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Noll Physiological Research Center, The Pennsylvania State University, University Park.
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Abstract |
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0.1% in middle-aged and younger skin, whereas a significant elevation in SkBF was only achieved with 10% CPZ in older skin. The elevated SkBF in the young and middle-aged groups at higher concentrations of CPZ was the result of both an increased area of vasodilation and an increased magnitude of vasodilation within the vasodilated areas. In the older group, the increase in cutaneous vascular conductance at the highest concentration of CPZ was entirely the result of an increased area of vasodilation. These results suggest impaired CSPA function in aged skin and age-specific differences in the ability of sensory nerves to modify local inflammatory SkBF responses. WHILE the primary function of sensory nerves in the skin is to transmit pain signals to the central nervous system and activate protective reflexes, during acute tissue trauma they also preserve homeostasis at the site of injury (1–4). Sensory nerves can modify the local cutaneous microcirculation and initiate neurogenic inflammation through the release of vasoactive neuropeptides (1). This local release of transmitter substances enables sensory nerves to exert a local control over the microvasculature by modifying cutaneous blood flow and vascular permeability. Consequently, sensory nerves are important for the promotion of wound healing and maintenance of tissue integrity in the skin.
Accordingly, the increased incidence and resistance to healing of leg ulcers in elderly persons may be partially explained by local impairment of sensory nerve function in aged skin (3,5). It has been shown that neurogenic vasodilation is markedly reduced in patients with varicose ulcers compared with controls, while other assessments of endothelial and vascular responsiveness remained unchanged (5). Furthermore, older individuals with varicose ulcers respond to chemical stimulation with a reduced neurogenic inflammatory response in their lower limbs, which may be associated with a progressively attenuated axon reflex vasodilator response with age (5). Aging studies in rats have suggested that changes occur at both pre- and post-terminal sites of the peripheral neurovascular apparatus in aged skin (3,4). Because decreases in neurogenic vasodilation associated with aging may have important health implications, gaining a better understanding of the underlying changes in sensory nerve function that affect neurogenic vasodilation in aged skin seems to be warranted.
Capsaicin, the pungent ingredient in a wide variety of hot peppers, is a useful pharmacological tool for investigating sensory nerve function. Application of capsaicin to the skin stimulates a population of capsaicin-sensitive primary afferents (CSPAs), which is characterized by a sensation of warmth, burning pain, cutaneous hypersensitivity, as well as a local vasodilation and flare (2,6–9). Capsaicin-sensitive primary afferents are generally classified as thin, primary afferent neurons, including most C-fiber polymodal nociceptors, some warm-sensitive thermoreceptors, and most type II A
-polymodal nociceptors (10,11). The resulting neurogenic vasodilation elicited by CSPA activation can be used as an index of sensory nerve function in the skin.
The purpose of the present study was to evaluate age-specific skin blood flow (SkBF) responses to acute capsaicin in order to assess sensory nerve function in aged skin. It was hypothesized that the SkBF responses to acute capsaicin would be progressively reduced with advancing age, i.e., there would be an impaired CSPA function in aged skin.
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30 kg/m2), hypertension (systolic > 140 mmHg and/or diastolic > 90 mmHg), underweight (body mass index < 20 kg/m2), smoking, any medication with the potential to alter cardiovascular or thermoregulatory control or response, allergies to hot peppers, and various dermatological conditions or diseases. Table 1 describes the physical characteristics of the subjects. All experimental procedures were explained to the participants, and they were given an opportunity to ask questions. Prior to participation, all participants signed an informed consent approved by the Institutional Research Board of The Pennsylvania State University.
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Preparations
Prior to the experiments, the right or left arm was selected randomly for each subject to serve as the experimental arm, and 8 application sites were randomized for the concentration of capsaicin to be used. A data acquisition system and Labview software program (National Instruments, Austin, TX) were prepared for data collection before arrival of the participant. A laser Doppler imaging (LDI) system (Moor Instruments, U.K.) with an associated data acquisition computer was configured to perform the necessary measurements.
Each experiment was performed in an environmental chamber where temperature and humidity were maintained at thermoneutral conditions (dry bulb temperature 
23°C, relative humidity 30%). Upon arrival at the laboratory, the experimental arm was gently washed and the participant placed himself comfortably on an adjustable hospital bed in a semireclined position. During an initial rest period of 5–10 minutes, each arm was supinated and set upon a separate, movable armrest to allow for access during the experimental procedures and maximal comfort of the subject. The inflatable cuff of a Finapres blood pressure monitor (Ohmeda, Madison, WI) was placed on the middle finger of the nonexperimental arm to estimate blood pressure and heart rate. This arm was placed at approximately heart level. Finapres output was monitored for stability before commencement of the experiments.
Protocol
The capsaicin application sites were marked on the ventral side of the forearm with a soft pen, according to the randomization map previously made. A template was used to ensure uniform size and placement of the application sites. All markings were outside of the area in which capsaicin was applied. Participants with excessive hair on their ventral forearms were asked to shave those areas at least 24 hours before the experiment. Lastly, the LDI system was positioned so that the laser apparatus was exactly 40 cm above the arm's surface.
Prior to any applications, an LDI scan was taken within 1 site to serve as a baseline measurement. After the initial scan, a series of acute capsaicin applications proceeded. Each application consisted of saturating the pad of a small bandage (14.5 cm2/2.25 in2) with a 300-ml capsaicin solution (Sigma Chemical Co., St. Louis, MO) and applying it to the skin at randomized sites. Each solution, except for the vehicle, contained a different concentration of capsaicin and inactive alcohol (95% ethanol [EtOH]). Seven different concentrations of capsaicin (0.001%–10%) were used, along with EtOH alone, which served as a vehicle. Thus, a total of 8 application pads were used. Each pad was prepared with solution immediately before application and added successively at 4-minute intervals. The application patches were removed in order after each patch had been applied for 32 minutes. Immediately after removal of each patch, an LDI scan was taken of the respective application site.
Several precautions were made to ensure that acute capsaicin applications were standardized. These included randomizing all application sites and using the same batches of capsaicin solutions for multiple subjects. In addition, capsaicin solutions underwent a uniform mixing procedure and were dispensed onto application patches in precise amounts using a micropipette. Other methods of topical capsaicin application such as painting liquid solutions or applying a cream onto the skin would have introduced mechanical stimulation to the application site, which could have interfered with the SkBF response.
LDI Data Analysis
The LDI configurations were set to the following: scan size = 1.6 x 1.6 cm; scan time = 1 minute, 48 seconds; scan speed = 50 ms/pixel. LDI used a visible red helium neon laser operating at a wavelength of 632.8 nm. This low-power laser beam scanned the surface of the skin in a raster pattern. A Doppler shift caused by moving blood in the microvasculature was processed to create a color-coded image of skin perfusion. At the size and distance chosen, a 1600 pixel (40 x 40 pixel) perfusion map was produced for each scan. Each pixel in the map had a unique flux value, proportional to SkBF. For each scan, the principal measurement of interest was the mean flux value. A mean flux value for an individual scan was found by averaging the mean flux values of 5 sampled regions of interest (ROIs) within the scan area. The LDI software calculated mean flux values for each ROI.
All LDI measurements were indexed to percentage of maximal cutaneous vascular conductance (%CVCmax) by dividing mean cutaneous vascular conductance (CVC) by CVCmax. Mean CVC was determined as the ratio of LDI flux to mean arterial pressure (MAP) at the time of the scan (CVC = LDI Flux/MAP). Maximal cutaneous vascular conductance was found by locally heating an area of the opposite arm with warm air to 42°C at a rate of 1°C/5 min and maintaining the local temperature of that site at 42°C for 30 minutes (12,13). Laser Doppler imaging scans were taken at 5-minute intervals, and the scan with the highest mean CVC value was used to determine CVCmax.
The percentage of the area that was vasodilated (%AVD) was also determined for scans taken at 0.001%, 0.01%, 0.1%, 1.0%, and 10.0% capsaicin sites. A threshold value that represented vasodilation (defined in this study as CVC > 25%CVCmax) was subtracted from each scan, and the LDI software calculated the percentage of valid pixels (those still remaining) to yield %AVD. Finally, %CVCmax of just those areas that were identified as being vasodilated also were determined.
Statistical Analysis
Comparisons of physical characteristics among the 3 age groups were done using a one-way analysis of variance (ANOVA) (Excel). When significant main effects of age were found, t tests were performed using Bonferonni corrections to determine between which groups the differences existed. Acute capsaicin measurements were compared using an ANOVA for detection of concentration differences, and a two-way ANOVA to probe for age differences among the results (SAS, SAS Institute Inc., Cary, NC). When appropriate, a post hoc analysis for multiple comparisons was performed. The significance level for all statistical tests was set at 
= 0.05. Data are presented as mean ± SE.
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Results |
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0.1% in middle-aged and younger skin, respectively. In older skin, %CVCmax was only significantly higher than that caused by the vehicle solution at the site with the highest concentration of capsaicin used (10.0%).
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Discussion |
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Examinations of %AVD and the magnitude of this vasodilation (Figure 2) reveal some possible explanations for the age differences that were observed. First, in Figure 2A, the higher %CVCmax of the young group at the lowest concentrations of capsaicin is primarily the result of a greater %AVD. Specifically, there were more areas of skin that had CVC values above a defined threshold for vasodilation. Since the %CVCmax of these vasodilated regions was nearly identical, it is likely that the larger %AVD in young skin was the result of an increased number of vasodilated vessels within the cutaneous vasculature. Another way of stating this is that %AVD reflects the number of activated neurovascular units. The anatomical properties of the neurovascular unit itself, including the size of the collateral nerve network and distribution of each innervated vessel in the skin, are implicated as contributing to this decline in vasodilated area with increasing age (6,15,16). Additionally, the relatively large intra- and interindividual variations in SkBF responses to acute capsaicin reported here and elsewhere are likely due to regional and individual differences in receptive field area and neurovascular unit arrangement (6,7,14,15).
There appears to be a threshold difference in the activation of these neurovascular units in the middle-aged and older individuals. Looking at Figure 2A, the approximate value of %AVD reached with a 0.001% capsaicin solution applied to younger skin is obtained with a 0.1% solution on middle-aged skin and a 10.0% solution on older skin. Furthermore, since the magnitude of SkBF within these vasodilated areas is very similar, the overall %CVCmax is nearly the same with a 0.001% capsaicin solution in young, a 0.1% capsaicin solution in middle-aged, and a 10.0% capsaicin solution in older individuals, respectively (Figure 1). This observation suggests a higher activation threshold of neurovascular units by acute capsaicin with advancing age. Thus, a higher concentration of capsaicin was necessary to produce a given area of neurogenic vasodilation in the skin of both middle-aged and older individuals.
A variation with age in the number of neurons within the collateral nerve network or the distribution of each innervated vessel would help to provide an explanation. Anatomical studies show no appreciable decline with age in the number of sensory nerve fibers (17). In contrast, a definite loss and disorganization of vessels within the cutaneous microcirculation is obvious in older skin (17–19). Therefore, a reduction in number and/or structural impairments of cutaneous blood vessels in older individuals may contribute to the results obtained in this study. In this scheme, a greater magnitude of stimulation would be required to vasodilate a damaged or sparse vasculature, resulting in the observed threshold shift. Several investigators have attributed the lower SkBF in aged skin during local heating to structural limitations including decreased functional capillary plexus units, damaged or obliterated vessels, and/or attenuated precapillary recruitment (20–23).
Maximal cutaneous vascular conductance is thought to reflect structural limitations in the vasculature of the skin, so that when values are expressed as %CVCmax, the index essentially controls for these differences among individuals (24). Therefore, the attenuations in %CVCmax of the older individuals suggest that in addition to structural characteristics, functional decrements are responsible for the reductions in SkBF during acute capsaicin stimulation. It is known that capsaicin causes neurogenic vasodilation in the skin by activation of the vanilloid receptor subtype 1 (VR1) complex on CSPA nerve endings, leading to an exocytotic release of vasoactive neuropeptides (2,25). Most notable among these neurotransmitters are Substance P, which predominantly affects plasma extravasation of the cutaneous microvasculature, and calcitonin gene-related peptide (CGRP), the primary mediator of neurogenic vasodilation in the skin. Accordingly, another interpretation of the threshold shift in middle-aged and older skin is that a diminished release of these neuropeptides from CSPAs occurs at a given concentration of capsaicin, while the cutaneous vessels retain a similar capacity to respond to the vasoactive stimulus.
Aging studies in rats that used electrical stimulation to elicit neurogenic vasodilation have suggested a decrease in neurotransmitter content in the sensory nerves of older rats (3,4). When capsaicin is used to stimulate CSPAs, a receptor-mediated activation takes place; therefore, a loss of receptors in the neuronal membrane or alterations in cellular signaling mechanisms may also account for the diminished responsiveness that has been described. While this distinction is beyond the scope of this discussion, it is interesting to note that while reductions in SkBF are seen in older rats during high-frequency electrical stimulation, with low-frequency stimulation, such decrements do not occur (26). This may imply preservation of CSPA capability, but also suggest limitations in cellular signaling processes with advanced age.
Diminished CSPA activity in the skin has important implications for repair processes associated with wound healing and the maintenance of tissue integrity in elderly skin. While the primary job of CSPAs is to receive and transmit information from the internal and external environment to preserve an organism's homeostasis, neurogenic inflammation prompted by CSPAs at the site of injury aids in maintaining local homeostasis (1–5). Given the results of the present study, it is not surprising that a resistance to healing of leg ulcers happens with greater incidence in the elderly, as this pathology is associated with a reduced neurogenic inflammatory response (5). For example, the neurogenic axon-reflex flare response to chemical stimulation is markedly reduced in patients with varicose ulcers when compared with controls (5). This implies that age-associated impairments in sensory nerve function likely compromise the promotion of wound healing in the skin of older individuals.
It should be noted that all subjects who participated in this study were men, and nearly all were Caucasian. Thus, caution should be taken when attempting to generalize the results from this study to women and different racial groups. Further study is needed to be able to confidently extend these findings to more diverse groups of individuals. Still, major deviations from the primary implications of this study should not be expected.
In summary, the results from this study demonstrate that cutaneous vasodilation caused by acute capsaicin applications was significantly attenuated in older skin compared with what occurred in the skin of younger individuals. Acute capsaicin stimulates a specific population of sensory nerves (CSPAs) in the skin that possess the capsaicin receptor, eliciting a local release of vasoactive neurotransmitters from their terminal nerve endings. Therefore, this assessment was used to evaluate CSPA function and provides evidence for impaired CSPA activity in aged skin. Results indicate that there was a higher activation threshold for neurovascular units within the skin of middle-aged and older individuals. Although it is speculative, reduced neuropeptide content and/or associated release mechanisms may be responsible for the decreased neurovascular reactivity observed in aged skin. Receptor availability and related signaling pathways also may contribute to the decrement in CSPA activity. These findings document functional changes in CSPAs and their ability to modify cutaneous vascular responses in older skin.
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Acknowledgments |
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Address correspondence to Thayne A. Munce, PhD, The Pennsylvania State University, 229 Noll Laboratory, University Park, PA 16802-6900. E-mail: tam275{at}psu.edu
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Footnotes |
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Received October 14, 2002
Accepted January 24, 2003
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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| All GSA journals | The Gerontologist |
| Journals of Gerontology Series B: Psychological Sciences and Social Sciences | |
Significantly different than Middle (p <.05)
