to handle and more specific for both afferent and efferent sensory fibers.32-33
Protein gene product 9.5 labeling identified nerves that originated from the dermal nerve trunk, entered the epidermis, and then divided into small fibers, the endings of which culminated near the surface of the skin as previously described.2-3 Previous studies have reported the spatial organization of epidermal innervation and more specifically the pattern of branching and the shape of the axon.18, 34 Other studies quantified neurons by computer analysis in different tissues.35-36
We evaluated sensory epidermal innervation of 4 different anatomical sites: upper eyelid, abdomen, and mammary and preauricular areas. Differences were observed between the sites with more densely innervated epidermis of the eyelid and the preauricular area. We also noticed that abdominal and mammary epidermis showed similar profiles of innervation. Previous studies reported data on topographic nerve density showing a rostral-caudal gradient.18-19 In these reports, nerve fiber density in human skin appeared to decrease from the trunk to the distal parts of the body. Conversely, Kawakami et al37 failed to detect epidermal innervation in several volunteers and, in those in whom epidermal innervation was detected, the highest nerve density was observed in the arm and the lowest in the back. In our study, we observed that facial skin (ie, the eyelid and preauricular area) was more densely innervated than the 2 truncal sites (ie, the mammary area and abdomen). We cannot rule out that increased facial innervation is the result of sun exposure, although the degree of photodamage varied among the different subjects. Toyoda et al38 reported a positive relationship between the degree of epidermal innervation and chronic photodamage, perhaps as a result of nerve growth factor (NGF) release by keratinocytes. Nerve growth factor is expressed, synthesized, and secreted by epidermal keratinocytes39-40 and plays a crucial role in cutaneous homeostasis. It favors the survival and differentiation of sensory neurons, regulates the expression of neurotransmitters, and induces connections between neurons and cellular cutaneous targets.41 It is also involved in cutaneous inflammation, wound healing, and pigmentation.42-43 Ultraviolet B irradiation is one factor which induces NGF secretion, at least in murine keratinocytes.44 It is possible that hypersecretion of NGF in sun-exposed areas increases epidermal nerve fiber density.
On the other hand, differences in intraepidermal nerve fiber density in different anatomical sites could be the result of a different origin of sensory innervation.45 The 2 facial areas examined in the present study are innervated by the facial and cranial nerves III, while the abdomen and mammary area are innervated by the thoracic ventral rami. Differences in the physiological or electrical nature of free nerve endings (eg, nociceptors, thermoreceptors, and polymodal receptors) could also explain the observed differences.46-47 This argument is in agreement with variations in spatial acuity of different body areas measured by Stevens and Choo.9 By evaluating the gap acuity, the authors observed that the face was the most discriminative site compared with the abdomen.9 The mechanism of these differences remains unclear.
In the second part of this study, we analyzed intraepidermal innervation as a function of aging. Other studies18, 48 have shown an age-associated decrease in skin innervation of the trunk of at least up to age 60 years. Also, age-associated loss of neuronal network has been reported around sweat glands8 as well as a decline in the number of dorsal root ganglion neurons and loss of small myelinated and unmyelinated fibers in the distal sensory sural nerve that innervates the skin.49 Our findings on facial skin are consistent with these reports, although, surprisingly, we observed increased nerve density with aging in mammary epidermis. These age-associated decreases may be explained by a selective loss of epidermal innervation, with a higher reduction in body areas that are basically more innervated. Age-associated decreased innervation may be the result of decreased synthesis of neurotrophic factors such as NGF. Although still controversial, such a decrease has been reported in the central nervous system of aging rats.50 Also, analogous to the central nervous system, it is possible that the level of neurotrophin receptors that mediate neuronal survival (those that belong to the trk family of receptors) decrease with aging compared with the level of the apoptotic NGF receptor, the 75-kd neurotrophin receptor.51
In summary, we confirmed and expanded previous findings showing different innervation density in different parts of the body. We also found an age-associated decrease in cutaneous innervation, at least of facial skin. Both aging and photoaging may play a role in the deterioration of the sensory innervation network.
AUTHOR INFORMATION
Accepted for publication July 27, 2002.
Corresponding author and reprints: Isabelle Besné, PhD, Direction des Science du Vivant, L'Oréal Recherche, 90 rue du général Roguet, 92583 Clichy, France (e-mail: ibesne{at}recherche.loreal.com).
From the Direction des Sciences du Vivant, L'Oréal Recherche, Clichy, France.
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