- Email: [email protected]
Changes in Tactile Spatial Discrimination and Cutaneous Coding Properties by Skin Hydration in the Elderly
Changes in Tactile Spatial Discrimination and Cutaneous Coding Properties by Skin Hydration in the Elderly Jean-Luc LeÂveÃque, Johanna Dresler, Edith Ribot-Ciscar,* Jean-Pierre Roll,* and Christine Poelman²
L'OreÂal-Recherche, Clichy, France; *Laboratoire de Neurobiologie Humaine UMR, CNRS 6562, Universite de Provence, Marseille, France; ²Faculte de Pharmacie, Universite de Paris 5, Paris, France
Neurosensory tactile functions were investigated in human subjects by two different and complementary experimental approaches. First, a conventional psychophysical method (two-point gap discrimination) was used to determine the tactile discrimination threshold by analyzing the subjects' ability to detect a gap of variable width between two contact points when a series of stimuli was applied to the skin. Using this method we con®rmed the marked degradation of tactile spatial acuity with age and showed that skin discriminative function was partially restored after hydration of the skin with a moisturizer. The second approach consisted of a micro-
neurographic recording of tactile afferent ®bers in response to two types of mechanical stimuli applied reproducibly to the corresponding receptive ®elds. With this method, we found that the afferent messages were depressed following hydration of the skin surface. Thus, partial restoration of tactile spatial acuity after hydration appears to be due to both a softening of the stratum corneum permitting better localization of the stimulus and a weaker transfer of the stimulus toward the sensory receptors. Key words: aging/hydration/tactile spatial discrimination. J Invest Dermatol 115:454±458, 2000
I
skin temperature, subject laterality, and sex (Verillo, 1979; Stevens and Choo, 1996). One of the widely used, standardized methods for assessing the spatial acuity of the skin is the two-point gap discrimination (TPGD) technique (Stevens and Choo, 1996). By means of this method, they ®nd that spatial acuity declines with age more on some body regions such as hands and feet than others. Some believe that the differential aging of skin spatial acuity may be explained by alterations in sensory receptors (Cauna and Montagna, 1965), but others suggest that local circulatory diseases may be the cause (Grif®n, 1990; Stevens and Patterson, 1995). Skin mechanoreceptors might be affected by changes in the biophysical properties of skin (Verillo et al, 1998), and in this work we have studied the effects of changes in stratum corneum hydration and the consequent changes in tactile spatial acuity. For this purpose, the unitary responses of slowly adapting pressure receptors and fast adapting super®cial units to calibrated mechanical stimuli were recorded by means of the microneurographic method. In parallel the effects of stratum corneum hydration on spatial acuity were investigated using the TPGD method in aged subjects. The results suggest that the changes in the cutaneous afferent messages observed might be responsible for the improvement in the subjects' tactile discrimination thresholds after their skin had been hydrated.
n addition to its protective and regulating functions as a body envelope, the skin is also a sense organ, as it is richly endowed with sensory nerve endings and receptors (Hilliges et al, 1995; Reilly et al, 1997). The information originating from the sensory receptors mediates body representation and makes it possible to explore the physical world. The neural basis of the cutaneous sensations arising from both glabrous and hairy skin has been thoroughly documented, thanks in particular to the use of percutaneous microelectrodes to record the activity of human peripheral afferent ®bers (Vallbo and Hagbarth, 1968). Detailed information about the various types of human tactile receptors, their properties, and the organization of tactile afferent messages is now available (Johansson and Vallbo, 1983) It is also possible to simultaneously perform psychophysical measurements of cutaneous sensations and record neural afferent activities from various receptor types in human subjects in order to correlate sensations with the corresponding perceptual events (KnibestoÈl and Vallbo, 1980). Experimental studies have shown that many factors may profoundly affect tactile sensations. Physical characteristics of skin itself are probably important determinants (Verrillo et al, 1996; 1998; Warner et al, 1988a, b). Moreover, cognitive or motor factors can also affect the transit and central processing of tactile information (Roll, 1987; Milne et al, 1988). The spatial acuity of the skin varies considerably from site to site, as demonstrated by ®ndings subsequently used to construct speci®c reference maps. These maps are also dependent on factors such as
MATERIALS AND METHODS Assessment of spatial acuity Spatial acuity was evaluated by the TPGD method with the minor modi®cations described by Stevens and Choo (1996). In short this method consists in analyzing the subject's perception of a gap of variable width between two contact points when a series of stimulators is applied to the skin. Clearly, the lower the TPGD, the higher the spatial acuity. Two experiments were carried out. The ®rst consisted in reproducing the results of Stevens and Choo demonstrating a decrease in spatial acuity in elderly compared with young subjects and a lower TPGD
Manuscript received June 30, 1999; revised May 24, 2000; accepted for publication May 25, 2000. Reprint requests to: Dr Jean-Luc LeÂveÃque, L'OreÂal-Recherche, 90 rue du GeÂneÂral Roguet, 92583 Clichy Cedex, France. Email: jlleveque@ recherche.loreal.com Abbreviation: TPGD, two-point gap discrimination. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 454
VOL. 115, NO. 3 SEPTEMBER 2000
when stimulators were applied to the cheek compared with the ventral surface of the forearm. In this ®rst experiment we also intended to investigate the in¯uence of laterality and preconditioning, i.e., the in¯uence of the day of measurement when two measurements are carried out on 2 d consecutively. The second experiment consisted in studying the effects of stratum corneum hydration using a cosmetic product on the TPGD of the cheek in a group of elderly subjects. These two experiments were carried out with the same stimulators and the same experimental procedure. The stimulators were carefully cut from a polyvinyl chloride plate, 5 mm thick. Thirty stimulators were used with a gap varying from 0.5 to 50 mm between the two prongs. One of them is illustrated in Fig 1. Although it is known that TPGD is independent of the pressure applied to the skin, especially in young individuals (Johnson and Phillips, 1981), the experimenter applied a calibrated, reproducible pressure of 200 6 50 g, identical to that used by Stevens and Choo (1996). The experimental procedure involved applying to the skin, successively and in a random manner, the two opposite sides of the stimulator: the ¯at side and the side with the two prongs. The subject had to say ``one'' or ``two'' depending on whether he perceived one or two points of contact. If the subject's response was wrong, the test was repeated with a stimulator gap between the two prongs increased by 2 mm. Conversely, if the subject's response was correct twice in succession, the distance between the two prongs was decreased by 2 mm. A correct response must always be duplicated. This procedure was repeated until there had been six transitions (from an increase to a decrease or vice versa). The same procedure was then repeated but stimulators with increases or decreases of only 1 mm in prong distance. After these new transitions, the sensitivity threshold was calculated as the geometric mean of the last six transitions. The mean time for a determination of TPGD was about 10 min. Experiment 1 Experiment 1 was conducted on two groups of 13 subjects: a young group (mean age 24 6 3 y), and an elderly group (mean age 66.5 6 5 y). Measurements were carried out on two body sites: the top of the cheek (probe parallel to the cephalocaudal axis), and the middle of the ventral side of the forearm (stimulus parallel to the axis of the forearm). The results from two groups of subjects were compared over two consecutive days (day 0 and day 1) on symmetrical sites: right and left cheeks (RC, LC) and right and left forearm (RF, LF). Subjects were asked not to apply
Figure 1. Representative stimulator punched out of a 5 mm thick polyvinyl chloride plate. Thirty stimulators, with the distance between the two prongs varying from 0.5 to 50 mm, were used.
Figure 2. Example of the two electromechanical devices used to apply calibrated and reproducible deformations on the skin when analyzing the nervous responses of mechanoreception afferents. (a) Vertical and deep deformation in the case of slowly adapting cutaneous receptors; (b) tangential super®cial deformation in the case of fast adapting cutaneous receptors.
SKIN TACTILE PROPERTIES AND HYDRATION
455
cosmetic products to the skin before the experiment. The statistical signi®cance of the results was assessed by an analysis of variance (SAS, mixed procedure) taking into account the time (day 0 or day 1), the side (right and left), the age of the subjects, and any interaction between these factors. Experiment 2 The effects of hydration were studied in 12 women in the elderly group (mean age 69.5 6 5 y). These subjects were instructed to apply a ¯uid moisturizing oil-in-water emulsion containing 5% glycerol (pH = 7) to one of their cheeks. Six subjects treated the right cheek and six the left cheek. Tactile discrimination thresholds were determined 30 min after application with measurements beginning randomly on one or the other cheek. The change in stratum corneum hydration was checked by measuring its electrical capacitance by means of a Corneometer CM 820 (Fluhr et al, 1999). Measurements were carried out on the same anatomic sites, just after the measurement of TPGD. The signi®cance of the results was tested by means of the Wilcoxon signed ranks test (p < 0.05 regarded as signi®cant). In both experiments (spatial acuity, in¯uence of hydration), subjects gave their signed informed consent. Analysis of cutaneous transductive properties A neurophysiologic study was conducted to investigate whether the transductive properties of cutaneous receptors are changed by skin hydration. Experiments were performed with six healthy subjects aged 22±33 y, all of whom gave their informed consent to the procedure. We also obtained the approval of the local ethics committee (CCP-PRB-Marseille I). The subjects were comfortably seated in a dentist's armchair with their legs positioned in cushioned grooves in a standardized relaxed position (knee joint angle 120°±130°). The unitary activities of four fast adapting (FA) and four slowly adapting (SA) cutaneous receptors were recorded with the microneurographic method introduced by Vallbo and Hagbarth in 1968 on the lateral peroneal nerve, which innervates the anterior part of the leg and the dorsal and lateral surfaces of the feet and toes. Single unit recordings were performed using insulated metal microelectrodes (tip diameter about 5 mm, impedance 1 MW tested at 1000 Hz; Frederick Haer, U.S.A.), which were manually inserted percutaneously at the level of the popliteal fossea. The bandpass of the recordings was limited to 300±3000 Hz to ensure an optimal signal-tonoise ratio. The microelectrode recordings were transmitted to an oscilloscope and a loudspeaker so that any multiunit activities indicating that the electrode was in the nerve could be detected. The skin on the anterior part of the leg, the foot, and the toes was stroked by the experimenter's hand to facilitate the recording of a single unitary activity by adjusting the position of the microelectrode in minute steps. The units were classi®ed on the basis of their adaptive and receptive ®eld properties (Johansson and Vallbo, 1983). Brie¯y, the units showing a sustained increase in their ®ring patterns in response to maintained forces were classi®ed as SA units, whereas those responding to light scratching of the innervated skin area, falling silent during maintained forces but responding during application of the stimulus, were taken to be FA units. The SA afferents were activated by means of a small device with a ¯at circular contact surface (diameter 5 mm; see Fig 2a). It was connected to both an electromechanical transducer through which the forces applied to the skin were recorded and a photo-cell allowing control of the displacement of the probe tip. The forces applied were sinusoidal with an amplitude of 2N. The FA afferents were activated by lightly stroking the skin by means of a paint-brush (see Fig 2b), which was also equipped with a force transducer to control the constancy of the stimulus before and after hydration. Depending on the afferent type, one of the two devices was ®xed to a jointed arm with which it could be placed at any point on the leg
456
LEÂVEÃQUE ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
or foot, depending on the receptive ®eld of interest. It was placed perpendicularly to the zone of maximum sensitivity of the receptive ®eld and locked in place. The experiments consisted in recording the response of the afferent both before and 3 min after stratum corneum hydration. The unitary nerve activity and electromechanical signals were ®rst stored on an analog tape recorder and then digitized by means of a computer. The spikes were converted into standard pulses using a level discriminator. The mean discharge frequency of the units was determined at each stroke of the skin in the case of the FA units, and for each skin indentation in the case of the SA ®bers. The signi®cance of the results was tested by means of paired t tests.
RESULTS TPGD Results of TPGD, depending on the group of subjects (young and elderly), the day of measurement (day 0 and day 1), and the side of application of the stimulator (left and right) are given in Table I for each body site investigated (forearm and cheek). Results are expressed as mean 6 SEM and their statistical signi®cance, obtained by the analysis of variance, is given. TPGD is not affected by the day of measurement nor by the laterality (right or left side). Only age emerges as a signi®cant factor to explain the differences in spatial acuity on a given anatomic site. Spatial acuity decreases by approximately 50% on the cheek (p < 0.008) and 30% on the forearm (p < 0.04) when the mean age of the subjects increases from 24 to 66 y. TPGD on the cheek is lower than TPGD on the forearm irrespective of the age of the volunteers (p < 0.001). The results are shown in Fig 3. The effects of hydration of the cheek stratum corneum on aged persons are illustrated in Fig 4 Hydration leads to an increase in skin electrical capacitance from 77 6 2 to 106 6 5 (mean 6 SEM,
arbitrary units). At the same time, TPGD decreases from 10.9 6 0.8 to 7.5 6 0.87 (mean 6 SEM, millimeters), which corresponds to a signi®cant 31% increase in spatial acuity. These results are shown in Fig 4(a) and 4(b) respectively as the median value and the 25th and 75th percentiles. Changes after treatment are highly signi®cant (p < 0.001 and p < 0.009, respectively). The Pearson correlation coef®cient (0.55) only expresses a weak intensity of correlation between the increase in capacitance and the decrease in TPGD (p = 0.07). Cutaneous coding properties Figure 5(a) gives, as an example, the response of an FA cutaneous afferent for two forward/backward movements of the paint-brush. The stimulus, which super®cially stroked the area of maximum sensitivity in the cutaneous receptive ®eld under investigation, was found to be an extremely effective means of activating receptors of this kind. The ®bers began to discharge at the onset of the stimulus, remained active throughout the stroking, and stopped discharging as soon as the paint-brush left the receptive ®eld. As can be seen in this ®gure, the units showed a preferred direction in which the stimulus was more effective in activating the unit, i.e., in the forward direction (from foot to thigh)
Figure 3. In¯uence of age on the two-point gap discrimination threshold measured on cheeks and forearms of two groups of volunteers. Means and standard deviations are represented: young group, n = 13, mean age 24 y; aged group, n = 13, mean age 66.5 y. The difference in TPGD between cheek and forearm is highly signi®cant (p < 0.001) whatever the age of the volunteers.
Table I. Two-point gap discrimination on forearms and cheeks Statistical results Factors
TPGD/forearms mean 6 SEM (mm)
TPGD/cheeks mean 6 SEM (mm)
Elderly (n = 13) Young (n = 13) Day 0 Day 1 Left side Right side
26.94 6 1.75 21.23 6 1.68 (p = 0.033) 23.86 6 1.43 24.31 6 1.42 (NS) 23.3 6 1.40 24.3 6 1.40 (NS)
9.98 6 0.89 6.65 6 0.86 (p = 0.0072) 8.74 6 0.75 7.89 6 0.75 (NS) 8.97 6 0.78 7.67 6 0.78 (NS)
Figure 4. In¯uence of hydration on capacitance and TPGD. (a) capacitance; (b) TPGD. These results were obtained on a group of 12 aged volunteers (mean age 69.5 y). Measurements were made alternately on the control and the treated cheek, 30 min after the application of an oil-inwater emulsion. Results are expressed as median value and 25th and 75th percentiles.
VOL. 115, NO. 3 SEPTEMBER 2000
Figure 5. In¯uence of skin hydration on the response of fast adapting cutaneous receptors (FA). (a) Example of the response of an FA cutaneous afferent to super®cial stroking of the skin; (b) mean responses of a population of FA units (n = 5) before (control) and 3 min after skin hydration.
the number of spikes composing the burst of activity was higher than in the opposite direction. The mean frequency of discharge was determined during both forward and backward movement of the stimulus for nine successive cycles of stimulus displacement. It was normalized as a percentage of the maximum frequency of discharge obtained for one of the cycles. Figure 5(b) gives the mean percentage of response for the units tested before (control) and 3 min after skin hydration. The ®gure shows that hydration gave rise to a highly signi®cant decrease in the response of the FA cutaneous receptors to skin stroking (t = 12.5, p < 0.001). The response of the SA cutaneous receptors to sinusoidal skin indentations is illustrated in Fig 6(a). The unit started discharging at certain threshold of skin indentation, increased its ®ring rate as the force was enhanced, and progressively stopped discharging as the probe was released. As previously described, the mean discharge frequency was calculated and normalized for all the units tested. As in the case of the FA receptors, the SA receptors were observed to be less responsive to the stimulus after stratum corneum hydration (see Fig 6b; t = 3.65, p < 0.001). DISCUSSION By combining psychophysic and neurophysiologic approaches, our results suggest that hydration of the stratum corneum improves the spatial discriminative power of the skin in aged subjects. One of the factors in such an improvement may take place at the peripheral level by modifying the transfer properties of the stimulus to the tactile sensory receptors. The neurosensory effects induced by the application of the stimuli to the skin during the TPGD study probably involve both rapidly and slowly adapting mechanoreceptors that are activated by the stimulus. Meissner and Pacinian corpuscles respond to the application and release of the stimulus whereas Ruf®ni endings and
SKIN TACTILE PROPERTIES AND HYDRATION
457
Figure 6. In¯uence of skin hydration on the response of slow adapting cutaneous receptors (SA). (a) Example of the reponse of an SA cutaneous afferent to sinusoidal indentations of the skin; (b) mean responses of a population of SA afferents (n = 5) before (control) and 3 min after skin hydration. The results are expressed as percentages of the maximum frequency of discharge obtained for one of the cycles in the control period for each unit.
Merkel cells exhibit a sustained discharge during the maintenance of a constant pressure (Vedel and Roll, 1982; Johanson and Vallbo, 1983; Ribot-Ciscar et al, 1989). A two-point discrimination then depends on population coding factors and on mechanisms of lateral inhibition, which are involved all along the ascending pathways and the corresponding cortical areas (Mountcastel, 1984). In this study, the stimuli were applied according to the tracking method described by Stevens and Choo (1996). The results con®rmed that tactile acuity decreases with age in a highly signi®cant manner in all body sites. Moreover, results are also in line with those of Weinstein (1968) in showing the absence of a difference in skin spatial acuity depending on the side of stimulation as well as on the day of test, suggesting no preconditioning of the subjects in estimating the tactile perceptual threshold. As represented on Fig 4(a, b), the application of a moisturizing emulsion on the cheek skin has two consequences: a marked increase in the skin electrical conductance and a decrease in the TPGD, which means an increase in the skin spatial acuity. The ®rst result illustrates a well-known phenomenon interpreted as an increase of the hydration of the stratum corneum (Fluhr et al, 1999). Hydrating the stratum corneum also increases its mechanical extensibility or compliance (de Rigal and LeÂveÁque, 1985). It is the ®rst time to our knowledge, however, that the positive in¯uence of hydration on skin spatial acuity is presented. The variations of these two parameters are weakly correlated (p = 0.07). This is probably due to both the use of a psychophysical method, which generates less accurate data than an objective method, and the relatively small number of observations. As discussed below, many biologic mechanisms have been proposed to explain why tactile acuity decreases with age. Our results suggest that dehydration of the skin surface, which is very often associated with aging, may be one of the causes.
458
LEÂVEÃQUE ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the barrier function, which is provided essentially by the super®cial keratinized layers of the epidermis. The epidermis also acts as a sensory organ by serving as a mediator between applied mechanical stimuli and the various tactile receptors it contains. It transmits this tactile information to ensure the sensory continuum of the tissue, all whilst enabling adequate local discrimination. The adequate water content would therefore be crucial for it to ful®ll all of its functions. Figure 7. Schematic deformations of the skin when applying a stimulator in the case of stratum corneum (a) dry and (b) hydrated. In (b), tactile receptors located between the two prongs of the stimulator would be less or not stretched.
This hypothesis was evaluated neurophysiologically by determination of cutaneous transductive properties following a reproducible series of mechanical stimuli applied on the skin surface. In both experimental conditions (periodic indentation of 1.5 mm and periodic super®cial stroking of the skin), the response of a localized receptor was decreased. It has to be remembered that the amplitude of the recorded signal corresponds to the mean frequency of the spikes in the afferent ®bers. The observed decreases in mean frequencies were highly signi®cant and most probably correspond to a decrease in the mechanical transfer function of the skin in response to the applied stimulus, due to a decrease in the modulus of elasticity of the hydrated stratum corneum. The depressed afferent messages do not fully explain the agerelated decrease in tactile spatial acuity, however. In fact, as noted in Methods, the responses in this psychophysical TPGD test are virtually independent of the pressure of the stimulus on the skin. The subject's response (``one'' or ``two'') depends in fact on the perception of a gap between the two prongs of the probe. When the softness of the skin is modi®ed by hydration, the deformation pro®le of the skin is modi®ed in turn, as schematically outlined in Fig 7. In such a case, the mechanoreceptors located between the two prongs have fewer or no constraints and therefore become silent. Furthermore, even a suf®cient contrast between the afferent messages emitted by the receptors placed under the prongs of the stimulator and those placed in the intermediate zone could allow a correct two-point discrimination. Numerous hypotheses have been advanced to explain the agerelated loss of tactile acuity, including a progressive decrease in the density of certain receptors (Bolton et al, 1965; Cauna, 1965), a gradual deterioration of central nervous system function, or degeneration of the peripheral microcirculation (Stevens and Patterson, 1995). A modi®cation of cutaneous compliance has also been proposed but has not been con®rmed experimentally (Woodward, 1993). In contrast to these studies, our ®ndings support the hypothesis that hydration increases tactile spatial acuity in the elderly by softening the stratum corneum. Consequently, the biomechanical properties of the skin, and more particularly of the epidermis, would deeply in¯uence the cutaneous discriminative function. It is worth noting that these results are in line with others, recently published, showing that skin hydration directly affects the subjective magnitude of the roughness of various standard grades of sand papers (Verrillo et al, 1998). To conclude, a large body of research accumulated over the years has clearly established the importance of water in the functional properties of the skin. This has been demonstrated in particular for
REFERENCES Bolton CF, Winkelmann RK, Dyck PJ: A quantitative study of Meisner's corpuscles in man. Neurology 16:1±9, 1965 Cauna N: The effects of aging on the receptor organs of the human dermis. In Montagna W (eds). Advances in Biology of Skin. Elmford, NY: Pergamon Press, 1965, pp 63±96 De Rigal J, LeÂveÁque JL: In vivo measurement of the stratum corneum elasticity. Bioeng Skin 1:13±23, 1985 Fluhr JW, Gloor M, Lazzerini S, Kleesz P, Gineshaber R, Berardesca E: Comparative study of ®ve instruments measuring stratum corneum hydration. Skin Res Technol 5:171±178, 1999 Grif®n MJ: Handbook of human vibration. London: Academic Press, 1990 Hilliges M, Wang L, Johansson O: Ultrastructural evidence for nerve ®bers within all vital layers of the human epidermis. J Invest Dermatol 104:134±137, 1995 Johansson RS, Vallbo AB: Tactile sensory coding in the glabrous skin of the human hand. Trends Neurosci 6:27±31, 1983 Johnson KO, Phillips JR: Tactile spatial resolution: I Two-point discrimination, gap detection, grating resolution, and letter recognition. J Neurophysiol 46: 1177±1191, 1981 KnibestoÈl M, Vallbo AB: Intensity of sensation related to activity of slowly adapting mechanoreceptive units in the human land. J Physiol Land 300:251±267, 1980 Milne RJ, Aniss AM, Kay NE, Gandevia SC: Reduction in perceived intensity of cutaneous stimuli during movement: a quantitative study. Exp Brain Res 70:569±576, 1988 Mountcastel VB. Central nervous mechanisms in mechanoreceptive sensibility. In: Darian-Smith I (ed.). Handbook of Physiology, Section 1: the Nervous System, Vol III Sensory Processes, Part 2, Bethesda, MD: American Physiological Society, 1984, pp 789±878 Reilly DM, Ferdinando D, Johnston C, Shaw C, Buchanan KD, Green MR: The epidermal nerve ®bre network: characterization of nerve ®bres in human skin by confocal microscopy and assessment of racial variations. Br J Dermatol 137:163±170, 1997 Ribot-Ciscar E, Vedel JP, Roll JP: Vibration sensitivity of slowly and rapidly adapting cutaneous mechanoreceptors in the human foot and leg. Neurosci Lett 104:130±135, 1989 Roll JP. Fonction de prise d'information et d'exploration. In: Piaget J, Nounond P, Branckart JP (eds). Psychologie, Encyclopedie de la PleõÈade. Paris: Gallimard, 1987, pp 1476±1535 Stevens JC, Choo KK: Spatial acuity of the body surface over the life span. Somatosens Mot Res 13:153±166, 1996 Stevens JC, Patterson MQ: Dimensions of spatial acuity in the touch sense: changes over the life span. Somatosens Mot Res 12:29±47, 1995 Vallbo AB, Hagbarth KE: Activity from skin mechanoreceptors recorded percutaneously in awake human subjects. Exp Neurol 21:270±289, 1968 Vedel JP, Roll JP: Response to pressure and vibration of slowly adapting cutaneous mechanoreceptors in the human foot. Neurosci Lett 34:289±294, 1982 Verillo RT: Comparison of vibrotactile threshold and suprathreshold responses in men and women. Percept Psychophys 26:20±24, 1979 Verrillo RT, Bolanowski SJ, Baran F, Smith PF: Effects of underwater environmental conditions on vibrotactile thresholds. J Acoust Soc Am 100:651±658, 1996 Verrillo RT, Bolanowski SJ, Checkosky CM, Glonefp MC: Effects of hydration on tactile sensation. Somatosens Mot Res 15:93±108, 1998 Warner RR, Myers MC, Taylor DA: Electron probe analysis of human skin: element concentration pro®les. J Invest Dermatol 90:78±85, 1988a Warner RR, Myers MC, Taylor DA: Electron probe analysis of human skin: determination of the water concentration pro®le. J Invest Dermatol 90:218±224, 1988b Weinstein S. Intensive and extensive aspects of tactile sensitivity as a function of body part, sex and laterality. In: Kenshalo DR (ed.). The Skin Senses, Spring®eld: Charles C Thomas, 1968, pp 195±222 Woodward KL: The relationship between skin compliance, age, gender and tactile discriminative thresholds in humans. Somatosens Mot Res 10:63±67, 1993
L'OreÂal-Recherche, Clichy, France; *Laboratoire de Neurobiologie Humaine UMR, CNRS 6562, Universite de Provence, Marseille, France; ²Faculte de Pharmacie, Universite de Paris 5, Paris, France
Neurosensory tactile functions were investigated in human subjects by two different and complementary experimental approaches. First, a conventional psychophysical method (two-point gap discrimination) was used to determine the tactile discrimination threshold by analyzing the subjects' ability to detect a gap of variable width between two contact points when a series of stimuli was applied to the skin. Using this method we con®rmed the marked degradation of tactile spatial acuity with age and showed that skin discriminative function was partially restored after hydration of the skin with a moisturizer. The second approach consisted of a micro-
neurographic recording of tactile afferent ®bers in response to two types of mechanical stimuli applied reproducibly to the corresponding receptive ®elds. With this method, we found that the afferent messages were depressed following hydration of the skin surface. Thus, partial restoration of tactile spatial acuity after hydration appears to be due to both a softening of the stratum corneum permitting better localization of the stimulus and a weaker transfer of the stimulus toward the sensory receptors. Key words: aging/hydration/tactile spatial discrimination. J Invest Dermatol 115:454±458, 2000
I
skin temperature, subject laterality, and sex (Verillo, 1979; Stevens and Choo, 1996). One of the widely used, standardized methods for assessing the spatial acuity of the skin is the two-point gap discrimination (TPGD) technique (Stevens and Choo, 1996). By means of this method, they ®nd that spatial acuity declines with age more on some body regions such as hands and feet than others. Some believe that the differential aging of skin spatial acuity may be explained by alterations in sensory receptors (Cauna and Montagna, 1965), but others suggest that local circulatory diseases may be the cause (Grif®n, 1990; Stevens and Patterson, 1995). Skin mechanoreceptors might be affected by changes in the biophysical properties of skin (Verillo et al, 1998), and in this work we have studied the effects of changes in stratum corneum hydration and the consequent changes in tactile spatial acuity. For this purpose, the unitary responses of slowly adapting pressure receptors and fast adapting super®cial units to calibrated mechanical stimuli were recorded by means of the microneurographic method. In parallel the effects of stratum corneum hydration on spatial acuity were investigated using the TPGD method in aged subjects. The results suggest that the changes in the cutaneous afferent messages observed might be responsible for the improvement in the subjects' tactile discrimination thresholds after their skin had been hydrated.
n addition to its protective and regulating functions as a body envelope, the skin is also a sense organ, as it is richly endowed with sensory nerve endings and receptors (Hilliges et al, 1995; Reilly et al, 1997). The information originating from the sensory receptors mediates body representation and makes it possible to explore the physical world. The neural basis of the cutaneous sensations arising from both glabrous and hairy skin has been thoroughly documented, thanks in particular to the use of percutaneous microelectrodes to record the activity of human peripheral afferent ®bers (Vallbo and Hagbarth, 1968). Detailed information about the various types of human tactile receptors, their properties, and the organization of tactile afferent messages is now available (Johansson and Vallbo, 1983) It is also possible to simultaneously perform psychophysical measurements of cutaneous sensations and record neural afferent activities from various receptor types in human subjects in order to correlate sensations with the corresponding perceptual events (KnibestoÈl and Vallbo, 1980). Experimental studies have shown that many factors may profoundly affect tactile sensations. Physical characteristics of skin itself are probably important determinants (Verrillo et al, 1996; 1998; Warner et al, 1988a, b). Moreover, cognitive or motor factors can also affect the transit and central processing of tactile information (Roll, 1987; Milne et al, 1988). The spatial acuity of the skin varies considerably from site to site, as demonstrated by ®ndings subsequently used to construct speci®c reference maps. These maps are also dependent on factors such as
MATERIALS AND METHODS Assessment of spatial acuity Spatial acuity was evaluated by the TPGD method with the minor modi®cations described by Stevens and Choo (1996). In short this method consists in analyzing the subject's perception of a gap of variable width between two contact points when a series of stimulators is applied to the skin. Clearly, the lower the TPGD, the higher the spatial acuity. Two experiments were carried out. The ®rst consisted in reproducing the results of Stevens and Choo demonstrating a decrease in spatial acuity in elderly compared with young subjects and a lower TPGD
Manuscript received June 30, 1999; revised May 24, 2000; accepted for publication May 25, 2000. Reprint requests to: Dr Jean-Luc LeÂveÃque, L'OreÂal-Recherche, 90 rue du GeÂneÂral Roguet, 92583 Clichy Cedex, France. Email: jlleveque@ recherche.loreal.com Abbreviation: TPGD, two-point gap discrimination. 0022-202X/00/$15.00
´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 454
VOL. 115, NO. 3 SEPTEMBER 2000
when stimulators were applied to the cheek compared with the ventral surface of the forearm. In this ®rst experiment we also intended to investigate the in¯uence of laterality and preconditioning, i.e., the in¯uence of the day of measurement when two measurements are carried out on 2 d consecutively. The second experiment consisted in studying the effects of stratum corneum hydration using a cosmetic product on the TPGD of the cheek in a group of elderly subjects. These two experiments were carried out with the same stimulators and the same experimental procedure. The stimulators were carefully cut from a polyvinyl chloride plate, 5 mm thick. Thirty stimulators were used with a gap varying from 0.5 to 50 mm between the two prongs. One of them is illustrated in Fig 1. Although it is known that TPGD is independent of the pressure applied to the skin, especially in young individuals (Johnson and Phillips, 1981), the experimenter applied a calibrated, reproducible pressure of 200 6 50 g, identical to that used by Stevens and Choo (1996). The experimental procedure involved applying to the skin, successively and in a random manner, the two opposite sides of the stimulator: the ¯at side and the side with the two prongs. The subject had to say ``one'' or ``two'' depending on whether he perceived one or two points of contact. If the subject's response was wrong, the test was repeated with a stimulator gap between the two prongs increased by 2 mm. Conversely, if the subject's response was correct twice in succession, the distance between the two prongs was decreased by 2 mm. A correct response must always be duplicated. This procedure was repeated until there had been six transitions (from an increase to a decrease or vice versa). The same procedure was then repeated but stimulators with increases or decreases of only 1 mm in prong distance. After these new transitions, the sensitivity threshold was calculated as the geometric mean of the last six transitions. The mean time for a determination of TPGD was about 10 min. Experiment 1 Experiment 1 was conducted on two groups of 13 subjects: a young group (mean age 24 6 3 y), and an elderly group (mean age 66.5 6 5 y). Measurements were carried out on two body sites: the top of the cheek (probe parallel to the cephalocaudal axis), and the middle of the ventral side of the forearm (stimulus parallel to the axis of the forearm). The results from two groups of subjects were compared over two consecutive days (day 0 and day 1) on symmetrical sites: right and left cheeks (RC, LC) and right and left forearm (RF, LF). Subjects were asked not to apply
Figure 1. Representative stimulator punched out of a 5 mm thick polyvinyl chloride plate. Thirty stimulators, with the distance between the two prongs varying from 0.5 to 50 mm, were used.
Figure 2. Example of the two electromechanical devices used to apply calibrated and reproducible deformations on the skin when analyzing the nervous responses of mechanoreception afferents. (a) Vertical and deep deformation in the case of slowly adapting cutaneous receptors; (b) tangential super®cial deformation in the case of fast adapting cutaneous receptors.
SKIN TACTILE PROPERTIES AND HYDRATION
455
cosmetic products to the skin before the experiment. The statistical signi®cance of the results was assessed by an analysis of variance (SAS, mixed procedure) taking into account the time (day 0 or day 1), the side (right and left), the age of the subjects, and any interaction between these factors. Experiment 2 The effects of hydration were studied in 12 women in the elderly group (mean age 69.5 6 5 y). These subjects were instructed to apply a ¯uid moisturizing oil-in-water emulsion containing 5% glycerol (pH = 7) to one of their cheeks. Six subjects treated the right cheek and six the left cheek. Tactile discrimination thresholds were determined 30 min after application with measurements beginning randomly on one or the other cheek. The change in stratum corneum hydration was checked by measuring its electrical capacitance by means of a Corneometer CM 820 (Fluhr et al, 1999). Measurements were carried out on the same anatomic sites, just after the measurement of TPGD. The signi®cance of the results was tested by means of the Wilcoxon signed ranks test (p < 0.05 regarded as signi®cant). In both experiments (spatial acuity, in¯uence of hydration), subjects gave their signed informed consent. Analysis of cutaneous transductive properties A neurophysiologic study was conducted to investigate whether the transductive properties of cutaneous receptors are changed by skin hydration. Experiments were performed with six healthy subjects aged 22±33 y, all of whom gave their informed consent to the procedure. We also obtained the approval of the local ethics committee (CCP-PRB-Marseille I). The subjects were comfortably seated in a dentist's armchair with their legs positioned in cushioned grooves in a standardized relaxed position (knee joint angle 120°±130°). The unitary activities of four fast adapting (FA) and four slowly adapting (SA) cutaneous receptors were recorded with the microneurographic method introduced by Vallbo and Hagbarth in 1968 on the lateral peroneal nerve, which innervates the anterior part of the leg and the dorsal and lateral surfaces of the feet and toes. Single unit recordings were performed using insulated metal microelectrodes (tip diameter about 5 mm, impedance 1 MW tested at 1000 Hz; Frederick Haer, U.S.A.), which were manually inserted percutaneously at the level of the popliteal fossea. The bandpass of the recordings was limited to 300±3000 Hz to ensure an optimal signal-tonoise ratio. The microelectrode recordings were transmitted to an oscilloscope and a loudspeaker so that any multiunit activities indicating that the electrode was in the nerve could be detected. The skin on the anterior part of the leg, the foot, and the toes was stroked by the experimenter's hand to facilitate the recording of a single unitary activity by adjusting the position of the microelectrode in minute steps. The units were classi®ed on the basis of their adaptive and receptive ®eld properties (Johansson and Vallbo, 1983). Brie¯y, the units showing a sustained increase in their ®ring patterns in response to maintained forces were classi®ed as SA units, whereas those responding to light scratching of the innervated skin area, falling silent during maintained forces but responding during application of the stimulus, were taken to be FA units. The SA afferents were activated by means of a small device with a ¯at circular contact surface (diameter 5 mm; see Fig 2a). It was connected to both an electromechanical transducer through which the forces applied to the skin were recorded and a photo-cell allowing control of the displacement of the probe tip. The forces applied were sinusoidal with an amplitude of 2N. The FA afferents were activated by lightly stroking the skin by means of a paint-brush (see Fig 2b), which was also equipped with a force transducer to control the constancy of the stimulus before and after hydration. Depending on the afferent type, one of the two devices was ®xed to a jointed arm with which it could be placed at any point on the leg
456
LEÂVEÃQUE ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
or foot, depending on the receptive ®eld of interest. It was placed perpendicularly to the zone of maximum sensitivity of the receptive ®eld and locked in place. The experiments consisted in recording the response of the afferent both before and 3 min after stratum corneum hydration. The unitary nerve activity and electromechanical signals were ®rst stored on an analog tape recorder and then digitized by means of a computer. The spikes were converted into standard pulses using a level discriminator. The mean discharge frequency of the units was determined at each stroke of the skin in the case of the FA units, and for each skin indentation in the case of the SA ®bers. The signi®cance of the results was tested by means of paired t tests.
RESULTS TPGD Results of TPGD, depending on the group of subjects (young and elderly), the day of measurement (day 0 and day 1), and the side of application of the stimulator (left and right) are given in Table I for each body site investigated (forearm and cheek). Results are expressed as mean 6 SEM and their statistical signi®cance, obtained by the analysis of variance, is given. TPGD is not affected by the day of measurement nor by the laterality (right or left side). Only age emerges as a signi®cant factor to explain the differences in spatial acuity on a given anatomic site. Spatial acuity decreases by approximately 50% on the cheek (p < 0.008) and 30% on the forearm (p < 0.04) when the mean age of the subjects increases from 24 to 66 y. TPGD on the cheek is lower than TPGD on the forearm irrespective of the age of the volunteers (p < 0.001). The results are shown in Fig 3. The effects of hydration of the cheek stratum corneum on aged persons are illustrated in Fig 4 Hydration leads to an increase in skin electrical capacitance from 77 6 2 to 106 6 5 (mean 6 SEM,
arbitrary units). At the same time, TPGD decreases from 10.9 6 0.8 to 7.5 6 0.87 (mean 6 SEM, millimeters), which corresponds to a signi®cant 31% increase in spatial acuity. These results are shown in Fig 4(a) and 4(b) respectively as the median value and the 25th and 75th percentiles. Changes after treatment are highly signi®cant (p < 0.001 and p < 0.009, respectively). The Pearson correlation coef®cient (0.55) only expresses a weak intensity of correlation between the increase in capacitance and the decrease in TPGD (p = 0.07). Cutaneous coding properties Figure 5(a) gives, as an example, the response of an FA cutaneous afferent for two forward/backward movements of the paint-brush. The stimulus, which super®cially stroked the area of maximum sensitivity in the cutaneous receptive ®eld under investigation, was found to be an extremely effective means of activating receptors of this kind. The ®bers began to discharge at the onset of the stimulus, remained active throughout the stroking, and stopped discharging as soon as the paint-brush left the receptive ®eld. As can be seen in this ®gure, the units showed a preferred direction in which the stimulus was more effective in activating the unit, i.e., in the forward direction (from foot to thigh)
Figure 3. In¯uence of age on the two-point gap discrimination threshold measured on cheeks and forearms of two groups of volunteers. Means and standard deviations are represented: young group, n = 13, mean age 24 y; aged group, n = 13, mean age 66.5 y. The difference in TPGD between cheek and forearm is highly signi®cant (p < 0.001) whatever the age of the volunteers.
Table I. Two-point gap discrimination on forearms and cheeks Statistical results Factors
TPGD/forearms mean 6 SEM (mm)
TPGD/cheeks mean 6 SEM (mm)
Elderly (n = 13) Young (n = 13) Day 0 Day 1 Left side Right side
26.94 6 1.75 21.23 6 1.68 (p = 0.033) 23.86 6 1.43 24.31 6 1.42 (NS) 23.3 6 1.40 24.3 6 1.40 (NS)
9.98 6 0.89 6.65 6 0.86 (p = 0.0072) 8.74 6 0.75 7.89 6 0.75 (NS) 8.97 6 0.78 7.67 6 0.78 (NS)
Figure 4. In¯uence of hydration on capacitance and TPGD. (a) capacitance; (b) TPGD. These results were obtained on a group of 12 aged volunteers (mean age 69.5 y). Measurements were made alternately on the control and the treated cheek, 30 min after the application of an oil-inwater emulsion. Results are expressed as median value and 25th and 75th percentiles.
VOL. 115, NO. 3 SEPTEMBER 2000
Figure 5. In¯uence of skin hydration on the response of fast adapting cutaneous receptors (FA). (a) Example of the response of an FA cutaneous afferent to super®cial stroking of the skin; (b) mean responses of a population of FA units (n = 5) before (control) and 3 min after skin hydration.
the number of spikes composing the burst of activity was higher than in the opposite direction. The mean frequency of discharge was determined during both forward and backward movement of the stimulus for nine successive cycles of stimulus displacement. It was normalized as a percentage of the maximum frequency of discharge obtained for one of the cycles. Figure 5(b) gives the mean percentage of response for the units tested before (control) and 3 min after skin hydration. The ®gure shows that hydration gave rise to a highly signi®cant decrease in the response of the FA cutaneous receptors to skin stroking (t = 12.5, p < 0.001). The response of the SA cutaneous receptors to sinusoidal skin indentations is illustrated in Fig 6(a). The unit started discharging at certain threshold of skin indentation, increased its ®ring rate as the force was enhanced, and progressively stopped discharging as the probe was released. As previously described, the mean discharge frequency was calculated and normalized for all the units tested. As in the case of the FA receptors, the SA receptors were observed to be less responsive to the stimulus after stratum corneum hydration (see Fig 6b; t = 3.65, p < 0.001). DISCUSSION By combining psychophysic and neurophysiologic approaches, our results suggest that hydration of the stratum corneum improves the spatial discriminative power of the skin in aged subjects. One of the factors in such an improvement may take place at the peripheral level by modifying the transfer properties of the stimulus to the tactile sensory receptors. The neurosensory effects induced by the application of the stimuli to the skin during the TPGD study probably involve both rapidly and slowly adapting mechanoreceptors that are activated by the stimulus. Meissner and Pacinian corpuscles respond to the application and release of the stimulus whereas Ruf®ni endings and
SKIN TACTILE PROPERTIES AND HYDRATION
457
Figure 6. In¯uence of skin hydration on the response of slow adapting cutaneous receptors (SA). (a) Example of the reponse of an SA cutaneous afferent to sinusoidal indentations of the skin; (b) mean responses of a population of SA afferents (n = 5) before (control) and 3 min after skin hydration. The results are expressed as percentages of the maximum frequency of discharge obtained for one of the cycles in the control period for each unit.
Merkel cells exhibit a sustained discharge during the maintenance of a constant pressure (Vedel and Roll, 1982; Johanson and Vallbo, 1983; Ribot-Ciscar et al, 1989). A two-point discrimination then depends on population coding factors and on mechanisms of lateral inhibition, which are involved all along the ascending pathways and the corresponding cortical areas (Mountcastel, 1984). In this study, the stimuli were applied according to the tracking method described by Stevens and Choo (1996). The results con®rmed that tactile acuity decreases with age in a highly signi®cant manner in all body sites. Moreover, results are also in line with those of Weinstein (1968) in showing the absence of a difference in skin spatial acuity depending on the side of stimulation as well as on the day of test, suggesting no preconditioning of the subjects in estimating the tactile perceptual threshold. As represented on Fig 4(a, b), the application of a moisturizing emulsion on the cheek skin has two consequences: a marked increase in the skin electrical conductance and a decrease in the TPGD, which means an increase in the skin spatial acuity. The ®rst result illustrates a well-known phenomenon interpreted as an increase of the hydration of the stratum corneum (Fluhr et al, 1999). Hydrating the stratum corneum also increases its mechanical extensibility or compliance (de Rigal and LeÂveÁque, 1985). It is the ®rst time to our knowledge, however, that the positive in¯uence of hydration on skin spatial acuity is presented. The variations of these two parameters are weakly correlated (p = 0.07). This is probably due to both the use of a psychophysical method, which generates less accurate data than an objective method, and the relatively small number of observations. As discussed below, many biologic mechanisms have been proposed to explain why tactile acuity decreases with age. Our results suggest that dehydration of the skin surface, which is very often associated with aging, may be one of the causes.
458
LEÂVEÃQUE ET AL
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
the barrier function, which is provided essentially by the super®cial keratinized layers of the epidermis. The epidermis also acts as a sensory organ by serving as a mediator between applied mechanical stimuli and the various tactile receptors it contains. It transmits this tactile information to ensure the sensory continuum of the tissue, all whilst enabling adequate local discrimination. The adequate water content would therefore be crucial for it to ful®ll all of its functions. Figure 7. Schematic deformations of the skin when applying a stimulator in the case of stratum corneum (a) dry and (b) hydrated. In (b), tactile receptors located between the two prongs of the stimulator would be less or not stretched.
This hypothesis was evaluated neurophysiologically by determination of cutaneous transductive properties following a reproducible series of mechanical stimuli applied on the skin surface. In both experimental conditions (periodic indentation of 1.5 mm and periodic super®cial stroking of the skin), the response of a localized receptor was decreased. It has to be remembered that the amplitude of the recorded signal corresponds to the mean frequency of the spikes in the afferent ®bers. The observed decreases in mean frequencies were highly signi®cant and most probably correspond to a decrease in the mechanical transfer function of the skin in response to the applied stimulus, due to a decrease in the modulus of elasticity of the hydrated stratum corneum. The depressed afferent messages do not fully explain the agerelated decrease in tactile spatial acuity, however. In fact, as noted in Methods, the responses in this psychophysical TPGD test are virtually independent of the pressure of the stimulus on the skin. The subject's response (``one'' or ``two'') depends in fact on the perception of a gap between the two prongs of the probe. When the softness of the skin is modi®ed by hydration, the deformation pro®le of the skin is modi®ed in turn, as schematically outlined in Fig 7. In such a case, the mechanoreceptors located between the two prongs have fewer or no constraints and therefore become silent. Furthermore, even a suf®cient contrast between the afferent messages emitted by the receptors placed under the prongs of the stimulator and those placed in the intermediate zone could allow a correct two-point discrimination. Numerous hypotheses have been advanced to explain the agerelated loss of tactile acuity, including a progressive decrease in the density of certain receptors (Bolton et al, 1965; Cauna, 1965), a gradual deterioration of central nervous system function, or degeneration of the peripheral microcirculation (Stevens and Patterson, 1995). A modi®cation of cutaneous compliance has also been proposed but has not been con®rmed experimentally (Woodward, 1993). In contrast to these studies, our ®ndings support the hypothesis that hydration increases tactile spatial acuity in the elderly by softening the stratum corneum. Consequently, the biomechanical properties of the skin, and more particularly of the epidermis, would deeply in¯uence the cutaneous discriminative function. It is worth noting that these results are in line with others, recently published, showing that skin hydration directly affects the subjective magnitude of the roughness of various standard grades of sand papers (Verrillo et al, 1998). To conclude, a large body of research accumulated over the years has clearly established the importance of water in the functional properties of the skin. This has been demonstrated in particular for
REFERENCES Bolton CF, Winkelmann RK, Dyck PJ: A quantitative study of Meisner's corpuscles in man. Neurology 16:1±9, 1965 Cauna N: The effects of aging on the receptor organs of the human dermis. In Montagna W (eds). Advances in Biology of Skin. Elmford, NY: Pergamon Press, 1965, pp 63±96 De Rigal J, LeÂveÁque JL: In vivo measurement of the stratum corneum elasticity. Bioeng Skin 1:13±23, 1985 Fluhr JW, Gloor M, Lazzerini S, Kleesz P, Gineshaber R, Berardesca E: Comparative study of ®ve instruments measuring stratum corneum hydration. Skin Res Technol 5:171±178, 1999 Grif®n MJ: Handbook of human vibration. London: Academic Press, 1990 Hilliges M, Wang L, Johansson O: Ultrastructural evidence for nerve ®bers within all vital layers of the human epidermis. J Invest Dermatol 104:134±137, 1995 Johansson RS, Vallbo AB: Tactile sensory coding in the glabrous skin of the human hand. Trends Neurosci 6:27±31, 1983 Johnson KO, Phillips JR: Tactile spatial resolution: I Two-point discrimination, gap detection, grating resolution, and letter recognition. J Neurophysiol 46: 1177±1191, 1981 KnibestoÈl M, Vallbo AB: Intensity of sensation related to activity of slowly adapting mechanoreceptive units in the human land. J Physiol Land 300:251±267, 1980 Milne RJ, Aniss AM, Kay NE, Gandevia SC: Reduction in perceived intensity of cutaneous stimuli during movement: a quantitative study. Exp Brain Res 70:569±576, 1988 Mountcastel VB. Central nervous mechanisms in mechanoreceptive sensibility. In: Darian-Smith I (ed.). Handbook of Physiology, Section 1: the Nervous System, Vol III Sensory Processes, Part 2, Bethesda, MD: American Physiological Society, 1984, pp 789±878 Reilly DM, Ferdinando D, Johnston C, Shaw C, Buchanan KD, Green MR: The epidermal nerve ®bre network: characterization of nerve ®bres in human skin by confocal microscopy and assessment of racial variations. Br J Dermatol 137:163±170, 1997 Ribot-Ciscar E, Vedel JP, Roll JP: Vibration sensitivity of slowly and rapidly adapting cutaneous mechanoreceptors in the human foot and leg. Neurosci Lett 104:130±135, 1989 Roll JP. Fonction de prise d'information et d'exploration. In: Piaget J, Nounond P, Branckart JP (eds). Psychologie, Encyclopedie de la PleõÈade. Paris: Gallimard, 1987, pp 1476±1535 Stevens JC, Choo KK: Spatial acuity of the body surface over the life span. Somatosens Mot Res 13:153±166, 1996 Stevens JC, Patterson MQ: Dimensions of spatial acuity in the touch sense: changes over the life span. Somatosens Mot Res 12:29±47, 1995 Vallbo AB, Hagbarth KE: Activity from skin mechanoreceptors recorded percutaneously in awake human subjects. Exp Neurol 21:270±289, 1968 Vedel JP, Roll JP: Response to pressure and vibration of slowly adapting cutaneous mechanoreceptors in the human foot. Neurosci Lett 34:289±294, 1982 Verillo RT: Comparison of vibrotactile threshold and suprathreshold responses in men and women. Percept Psychophys 26:20±24, 1979 Verrillo RT, Bolanowski SJ, Baran F, Smith PF: Effects of underwater environmental conditions on vibrotactile thresholds. J Acoust Soc Am 100:651±658, 1996 Verrillo RT, Bolanowski SJ, Checkosky CM, Glonefp MC: Effects of hydration on tactile sensation. Somatosens Mot Res 15:93±108, 1998 Warner RR, Myers MC, Taylor DA: Electron probe analysis of human skin: element concentration pro®les. J Invest Dermatol 90:78±85, 1988a Warner RR, Myers MC, Taylor DA: Electron probe analysis of human skin: determination of the water concentration pro®le. J Invest Dermatol 90:218±224, 1988b Weinstein S. Intensive and extensive aspects of tactile sensitivity as a function of body part, sex and laterality. In: Kenshalo DR (ed.). The Skin Senses, Spring®eld: Charles C Thomas, 1968, pp 195±222 Woodward KL: The relationship between skin compliance, age, gender and tactile discriminative thresholds in humans. Somatosens Mot Res 10:63±67, 1993