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1 National Ageing Research Institute, The University of Melbourne, Victoria, Australia.
2 Monash Institute of Health Services Research, Monash University, Victoria, Australia.
Address correspondence to Zeinab Khalil, National Ageing Research Institute, The University of Melbourne, Poplar Road Parkville, Melbourne Victoria, 3052. E-mail: z.khalil{at}nari.unimelb.edu.au
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Abstract |
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Methods. The effects of a sensory-independent vasodilator (acetylcholine) and a sensory-dependent vasodilator (capsaicin) on microvascular blood flow and oxygen perfusion in persons of different ages were measured. Laser Doppler flowmetry and a commercially available transcutaneous oxygen monitor (with sensors set at 39°C and 44°C) were used. Healthy volunteers were recruited: 11 young, 14 middle aged, and 19 older.
Results. Under basal conditions (skin temperature, 37°C to 39°C), both basal blood flow and tcPO2 increased with increasing age. However, with the sensor set at 44°C, tcPO2 showed a significant decrease with age. Acetylcholine increased blood flow approximately equally in the three age groups. Capsaicin increased blood flow and tcPO2 in all age groups, with the young showing a greater increase compared with the older participants.
Conclusions. The age-associated changes in basal and stimulated microvascular blood flow and tcPO2 could be attributed in part to altered neuronal function. Measuring tcPO2 at 39°C showed a trend toward an increase with age. In contrast, a decrease with age was observed when tcPO2 was measured at 44°C, a temperature sufficient to activate sensory nerve endings. The results may reflect a decline in sensory nerve function with age rather than a decrease in oxygen delivery for vascular reasons. This is supported by the complementary data showing a significant age-related decrease in stimulated blood flow in response to capsaicin, with no change in the response to the sensory-independent vasodilator acetylcholine. Thus, for clinical purposes, data obtained using the tcPO2 monitor should be interpreted with full knowledge of the conditions under which the measurements were made. Furthermore, for scientific purposes, the tcPO2 monitor could be used to assess sensory nerve function when sensors are heated to 44°C.
In older persons, sympathetic nervous system activity is decreased, with downregulation of 
-adrenoceptors (4), reducing vasoconstrictor tone in the skin. A decline in sensory nerve (C fiber) function is an important contributor to the age-related deterioration of blood flow and cutaneous vasodilation (5–7). Decreased blood cell velocity in older compared with young participants was demonstrated in studies more than 70 years ago (8). Investigators have observed a 40%–70% difference in cutaneous blood flow in 20- and 70-year-olds after heat or electric stimulation sufficient to stimulate sensory nerve endings (6,9–11). Furthermore, sensory nerve activity under inflammatory conditions decreases with age, resulting in a reduction in vascular inflammatory responses and delayed tissue repair (12,13). However, the effect of these neuronal changes with age on transcutaneous oxygen tension (tcPO2) has not received similar attention. In particular, studies have not directly assessed the relationship between oxygen perfusion and basal or stimulated sensory nerve conditions. Nor has the use of a transcutaneous oxygen monitor (TCM) with sensors heated to 44°C, at which temperature they incite sensory thermal stimulation, been described in terms of sensory nerve involvement.
The clinical use of TCMs is increasing. Their initial clinical application was to measure neonatal arterial oxygen tension. Currently, advocates promote oxygen tension measurement around wounds (14), in the skin of persons with venous edema (15), and in ischemic limbs to determine appropriate amputation levels (16).
It is important to distinguish basal from stimulated conditions when microvascular blood flow, or tcPO2, is assessed. A commercially available TCM uses sensors heated to 44°C. The manufacturer suggests that at this temperature, maximum vasodilatation occurs and the increase in oxygen due to the increase in blood flux is greater than the metabolic requirements of local tissue (17). Oxygen diffuses out of the cutaneous vessels and, after overcoming the diffusional resistances of the dermis and epidermis, passes into the sensor. However, we raise the notion that this temperature is high enough for the body to perceive it as a noxious stimulus and, as a result, the sensory nerves may become activated and produce a neurogenic inflammatory response (18). Thus, the TCM seems to monitor the effect of neurogenic inflammation on oxygen tension rather than on basal skin oxygen tension.
In support of our argument, other investigators have noted that skin oxygen tension responses at 37°C and 42°C are not the same, although the mechanisms responsible for these observations have not been specifically addressed (2,19–21). When TCM sensors are heated to physiologic levels (e.g., to 37°C), skin oxygen tension is increased in older patients (19). When the sensors are heated to more than 42°C, oxygen tension is decreased in older persons (2), in persons with diabetes (21), and in those with diabetic peripheral neuropathy (20). The mechanisms underlying this interesting, and perhaps clinically relevant, phenomenon have not been reported.
Our aim, therefore, was to investigate the age-related changes in the relationship between microvascular blood flow and skin oxygen tension under basal (non-noxious) and stimulated (noxious) conditions. We achieved the non-noxious (sensory independent) condition using the direct endothelial vasodilator, acetylcholine, whereas we stimulated the noxious (sensory dependent) condition using capsaicin, which is a selective stimulator of C fibers.
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METHODS |
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In the second set of experiments, we measured the effect of a noxious selective activator of C fibers (capsaicin) on microvascular blood flow. We tested skin oxygen tension using sensors at 39°C, a temperature that does not activate sensory nerve endings.
Outcome Measures
Vascular responses.--
We used noninvasive laser Doppler flowmetry (LDF) to measure capillary blood flow. This technique is reliable and valid (22). The Periflux LDF (PF2B; Perimed AB, Stockholm, Sweden), with a wavelength of 633 nm (visible red light), penetrates approximately 1 mm into the skin. The laser does not produce heat on the skin. By the Doppler effect, it measures changes in blood flux in superficial capillaries, which are recorded on a chart recorder (Unicorder Pantos U-228; Nippon Denshi Kagaku, Tokyo, Japan). We measured the area under the response curve (in square centimeters) using a digital planimeter (Planix Tamaya digital planimeter; Tokyo, Japan).
We inserted the laser probe into a circular perspex chamber, with a well on the base and a port in the middle, which was fixed to the skin with a ring of double-sided adhesive tape. The LDF was set at a bandwidth of 12 kHz and a gain of 10, with the time constant set at 1.5 seconds. This procedure was established in the National Ageing Research Institute (NARI) Biology Laboratory (11).
Transcutaneous oxygen tension.-- We measured tcPO2 using the TCM 400 (Radiometer, Copenhagen, Denmark) according to the manufacturer's instructions. We secured self-adhesive fixation rings (Radiometer) to the participants' skin. Three drops of contact liquid (S44416; Radiometer) were deposited into each fixation ring. We screwed the tcPO2 electrodes into the fixation rings on the participants' arms and chests. The oxygen sensors heat the skin that is in contact with the sensors to an operator-set temperature between 37°C and 45°C. The sensors detect current generated by the breakdown of free oxygen molecules during chemical interaction with hydroxyl ions in an aqueous electrolyte solution (17). The partial pressure of oxygen is calculated by microcomputer from the resulting current.
Participants
Healthy adult volunteers were recruited from among the staff and students of the NARI and the University of Melbourne and from the NARI database of older volunteers. We excluded anyone with known hypercholesterolemia, diabetes, or clinically apparent atherosclerosis and peripheral vascular disease.
Group 1. Sensory-independent, endothelial-mediated microvascular response (acetylcholine group).-- Before the experiments, the volunteers were separated into three groups according to age. Group 1.1 included 9 women and 2 men who were aged 20–39 years (mean age, 26.7 ± 1 years). Group 1.2 included 8 women and 6 men who were aged 40–59 years (mean age, 49.7 ± 1.8 years). Group 1.3 included 5 women and 6 men who were aged 60 years or older (mean age, 72.5 ± 2.3 years).
Group 2. Sensory-dependent (Capsaicin) group.-- The volunteers in group 1 were invited to participate in the second study. All except six did so. Group 2.1 included 5 women and 6 men who were aged 20–39 years (mean age, 26.7 ± 1.4 years). Group 2.2 included 3 women and 7 men who were aged 40–59 years (mean age, 45.7 ± 1.8 years). Group 2.3 included 9 women and 10 men who were aged 60 years or older (mean age, 74.7 ± 1.4 years).
Procedures
We performed all tests in a climate-controlled environment, with room temperature maintained between 20°C to 23°C. We measured cutaneous blood flow.
Volunteers abstained from coffee, cigarettes, and vigorous exercise for at least 2 hours before testing. During testing, participants were seated in a reclined armchair with a pillow placed under the left arm. The midvolar surface of the left forearm was used for both capillary blood flow and tcPO2 measurements under both basal and stimulated conditions. For comparison, we also measured tcPO2 on the chest. We shaved the skin sites where the LDF probe chamber and TCM sensor fixation rings were to be applied using a dry razor if necessary. The sites were exfoliated using Nuprep (D. O. Weaver & Co., Aurora, CO), and cleaned with an alcohol swab. This removed dead, dry, impermeable layers of the epidermis that impair measurements.
Procedure 1: Acetylcholine study.-- We attached two specially built perspex chambers to the middle third of the volar surface of the arm. The chambers accommodate the laser fiberoptic cable. They also provide a means to deliver test substances in solution to the skin. We attached two TCM electrodes (E5 250) on either side of the LDF probe chamber. We recorded forearm skin erythrocyte flux until a stable baseline was achieved. The temperature of the TCM 400 electrodes was set to 44°C and calibrated with atmospheric air. We recorded measurements after the oxygen tension had been stable for 15–20 minutes. We measured the vascular response for 5 minutes after stimulation using the LDF. The acetylcholine response is characterized by a gradual desensitization, so 5 minutes provides an accurate representation of skin response to this substance.
When baseline recordings were completed, the test substances could be applied. We filled one perspex chamber's delivery compartment with 100 µl of 20% acetylcholine (Sigma Chemical, St. Louis, MO) dissolved in distilled water. The other chamber contained the apparently pharmacologically inert vehicle solution. Introduction of the acetylcholine to the skin requires electrophoresis for an adequate response to be produced. We used the inert solution to provide a base result, allowing correction of any changes in blood flow as a result of the electrophoresis process itself when we assessed the data. Therefore, we present results as a ratio to relate skin response to acetylcholine to the vehicle as an internal control. We connected a battery-powered electrophoresis unit (Phoresor II, model pm 700; Salt Lake City, UT) at random to either of the two chambers to provide a direct current for drug electrophoresis (11). The blood flux was recorded continuously. We connected the electrophoresis unit to the chambers to deliver either vehicle or acetylcholine solution in a random manner, with a waiting period of approximately 20 minutes between the two stimuli.
Procedure 2: Capsaicin study.-- We performed the capsaicin study at a separate session. We set the TCM 400 electrodes to 39°C and calibrated them with atmospheric air. We placed sensors on the participants' forearms and chests, as previously described.
When blood flow and tcPO2 measurements were stable, we injected the capsaicin (0.4 ml of 5 mg/ml solution dissolved in 70% methylated alcohol) into the drug delivery port of the perspex chamber. The capsaicin diffused into the skin for 20 minutes while blood flow and tcPO2 were recorded continuously. Capsaicin, unlike acetylcholine, can produce a response without electrophoresis.
We considered readings made at an average of 2 minutes before capsaicin treatment as the baseline values for both LDF and tcPO2. We chose the area under the curve on the LDF tracing of the last 2 minutes of the 20-minute treatment period a priori based on previously obtained postcapsaicin values. Similarly, we considered the tcPO2 of the last 2 minutes of the 20-minute treatment period as the postcapsaicin value, allowing sufficient response time to provide accurate representation of the capsaicin effect.
Statistical Analyses
We analyzed our results using repeated measures analysis of variance, followed by post hoc Student and paired samples t tests. We also used chi-squared analysis and Pearson correlation coefficients.
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RESULTS |
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An independent t test confirmed that the effect of age in the difference scores among the groups was significant between the young and older (t(27) = –3.4, p <.05), but not between the young and middle age (t(20) = 1.4, p =.2) nor the middle age and older participant groups (t(26) = –1.8, p =.1), as indicated in Figure 6.
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DISCUSSION |
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adrenoceptors with age (4,10,11,23–25) could decrease basal vasoconstrictor tone in the skin of older persons and thus could explain the increase in basal blood flow we observed with age under physiologic conditions (Figure 1). This proposition could also explain the positive correlation we observed with age when we used the TCM with the sensors set at normal skin temperature (Figure 4), which could be attributed to an increase in oxygen availability resulting from the increased basal blood flow that accompanies aging. When we heated the sensors to 44°C, oxygen tension decreased in the older participants (Figure 2). Heating the sensors to more than 42°C acts as a noxious stimulus and will stimulate C fibers, inducing a neurogenic inflammatory response (18). This inflammatory response decreases with age (12). Therefore, the TCM with the sensors heated to 44°C may be monitoring the effect of neurogenic inflammation on oxygen tension rather than monitoring basal oxygen tension. This could explain the observed age-related reduction in oxygen tension when using the TCM with the sensors heated to 44°C as opposed to increased oxygen tension with age when the sensors were used at basal temperatures.
These findings indicate that sympathetic vasoconstrictor tone predominates in controlling microvascular blood flow under basal conditions, whereas C fiber vasodilator activity predominates in the presence of noxious stimuli from heat and chemicals. With capsaicin stimulation, both blood flow and tcPO2 increased in all the participant groups. However, the increase was significantly less in the older volunteers, supporting previous evidence of an age-induced decrease in C-fiber function (12). Furthermore, the ratio of the increase between these factors was equal within groups, suggesting a relationship between blood flow and tcPO2. This observation could be clinically relevant for wound healing. The demonstrated reduced C fiber function could decrease the amount of blood and oxygen reaching the skin when a cutaneous injury occurs. Indeed, blood flow is one of the most important factors involved in the efficient delivery of oxygen to tissues (19,26).
Because C fibers release sensory neuropeptides with vascular and growth-promoting effects, a decrease in this function would subsequently decrease the blood supply to injured tissues and reduce the transport of immune cells and growth factors to the damaged region. This could be a mechanism by which repair is slowed. The current study corroborates the findings of previous research. Gaylarde and colleagues (27) found that the feet of patients with diabetic peripheral neuropathy have increased oxygen tension when compared with controls at a sensor temperature of 37°C. However, once the sensor temperature is increased to 44°C, the oxygen tension of controls is greater than that in persons with diabetic peripheral neuropathy. The skin in persons with this condition has reduced sympathetic control compared with the skin of those without diabetic peripheral neuropathy, thus explaining the increased oxygen tension found by the sensors at 37°C. At the higher temperatures, the TCM sensors produce a noxious effect, triggering the sensory nerves to produce a neurogenic inflammatory response that is decreased in the presence of diabetes. Similarly, Sindrup and coworkers (28) and Stucker and colleagues (29) showed significantly decreased oxygen perfusion in ulcerated legs compared with contralateral legs only when patients with venous leg ulcers were assessed with a TCM set at 44°C.
Our data and that of others, which reveal age-associated changes in oxygen tension depending on the temperature of the TCM sensors, indicate that TCM sensors, when applied at 44°C, can reflect the function of sensory nerves. The clinical significance of this proposition is related to the importance of these nerves in tissue repair.
The investigations we cited do not mention the possibility that the reported tcPO2 measurements were, in fact, a reflection of sensory nerve activity rather than basal oxygen tension. The novelty of our current findings is the fact that aging had two opposite effects on skin oxygen tension when TCM sensors were used at 39°C and 44°C. The only plausible explanation for this differential effect is that the TCM sensors measure the contribution of different factors modulating microvascular blood flow and oxygen tension occurring under basal and stimulated conditions. To our knowledge, the current study is the first to assess age-related changes in tcPO2 in healthy volunteers under these different conditions.
It is of interest to note that age appears to affect blood flow and tcPO2 equally after stimulation with topical capsaicin (compare Figures 5 and 6). In the young participant group, we found a fourfold increase in blood flow, whereas it doubled in the older volunteers, yielding a 2:1 ratio of young versus older participants. We found a similar 2:1 ratio increase in tcPO2 in response to capsaicin. The relationship was therefore maintained, with the middle age group closely following the effect of the young age group. This supports the association between changes in blood flow and that of oxygen tension and shows that both phenomena are controlled by the activity of sensory nerves under stimulated conditions.
Our data also support our previous finding (13) that sensory nerve activity, as monitored by the vascular response to capsaicin stimulation, decreases significantly with age. The data also indicate that endothelial cell function does not change with age (12,30), as demonstrated by the absence of a significant difference between different age groups when their responses to acetylcholine were assessed. However, the significant increase with age in basal blood flow could have perhaps masked a possible decline in endothelial cell function with age, because both measurements were taken under basal physiologic condition. This proposition needs further investigation.
Conclusion
We found that under basal conditions (sensor temperature, 37°C to 39°C), endothelial cell responses do not change with age at the level of skin microvasculature and that blood flow and tcPO2 were greater in older compared with young participants. We attribute the latter finding to a lower vasoconstrictor tone with increasing age, an assumption that is supported by the findings of previous studies showing decreased sympathetic activity with age. This positive relationship with age was reversed when blood flow and tcPO2 were tested under conditions sufficient to stimulate sensory nerve endings. When the sensory nerves were stimulated by capsaicin, blood flow and tcPO2 increased in all age groups. However, the response was lower in older persons. Furthermore, tcPO2 decreased significantly with age when tissue oxygen tension was measured using a sensor temperature of 44°C. The sensor temperature was not noxious, although it was sufficient to activate peripheral terminals of unmyelinated primary afferents, thus increasing blood supply to the area and consequently oxygen tension.
Because C fiber function declines with age, the observed decrease in tcPO2 with age most likely represents a decline in sensory nerve function rather than a true physiologic decrease in tcPO2 with age. Table 1 shows the differences in oxygen perfusion between sensor temperatures and before and after capsaicin stimulation. The data raise the important issue that results obtained using a tcPO2 monitor should be interpreted with caution depending on the experimental condition. Overall, the data support evidence of an age-related decline in C fiber function and suggest that the tcPO2 monitor could be used to assess sensory nerve function.
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Acknowledgments |
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Footnotes |
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Received August 26, 2003
Accepted September 3, 2003
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