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. 2003 Apr 17;549(Pt 3):855–863. doi: 10.1113/jphysiol.2002.037994

Oxygen partial pressure in outer layers of skin of human finger nail folds

W Wang *, C P Winlove †, C C Michel ‡
PMCID: PMC2342999  PMID: 12702744

Abstract

To gain insight into oxygen transport by the cutaneous microcirculation, we have developed oxygen-sensitive microelectrodes (tip diameter ∼5 μm) to measure the distribution of PO2 in dermal papillae of the finger nail folds of healthy human subjects. Oxygen entry into the tissue was minimised by covering the skin with a layer of paraffin oil. The finger was held under a dissecting microscope and microelectrodes were guided into position. PO2 varied from 5–25 % of its atmospheric value, Pair (∼160 mmHg), depending on the location within the papilla. Along the axis of a papillary loop, PO2 decreased from 40.0 ± 4.8 mmHg (mean ± s.e.m., n = 6) at the base to 30.4 ± 5.2 mmHg (n = 6) at the tip. The lowest values of PO2, in the range of 5 % of Pair, were measured in the epidermis where the metabolism of cells was highest and the steepest PO2 gradients were recorded in the vicinity of the epidermal–dermal boundary. When the local circulation was abruptly reduced or stopped, PO2 fell exponentially with time, with a time constant of 8.4 ± 1.5 s (n = 7). When flow was reinstated, PO2 rose exponentially to a new value with a time constant of 4.8 ± 0.8 s (n = 6). The steady state PO2 following reperfusion was ∼23 % higher than the pre-occlusion value (P < 0.05, ANOVA and two-tailed Student's t test) indicating localised reactive hyperaemia.

The cutaneous microcirculation has a unique anatomical arrangement that accommodates different and sometimes conflicting functions - the supply of nutrients, clearance of waste products and control of heat exchange (Wheater et al. 1979). The arterial supply and venous drainage to the skin are located deep in the hypodermis. They give rise to arterioles and venules to form two important plexuses in the dermis: the deeper cutaneous plexus at the junction of the hypodermis and the dermis and the superficial sub-papillary plexus just beneath the dermal papillae (Braverman, 1997). The sub-papillary plexus supplies the upper layer of the dermis and gives rise to a capillary loop in each dermal papilla. In the epidermis, cells produced by mitosis in the germinal layer undergo different stages of maturation and move progressively to the outer layers. The cornified layer in the outermost region of the skin consists of flattened cell remnants that are constantly shed.

In this paper we are concerned with the transport of oxygen to tissues of the papillary layer of the dermis and the epidermis. This is the region between the sub-papillary plexus and the surface of the skin. While there is recent evidence that oxygen entering the skin from the atmosphere may meet the oxygen requirements of the cells in these areas (Stücker et al. 2002), we are interested in whether capillary loops in the papilla can contribute significantly to oxygen delivery to the epidermis in view of their counter-current arrangement (Wang et al. 1996; Wang, 2000). While there have been many studies on the mechanisms of oxygen transport within tissues including the skin (Scheuplein & Blank, 1971; see reviews by Popel, 1989; Pittman, 1995), much of the experimental work on skin has been limited to estimates of mean tissue PO2 from measurements of transcutaneous PO2 or has used needle electrodes that were large relative to the dermal papilla (e.g. Evans & Naylor, 1966/1967; Spence & Walker, 1976; Lübbers, 1987; Opitz & Lübbers, 1987; Bader, 1990; Jaszczak, 1991; Harrison et al. 2002). There have been few attempts to relate the spatial distribution of PO2 to the arrangement of the cutaneous microcirculation.

In the present study, we have investigated the distribution of PO2 in the epidermis and dermal papillae in the skin of the finger nail folds of healthy human subjects when entry of O2 from the atmosphere has been minimised by covering the skin surface with oil. PO2 was measured using O2-sensitive microelectrodes with overall tip diameters of approximately 5 μm. In the nail fold, the microvascular architecture can be visualised more easily as the cornified epithelium is thin and most distal papillary loops lie almost parallel to the skin surface (Fig. 1). As a consequence it has been possible to determine the PO2 not only at different depths from the skin surface but also at points at the same depth, close to and distant from the capillaries. We have also investigated the changes in PO2 in the tissue when the blood flow to adjacent capillaries was stopped and later re-started.

Figure 1. Diagrammatic representation of papillary loops in the dermal papillae of human nail-fold skin seen in sagittal section.

Figure 1

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On the left of the diagram, away from the limiting boundary of the nail fold, the axes of the capillary loops lie perpendicular to the surface as in other areas of skin. The axes of the loops closest to the limiting boundary (on the right) lie almost parallel to the skin surface so that entire loops can be viewed through a microscope placed above them. The arrow on the right of the diagram shows the angle of approach used to drive the O2-sensitive microelectrodes through the epidermis and papillary dermis of the nail fold. Not shown are the extensive connections of the sub-papillary plexuses that occur above and below the plane of the diagram.

The work was carried out in accordance with the Declaration of Helsinki. Preliminary reports of our findings have been presented to the British Microcirculation Society (Wang et al. 2001).

METHODS

Fabrication, testing and calibration of microelectrodes

The oxygen-sensitive microelectrodes were made using platinum– iridium wire (Fig. 2). A short length of 25 μm diameter Pt-Ir wire (Goodfellow, Cambridge) was electrochemically etched in saturated nitric acid solution to a slender profile with a tip diameter less than 1 μm. It was soldered to a conducting wire and then fixed inside a 1.5 mm standard-walled glass pipette (Clark, Reading) using epoxy resin Araldite. The glass pipette was mounted on a micropipette puller (Narishige, Japan), which pulled the electrode to the desired tip size and profile. The pulling conditions were adjusted so that the glass sealed around the top of the wire. The tip of the electrode was bevelled using a micropipette grinder (Narishige, Japan) to enable easy tissue penetration. The surface of the metal electrodes was cleaned by sonication in distilled deionised water and was coated with Nafion perfluorinated ion exchange resin (Sigma-Aldrich, Dorset).

Figure 2. Diagrams of O2-sensitive microelectrodes.

Figure 2

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Left, the diagram shows the principle components of the standard microelectrode used in the study. Etched Pt–Ir wire is soldered to a conducting wire, sealed in a glass pipette using Araldite and drawn out into the microelectrode tip. Centre, en-face view of the bevelled tip which is coated with Nafion. Right, diagram of a Φ-shaped electrode developed for the microcirculation occlusion experiments.

To investigate the effects of occluding the local microcirculation on the tissue PO2, some electrodes were made with a special Φ-shaped tip by repeated heating of the tip of the pulled electrode, using the heating element of the micropipette puller and fusing a small glass bead to the shaft approximately 200 μm above the tip. These microelectrodes behaved identically to conventional ones as they penetrated the tissue up to the depth at which the bulbous region of the glass shield came into contact with the skin surface. Further advance of the electrode now compressed the underlying tissue, occluding the local microcirculation. This provided a convenient method for investigating the transient effects of microcirculatory arrest upon tissue PO2.

All electrodes were tested for their electrochemical characteristics using a potentiostat (CV37, BAS, Cheshire, UK), e.g. I-V (current- voltage) curves (Fig. 3). The plateau in the curve (circled in the figure) at around −0.75 V corresponded to the so-called diffusion-limited current, which was independent of the applied potentials. This was a window of great analytical utility since the flux of the reactant, O2, was directly proportional to its concentration in the bulk fluid, as the concentration of O2 at the surface of the electrode was effectively zero due to rapid electron transfer (Winlove & O'Hare, 1993). In all our measurements, the potential was chosen at −0.75 V. Electrodes were kept in saline for storage before and after testing and measurements. Calibration of electrodes was carried out before and after each experiment in saline bubbled with 100 % N2, air, and 97 % O2+ 3 % CO2, respectively. We observed a slight drop (< 5 %) in the current after electrodes were exposed to the tissue, which may be due to the contamination of the surface of the electrodes. The electrode's response time to a change in PO2 is very short (∼0.01–0.05 s). This allows us to investigate temporal variations of the PO2 when blood flow conditions are altered.

Figure 3. Current-voltage curves for Pt-Ir oxygen-sensitive microelectrodes.

Figure 3

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Typical current–voltage curves from microelectrodes are shown. Estimations of PO2 are made with the voltage set to bring the current onto the plateau region of the curve (circled region).

General protocol in PO2 measurement

Approval was granted by St Mary's Hospital Ethical Committee for the experiments to be carried out in the microcirculation laboratory at Imperial College. Nine subjects (two males and seven females, aged between 25 and 45 years) were recruited and informed written consent was given by all volunteers. Volunteers spent approximately 25–30 min in the laboratory before any measurement started. All the results shown were recorded at room temperature, which was ∼22 °C. A number of measurements were made on each of the subjects and they were all treated as independent measurements. Because the emphasis of this investigation was on the spatial and temporal variations of the PO2 in the epidermis and the dermal papillae, we did not distinguish data from subjects of different age and gender groups in the results, as we had no intention of investigating their effects on the skin oxygenation. Damage to the O2 electrodes during one of the three stages of an experiment (the initial calibration, the measurements of skin PO2 and the final calibration) meant that we were unable to obtain complete sets of data from three subjects. The most common cause of these accidents was unconscious movements of the finger during experiments. Penetration of skin and advance of electrodes in tissue were also occasionally responsible. If an electrode broke while in the tissue, the area was viewed under the microscope and any broken remnants of electrode removed. Subjects often had no sensation of the penetration and the movement of the electrode in the skin because of the small tip size of the electrode and the fact that the measurement was carried out in the upper layers of the finger nail folds.

To remove the stratum corneum for easier electrode penetration into the nail-fold skin, adhesive tape was applied to the area and removed six times before the start of an experiment. The lower arm and hand of the subject were taped onto a steel plate on the table to minimise excessive movement of the hand. Blu-Tack (Bostik, Stafford, UK) adherent to the plate was moulded around the finger on which observations were to be made to immobilise it. The reference electrode (Ag-AgCl ECG electrode) was placed on the skin near the finger. The microelectrode was mounted on a three-dimensional remote hydraulic micromanipulator (Narishige, Japan), which in turn was mounted on a three-axial coarse micromanipulator (Narishige, Japan). Both the working and the reference electrodes were connected to the potentiostat (CV37, BAS). Currents from the potentiostat at −0.75 V were recorded on a chart recorder.

Skin was illuminated using a cool light source. Paraffin oil was superfused over the finger to minimise transport of oxygen from the air and to reduce light reflections from the surface of the skin. A Wild M10 stereomicroscope (Leica) was used to visualise nail-fold capillaries and flows in these capillaries. No measurements of blood flow velocity were made, but red cells were used as markers to indicate flow conditions (e.g. flow stopped, partially stopped, reinstated). The coarse micromanipulator was used to position the electrode just above the site of the measurement, while the remote hydraulic micromanipulator was used for the penetration of the skin and movement of the electrode within the tissue.

The depth of the tip of the electrode was estimated from: (1) the surface of the skin, (2) the depth at which the papillary loops exist, and (3) the angle of attack between the skin surface and the electrode as it was advanced by the fine adjustment on the hydraulic micromanipulator. The electrodes were positioned at approximately 30 deg to the surface of the skin, so that when an electrode moved in its axial direction by a distance of L, the tip of the electrode travel led by a distance of L/2 in the vertical direction. To minimise tissue deformation in our measurements as the electrode was advanced, it was driven slightly further (∼1–2 μm) than its intended destination and then withdrawn by a few micrometres to bring the tip of the electrode into the desired position. An equivalent manoeuvre was performed as the electrode was withdrawn. This helped to release the tension in nearby tissue caused by the relative movement between the electrode and the tissue and to minimise tissue deformation introduced by the insertion and withdrawal of the electrode.

Statistical analysis

Data acquired in each experiment were exported into Microsoft Excel. Average values and standard deviations were calculated for each set of data using mathematical functions in Excel. Rankits method was used to assess the normality of the distribution of the data. Analysis of variance (ANOVA), which compares the within-group variance to the between-group variance of data, was carried out when there were more than two sets of data for comparison. F values and the associated probability were calculated using a Fortran program written in our laboratory. Student's t tests were then used to investigate statistical significance between any two data sets.

RESULTS

Spatial variations of PO2

Figure 4 shows the variations of PO2 with the depth into the skin as a PO2 microelectrode was advanced from the surface into the dermal papilla of one subject. Three consecutive electrode penetrations were made in this experiment: first towards the tip, then to the middle and finally to the base of the papillary loop. It is seen that the PO2 increased with depth from the surface of the skin to the dermis. With paraffin oil applied on top of the nail fold, PO2 was close to zero at the surface of the skin. The value increased to between 35 and 45 mmHg in the dermis at a depth between 100 and 120 μm, just above the sub-papillary plexus. There seem to be two different regions on each curve for the gradient of the PO2 with the depth. In the superficial region between the surface and 20–30 μm, where the germinal layers are deduced to exist, PO2 gradients are approximately 2–3 times greater than those in the deeper region between 30 and 120 μm. The profiles are consistent with the difference in the oxygen consumption rates in the dermal papillae and the germinal layers. The overall pattern of the change in PO2 with the depth into the skin was similar at the different sites along the papillary loop but there were some quantitative differences. For example, PO2 values seemed to be lower near the tip of the papillary loop compared to those near the base. Although recordings from three successive electrode penetrations of the skin were only rarely accomplished in a single experiment, it was found that in all subjects PO2 increased with depth and the rate of increase was greater during penetration of the epidermis than of the dermis.

Figure 4. Single experiments on the variation of PO2 with the depth from the skin surface of finger nail folds.

Figure 4

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The three curves show three single experiments in one subject, when the electrode was driven towards the tip, base and middle of a papillary loop. In all three profiles the gradient of PO2 is 2–3 times steeper over the most superficial 30 μm than between 30 and 120 μm depth.

Mean values for the spatial variations of the PO2 with depth in the outer skin are presented in Fig. 5. In Fig. 5A, mean PO2 has been plotted for the superficial (5–10 μm), the middle (45–65 μm) and the deep (100–120 μm) regions in the dermal papilla of finger nail folds. In the superficial region of the skin, PO2 was approximately 8.0 ± 3.2 mmHg (n = 6). The value increased to 24.0 ± 6.4 mmHg (n = 8) in dermal papillae and at the depth just above the sub-papillary plexus, PO2 was approximately 35.2 ± 8.0 mmHg (n = 9). ANOVA of the three sets of data gave the F value 34.90. With the degrees of freedom in our data, v1 = 2 (i.e. 3 - 1) and v2 = 20 (i.e. 23 - 3), it is highly unlikely (P < 10−6) that the three data sets are random samples from the same population. A two-tailed Student's t test showed that these results differed very significantly: between the superficial and the middle regions to a level of P < 0.00005, and between the middle and the deep regions to a level of P < 0.005.

Figure 5. Spatial variations of PO2 in outer layers of nail-fold skin.

Figure 5

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A, mean values for PO2 at depths of 5–10 μm (surface), 45–65 μm (middle) and 100–120 μm (deep) in all areas of nail-fold skin. The PO2 at the surface and in the deep regions differ very significantly from those in the middle region (P < 0.00005 and P < 0.005, respectively). B, mean values for PO2 with depth in tissue close to the axis of the capillary loops of the papillae. While PO2 at the tip differs significantly from its value at the base (P < 0.01), the gradient is too small for significant differences to be detected between the PO2 of the tissues around the middle of the capillary loops and the base (P < 0.13).

We also measured PO2 along the axis of papillary loops. These measurements were carried out with the tip of the microelectrode at points in the tissue that were close to the most distal (and hence most horizontal) capillary loops of the nail fold. As shown in Fig. 5B, PO2 generally decreased from the base to the tip of the papillary loop. At the base of the loop, PO2 was approximately 40 ± 4.8 mmHg (n = 6); the value dropped to 35.2 ± 3.2 mmHg in the middle (n = 6) and was approximately 30.4 ± 5.2 mmHg near the tip of the loop (n = 6). ANOVA of the three data sets yielded F = 6.01. Given the degrees of freedom in the data (v1 = 2 and v2 = 15), the probability that the data sets have the same mean values is very small (P < 0.02). This confirms that there are statistically significant variations in the PO2 along the axis of papillary loops. Student's t test (two-tailed heteroscedastic test) revealed a significance of P < 0.01 between data at the base and the tip of the papillary loop; however, the differences between values at the base and the middle of the papillary loop, or between the middle and the tip of the loop, are not significant (P < 0.13 and P < 0.10, respectively).

Transient variations of PO2 following micro-occlusion

We used the special Φ-shaped electrodes to investigate the transient changes in skin PO2 when the microcirculation is suddenly arrested and restored.

Figure 6 shows the temporal variations of the tissue PO2 near papillary loops following the altered flow conditions in nail-fold capillaries. In the experiments shown in Fig. 6A, the tips of the microelectrodes were positioned in the lower half of the papilla, close to the capillary loop, and flow in the capillary and underlying sub-papillary plexus was either reduced or stopped at t = 0. It is seen that PO2 in the dermis decreased with time in all cases by 16–24 mmHg. In several cases there were initial short-lived increases, or jumps, in PO2. These were most probably caused by interference from the slight movement of the electrode in the tissue. New steady values in PO2 were reached within 30–60 s following alterations in capillary flow. In comparison, the response time of the electrode to a change in PO2 is very short (approximately 0.01–0.05 s). The dynamic changes in PO2 with time can be described by a single exponential decay function,

graphic file with name tjp0549-0855-mu1.jpg

where P is the value of the PO2 during the transient, τ is the time constant, a is a constant representing the difference between the initial and final values of PO2 and b is the final value of the PO2. Continuous lines in all panels at t≥ 0 represent the exponential function with parameters a, b and τ calculated using the least squares method.

Figure 6. Single experiments on temporal variations in dermal PO2.

Figure 6

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Each panel shows the results of a single experiment. The continuous curves are mono-exponential functions that have been fitted to the data. A, seven experiments on step reductions on capillary blood flow. B, five experiments on restorations of flow. In the experiment shown in the last panel, blood flow was restored in two stages and similar exponential increases in PO2 accompanied each increase in flow.

After a period of microcirculatory arrest lasting between 1 and 5 min, flow was restored and the changes in PO2 are shown in Fig. 6B. The increase in PO2 can be described by a similar single exponential function,

graphic file with name tjp0549-0855-mu2.jpg

with τ′ being the time constant for the exponential rise. a′ and b′ are constant parameters representing the range and the initial value of the PO2. In the last panel of the figure, blood flow was restored in two stages and similar exponential increases in PO2 accompanied each increase in flow.

The time constants for the exponential decays in reduced capillary flows and rises in reperfusion of the PO2 are shown in Fig. 7A. It was found that the mean time constant for decays, τ = 8.44 ± 1.53 s (n = 7), was consistently greater than that for the exponential rises, τ′ = 4.75 ± 0.82 s (n = 6). A two-tailed t test was applied to the data and revealed a significant difference between the time constants (P < 0.0005).

Figure 7. Temporal characteristics in localised occlusion and reperfusion of microvessels.

Figure 7

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A, time constants of mono-exponential decays of the PO2 during microvessel occlusion differed significantly from those of exponential rises in reperfusion (P < 0.0005). B, mean values for PO2 before, during and after local reduction in blood flow. Following occlusion of local capillaries, mean PO2 was very significantly reduced (P < 0.0003). After restoration of flow, PO2 was increased to levels significantly above the initial control values (P < 0.05).

In Fig. 7B, we compare the steady values of PO2 following reduced capillary flow and after reperfusion with their values prior to arrest of the microcirculation (the control state). There was a significant decrease in the PO2 following arrest of the local microcirculation. PO2 fell from a mean of 35.2 ± 4.8 mmHg (n = 6) to 19.2 ± 6.4 mmHg (n = 6), i.e. a fall in PO2 of 16 mmHg. When blood flow was restored, PO2 increased to 43.2 ± 6.4 mmHg (n = 6), an approximately 125 % increase in comparison to the value in ischaemia. This value, interestingly, was also 23 % greater than the control. Analysis of variance (ANOVA) of data from the three conditions revealed that it was very unlikely that data were from the same population, (P < 0.00001, for F = 32, v1 = 2 and v2 = 15). Further investigation using Student's t test (two-tailed heteroscedastic) was carried out between any two chosen sets of data. Differences in PO2 between occlusion and control, or between occlusion and reperfusion were highly significant (P < 0.0003 and P < 0.00004, respectively). There was also a significant difference in the mean values of the PO2 between the control and reperfusion (P < 0.05).

DISCUSSION

Variations of PO2 with depth in the epidermis and superficial dermis

We have found that when the skin of the human finger nail fold is covered with paraffin oil, the PO2 in the tissue increases with depth from values close to zero at the surface to about 40–50 mmHg close to the sub-papillary plexus. The gradient appears to be steepest through the outer 30 μm that corresponds to the epidermis. Our values for PO2 in the superficial dermis agree with previous experimental and theoretical estimates of PO2 in this part of the skin (e.g. Grossmann, 1982; Baumgärtl et al. 1987; Jaszczak, 1991; Stücker et al. 2000). This was in spite of our measurements being made on the epidermis and superficial dermis of nail-fold skin, where the papillae lie almost parallel to the skin surface.

Our observation that PO2 increases with depth would be predicted if all O2 is delivered to the papillary dermis and epidermis from the sub-papillary plexus. Under physiological conditions, however, O2 can enter the outermost layers of the skin from the atmosphere and recent measurements by Stücker et al. (2002) have shown this may be as much as 5.3 ml cm−2 min−1 and would be sufficient to meet the needs of the epidermis. Under these conditions the PO2 profile with depth beneath the skin surface might be expected to differ considerably from that reported here. Interestingly, the only measurements that we are aware of that are comparable with our own were made by Baumgärtl et al. (1987), under conditions of O2 entry from the atmosphere into the skin of the lower leg of a human subject. While there are striking differences between their results and ours, there are also similarities. Baumgärtl et al. (1987) measured PO2 with a microelectrode, but because of technical difficulties, a duplicate set of measurements was possible only on a single subject. In their experiment, the skin was first pierced with a sharpened microcannula that was removed, and the O2 microelectrode was inserted through the pre-formed opening to a depth of 1700 μm. The electrode was then withdrawn in a series of steps and PO2 measured at each step. When the electrode reached the surface, it was re-inserted to a depth of 500 μm and the measurements repeated as it was withdrawn once again. Two profiles of PO2 with depth in the skin showed the same general pattern. At depths greater than 300–500 μm below the surface, PO2 was in the range of 40–50 mmHg and varied little with depth. These regions of the dermis were beneath the sub-papillary plexus which the authors suggest is 250–400 μm below the surface in the skin of the lower leg. As the electrode was withdrawn further, PO2 fell steadily between 300 and 100 μm, a region that presumably corresponds to papillary dermis superficial to the plexus. PO2 rose sharply as the electrode was withdrawn through the most superficial 50 μm, reaching 80 mmHg at the surface where the tip was in contact with water that covered the skin.

Bearing in mind that the sub-papillary plexus in the nail fold is only 150 μm below the surface (compared with 250–400 μm in the skin of the lower leg) our observation of a rise in PO2 with depth in the papillae corresponds to the fall in PO2 which Baumgärtl et al. (1987) recorded as they withdrew their electrode between 300 and 100 μm. The rise of PO2 which Baumgärtl et al. (1987) recorded as their electrode was withdrawn through the most superficial 50 μm can be accounted for by the entry of O2 into the epidermis from the atmosphere (Stücker et al. 2002). In our experiments, the skin was covered with paraffin oil that acted as an effective diffusion barrier to O2 so that entry of O2 into the epidermis was virtually zero.

While our finding that PO2 increases with depth at the level of the sub-papillary plexus is consistent with both the observations of Baumgärtl et al. (1987) and the more extensive but less direct measurements of Stücker et al. (2000), the early report of Evans & Naylor (1966/1967) failed to detect such a correlation. In this study, however, the O2 electrodes were large (diameters ∼0.5 mm) and the measurements were made at depths of 1–3 mm beneath the surface (Evans & Naylor, 1966/1967). This would be well below the sub-papillary plexus in a region where PO2 varies little (Baumgärtl et al. 1987).

Whereas the correlation between the PO2 and depth that we observed is consistent with O2 being delivered to the papillary tissue from the sub-papillary plexus, a contribution is also being made by the capillary loops. This can be seen from the smaller changes in PO2 with depth that occur in the vicinity of the capillary loops than occur in the papillary tissue as a whole (Figs 5A and B). Whereas the values for PO2 at the base of the papilla (Fig. 5B) were similar to those found ‘deep’ in the tissues (Fig. 5A) close to the sub-papillary plexus, the mean values for PO2 in the middle region and near the tip of the papilla were considerably greater than the mean values for the middle depth of the skin as a whole. While these findings are to be expected, this is the first time, to our knowledge, that they have been demonstrated. Steeper gradients of PO2 between the papillae probably reflect both the absence of the capillary loops (and hence a lower rate of O2 delivery) and also the greater thickness of the epidermal layer in these areas. One might anticipate that the PO2 within a papilla would be increased and the axial PO2 gradient would be reduced still further as the blood flow through the capillary loop is increased. Unfortunately we were not able to demonstrate this.

Although there is now strong evidence that O2 may enter the skin from the atmosphere, our measurements demonstrate that when this is prevented, oxygen supply to the epidermis and superficial dermis can be maintained by the papillary microcirculation. There is also a suggestion that the papillary microcirculation is regulated to meet the O2 requirements of the tissue at a very local level.

Changes in papillary tissue PO2 following arrest and restoration of capillary blood flow

When flow was stopped, PO2 fell exponentially from a mean value of ∼35 to ∼19 mmHg with a mean time constant of just less than 8.5 s. In six of the seven experiments shown in Fig. 6A the recording electrode was close to the sub-papillary plexus, as indicated by relatively high pre-occlusion values of the PO2 (∼36–60 mmHg). When flow was stopped in vessels close to points near the base of the papillae, PO2 fell to 20–30 mmHg, indicating that the supply of O2 could be maintained, presumably by diffusion from the surrounding vessels where microvascular flow was undisturbed. In the experiment shown in the lower right hand panel of Fig. 6A, however, the electrode tip was at an intermediate depth in the tissue. Here, the arrest of local capillary flow brought PO2 close to zero. This one observation suggests that O2 supply to the outer papillary dermis and epidermis is dependent on flow in the closest vessels when oxygen supply from the air is prevented. A further observation can be made from the data shown in Fig. 6A consistent with this conjecture. The fall in PO2 upon occlusion was approximately 16 mmHg in all experiments. Since PO2 in the epidermis and outer regions of the dermis is roughly 16 mmHg prior to occlusion when the skin surface is covered by paraffin oil, a fall of 16 mmHg would bring PO2 to zero here.

The exponential decline in PO2 when flow is stopped would be consistent with the discharge of O2 from stores in the tissue. The most likely site of these O2 stores is the haemoglobin of the red cells in the capillaries where flow has been arrested. The oxyhaemoglobin dissociation curve is approximately linear over the range of PO2 between 40 and 15 mmHg, and the unloading of O2 over this range might be expected to approximate to a single exponential function.

The rise of PO2 to a new steady state as flow is restored is determined by the rate at which the PO2 in capillaries is returned to its pre-occlusion value. This is primarily dependent on the blood flow velocity in the reperfused capillaries. If we consider the tissue at the tip of the microelectrode being principally supplied by O2 from a point along a nearby capillary, the rate at which capillary PO2 will rise at this point will depend on how rapidly blood with a higher PO2 reaches that point and how much the blood PO2 falls between leaving the arteries and arriving at this point. Providing the O2 consumption of the tissue remains constant, the extraction (i.e. the O2 lost from a unit volume of blood) should be largely dependent on the blood flow. If the post-occlusion flow is greater than in the pre-occlusion state, not only will the PO2 rise rapidly, but it should rise to a level higher than it was initially. The tissue PO2 should then remain above its initial value for as long as the flow is elevated. This is just what has been observed. When blood flow is restored, tissue PO2 rises to a value that is ∼23 % higher than its pre-occlusion value. This is consistent with either a decrease in tissue O2 consumption or a significant degree of local reactive hyperaemia. Assuming that tissue O2 consumption is the same as in the pre-occlusion state, the 23 % increase in PO2 is equivalent to an increase in O2 saturation in the blood of the nearest capillaries from 70 % to 85 %. If the saturation of the arterial blood is 95 %, the increase in blood flow equivalent to the rise in tissue PO2 is of the order of 2.5-fold. This calculation indicates that local reactive hyperaemia occurs with a high spatial resolution in skin and this would be consistent with the very localised hyperaemic responses seen in some other tissues (Burton & Johnson, 1972). Although the rise in PO2 accompanying reperfusion can be described approximately by a single exponential, the data in Fig. 6B are less regular than those describing the decline of PO2 with vascular occlusion. This may represent fluctuations in blood flow velocity or red cell flux with the restoration in flow (Johnson & Wayland, 1967).

In conclusion, using O2-sensitive microelectrodes, we have demonstrated that when oxygen is prevented from entering the skin from the atmosphere, the PO2 in the superficial layers of the skin increases with depth down to the level of the sub-papillary plexus. Within papillae, the PO2 is raised by the blood flow through the capillary loops. When microvascular blood flow in the immediate vicinity of a point in the tissue is occluded, PO2 falls exponentially to a value 10–20 mmHg below its pre-existing value. From this it would appear that the O2 supply to the epidermis is vulnerable to very local reductions in perfusion when the O2 supply from the air is eliminated.

Acknowledgments

This work was supported by the Wellcome Trust (051015).

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