MATERIALS AND METHODS

Subject selection

Since 1983 we have collected a body of material on patients with EM. A review with clinical and epidemiologic characteristics of 87 patients has previously been reported (^{Kalgaard et al, 1997}). In this study patients from this body of material were selected according to the following inclusion criteria: primary adult EM (no underlying disease, age > 18 y), severity group 2–3 with good mental and physical health. Severity group 2 indicates a patient having periods of uncomfortably warm hands and feet, but with ability to find pain relief through walking barefoot on cold floors, etc. Group 3 indicates periods of burning pain and a patient immersing the feet in cold water < 1 h per d (^{Kalgaard et al, 1997}). Subjects were excluded if they had a history of any disease or condition that, in the opinion of the investigator, might interfere with the results of the study, were currently using vasoactive drugs, had undergone treatment with any other investigational drug during the 3 mo preceding the study, or had a history of drug or alcohol abuse. EM patients were compared with age- and sex-matched volunteers. None of the controls had a history of cardiovascular or other serious diseases. The procedure and purpose of the examination was fully explained to all participants, who gave their written, informed consent.

Capillary microscopy and temperature measurements

Skin capillary density has for many years been studied by intravital light microscopy (^{Gilje, 1953}). Combining modern microscope techniques and information technology, a package able to facilitate the extraction of capillary parameters from video microscopy skin images has been developed, analyzing capillary density and capillary size (^{Zhong et al, 2000}). In addition, the images can be analyzed further. The software can calculate the average distance between first, second, and third neighboring capillaries in the imaging field. Another parameter, mutual distance, is derived by creating a triangulation map of the capillaries in the image and gives the average length of the limbs of the triangles. The final parameter, distribution uniformity, giving a measure of the heterogeneity of the capillary distribution in the field of view, is defined by the formula (1 - SD of mutual distance)/mutual distance. The hardware in the microscopy system comprises a video capillary microscope and a computer workstation. The capillary microscope is a portable video microscopy system consisting of a video signal control unit and a handheld mini-CCD camera (Microvision MV2100, Finlay Microvision, Warwickshire, U.K.). Magnification of the system was set to 200 to enable the investigation of an ensemble of approximately 100 capillaries in a field of view of 1.7 mm². The color images captured by the system show capillary ensembles in which the capillaries appear as dark red spots with comma-like shapes. These spots are actually the apexes of the blood-filled capillary loops perpendicular to the skin surface. This technique does not give information on blood flow dynamics.

Skin temperature was evaluated in the pulp of the first left toe using an Anritsu HFT-80 Digital Handheld Thermometer (Anritsu Meter, Japan) with a 5 mm flat leaf probe, model 540E/540K.

Laboratory procedures

The subjects were instructed not to eat, drink coffee, tea or alcohol, or smoke for a minimum of 3 h before measurements. The experiments were performed during the coldest period of the year (mean outdoor temperature -6.2°C during the test period according to the Norwegian Meteorological Institute). Twenty minutes prior to measurements the subjects were recumbent in a room with an ambient temperature of 23°C (22°C-24°C) without physical or psychologic stress. In order to induce EM attacks, the subjects were covered with multiple duvets except for the left foot and head. Pain intensity was scored using a visual analog scale (VAS 0–100) before and after heating (^{Scott and Huskisson, 1976}). Central body heating was terminated when patients suffered EM symptoms (pain VAS score 50), reached a toe temperature > 32°C, or if the heating period exceeded 120 min. Two test sites were chosen for the capillary microscopy. The first measurement site was on the terminal phalanx of the first toe, just proximal to the nail bed. In this region the capillary loops are perpendicular to the skin surface and the vasculature contains numerous AV shunts (^{Grant and Bland, 1931;Sherman, 1963}). The other site was in the arch of the foot containing fewer AV anastomoses. Two microscopy images were captured at baseline and after whole body heating at tiny spots marked with black ink. The capillary parameters were extracted from an average of two images. To make the skin more transparent and to minimize reflection from the surface, measurement sites were prepared with immersion oil. The handheld video microscopy unit was perpendicular to the skin surface and hardly touching it so as to prevent application of pressure to the underlying tissue. The intensity of the light source was adjusted to maximize the visual contrast in the image. Captured images were stored in the internal database of the image capillary analysis program for immediate or later processing.

Statistical analyses

The results are expressed as medians with total range. Data calculated in the capillary analysis software are given as mean with SD. Significance levels reported are two-tailed and the results were tested at a 5% level of significance. The Kruskal–Wallis test was used to determine differences between more than two different groups and the Mann–Whitney test for comparison of two groups. Differences between pre and post heating were analyzed by the Wilcoxon signed rank test and Fisher's exact test was applied for demographic data. All analyses were performed using SPSS 10.0 software (SPSS, 233S Wacher Drive, Chicago, IL).

RESULTS

Fourteen EM patients fulfilling the inclusion and exclusion criteria and 10 control subjects were studied. None of the patients had EM symptoms prior to baseline measurements. EM attacks were induced in eight patients (symptomatic EM) after whole body heating whereas no discomfort was experienced in the control group. Maximal VAS pain score was 65 (50–100) in patients with EM symptoms. The asymptomatic EM group was significantly older [61 y (29–71)] compared to symptomatic EM patients [33 y (20–62); p <0.05] and the healthy control subjects [29 y (24–62); p <0.05]. No statistical difference was found for gender (symptomatic EM male/female 2/6 versus asymptomatic EM male/female 1/5), duration of EM [symptomatic EM 9 y (6–16) versus asymptomatic EM 11 y (2–21)] or prestudy severity of EM [2.5 (2–3) for both groups].

In EM patients the capillary density in the nail bed area decreased significantly after central body heating (p =0.01) in contrast to density in the foot arch (Table I). In the subset of symptomatic EM patients this reduction in the nail bed was even more pronounced. In this group there was also a nonsignificant trend towards reduced number of capillaries in the foot arch (p =0.16). For asymptomatic patients and healthy control subjects no changes were observed in either of the two measuring sites. The reduction in capillary density of EM patients was significantly larger (p <0.05) compared to the control group (Figure 2). Symptomatic patients had a more pronounced reduction in density and it was significantly larger compared to asymptomatic patients (p <0.05) and controls (p <0.01) (Figure 3), but no such difference was found in the foot arch. The sizes of the capillaries, distance to the first, second, and third neighbor, and mutual distance were significantly reduced in the nail bed of symptomatic EM patients after warming (Table II). No significant changes were demonstrated for the same parameters for asymptomatic EM patients and control subjects before and after heating. The changes in the same parameters after heating were not significantly different between the groups. Distribution uniformity was not significantly changed within or between the groups. The EM patients had a tendency towards lower skin temperature at baseline compared with the control group (p =0.15), but increase in skin temperature was significantly higher in symptomatic patients after heat provocation compared to asymptomatic EM (p <0.05) and control persons (p <0.05) (Table III).

Figure 2.

Capillary density was significantly reduced in EM patients after central body heating in the nail bed area with the highest concentration of AV shunts. (a) EM patients and (b) healthy control group. The reduction in capillary density was significantly higher in EM patients in the nail bed area () compared to the control group (*p <0.05), in contrast to the foot arch ().

Full figure and legend (16K)

Figure 3.

The symptomatic subset of EM patients had more pronounced reduction in capillary density in the nail bed. (a) Symptomatic EM patients; (b) asymptomatic EM patients; and (c) healthy control group. The reduction in capillary density was significantly higher in symptomatic EM patients in the nail bed area () compared to asymptomatic patients and controls after central body heating. No such difference was found between the three groups in the foot arch, () but the same nonsignificant trend was observed. Distribution of data is illustrated as box plots, where the boxes show median with quartiles. *p <0.05 and **p <0.01 compared to symptomatic EM in the nail bed area.

Full figure and legend (18K)

Table I - Capillary density in the nail bed region decreased significantly compared with foot arch in symptomatic EM, but not in asymptomatic EM and healthy control subjects.

Full table

Table II - Capillary size, distance to the neighboring capillaries, and mutual distance are significantly reduced in the nail bed of symptomatic EM patients.

Full table

Table III - Increase in skin temperature of symptomatic EM patients is larger compared with asymptomatic patients and healthy control subjects.

Full table

DISCUSSION

In EM patients the reduction in capillary density in the nail bed area was significantly larger after central body heating compared to the control group. This change was more pronounced in symptomatic EM patients.

The selection of patients was based on a standardized definition of EM and predefined inclusion and exclusion criteria. The patients in this study were primary cases with medium severity and duration of approximately 10 y from onset of EM symptoms. We could not find any clinical features that separated the symptomatic and asymptomatic EM patients.

The skin perfusion is far greater than needed for epidermal and dermal nutrition and the excess is related to thermoregulation. Nutritional needs of superficial skin are mainly supplied by the perpendicular capillary loops in the papillary dermis. In the acral skin areas, thermoregulatory perfusion bypasses these capillaries by shunting arterial blood through microvascular AV anastomoses into deep dermal venous plexa (^{Braverman, 2000}). According to the shunt hypothesis of EM, the increased skin flow during attacks passes through anatomical shunts in patients with primary EM and functional shunts in secondary EM (^{Kvernebo, 1998;Mørk et al, 2000}). Assessing primary EM in this study, the choice of measurement sites was based on the localization of the AV anastomoses. The highest concentration of anastomoses in the foot is in the nail bed, followed in decreasing order by the toe tips, plantar surface of the first toe, and plantar aspect of the foot. No anastomoses are found on the dorsal aspect of the foot (^{Grant and Bland, 1931;Sherman, 1963}). Capillaries may be difficult to see through a thick stratum corneum, which limits the choice of measurement sites in this study. We therefore chose two test sites: the nail bed area (numerous AV anastomoses) and the foot arch (fewer AV anastomoses) with thin stratum corneum compared to the weight bearing parts of the sole.

Central body heating was used to induce EM attacks as central heat stimulation increases skin blood flow by a synchronous opening of the AV shunts controlled by the central nervous system. This is in contrast to local heating, which mainly affects the skin capillary blood flow, independent of sympathetic nervous activity (^{Hales et al, 1978a;1978b;1993}).

Skin capillaries work intermittently; some are closed whereas others are perfused (^{Antonios et al, 1999}). The computer-assisted video microscopy used in this study detects capillaries that are filled with blood cells and does not give the dynamics of blood flow. In thermoneutral subjects AV anastomoses open and close simultaneously at a frequency of 2–3 cycles per minute (^{Thoresen and Walløe, 1980}). When the anastomoses are intermittently open they represent a low resistance alternative to blood flow. On the other hand, when the shunts are closed, flow must take place through capillaries. In centrally heated subjects, the anastomoses stay open most of the time. In symptomatic patients, we have demonstrated a reduction in capillary density parameters (number of perfused capillaries, distance to the first, second, and third neighbor, and mutual distance) in areas with the highest concentration of AV anastomoses (nail bed region) after central warming. Our interpretation is that the AV anastomoses stay permanently open, causing a steal of blood flow from the capillaries, resulting in a relative lack of capillary perfusion and a corresponding skin hypoxia. In the foot arch, with fewer AV shunts, we observed the same trend towards reduced number of visible capillaries (Figure 2). The finding of reduced capillary size in symptomatic patients can also be a consequence of a steal phenomenon. The capillary wall consists of a basal membrane and an endothelial lining. The wall structure is distensible and the size is therefore proportional to the intracapillary pressure, and steal through shunts implies reduced intracapillary pressure. The lack of changes in the distribution uniformity parameter implies that the reduction in capillary density parameters was not accompanied by increase in the heterogeneity of distribution.

A tendency towards lower skin temperature in EM patients between attacks has been demonstrated previously (^{Littleford et al, 1999;Mørk et al, 2002}). In this study, symptomatic EM patients showed a greater increase in pulp temperature (many AV anastomoses) in response to central body heating compared to the two other groups. This temperature increase will accelerate the metabolism of the skin, and thereby increase the nutritional needs (Figure 1). But as the number of perfused capillaries decreases, skin hypoxia is likely in affected areas (areas with AV shunts) during EM attacks. It is a well-known clinical feature that primary EM patients experience more pain in the nail bed area and pulp region than in the foot arch, whereas they seldom experience pain in areas without shunts.

Laser Doppler perfusion imaging measures capillary and thermoregulatory skin perfusion, but the main contribution to the laser signal comes from the deeper layers of the skin vasculature (^{Wårdell et al, 1993;Arildsson et al, 2000}). Our group has demonstrated increased skin perfusion in areas with AV anastomoses during EM attacks, using this method (^{Mørk et al, 2000}). The interpretation of the study was that the perfusion increase took place in AV shunts and deep dermal plexa. In this study we have shown a corresponding decrease in capillary perfusion in superficial skin layers, further supporting the interpretation that increased laser Doppler perfusion took place in shunts in deep dermis.

Tissue hypoxia resulting from diminished oxygen delivery normally results in a combination of increased tissue perfusion and increased number of open capillaries (recruitment) (^{Parthasarathi and Lipowsky, 1999}). We could not demonstrate recruitment in control subjects or asymptomatic EM (Table I), but we cannot draw conclusions about perfusion of the microscopically demonstrated capillaries because the method assesses morphology and not perfusion parameters (^{Kvernebo, 1998;Mørk et al, 2000}). Skin hypoxia has previously been postulated based on clinical and laboratory findings. In our patient material, we have observed spontaneous foot ulcers and gangrene, and three limbs have been amputated in spite of open limb arteries in patients with severe EM. We have observed Beau lines in the nails of the feet after periods of severe EM attacks that could be interpreted as growth disturbances secondary to hypoxia. Pain relief by cooling could be explained by reversal of the sequences of events shown in Figure 1, secondary to reduced metabolism. Coexistence of hyperaemia (laser Doppler measurements) and hypoxia (transcutaneous oxygen tensiometry) has been demonstrated in severely affected EM skin (^{Kvernebo, 1998}). Reduced symptoms with a corresponding increase in oxygen tension and reduction in perfusion have also been observed after treatment with vasodilators in selected cases (^{Kvernebo, 1998}).

References

References