- Email: [email protected]
Time course of UVA- and UVB-induced inflammation and hyperalgesia in human skin
European Journal of Pain (1999) 3: 131–139
Time course of UVA- and UVB-induced inflammation and hyperalgesia in human skin R.T. Hoffmann and M. Schmelz Department of Physiology I, University of Erlangen/Nümberg, D-91054 Erlangen, Germany
Dose-dependency and time course of hyperalgesia and erythema following UVA (16.8 and 36 J/cm2) and UVB (one and three times the minimum erythema threshold) irradiation was investigated in 10 healthy human subjects. Skin patches (1.5 cm in diameter) on the ventral side of the upper leg were irradiated with UVA or UVB light. Hyperaemia (Laser Doppler flowmetry, infrared thermography), thermal hyperalgesia to radiant heat stimuli, and mechanical hyperalgesia to controlled impact stimuli were tested at 1, 6, 12, 24, 48 and 96 h after irradiation. Dose-dependent delayed hyperaemia and hyperalgesia was induced only by UVB irradiation. UVB-induced increase in blood flow peaked at 12 h after irradiation and normalized by 96 h. Although superficial blood flow, as measured by Laser Doppler flowmetry, increased up to eight-fold, no significant increase of skin temperature was detected using infrared thermography. Development of mechanical and thermal hyperalgesia was delayed and reached a plateau between 24 and 48 h. In contrast to UVB, UVA irradiation of up to 36 J/cm2, sufficient to produce intense tanning of the skin, did not induce significant hyperalgesia or delayed hyperaemia. It is concluded that UVB- but not UVA-irradiation is a suitable experimental model of subacute thermal and mechanical hyperalgesia. The different time courses of erythema and hyperalgesia indicate that inflammatory mediators responsible for vasodilatation are not identical with those inducing hyperalgesia. KEYWORDS: pain, nociception, sunburn.
INTRODUCTION UVA and UVB are differently effective in the induction of inflammatory skin reactions. While UVB (290–320 nm) is largely absorbed in the epidermis, UVA (320–400 nm) penetrates into the deeper layers of the dermis. The sunburn reaction following UVB-irradiation, which includes hyperaemia and hyperalgesia, has been studied extensively. However, the role of UVA irradiation in producing a sunburn reaction is less clear (Gilchrest et al., 1983). In general, the doses necessary for induction of an UVA erythema are found to be very high, i.e. more than 1000-fold
the required UVB doses (Warin, 1978). However, with higher UVA intensities, more-pronounced unspecific thermal effects are induced, which also differ histologically from UVB-induced changes (Willis & Cylus, 1977; Rosario et al., 1979). The aim of our study was to assess dose–response curves for hyperaemia and hyperalgesia following UVA and UVB-irradiation. In addition, the time course and pattern of hyperalgesia following UVA- and UVB-irradiation was evaluated and compared with objective signs of inflammation, i.e. skin temperature and superficial blood flow.
MATERIALS AND METHODS Paper received 1 June 1998 and accepted in revised form 20 December 1998. Correspondence to: Dr Martin Schmelz, Institut für Physiologie und Experimentelle Pathophysiologie, Universitätsstr. 17, 91054 Erlangen, Germany. Email: schmelz@physiologie 1.uni-erlangen.de
Subjects Ten volunteers, five male, five female (20–25 years of age), participated in the study. All
1090-3801/99/020131 + 09 $12.00/0 © 1999 European Federation of Chapters of the International Association for the Study of Pain
132
subjects gave their informed consent according to the Declaration of Helsinki, and the study was approved by the local ethics committee. Only subjects showing no neurological or dermatological abnormalities were included. The study was performed during winter and early spring and all subjects were instructed to avoid additional UV-exposure of their thighs. Hyperalgesia models
UV-erythema model Mode of UV application One week before the first
experimental session, the minimum erythema dose for UVA-(320–400 nm wavelength) and UVB-irradiation was assessed using a calibrated UV source (Saalmann multitester® SBB LT 400, Saalmann Medizintechnik, Herford, Germany). Five circular spots on the thigh with a diameter of 1.5 cm and a constant distance of 2.5 cm were irradiated with increasing doses of UVA (20 min; 16.8–36 J/cm2) and UVB light (10 s; 0.01–0.05 J/cm2). UV-induced erythema has been shown to be reciprocal (Meanwell & Diffey, 1989); thus, total energy but not duration of the irradiation is the main factor for the sunburn reaction. The minimum dose which induced a visible erythema 24 h after irradiation was determined as minimal erythema dose (MED). One week after the determination, the subjects were irradiated with one and three times the individual MED, and time course of hyperalgesia and inflammation was assessed. In the following, we refer to these dosages as UVB-1 and UVB-3. The skin reaction to UVA was different. In pilot experiments, UVA application led to warming of the skin, and UVA doses above 36 J/cm2 induced increasing heat pain. To minimise sideeffects from heating, it was necessary to stop the irradiation before heat pain occurred. Therefore, we chose to apply UVA in doses of 16.8 and 36 J/cm2, which were strong enough to induce clear tanning of the skin without being painful during irradiation. In this manuscript, we refer to these two dosages as UVA-1 (low) and UVA-h (high). After UVA-l and UVA-h irradiation (about 500× higher than the UVB doses used), the skin showed an immediate erythema reaction, which declined within a few hours. European Journal of Pain (1999), 3
R.T. H OFFMANN
AND
M. S CHMELZ
Thermography For thermography, an infrared camera was used (Agema Thermovision® 900, Sweden), and recordings were stored on hard disk for off-line analysis. Thirty-two successive recordings (3 Hz) were averaged, and the average picture was stored on hard disk for later analysis. The distance between camera and upper leg of each volunteer was 1 m. Average skin temperature of the four irradiated areas and a non-irradiated control area was determined by dedicated software (Erika Thermovision, Agema, Sweden). Laser Doppler flow measurements Superficial blood flow on the test spots was measured by laser Doppler flowmetry immediately before the algesimetric session. For this purpose, a laser Doppler probe (Periflux PF2, Perimed, Sweden) was applied to the skin for at least 20 s on the previously irradiated spots and a control spot. At each spot, mean values of at least two different probe positions were obtained. Heat pain thresholds Radiant heat stimuli were
delivered from a halogen bulb focused on the skin (diameter 1 cm) and feed-back was controlled from a thermocouple gently attached to the skin in the centre of the light beam (Beck et al., 1974). The skin temperature was slowly increased by 0.67°C/s, from an adapting temperature of 32°C. Subjects were instructed to stop the heating by pressing a button as soon as the heat became painful. The cutoff temperature of 52°C was not reached in any of the cases. The UVA-l, UVA-h, UVB-1, UVB-3, and a control spot on untreated skin between the irradiated spots were tested in a randomised order, unpredictable for the subject. Mechanical impact stimulation Mechanical hyperal-
gesia was assessed by delivering impact stimuli. For this purpose, a plastic cylinder (0.5 g, diameter 0.5 cm) was driven at a controlled speed through a barrel against the skin by a pressurised air-driven stimulator, as described elsewhere in detail (Kohllöffel et al., 1991). The UV-treated skin patches and the control spot on untreated skin were tested twice in random order with an impact velocity of 5, 9 and 12 m/s, applied in ascending order. Subjects were instructed to give pain ratings after each impact. A rating of zero indicated ‘no
UVA-
AND
UVB- INDUCED
133
INFLAMMATION AND HYPERALGESIA
sensation’ and a rating of 100 should represent a sensation being just painful. For sensations of double that intensity ratings should be duplicated etc. The rationale of this kind of rating scale has been discussed elsewhere (Koltzenburg & Handwerker, 1994). The obtained ratings were in the range of 5–180. The values from the two stimulus repetitions on each spot were averaged.
Experimental protocol Dose-dependency of the irradiation and the minimal erythema dose of each volunteer were determined by measurements 24 h after application of fixed doses of UVA and UVB. Pain thresholds and degree of inflammation were assessed at 1, 6, 12, 24, 48 and 96 h after application of UV radiation. To reduce circadian fluctuations, irradiations were performed at 8.00 a.m. for each subject. Additional measurements were performed after 132 h if blood flow after 96 h had not returned to baseline. At each time, radiation effects were assessed in the same order, starting with infrared thermography and Laser-Doppler flux measurement. After these non-invasive measurements, heat pain thresholds were determined and, finally, mechanical impact stimuli were applied. Thus, unwanted changes of superficial blood flow and temperature by the test stimuli were minimised.
Statistical procedures
Dose dependency Blood flux values, mean temperatures at the irradiation spots, pain ratings and heat pain thresholds were analysed by ANOVA with dose of irradiation (5 levels) and type of irradiation (UVA and UVB) as independent variables.
Time course Blood flux, skin temperature, pain ratings and heat pain thresholds were analysed by ANOVA with time of session (1, 6, 12, 24, 48 and 96 h), type of UV irradiation (UVA and UVB), and intensity of irradiation (1MED and 3MED) as independent variables (repeated measurement).
Post-hoc Scheffé tests were performed on significant factors, where suitable. All statistical analysis was done using the STATISTICA 5.0® software package (Statsoft, Tulsa, USA). The significance level was set at p<0.05.
RESULTS Acute effects and tanning
UVA irradiation UVA irradiation induced warm or slightly hot sensations in all subjects, but was not felt as painful. Immediately after 20 min of UVA irradiation, the skin appeared reddened and temperature was at about 40°C. The skin reddening declined and normalised within a few hours. UVA irradiation produced a pronounced tanning of the irradiated skin which lasted for several weeks. No blisters or permanent marks were induced by the UVA light.
UVB irradiation The UVB exposed skin areas showed no alterations immediately after UV-expositions. No spontaneous ongoing pain was reported from either spot, an erythema developed in the course of the following 12 h. No permanent marks or blisters were induced in any subject. One day after irradiation, UVB-irradiated skin areas exhibited a light brown tanning, which lasted for several weeks. Blisters or permanent marks were not induced in any of the subjects.
Dose-dependent effects of UV radiation
Erythema Dose–response curves for UVA and UVB light were obtained by measuring the effects 24 h after irradiation. Even the most intense UVA irradiation (36.0 J/cm2) did not induce any significant increase in superficial blood flow as compared to the control area. In contrast, UVB-irradiation caused increased blood flow even at the lowest dose (0.01 J/cm2), which was significant for skin sites irradiated with more than 0.02 J/cm2.
European Journal of Pain (1999), 3
134
R.T. H OFFMANN
Visual inspection of the irradiated skin sites was less sensitive to detect increased blood flow (mean MED: 0.015 J/cm2). With increasing UVB intensity to 0.05 J/cm2 a linear increase of blood flow was measured, which was paralleled by more intense reddening of the irradiated skin (Fig. 1).
Skin temperature Despite the significant blood flow changes at the UVB spots, there was virtually no difference in skin temperature between the irradiated areas and the control spot for both UVA and UVB irradiation. The maximum difference to control was only 0.24°C for the UVA measurements and 0.84°C for the UVB application, which did not reach a significant level [UVB (0.05 J/cm2) vs control: p=0.44; ANOVA; Scheffé post hoc test] (Fig.1).
Mechanical hyperalgesia Mechanical hyperalgesia was tested by using impact stimuli with three different velocities (5, 9 and 12 m/s). In control skin, the light impact was felt as touch and the strongest as just painful (Fig.2). UVA irradiation did not induce mechanical hyperalgesia. In the tested range of impact intensities (5–12 m/s), no significant difference to control
AND
M. S CHMELZ
skin was observed. In contrast, UVB irradiation caused a dose-dependent mechanical hyperalgesia (Fig. 2). Even the weaker impact stimuli were felt stronger in skin irradiated with more than 0.3 J/cm2. For the higher velocity, irradiation above 0.2 J/cm2 already induced significant mechanical hyperalgesia. At the 0.5 J/cm2 irradiated spots, pain ratings were about 50% higher than on control skin (Fig. 2).
Thermal hyperalgesia Heat pain thresholds were found only slightly decreased on the UVA spots. The threshold was reduced between 0.8 and 1.9°C compared to the control spot (dose range 16.8–31.2 J/cm2), and reached a minimum on the spot irradiated with a dose of 36 J/cm2; this reduction (2.8°C) reached statistical significance (ANOVA, planned comparison; p=0.02) (Fig. 3). In contrast, heat pain thresholds were profoundly reduced by UVB irradiation. Even the weakest dose of 0.01 J/cm2 caused a significant decrease in heat pain threshold by 3.27°C as compared to the non-irradiated control. Irradiation with doses of 0.04 and 0.05 J/cm2 caused a decrease of the heat pain threshold to a plateau temperature of 36.5°C, which is about 8°C below heat pain thresholds in control skin.
200 150 100
34
50 32 0
0.01 Control 16.8
0.02 21.6
0.03 26.4
0.04 31.4
0.05 36.0
Skin temperature (C°)
LDF (relative units)
250
UVB UVA
Dose of irradiation (J/cm2)
Fig. 1. Dose–response curves of UVA (■) and UVB (●) irradiation for the induction of increased superficial blood flow (LDF) and increase in skin temperature at 24 h after irradiation. Mean±SEM, n=10; **, p<0.01, ANOVA, Scheffé test.
European Journal of Pain (1999), 3
Impact pain rating
160 140 120 100 80 60 40 20 0
0.01 Control 16.8
0.02 21.6
0.03 26.4
0.04 31.4
0.05 UVB 36.0 UVA
Dose of irradiation (J/cm2)
Fig. 2. Dose–response curves of UVA (●) and UVB (■) irradiation for the induction of mechanical hyperalgesia at 24 h after irradiation. Mechanical impact stimuli were delivered at a speed of i=5 (open symbols) and 12 m/s (closed symbols). Mean±SEM, n=10; *; 0.05>p>0.01, ANOVA Scheffé test; **, p<0.01, ANOVA, Scheffé test.
AND
UVB- INDUCED
135
INFLAMMATION AND HYPERALGESIA
LDF (relative units)
Decrease of heat pain threshold (∆T; °C)
UVA-
0 –2 –4 –6
200 150 100 50 0
–8 –10 Control
0.01 16.8
0.02 21.6
0.03 26.4
0.04 31.4
0.05 UVB 36.0 UVA
Dose of irradiation (J/cm2)
Fig. 3. Dose–response curves of UVA (■) and UVB (●) irradiation for the induction of thermal hyperalgesia at 24 h after irradiation. Pain threshold to radiant heat are given as difference between irradiated skin areas and control skin. Mean±SEM, n=10; **, 0.05>p>0.01; **, p<0.01, ANOVA, Scheffé test.
Time course of inflammation and hyperalgesia
Erythema Fixed doses of UVA (16.8 and 36.0 J/cm2) and UVB irradiation (one and three times the individual minimal erythema dose) were used to determine time course of blood flow changes in superficial skin layers during the first 96 h after irradiation. Blood flow changes at the low dose UVA spot (UVA-I) did not reach statistical significance (ANOVA, planned comparison; p=0.24). Superficial blood flow at the high dose UVA spot (UVA-h) showed a clear increase 1 h after irradiation, and returned to baseline within 6 h after irradiation (see Fig.4). In contrast, time course of hyperemia after UVB-irradiation was different. Superficial blood flow at the UVB-1 spot doubled 6 h after application, and peaked at 24 h after irradiation. Forty-eight hours after irradiation flow, values were still 1.5-fold higher than measurements on control spot. UVB-3 treatment already caused a significant increase after 1 h and during the period to 48 h after irradiation (Fig.4). Peak hyperaemia was reached 12 h after irradiation, with flux values 8.5-fold higher than in the control area. Blood flow remained elevated after
20
40 60 Time (h)
80
100
Fig. 4. Time course of erythema (LDF) for 96 h after UVA- and UVB-irradiation. The dose used for UVA irradiation was fixed at 16.8 and 36.0 J/cm2 for each volunteer; UVB dosage was the individual minimal erythema dose (MED) and three times the MED. Mean±SEM, n=10; **, 0.05>p>0.01, **, p<0.01, ANOVA, Scheffé test. (■ ■) UVA (h); (■) UVA (l); (● ●) uvB-1; (●) UVB-3; (◆ ◆) control.
96 h, and did not completely return to baseline until 132 h after irradiation.
Skin temperature Only UVA irradiation caused immediate increase in skin temperature, which had virtually normalized by 1 h. The difference to the control area reached a maximum (+1.92°C) 5 min after UVA-h irradiation. In contrast, UVB irradiation did not cause a significant increase in skin temperature at any time. Even at 12 h after irradiation, when superficial blood flow was maximally enhanced at the UVB-3 spot, skin temperature did not differ significantly from control skin.
Mechanical hyperalgesia UVA irradiation at both doses did not induce any mechanical hyperalgesia to impact stimulation. In contrast, at the UVB-3 irradiated skin, mechanical hyperalgesia developed by 6 h and reached a plateau between 1 and 48 h. Pain ratings gradually decreased thereafter and reached baseline after 132 h. The mechanical hyperalgesia reached statistical significance between 12 and 48 h. At the UVB-1 spot, the time course of hyperalgesia was similar, but even at its maximum the effect did not reach statistical significance in the Scheffé post-hoc test (p=0.08) (Fig.5). European Journal of Pain (1999), 3
R.T. H OFFMANN
Impact pain rating
140 130 120 110 90 80 0 10 20
40
60
80
100
Time (h)
Fig. 5. Time course of mechanical hyperalgesia for 96 h after UVA- and UVB-irradiation. Pain ratings to mechanical impact stimuli which were delivered to the irradiated skin at a speed of 5 and 12 m/s are presented. The dose used for UVA irradiation was fixed at 16.8 and 36.0 J/cm2 for every volunteer; UVB dosage was the individual minimal erythema dose (MED) and three times the MED. Mean±SEM, n=10; *, 0.05>p>0.01; **, p<0.01, ANOVA, Scheffé test. (■ ■) UVA (h); (■) UVA (l); (● ●) UVB-1; (●) UVB-3; (◆ ◆) control; •; v = 12 m/s.
Thermal hyperalgesia Thermal hyperalgesia was also found to be dependent on quality and dose of radiation. Heat pain thresholds at control spots (44±0.4°C) corresponded to previous reports (Bickel et al., 1998). At the UVA spots, heat pain thresholds were slightly lower between 6 and 12 h afterwards, without reaching statistical significance (Fig.6). In contrast, UVB-irradiation induced a pronounced thermal hyperalgesia. Heat pain thresholds at the UVB-1 spots decreased gradually to a minimum of 3.8°C below control after 24 h (p=0.05, ANOVA, Scheffe post hoc test) and returned to normal after 96 h. UVB-3 irradiation caused thermal hyperalgesia with a similar time course, with a maximum drop in heat pain threshold of 8.5°C at 24 h after irradiation. Heat pain thresholds were significantly decreased during the period from 6 to 96 h after irradiation (Fig.6).
DISCUSSION Aspects of UV-induced inflammatory skin reactions have been investigated in humans before. European Journal of Pain (1999), 3
Decrease of heat pain threshold (∆T; °C)
136
AND
M. S CHMELZ
0 –2 –4 –6 –8 –10 0
10 20
40
60
80
100
Time (h)
Fig. 6. Time course of thermal hyperalgesia for 96th after UVA- and UVB-irradiation. Pain thresholds to radiant heat are given as difference between irradiated and control skin. The dose used for UVA irradiation was fixed at 16.8 and 36.0 J/cm2 for every volunteer; UVB dosage was one and three times the individual minimal erythema dose Mean±SEM, n=10; *, 0.05>p>0.01; **; p<0.01, ANOVA, Scheffé test. (■ ■) UVA (l); (■) UVA (h); (● ●) UVB-1; (●) UVB-3.
However, most investigators concentrated on measurements of superficial blood flow using reflectance instruments, laser Doppler flowmetry or scaling of skin redness. Hyperalgesia was either not tested or was reported casually based on complaints of patients (Snyder, 1975; Stern & Dodson, 1985). A systematic study about time course of thermal and mechanical hyperalgesia after UVB irradiation in humans has been reported only in abstract form before (Benrath et al., 1993). There is evidence that the early component (4–24 h) of the UVB-induced erythema is partly prostaglandin dependent: prostaglandins were found to be increased after UVB irradiation (Gresham et al., 1996), and cyclooxygenase inhibitors given topically or systemically suppress skin erythema in this period (Snyder, 1975; Anderson et al., 1989; Hughes et al., 1992; Juhlin & Shroot, 1992). However, the late phase of the erythema (>24 h) does not respond to cyclooxygenase inhibitors like indomethacin or piroxicam (Farr & Diffey, 1986; Juhlin & Shroot, 1992). Other mediators which could play a role in UV-induced inflammation include neuropeptides (Benrath et al., 1995), histamine (Gilchrest et al., 1981; Malaviya et al., 1996),
UVA-
AND
UVB- INDUCED
137
INFLAMMATION AND HYPERALGESIA
interleukins (Kennedy et al., 1997), nitric oxide (Goldsmith et al., 1996; Deliconstantinos et al., 1997) and oxygen radicals (Hruza & Pentland, 1993). Although hyperalgesia of sunburned skin is a common experience, and in numerous studies release of prostanoids has been found to be related to the UV-induced hyperaemia (Gilchrest et al., 1981; Kimura & Doi, 1998), UV-sunburn has rarely been used as an experimental hyperalgaesia model in humans (Bickel et al., 1998).
UVB-induced hyperaemia Dose dependency and time course of UVBinduced erythema were found in accordance with previous reports (Frödin et al., 1988; Hughes et al., 1992). Peak hyperaemia was detected between 6 and 24 h after irradiation, and had decreased by about 50% at 48 h. We did not observe a second peak of erythema as found by Benrath et al. (1993). Although an increase of up to eight-fold in superficial blood flow was measured by Laser Doppler flowmetry after UVB irradiation, no significant increase in skin temperature was detected using infrared thermography. A lack of correlation between superficial blood flow and skin temperature has already been described before (Seifalian et al., 1994). This discrepancy can be explained by the different depth of vasodilation. The laser Doppler detects increased blood flow preferentially in capillaries of the upper layers of the skin (<1 mm) which do not allow convective warmth transport. In contrast, vasodilation in arteriovenous shunts or larger arterioles directing warmer blood to the skin surface will increase skin temperature. The differential organisation of the superficial and deeper neuronal blood control in the skin has already been described (Greiner et al., 1995). As can be expected from histological sections, UVBinduced inflammatory changes are very superficial (Rosario et al., 1979), which explains the prominent superficial hyperaemia. Only inflammation reaching deeper skin layers can induce hyperaemia in superficial and deeper skin layers, leading to concomitant reddening and warming of the skin (Kelfkens et al., 1990).
UVB-induced hyperalgesia Thermal and mechanical hyperalgesia developed in all subjects after UVB irradiation with three times the minimal erythema dose. We did not encounter two peaks of the hyperalgesia at 6 and 36 h as observed by Benrath et al. (1993). Instead, a plateau of maximum hyperalgesia between 12 and 48 h after irradiation was found. However, our study confirms the delayed development of hyperalgesia as compared to hyperaemia described in their study (Benrath et al., 1993). The early component of hyperaemia (≤ 24 h) and hyperalgesia could be partly suppressed using cyclooxygenase-inhibitors (Snyder, 1975; Greaves et al., 1977; Anderson et al., 1989; Hughes et al., 1992; Juhlin & Shroot, 1992). In accordance with these results, the inducible cyclooxygenase (COX2) has recently been described to be upregulated in human skin following UVB irradiation (Buckman et al., 1998). However, the late erythema was unaffected by cyclooxygenase-inhibitors (Farr & Diffey, 1986; Juhlin & Shroot, 1992). Also, topical steroids did not suppress the late erythema (Kaidbey & Kurban, 1976; Hughes et al., 1992). The release of nitric oxide (Goldsmith et al., 1996; Deliconstantinos et al., 1997) or neuropeptides (Benrath et al., 1995) following UVB-irradiation could explain at least part of the cyclooxygenaseindependent hyperaemia. It is unclear to date which mediator could be responsible for the delayed hyperalgesia. Prostaglandin E2 and histamine were found to be elevated only in the early phase after UV irradiation (Gilchrest et al., 1981; Hawk et al., 1983). Also, pro-inflammatory cytokines like interleukin (IL) 1, 6, 8 and tumour necrosis factor α have been found to be elevated in human skin and keratinocytes after UV irradiation (Wlaschek et al., 1994; Sjolin et al., 1995; Strickland et al., 1997). Metabotropic glutamate receptors (mGluR), which contribute to mechanical hyperalgesia (Budai & Larson, 1998), could play a role in UV-induced hyperalgesia. UVB irradiation has been shown to enhance expression of the mGluR3 messenger RNA (Boxall et al., 1998) in the spinal cord. Interestingly, mGluR 2/3 have also been found on primary afferent neurons (Hargett et al., 1998), which
European Journal of Pain (1999), 3
138
could provide a basis for peripheral sensitisation of nociceptors by UVB irradiation.
Effects of UVA irradiation Long-wave UV irradiation (UVA: 320–400 nm) induces erythema at more than 1000-fold higher exposure doses compared to UVB. In contrast to UVB irradiation, these high dosages increase skin temperature, and induce pain and immediate erythema (Kaidbey & Kligman, 1979). The UVA erythema could not be diminished by cyclooxygenase inhibitors given topically, intradermally or systemically (Morison et al., 1977; Soter, 1990). In addition, clear histological differences between UVA and UVB-induced changes have been described: UVA has a greater effect on the dermis with an immediate onset, recruits neutrophils in the skin and does not induce typical ‘sunburn’ cells (Willis & Cylus, 1977; Rosario et al., 1979; Gilchrest et al., 1983). In our study, intensity of UVA irradiation was limited in order to prevent unspecific heat effects which could themselves cause inflammation and hyperalgesia (Møiniche et al., 1993). On the other hand, the intensity used (36 J/cm2) was effective to produce rapid tanning of the skin. No hyperalgesia or delayed erythema was observed using this dosage. At higher doses, UVA irradiation induces delayed hyperaemia and leukocyte infiltration, with a maximum at 3 h post irradiation (Gilchrest et al., 1983). This time course fits nicely to the time course of thermal and mechanical hyperalgesia seen after heat injury (Møiniche et al., 1993). Thus, thermal injury may well contribute to UVA-induced hyperaemia and hyperalgesia. In conclusion, UVA irradiation sufficient to induce tanning of the skin without thermal lesions does not produce delayed erythema or hyperalgesia. In contrast, UVB-induced sunburn provides a reliable and well-controlled experimental model of mechanical and thermal hyperalgesia. Erythema and hyperalgesia have different time courses and presumably are induced by different mediators. While prostaglandins are responsible for at least part of the early component of the UVB erythema, mediators for the late component and the concomitant hyperalgesia are yet to be identified. European Journal of Pain (1999), 3
R.T. H OFFMANN
AND
M. S CHMELZ
REFERENCES Anderson TF, Peterson C, Hamilton T. Meclofenomate inhibition of UV-induced erythema—a randomized placebocontrolled, double-blind study. Photodermatology 1989; 6: 63–68. Beck PW, Handwerker HO, Zimmermann M. Nervous outflow from the cat’s foot during noxious radiant heat stimulation. Brain Res 1974; 67: 373–386. Benrath J, Gillardon F, Zimmermann M. Differential time courses of hyperalgesia and inflammation in human skin after ultraviolet irradiation. Neuropeptides 1993; 24: 205. Benrath J, Eschenfelder C, Zimmermann M, Gillardon F. Calcitonin gene related peptide, substance P and nitric oxide are involved in cutaneous inflammation following ultraviolet irradiation. Eur J Pharmacol-Environ Toxic 1995; 293: 87–96. Bickel A, Dorfs S, Schmelz M, Forster C, Uhl W, Handwerker HO. Effects of antihyperalgesic drugs on experimentally induced hyperalgesia in man. Pain 1998; 76: 317–325. Boxall SJ, Berthele A, Laurie DJ, Sommer B, Zieglgänsberger W, Urban L, Tölle TR. Enhanced expression of metabotropic glutamate receptor 3 messenger RNA in the rat spinal cord during ultraviolet irradiation induced peripheral inflammation. Neuroscience 1998; 82: 591–602. Buckman SY, Gresham A, Hale P, Hruza G, Anast J, Masferrer J, Pentland AP. COX-2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer. Carcinogenesis 1998; 19: 723–729. Budai D, Larson AA. The involvement of metabotropic glutamate receptors in sensory transmission in dorsal horn of the rat spinal cord. Neuroscience 1998; 83: 571–580. Deliconstantinos G, Villiotou V, Stavrides JC. Inhibition of ultraviolet B-induced skin erythema by N-nitro-L-arginine and N-monomethyl-L-arginine. J Dermatol Sci 1997; 15: 23–35. Farr PM, Diffey BL. A quantitative study of the effect of topical indomethacin on cutaneous erythema induced by UVB and UVC radiation. Br J Dermatol 1986; 115: 453–466. Frödin T, Molin L, Skogh M. Effects of single doses of UVA, UVB, and UVC on skin blood flow, water content, and barrier function measured by laser-Doppler flowmetry, optothermal infrared spectrometry, and evaporimetry. Photodermatology 1988; 5: 187–195. Gilchrest BA, Soter NA, Stoff JS, Mihm MC, Jr. The human sunburn reaction: histologic and biochemical studies. J Am Acad Dermatol 1981; 5: 411–422. Gilchrest BA, Soter NA, Hawk JL, Barr RM, Black AK, Hensby CN, Mallet AI, Greaves MW, Parrish JA. Histologic changes associated with ultraviolet A-induced erythema in normal human skin. J Am Acad Dermatol 1983; 9: 213–219. Goldsmith PC, Leslie TA, Hayes NA, levell NJ, Dowd PM, Foreman JC. Inhibitors of nitric oxide synthase in human skin. J Invest Dermatol 1996; 106: 113–118. Greaves MW, Hensby CN, Black AK. Inflammation in human skin induced by ultraviolet irradiation. Postgrad Med J 1977; 53: 656–657. Greiner T, Nischik M, Schmelz M, Forster C, Handwerker HO. Neurogenic flare responses are heterogeneous in
UVA-
AND
UVB- INDUCED
INFLAMMATION AND HYPERALGESIA
superficial and deep layers of human skin. Neurosci Lett 1995; 185: 33–36. Gresham A, Masferrer J, Chen X, Leal KS, Pentland AP. Increased synthesis of high-molecular-weight cPLA2 mediates early UV-induced PGE2 in human skin. Am J Physiol 1996; 270: C1037–C1050 Hargett GL, Coggeshall RE, Carlton SM. Metabotropic glutamate receptors are differentially distributed on primary afferent terminals in the dorsal horn. Soc Neurosci Abstr 1998; 24: 1869. Hawk JL, Black AK, Jaenicke KF, Barr RM, Soter NA, Mallett AI, Gilchrest BA, Hensby CN, Parrish JA, Greaves MW. Increased concentrations of arachidonic acid, prostaglandins E2, D2, and 6-oxo-F1 alpha, and histamine in human skin following UVA irradiation. J Invest Dermatol 1983; 80: 496–499. Hruza LL, Pentland AP. Mechanisms of UV-induced inflammation. J Invest Dermatol 1993; 100: 35S–41S. Hughes GS, Francom SF, Means LK, Bohan DF, Caruana C, Holland M. Synergistic effects of oral nonsteroidal drugs and topical corticosteroids in the therapy of sunburn in humans. Dermatology 1992; 184: 54–58. Juhlin L, Shroot B. Effect of drugs on the early and late phase UV erythema. Acta Derm Venereol (Stockh) 1992; 72: 222–223. Kaidbey KH, Kligman AM. The acute effects of long-wave ultraviolet radiation on human skin. J Invest Dermatol 1979; 72: 253–256. Kaidbey KH, Kurban AK. The influence of corticosteroids and topical indomethacin on sunbum erythema. J Invest Dermatol 1976; 66: 153–156. Kelfkens G, vanHelden AC, van-der-Leun JC. Skin temperature changes induced by ultraviolet A exposure: implications for the mechanism of erythemogenesis. Photodermatol Photoimmunol Photomed 1990; 7: 178–182. Kennedy M, Kim KH, Harten B, Brown J, Planck S, Meshul C, Edelhauser H, Rosenbaum JT, Armstrong CA, Ansel JC. Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci 1997; 38: 2483–2491. Kimura T, Doi K. Effects of indomethacin on sunburn and suntan reactions in hairless descendants of Mexican hairless dogs. Histol Histopathol 1998; 13: 29–36. Kohllöffel LU, Koltzenburg M, Handwerker HO. A novel technique for the evaluation of mechanical pain and hyperalgesia. Pain 1991; 46: 81–87. Koltzenburg M, Handwerker HO. Differential ability of human cutaneous nociceptors to signal mechanical pain
139
and to produce vasodilatation. J Neurosci 1994; 14: 1756–1765. Malaviya R, Morrison AR, Pentland AP. Histamine in human epidermal cells is induced by ultraviolet light injury. J Invest Dermatol 1996; 106: 785–789. Meanwell EF, Diffey BL. Reciprocity of ultraviolet erythema in human skin. Photodermatology 1989; 6: 146–148. Møiniche S, Dahl JB, Kehlet H. Time course of primary and secondary hyperalgesia after heat injury to the skin. Br J Anaesth 1993; 71: 201–205. Morison WL, Paul BS, Parrish JA. The effects of indomethacin on long-wave ultraviolet-induced delayed erythema. J Invest Dermatol 1977; 68: 130–133. Rosario R, Mark GJ, Parrish JA, Mihm MC. Jr. Histological changes produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and UV-A with psoralens. Br J Dermatol 1979; 101: 299–308. Seifalian AM, Stansby G, Jackson A, Howell K, Hamilton G. Comparison of laser Doppler perfusion imaging, laser Doppler flowmetry, and thermographic imaging for assessment of blood flow in human skin. Eur J Vasc Surg 1994; 8: 65–69. Sjolin FG, Berne B, Eggelte TA, Karlsson PA. In situ localization of chloroquine and immunohistological studies in UVB-irradiated skin of photosensitive patients. Acta Derm Venereol (Stockh) 1995; 75: 228–231. Snyder DS. Cutaneous effects of topical indomethacin, an inhibitor of prostaglandin synthesis, on UV-damaged skin. J Invest Dermatol 1975; 64: 322–325. Soter NA. Acute effects of ultraviolet radiation on the skin. Semin Dermatol 1990; 9: 11–15. Stern RS, Dodson TB. Ibuprofen in the treatment of UV-Binduced inflammation. Arch Dermatol 1985; 121: 508–512. Strickland I, Rhodes LE, Flanagan BF, Friedmann PS. TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selection expression. J Invest Dermatol 1997; 108: 763–768. Warin AP. The ultraviolet erythemas in man. Br J Dermatol 1978; 98: 473–477. Willis I, Cylus L. UVA erythema in skin: is it a sunburn? J Invest Dermatol 1977; 68: 128–129. Wlaschek M, Heinen G, Poswig A, Schwarz A, Krieg T, Scharffetter KK. UVA-induced autocrine stimulation of fibroblast-derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol 1994; 59: 550–556.
European Journal of Pain (1999), 3
Time course of UVA- and UVB-induced inflammation and hyperalgesia in human skin R.T. Hoffmann and M. Schmelz Department of Physiology I, University of Erlangen/Nümberg, D-91054 Erlangen, Germany
Dose-dependency and time course of hyperalgesia and erythema following UVA (16.8 and 36 J/cm2) and UVB (one and three times the minimum erythema threshold) irradiation was investigated in 10 healthy human subjects. Skin patches (1.5 cm in diameter) on the ventral side of the upper leg were irradiated with UVA or UVB light. Hyperaemia (Laser Doppler flowmetry, infrared thermography), thermal hyperalgesia to radiant heat stimuli, and mechanical hyperalgesia to controlled impact stimuli were tested at 1, 6, 12, 24, 48 and 96 h after irradiation. Dose-dependent delayed hyperaemia and hyperalgesia was induced only by UVB irradiation. UVB-induced increase in blood flow peaked at 12 h after irradiation and normalized by 96 h. Although superficial blood flow, as measured by Laser Doppler flowmetry, increased up to eight-fold, no significant increase of skin temperature was detected using infrared thermography. Development of mechanical and thermal hyperalgesia was delayed and reached a plateau between 24 and 48 h. In contrast to UVB, UVA irradiation of up to 36 J/cm2, sufficient to produce intense tanning of the skin, did not induce significant hyperalgesia or delayed hyperaemia. It is concluded that UVB- but not UVA-irradiation is a suitable experimental model of subacute thermal and mechanical hyperalgesia. The different time courses of erythema and hyperalgesia indicate that inflammatory mediators responsible for vasodilatation are not identical with those inducing hyperalgesia. KEYWORDS: pain, nociception, sunburn.
INTRODUCTION UVA and UVB are differently effective in the induction of inflammatory skin reactions. While UVB (290–320 nm) is largely absorbed in the epidermis, UVA (320–400 nm) penetrates into the deeper layers of the dermis. The sunburn reaction following UVB-irradiation, which includes hyperaemia and hyperalgesia, has been studied extensively. However, the role of UVA irradiation in producing a sunburn reaction is less clear (Gilchrest et al., 1983). In general, the doses necessary for induction of an UVA erythema are found to be very high, i.e. more than 1000-fold
the required UVB doses (Warin, 1978). However, with higher UVA intensities, more-pronounced unspecific thermal effects are induced, which also differ histologically from UVB-induced changes (Willis & Cylus, 1977; Rosario et al., 1979). The aim of our study was to assess dose–response curves for hyperaemia and hyperalgesia following UVA and UVB-irradiation. In addition, the time course and pattern of hyperalgesia following UVA- and UVB-irradiation was evaluated and compared with objective signs of inflammation, i.e. skin temperature and superficial blood flow.
MATERIALS AND METHODS Paper received 1 June 1998 and accepted in revised form 20 December 1998. Correspondence to: Dr Martin Schmelz, Institut für Physiologie und Experimentelle Pathophysiologie, Universitätsstr. 17, 91054 Erlangen, Germany. Email: schmelz@physiologie 1.uni-erlangen.de
Subjects Ten volunteers, five male, five female (20–25 years of age), participated in the study. All
1090-3801/99/020131 + 09 $12.00/0 © 1999 European Federation of Chapters of the International Association for the Study of Pain
132
subjects gave their informed consent according to the Declaration of Helsinki, and the study was approved by the local ethics committee. Only subjects showing no neurological or dermatological abnormalities were included. The study was performed during winter and early spring and all subjects were instructed to avoid additional UV-exposure of their thighs. Hyperalgesia models
UV-erythema model Mode of UV application One week before the first
experimental session, the minimum erythema dose for UVA-(320–400 nm wavelength) and UVB-irradiation was assessed using a calibrated UV source (Saalmann multitester® SBB LT 400, Saalmann Medizintechnik, Herford, Germany). Five circular spots on the thigh with a diameter of 1.5 cm and a constant distance of 2.5 cm were irradiated with increasing doses of UVA (20 min; 16.8–36 J/cm2) and UVB light (10 s; 0.01–0.05 J/cm2). UV-induced erythema has been shown to be reciprocal (Meanwell & Diffey, 1989); thus, total energy but not duration of the irradiation is the main factor for the sunburn reaction. The minimum dose which induced a visible erythema 24 h after irradiation was determined as minimal erythema dose (MED). One week after the determination, the subjects were irradiated with one and three times the individual MED, and time course of hyperalgesia and inflammation was assessed. In the following, we refer to these dosages as UVB-1 and UVB-3. The skin reaction to UVA was different. In pilot experiments, UVA application led to warming of the skin, and UVA doses above 36 J/cm2 induced increasing heat pain. To minimise sideeffects from heating, it was necessary to stop the irradiation before heat pain occurred. Therefore, we chose to apply UVA in doses of 16.8 and 36 J/cm2, which were strong enough to induce clear tanning of the skin without being painful during irradiation. In this manuscript, we refer to these two dosages as UVA-1 (low) and UVA-h (high). After UVA-l and UVA-h irradiation (about 500× higher than the UVB doses used), the skin showed an immediate erythema reaction, which declined within a few hours. European Journal of Pain (1999), 3
R.T. H OFFMANN
AND
M. S CHMELZ
Thermography For thermography, an infrared camera was used (Agema Thermovision® 900, Sweden), and recordings were stored on hard disk for off-line analysis. Thirty-two successive recordings (3 Hz) were averaged, and the average picture was stored on hard disk for later analysis. The distance between camera and upper leg of each volunteer was 1 m. Average skin temperature of the four irradiated areas and a non-irradiated control area was determined by dedicated software (Erika Thermovision, Agema, Sweden). Laser Doppler flow measurements Superficial blood flow on the test spots was measured by laser Doppler flowmetry immediately before the algesimetric session. For this purpose, a laser Doppler probe (Periflux PF2, Perimed, Sweden) was applied to the skin for at least 20 s on the previously irradiated spots and a control spot. At each spot, mean values of at least two different probe positions were obtained. Heat pain thresholds Radiant heat stimuli were
delivered from a halogen bulb focused on the skin (diameter 1 cm) and feed-back was controlled from a thermocouple gently attached to the skin in the centre of the light beam (Beck et al., 1974). The skin temperature was slowly increased by 0.67°C/s, from an adapting temperature of 32°C. Subjects were instructed to stop the heating by pressing a button as soon as the heat became painful. The cutoff temperature of 52°C was not reached in any of the cases. The UVA-l, UVA-h, UVB-1, UVB-3, and a control spot on untreated skin between the irradiated spots were tested in a randomised order, unpredictable for the subject. Mechanical impact stimulation Mechanical hyperal-
gesia was assessed by delivering impact stimuli. For this purpose, a plastic cylinder (0.5 g, diameter 0.5 cm) was driven at a controlled speed through a barrel against the skin by a pressurised air-driven stimulator, as described elsewhere in detail (Kohllöffel et al., 1991). The UV-treated skin patches and the control spot on untreated skin were tested twice in random order with an impact velocity of 5, 9 and 12 m/s, applied in ascending order. Subjects were instructed to give pain ratings after each impact. A rating of zero indicated ‘no
UVA-
AND
UVB- INDUCED
133
INFLAMMATION AND HYPERALGESIA
sensation’ and a rating of 100 should represent a sensation being just painful. For sensations of double that intensity ratings should be duplicated etc. The rationale of this kind of rating scale has been discussed elsewhere (Koltzenburg & Handwerker, 1994). The obtained ratings were in the range of 5–180. The values from the two stimulus repetitions on each spot were averaged.
Experimental protocol Dose-dependency of the irradiation and the minimal erythema dose of each volunteer were determined by measurements 24 h after application of fixed doses of UVA and UVB. Pain thresholds and degree of inflammation were assessed at 1, 6, 12, 24, 48 and 96 h after application of UV radiation. To reduce circadian fluctuations, irradiations were performed at 8.00 a.m. for each subject. Additional measurements were performed after 132 h if blood flow after 96 h had not returned to baseline. At each time, radiation effects were assessed in the same order, starting with infrared thermography and Laser-Doppler flux measurement. After these non-invasive measurements, heat pain thresholds were determined and, finally, mechanical impact stimuli were applied. Thus, unwanted changes of superficial blood flow and temperature by the test stimuli were minimised.
Statistical procedures
Dose dependency Blood flux values, mean temperatures at the irradiation spots, pain ratings and heat pain thresholds were analysed by ANOVA with dose of irradiation (5 levels) and type of irradiation (UVA and UVB) as independent variables.
Time course Blood flux, skin temperature, pain ratings and heat pain thresholds were analysed by ANOVA with time of session (1, 6, 12, 24, 48 and 96 h), type of UV irradiation (UVA and UVB), and intensity of irradiation (1MED and 3MED) as independent variables (repeated measurement).
Post-hoc Scheffé tests were performed on significant factors, where suitable. All statistical analysis was done using the STATISTICA 5.0® software package (Statsoft, Tulsa, USA). The significance level was set at p<0.05.
RESULTS Acute effects and tanning
UVA irradiation UVA irradiation induced warm or slightly hot sensations in all subjects, but was not felt as painful. Immediately after 20 min of UVA irradiation, the skin appeared reddened and temperature was at about 40°C. The skin reddening declined and normalised within a few hours. UVA irradiation produced a pronounced tanning of the irradiated skin which lasted for several weeks. No blisters or permanent marks were induced by the UVA light.
UVB irradiation The UVB exposed skin areas showed no alterations immediately after UV-expositions. No spontaneous ongoing pain was reported from either spot, an erythema developed in the course of the following 12 h. No permanent marks or blisters were induced in any subject. One day after irradiation, UVB-irradiated skin areas exhibited a light brown tanning, which lasted for several weeks. Blisters or permanent marks were not induced in any of the subjects.
Dose-dependent effects of UV radiation
Erythema Dose–response curves for UVA and UVB light were obtained by measuring the effects 24 h after irradiation. Even the most intense UVA irradiation (36.0 J/cm2) did not induce any significant increase in superficial blood flow as compared to the control area. In contrast, UVB-irradiation caused increased blood flow even at the lowest dose (0.01 J/cm2), which was significant for skin sites irradiated with more than 0.02 J/cm2.
European Journal of Pain (1999), 3
134
R.T. H OFFMANN
Visual inspection of the irradiated skin sites was less sensitive to detect increased blood flow (mean MED: 0.015 J/cm2). With increasing UVB intensity to 0.05 J/cm2 a linear increase of blood flow was measured, which was paralleled by more intense reddening of the irradiated skin (Fig. 1).
Skin temperature Despite the significant blood flow changes at the UVB spots, there was virtually no difference in skin temperature between the irradiated areas and the control spot for both UVA and UVB irradiation. The maximum difference to control was only 0.24°C for the UVA measurements and 0.84°C for the UVB application, which did not reach a significant level [UVB (0.05 J/cm2) vs control: p=0.44; ANOVA; Scheffé post hoc test] (Fig.1).
Mechanical hyperalgesia Mechanical hyperalgesia was tested by using impact stimuli with three different velocities (5, 9 and 12 m/s). In control skin, the light impact was felt as touch and the strongest as just painful (Fig.2). UVA irradiation did not induce mechanical hyperalgesia. In the tested range of impact intensities (5–12 m/s), no significant difference to control
AND
M. S CHMELZ
skin was observed. In contrast, UVB irradiation caused a dose-dependent mechanical hyperalgesia (Fig. 2). Even the weaker impact stimuli were felt stronger in skin irradiated with more than 0.3 J/cm2. For the higher velocity, irradiation above 0.2 J/cm2 already induced significant mechanical hyperalgesia. At the 0.5 J/cm2 irradiated spots, pain ratings were about 50% higher than on control skin (Fig. 2).
Thermal hyperalgesia Heat pain thresholds were found only slightly decreased on the UVA spots. The threshold was reduced between 0.8 and 1.9°C compared to the control spot (dose range 16.8–31.2 J/cm2), and reached a minimum on the spot irradiated with a dose of 36 J/cm2; this reduction (2.8°C) reached statistical significance (ANOVA, planned comparison; p=0.02) (Fig. 3). In contrast, heat pain thresholds were profoundly reduced by UVB irradiation. Even the weakest dose of 0.01 J/cm2 caused a significant decrease in heat pain threshold by 3.27°C as compared to the non-irradiated control. Irradiation with doses of 0.04 and 0.05 J/cm2 caused a decrease of the heat pain threshold to a plateau temperature of 36.5°C, which is about 8°C below heat pain thresholds in control skin.
200 150 100
34
50 32 0
0.01 Control 16.8
0.02 21.6
0.03 26.4
0.04 31.4
0.05 36.0
Skin temperature (C°)
LDF (relative units)
250
UVB UVA
Dose of irradiation (J/cm2)
Fig. 1. Dose–response curves of UVA (■) and UVB (●) irradiation for the induction of increased superficial blood flow (LDF) and increase in skin temperature at 24 h after irradiation. Mean±SEM, n=10; **, p<0.01, ANOVA, Scheffé test.
European Journal of Pain (1999), 3
Impact pain rating
160 140 120 100 80 60 40 20 0
0.01 Control 16.8
0.02 21.6
0.03 26.4
0.04 31.4
0.05 UVB 36.0 UVA
Dose of irradiation (J/cm2)
Fig. 2. Dose–response curves of UVA (●) and UVB (■) irradiation for the induction of mechanical hyperalgesia at 24 h after irradiation. Mechanical impact stimuli were delivered at a speed of i=5 (open symbols) and 12 m/s (closed symbols). Mean±SEM, n=10; *; 0.05>p>0.01, ANOVA Scheffé test; **, p<0.01, ANOVA, Scheffé test.
AND
UVB- INDUCED
135
INFLAMMATION AND HYPERALGESIA
LDF (relative units)
Decrease of heat pain threshold (∆T; °C)
UVA-
0 –2 –4 –6
200 150 100 50 0
–8 –10 Control
0.01 16.8
0.02 21.6
0.03 26.4
0.04 31.4
0.05 UVB 36.0 UVA
Dose of irradiation (J/cm2)
Fig. 3. Dose–response curves of UVA (■) and UVB (●) irradiation for the induction of thermal hyperalgesia at 24 h after irradiation. Pain threshold to radiant heat are given as difference between irradiated skin areas and control skin. Mean±SEM, n=10; **, 0.05>p>0.01; **, p<0.01, ANOVA, Scheffé test.
Time course of inflammation and hyperalgesia
Erythema Fixed doses of UVA (16.8 and 36.0 J/cm2) and UVB irradiation (one and three times the individual minimal erythema dose) were used to determine time course of blood flow changes in superficial skin layers during the first 96 h after irradiation. Blood flow changes at the low dose UVA spot (UVA-I) did not reach statistical significance (ANOVA, planned comparison; p=0.24). Superficial blood flow at the high dose UVA spot (UVA-h) showed a clear increase 1 h after irradiation, and returned to baseline within 6 h after irradiation (see Fig.4). In contrast, time course of hyperemia after UVB-irradiation was different. Superficial blood flow at the UVB-1 spot doubled 6 h after application, and peaked at 24 h after irradiation. Forty-eight hours after irradiation flow, values were still 1.5-fold higher than measurements on control spot. UVB-3 treatment already caused a significant increase after 1 h and during the period to 48 h after irradiation (Fig.4). Peak hyperaemia was reached 12 h after irradiation, with flux values 8.5-fold higher than in the control area. Blood flow remained elevated after
20
40 60 Time (h)
80
100
Fig. 4. Time course of erythema (LDF) for 96 h after UVA- and UVB-irradiation. The dose used for UVA irradiation was fixed at 16.8 and 36.0 J/cm2 for each volunteer; UVB dosage was the individual minimal erythema dose (MED) and three times the MED. Mean±SEM, n=10; **, 0.05>p>0.01, **, p<0.01, ANOVA, Scheffé test. (■ ■) UVA (h); (■) UVA (l); (● ●) uvB-1; (●) UVB-3; (◆ ◆) control.
96 h, and did not completely return to baseline until 132 h after irradiation.
Skin temperature Only UVA irradiation caused immediate increase in skin temperature, which had virtually normalized by 1 h. The difference to the control area reached a maximum (+1.92°C) 5 min after UVA-h irradiation. In contrast, UVB irradiation did not cause a significant increase in skin temperature at any time. Even at 12 h after irradiation, when superficial blood flow was maximally enhanced at the UVB-3 spot, skin temperature did not differ significantly from control skin.
Mechanical hyperalgesia UVA irradiation at both doses did not induce any mechanical hyperalgesia to impact stimulation. In contrast, at the UVB-3 irradiated skin, mechanical hyperalgesia developed by 6 h and reached a plateau between 1 and 48 h. Pain ratings gradually decreased thereafter and reached baseline after 132 h. The mechanical hyperalgesia reached statistical significance between 12 and 48 h. At the UVB-1 spot, the time course of hyperalgesia was similar, but even at its maximum the effect did not reach statistical significance in the Scheffé post-hoc test (p=0.08) (Fig.5). European Journal of Pain (1999), 3
R.T. H OFFMANN
Impact pain rating
140 130 120 110 90 80 0 10 20
40
60
80
100
Time (h)
Fig. 5. Time course of mechanical hyperalgesia for 96 h after UVA- and UVB-irradiation. Pain ratings to mechanical impact stimuli which were delivered to the irradiated skin at a speed of 5 and 12 m/s are presented. The dose used for UVA irradiation was fixed at 16.8 and 36.0 J/cm2 for every volunteer; UVB dosage was the individual minimal erythema dose (MED) and three times the MED. Mean±SEM, n=10; *, 0.05>p>0.01; **, p<0.01, ANOVA, Scheffé test. (■ ■) UVA (h); (■) UVA (l); (● ●) UVB-1; (●) UVB-3; (◆ ◆) control; •; v = 12 m/s.
Thermal hyperalgesia Thermal hyperalgesia was also found to be dependent on quality and dose of radiation. Heat pain thresholds at control spots (44±0.4°C) corresponded to previous reports (Bickel et al., 1998). At the UVA spots, heat pain thresholds were slightly lower between 6 and 12 h afterwards, without reaching statistical significance (Fig.6). In contrast, UVB-irradiation induced a pronounced thermal hyperalgesia. Heat pain thresholds at the UVB-1 spots decreased gradually to a minimum of 3.8°C below control after 24 h (p=0.05, ANOVA, Scheffe post hoc test) and returned to normal after 96 h. UVB-3 irradiation caused thermal hyperalgesia with a similar time course, with a maximum drop in heat pain threshold of 8.5°C at 24 h after irradiation. Heat pain thresholds were significantly decreased during the period from 6 to 96 h after irradiation (Fig.6).
DISCUSSION Aspects of UV-induced inflammatory skin reactions have been investigated in humans before. European Journal of Pain (1999), 3
Decrease of heat pain threshold (∆T; °C)
136
AND
M. S CHMELZ
0 –2 –4 –6 –8 –10 0
10 20
40
60
80
100
Time (h)
Fig. 6. Time course of thermal hyperalgesia for 96th after UVA- and UVB-irradiation. Pain thresholds to radiant heat are given as difference between irradiated and control skin. The dose used for UVA irradiation was fixed at 16.8 and 36.0 J/cm2 for every volunteer; UVB dosage was one and three times the individual minimal erythema dose Mean±SEM, n=10; *, 0.05>p>0.01; **; p<0.01, ANOVA, Scheffé test. (■ ■) UVA (l); (■) UVA (h); (● ●) UVB-1; (●) UVB-3.
However, most investigators concentrated on measurements of superficial blood flow using reflectance instruments, laser Doppler flowmetry or scaling of skin redness. Hyperalgesia was either not tested or was reported casually based on complaints of patients (Snyder, 1975; Stern & Dodson, 1985). A systematic study about time course of thermal and mechanical hyperalgesia after UVB irradiation in humans has been reported only in abstract form before (Benrath et al., 1993). There is evidence that the early component (4–24 h) of the UVB-induced erythema is partly prostaglandin dependent: prostaglandins were found to be increased after UVB irradiation (Gresham et al., 1996), and cyclooxygenase inhibitors given topically or systemically suppress skin erythema in this period (Snyder, 1975; Anderson et al., 1989; Hughes et al., 1992; Juhlin & Shroot, 1992). However, the late phase of the erythema (>24 h) does not respond to cyclooxygenase inhibitors like indomethacin or piroxicam (Farr & Diffey, 1986; Juhlin & Shroot, 1992). Other mediators which could play a role in UV-induced inflammation include neuropeptides (Benrath et al., 1995), histamine (Gilchrest et al., 1981; Malaviya et al., 1996),
UVA-
AND
UVB- INDUCED
137
INFLAMMATION AND HYPERALGESIA
interleukins (Kennedy et al., 1997), nitric oxide (Goldsmith et al., 1996; Deliconstantinos et al., 1997) and oxygen radicals (Hruza & Pentland, 1993). Although hyperalgesia of sunburned skin is a common experience, and in numerous studies release of prostanoids has been found to be related to the UV-induced hyperaemia (Gilchrest et al., 1981; Kimura & Doi, 1998), UV-sunburn has rarely been used as an experimental hyperalgaesia model in humans (Bickel et al., 1998).
UVB-induced hyperaemia Dose dependency and time course of UVBinduced erythema were found in accordance with previous reports (Frödin et al., 1988; Hughes et al., 1992). Peak hyperaemia was detected between 6 and 24 h after irradiation, and had decreased by about 50% at 48 h. We did not observe a second peak of erythema as found by Benrath et al. (1993). Although an increase of up to eight-fold in superficial blood flow was measured by Laser Doppler flowmetry after UVB irradiation, no significant increase in skin temperature was detected using infrared thermography. A lack of correlation between superficial blood flow and skin temperature has already been described before (Seifalian et al., 1994). This discrepancy can be explained by the different depth of vasodilation. The laser Doppler detects increased blood flow preferentially in capillaries of the upper layers of the skin (<1 mm) which do not allow convective warmth transport. In contrast, vasodilation in arteriovenous shunts or larger arterioles directing warmer blood to the skin surface will increase skin temperature. The differential organisation of the superficial and deeper neuronal blood control in the skin has already been described (Greiner et al., 1995). As can be expected from histological sections, UVBinduced inflammatory changes are very superficial (Rosario et al., 1979), which explains the prominent superficial hyperaemia. Only inflammation reaching deeper skin layers can induce hyperaemia in superficial and deeper skin layers, leading to concomitant reddening and warming of the skin (Kelfkens et al., 1990).
UVB-induced hyperalgesia Thermal and mechanical hyperalgesia developed in all subjects after UVB irradiation with three times the minimal erythema dose. We did not encounter two peaks of the hyperalgesia at 6 and 36 h as observed by Benrath et al. (1993). Instead, a plateau of maximum hyperalgesia between 12 and 48 h after irradiation was found. However, our study confirms the delayed development of hyperalgesia as compared to hyperaemia described in their study (Benrath et al., 1993). The early component of hyperaemia (≤ 24 h) and hyperalgesia could be partly suppressed using cyclooxygenase-inhibitors (Snyder, 1975; Greaves et al., 1977; Anderson et al., 1989; Hughes et al., 1992; Juhlin & Shroot, 1992). In accordance with these results, the inducible cyclooxygenase (COX2) has recently been described to be upregulated in human skin following UVB irradiation (Buckman et al., 1998). However, the late erythema was unaffected by cyclooxygenase-inhibitors (Farr & Diffey, 1986; Juhlin & Shroot, 1992). Also, topical steroids did not suppress the late erythema (Kaidbey & Kurban, 1976; Hughes et al., 1992). The release of nitric oxide (Goldsmith et al., 1996; Deliconstantinos et al., 1997) or neuropeptides (Benrath et al., 1995) following UVB-irradiation could explain at least part of the cyclooxygenaseindependent hyperaemia. It is unclear to date which mediator could be responsible for the delayed hyperalgesia. Prostaglandin E2 and histamine were found to be elevated only in the early phase after UV irradiation (Gilchrest et al., 1981; Hawk et al., 1983). Also, pro-inflammatory cytokines like interleukin (IL) 1, 6, 8 and tumour necrosis factor α have been found to be elevated in human skin and keratinocytes after UV irradiation (Wlaschek et al., 1994; Sjolin et al., 1995; Strickland et al., 1997). Metabotropic glutamate receptors (mGluR), which contribute to mechanical hyperalgesia (Budai & Larson, 1998), could play a role in UV-induced hyperalgesia. UVB irradiation has been shown to enhance expression of the mGluR3 messenger RNA (Boxall et al., 1998) in the spinal cord. Interestingly, mGluR 2/3 have also been found on primary afferent neurons (Hargett et al., 1998), which
European Journal of Pain (1999), 3
138
could provide a basis for peripheral sensitisation of nociceptors by UVB irradiation.
Effects of UVA irradiation Long-wave UV irradiation (UVA: 320–400 nm) induces erythema at more than 1000-fold higher exposure doses compared to UVB. In contrast to UVB irradiation, these high dosages increase skin temperature, and induce pain and immediate erythema (Kaidbey & Kligman, 1979). The UVA erythema could not be diminished by cyclooxygenase inhibitors given topically, intradermally or systemically (Morison et al., 1977; Soter, 1990). In addition, clear histological differences between UVA and UVB-induced changes have been described: UVA has a greater effect on the dermis with an immediate onset, recruits neutrophils in the skin and does not induce typical ‘sunburn’ cells (Willis & Cylus, 1977; Rosario et al., 1979; Gilchrest et al., 1983). In our study, intensity of UVA irradiation was limited in order to prevent unspecific heat effects which could themselves cause inflammation and hyperalgesia (Møiniche et al., 1993). On the other hand, the intensity used (36 J/cm2) was effective to produce rapid tanning of the skin. No hyperalgesia or delayed erythema was observed using this dosage. At higher doses, UVA irradiation induces delayed hyperaemia and leukocyte infiltration, with a maximum at 3 h post irradiation (Gilchrest et al., 1983). This time course fits nicely to the time course of thermal and mechanical hyperalgesia seen after heat injury (Møiniche et al., 1993). Thus, thermal injury may well contribute to UVA-induced hyperaemia and hyperalgesia. In conclusion, UVA irradiation sufficient to induce tanning of the skin without thermal lesions does not produce delayed erythema or hyperalgesia. In contrast, UVB-induced sunburn provides a reliable and well-controlled experimental model of mechanical and thermal hyperalgesia. Erythema and hyperalgesia have different time courses and presumably are induced by different mediators. While prostaglandins are responsible for at least part of the early component of the UVB erythema, mediators for the late component and the concomitant hyperalgesia are yet to be identified. European Journal of Pain (1999), 3
R.T. H OFFMANN
AND
M. S CHMELZ
REFERENCES Anderson TF, Peterson C, Hamilton T. Meclofenomate inhibition of UV-induced erythema—a randomized placebocontrolled, double-blind study. Photodermatology 1989; 6: 63–68. Beck PW, Handwerker HO, Zimmermann M. Nervous outflow from the cat’s foot during noxious radiant heat stimulation. Brain Res 1974; 67: 373–386. Benrath J, Gillardon F, Zimmermann M. Differential time courses of hyperalgesia and inflammation in human skin after ultraviolet irradiation. Neuropeptides 1993; 24: 205. Benrath J, Eschenfelder C, Zimmermann M, Gillardon F. Calcitonin gene related peptide, substance P and nitric oxide are involved in cutaneous inflammation following ultraviolet irradiation. Eur J Pharmacol-Environ Toxic 1995; 293: 87–96. Bickel A, Dorfs S, Schmelz M, Forster C, Uhl W, Handwerker HO. Effects of antihyperalgesic drugs on experimentally induced hyperalgesia in man. Pain 1998; 76: 317–325. Boxall SJ, Berthele A, Laurie DJ, Sommer B, Zieglgänsberger W, Urban L, Tölle TR. Enhanced expression of metabotropic glutamate receptor 3 messenger RNA in the rat spinal cord during ultraviolet irradiation induced peripheral inflammation. Neuroscience 1998; 82: 591–602. Buckman SY, Gresham A, Hale P, Hruza G, Anast J, Masferrer J, Pentland AP. COX-2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer. Carcinogenesis 1998; 19: 723–729. Budai D, Larson AA. The involvement of metabotropic glutamate receptors in sensory transmission in dorsal horn of the rat spinal cord. Neuroscience 1998; 83: 571–580. Deliconstantinos G, Villiotou V, Stavrides JC. Inhibition of ultraviolet B-induced skin erythema by N-nitro-L-arginine and N-monomethyl-L-arginine. J Dermatol Sci 1997; 15: 23–35. Farr PM, Diffey BL. A quantitative study of the effect of topical indomethacin on cutaneous erythema induced by UVB and UVC radiation. Br J Dermatol 1986; 115: 453–466. Frödin T, Molin L, Skogh M. Effects of single doses of UVA, UVB, and UVC on skin blood flow, water content, and barrier function measured by laser-Doppler flowmetry, optothermal infrared spectrometry, and evaporimetry. Photodermatology 1988; 5: 187–195. Gilchrest BA, Soter NA, Stoff JS, Mihm MC, Jr. The human sunburn reaction: histologic and biochemical studies. J Am Acad Dermatol 1981; 5: 411–422. Gilchrest BA, Soter NA, Hawk JL, Barr RM, Black AK, Hensby CN, Mallet AI, Greaves MW, Parrish JA. Histologic changes associated with ultraviolet A-induced erythema in normal human skin. J Am Acad Dermatol 1983; 9: 213–219. Goldsmith PC, Leslie TA, Hayes NA, levell NJ, Dowd PM, Foreman JC. Inhibitors of nitric oxide synthase in human skin. J Invest Dermatol 1996; 106: 113–118. Greaves MW, Hensby CN, Black AK. Inflammation in human skin induced by ultraviolet irradiation. Postgrad Med J 1977; 53: 656–657. Greiner T, Nischik M, Schmelz M, Forster C, Handwerker HO. Neurogenic flare responses are heterogeneous in
UVA-
AND
UVB- INDUCED
INFLAMMATION AND HYPERALGESIA
superficial and deep layers of human skin. Neurosci Lett 1995; 185: 33–36. Gresham A, Masferrer J, Chen X, Leal KS, Pentland AP. Increased synthesis of high-molecular-weight cPLA2 mediates early UV-induced PGE2 in human skin. Am J Physiol 1996; 270: C1037–C1050 Hargett GL, Coggeshall RE, Carlton SM. Metabotropic glutamate receptors are differentially distributed on primary afferent terminals in the dorsal horn. Soc Neurosci Abstr 1998; 24: 1869. Hawk JL, Black AK, Jaenicke KF, Barr RM, Soter NA, Mallett AI, Gilchrest BA, Hensby CN, Parrish JA, Greaves MW. Increased concentrations of arachidonic acid, prostaglandins E2, D2, and 6-oxo-F1 alpha, and histamine in human skin following UVA irradiation. J Invest Dermatol 1983; 80: 496–499. Hruza LL, Pentland AP. Mechanisms of UV-induced inflammation. J Invest Dermatol 1993; 100: 35S–41S. Hughes GS, Francom SF, Means LK, Bohan DF, Caruana C, Holland M. Synergistic effects of oral nonsteroidal drugs and topical corticosteroids in the therapy of sunburn in humans. Dermatology 1992; 184: 54–58. Juhlin L, Shroot B. Effect of drugs on the early and late phase UV erythema. Acta Derm Venereol (Stockh) 1992; 72: 222–223. Kaidbey KH, Kligman AM. The acute effects of long-wave ultraviolet radiation on human skin. J Invest Dermatol 1979; 72: 253–256. Kaidbey KH, Kurban AK. The influence of corticosteroids and topical indomethacin on sunbum erythema. J Invest Dermatol 1976; 66: 153–156. Kelfkens G, vanHelden AC, van-der-Leun JC. Skin temperature changes induced by ultraviolet A exposure: implications for the mechanism of erythemogenesis. Photodermatol Photoimmunol Photomed 1990; 7: 178–182. Kennedy M, Kim KH, Harten B, Brown J, Planck S, Meshul C, Edelhauser H, Rosenbaum JT, Armstrong CA, Ansel JC. Ultraviolet irradiation induces the production of multiple cytokines by human corneal cells. Invest Ophthalmol Vis Sci 1997; 38: 2483–2491. Kimura T, Doi K. Effects of indomethacin on sunburn and suntan reactions in hairless descendants of Mexican hairless dogs. Histol Histopathol 1998; 13: 29–36. Kohllöffel LU, Koltzenburg M, Handwerker HO. A novel technique for the evaluation of mechanical pain and hyperalgesia. Pain 1991; 46: 81–87. Koltzenburg M, Handwerker HO. Differential ability of human cutaneous nociceptors to signal mechanical pain
139
and to produce vasodilatation. J Neurosci 1994; 14: 1756–1765. Malaviya R, Morrison AR, Pentland AP. Histamine in human epidermal cells is induced by ultraviolet light injury. J Invest Dermatol 1996; 106: 785–789. Meanwell EF, Diffey BL. Reciprocity of ultraviolet erythema in human skin. Photodermatology 1989; 6: 146–148. Møiniche S, Dahl JB, Kehlet H. Time course of primary and secondary hyperalgesia after heat injury to the skin. Br J Anaesth 1993; 71: 201–205. Morison WL, Paul BS, Parrish JA. The effects of indomethacin on long-wave ultraviolet-induced delayed erythema. J Invest Dermatol 1977; 68: 130–133. Rosario R, Mark GJ, Parrish JA, Mihm MC. Jr. Histological changes produced in skin by equally erythemogenic doses of UV-A, UV-B, UV-C and UV-A with psoralens. Br J Dermatol 1979; 101: 299–308. Seifalian AM, Stansby G, Jackson A, Howell K, Hamilton G. Comparison of laser Doppler perfusion imaging, laser Doppler flowmetry, and thermographic imaging for assessment of blood flow in human skin. Eur J Vasc Surg 1994; 8: 65–69. Sjolin FG, Berne B, Eggelte TA, Karlsson PA. In situ localization of chloroquine and immunohistological studies in UVB-irradiated skin of photosensitive patients. Acta Derm Venereol (Stockh) 1995; 75: 228–231. Snyder DS. Cutaneous effects of topical indomethacin, an inhibitor of prostaglandin synthesis, on UV-damaged skin. J Invest Dermatol 1975; 64: 322–325. Soter NA. Acute effects of ultraviolet radiation on the skin. Semin Dermatol 1990; 9: 11–15. Stern RS, Dodson TB. Ibuprofen in the treatment of UV-Binduced inflammation. Arch Dermatol 1985; 121: 508–512. Strickland I, Rhodes LE, Flanagan BF, Friedmann PS. TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selection expression. J Invest Dermatol 1997; 108: 763–768. Warin AP. The ultraviolet erythemas in man. Br J Dermatol 1978; 98: 473–477. Willis I, Cylus L. UVA erythema in skin: is it a sunburn? J Invest Dermatol 1977; 68: 128–129. Wlaschek M, Heinen G, Poswig A, Schwarz A, Krieg T, Scharffetter KK. UVA-induced autocrine stimulation of fibroblast-derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6. Photochem Photobiol 1994; 59: 550–556.
European Journal of Pain (1999), 3