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Skin suction blister wound exposed to u.v. irradiation: a burn wound model for use in humans
Bums (1991) 17, (I), 41-46
Prinkd in Great Britain
41
Skin suction blister wound exposed to U.V. irradiation: a burn wound model for use in humans C. Svedmanl, C. Hammarlundz, N. Kutlu’ and P. Svedman’ ‘Department Helsingborg
of Plastic and Reconstructive Surgery, Allmba Sjukhuset, MalmG and 2Department Hospital, Helsingborg, University of Lund, Lund, Sweden
The effects of exposing blister wounds to U.V.irradiation were assessed in 14 male voluntem, on whose foream blister wounds (diameter 5mm, suctionset at 200 mm@ below atmospheric for2 h 15 min, blister roof cuf at base), and irradiated blister wounds (as above, in addition xv. irradiation given selectively for 30 min from a distance of 10 MI), were produced. In non-xv.-irradiated wounds, flow cessation, asxssed by video microscopy (n = 6), was obsemti in a small proportion of the papillary loop ve5sels. The oedema adjacent to the wound was poorly &eloped. Laser Doppler linear scans (n = 8) demonstrated a pronounced hyperaemia in the wound bed and also in the adjacent skin, the reaction subsiding over a few days. The exudation rate, deknined by weighing the hydrocolloid dressingsapplied to the wound, was makmal on day I ana’then rapidly decreased. Epithelialization, assessed evapoketkally as the time taken for reinstatement of the epidetmal water barn& was complete in 5.1 hys. In the xv.-irradiated wounds Hz blood flow had ceased in all the papilkzry hyperaemia at loop vessels by day I, and increased oedema, exudation ana’ the wound edges were observed Epithelializalion was not significantly retarded by the irradiation injury. After 6 months, slight discolouration of the skin was occasionally observed, but no cosmetically disturbing scars. This benign and standardized wound model in humans - based on a combination of a mechanical strctioninjury ana’a supe$cial radiation burn - may prove to be useful, for instance when studykg the effects of bums treatment.
Introduction Wound models in animals are indispensable for studying wound healing since human wounds are complex and variable and are difficult to sample and measure. At present, findings from research in experimental animals, such as the stimulating effects of oxygen (Hunt and Pai, 1972), and of nutritional (Niinikoski et al., 1977) and growth factors (Brown, 1986) on healing need to be corroborated in human patients. It is also important to study the way diabetes and peripheral vascular diseases affect the healing processes in humans, as well as how the same processes are influenced by the common drugs in clinical use. Furthermore, many therapy modes are advocated for treating wounds and burns without adequate clinical testing. Thus measurement of wound healing in patients is a necessity. This challenge can be met by developing standardized, benign and miniaturized wound models by which healing can be measured quantitatively, preferably with non0 1991 Butterworth-Heineman Ltd 0305/4179/91/010041-06
of Anaesthesiology,
invasive techniques (Svedman et al., 1989). By means of evaporimetry and laser Doppler flowmetry, we have studied the blister wound, formed painlessly after sucking loose and excising the epidermis within a small area of skin (Svedman et al., 1990). Initially, wound fluid evaporated quickly in the absence of the epidermal barrier, but within a week the rate normalized as the barrier reformed. A simple model for determining the end-point of epithelialization in humans was thus devised. The flowmetry revealed a strong hyperaemic reaction to the dermal injury caused by the suction. In this investigation suction blister wounds in healthy volunteers were exposed to U.V.irradiation, creating an irradiation injury in addition to the original mechanical one. The damage to the superficial wound bed of both non-irradiated and irradiated lesions was assessed by video microscopy. During the initial days after the injury the total diameter including the oedema zone - was measured, and the epithelialization was assessed evaporimetrically. The microcirculatory events of the healing process were studied by laser Doppler flowrnetry, and by determining the rate of exudation from the wound.
Materials and methods Fourteen healthy, non-smoking male volunteers were studied, their ages ranging from 24 to 29 (median = 26.5) years. The volunteers wore normal, indoor clothing, and were sitting comfortably, resting their lower arm with its exposed measuring sites at heart level. The measurements were begun after 10 min of rest. Conversation was avoided. The room temperature was controlled with a thermostat and maintained at 22-23°C. Video microscopy A stereomicroscope (M3 C Kombistereoscope, Wild Leitz, Switzerland) equipped with a TV camera (Panasonic WV 1850/G, Japan), and connected to a video monitor (Panasonic WV 5360/G), a recorder (Goldstar GI-IV 1323 P, South Korea) and a video printer (Sony, UP 850, Japan) was used. The wound was illuminated with cold light (Intralux 6000, Volpi AG, Switzerland) that had been passed through a blue filter (Volpi AC). The microscope had a three-stage magnification changer. The total magnifications obtained on the monitor were 40 x , 120 x and 450 x . The forearm
Bums (1991) Vol. 17/No. 1
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with the blister wound was laid in a rigid, lightly padded rest which could be moved in a controlled way in the horizontal plane (Svedman and Kutlu, 1990). When present, fibrin clots on the wounds surface were removed under magnification (2.5 x ) using ophthalmological tweezers. During the examination the wound was covered with a transparent polyester film (Mylar, thickness 6 pm; BioRad, UK). The appearance of the superficial dermal microvascular network was described from video images, and the presence or absence of moving blood cells in the vessels was noted. Video-prints were made from the filmed material.
Evaporimetry The evaporation rate (ER) was measured using an evaporimeter (EPI, Servomed AB, Stockholm, Sweden) with a digital output (Nilsson, 1977). The open cylinder probe contains two sets of sensors, each consisting of a capacitive film and a thermistor. The water vapour pressure gradient in the volume of air between the two sets of sensors is estimated, and from this estimate the ER value, determined as the approximate mean of values displayed during a minimum of 2 min after stabilization, was determined in g/m2/h.
Measurement of blood flow A laser Doppler flowmeter (Periflux PF 3, Perimed KB, Sweden) was used (Nilsson et al., 1980a,b). Light (632.8 nm) from the flowmeter’s laser (He-Ne, 2mW) is brought to transilluminate the measuring volume by an optic fibre within a conductor. The backscattered light is transmitted through a separate pair of optical fibres to a photodetector system. Photons scattered in moving blood cells change wavelength according to the Doppler principle, and the laser Doppler flowmetry (LDF) output is related to the number of Doppler-shifted beams and the average Doppler frequency shift generated by movement of the blood cells. The measuring volume is thought to be hemispherical, with a radius of about 1 mm (Nilsson et al., 1980b; Kolani, 1985). The cut-off frequency of the flowmeter filter was set at 12 kHz, except for measurement on intact skin sites when it was set at 4 kHz. The time constant of the output amplifier was set at 3 s. The LDF values were displayed digitally and read in perfusion units (PU) which were defined by calibration against a motility standard (Perimed) giving a reading of 250PU (2.5 V, gain x 1) at 12 kHz. Each LDF reading was determined as the approximate mean of values displayed during a minimum of 2 min after stabilization. The measurements were made through a freshly applied transparent film of polyurethane (Tegaderm, 3M, USA). The conductor tip was secured in a holder within a rigid frame which in turn was taped to the skin adjacent to the wound (Svedman et al., 1990). The holder could be moved on the frame in a controlled way by turning a screw. This allowed linear advancement of the conductor tip along a line passing through the centre of the wound onto the skin on either side. The conductor tip was kept at roughly the same minimal distance from the underlying polyurethane film. Each LDF scan included I I separate measurements made at sites l.Omm apart. The sixth site was positioned approximately over the centre of the wound. At the end of each experiment the LDF value was measured randomly on the forearm during arterial occlusion produced by a bra&al cuff (2OOmmHg). These values were in the range 0.8-2.5 PU.
Maximum length of the lesions The maximum distance between the outer edges of the lesion was measured under magnification (2.5 X) using a ruler (scale unit = 0.1 mm). Oedema adjacent to the wound was included in the lesion. Measurement of exudation Hydrocolloid dressings (2 x 2 cm, Metoderm, Molnlycke Health Care AB, Sweden) were used; for each assessment one dressing was applied to the wound, and another on adjacent, intact skin. The dressings were weighed (Mettler P 163N, Mettler Instrument AC, Switzerland) before and after use. The rate of exudation &g/24 h) from the wound was determined by calculating the difference in weight increase between the two. Infliction of the lesions Rigid, transparent rectangular boxes, with either two or six circular perforations (diameter = 5 mm, spaced 15 mm apart) on one side, were connected by pliable tubing to a suction pump (Junior DK, Fricat AG, Switzerland). A mineral-light lamp (Mineral-light lamp, model S-52-T, Ultra Violet Products Inc., USA) was also used, its radiation wavelengths ranging between 200 and 400 nm with a peak at 254 nm. The volunteer’s trunk and extremities were covered with a blanket. The forearm was exposed, and the skin on the volar part was cleansed with ethyl alcohol (70 per cent). Five minutes later the perforated side of the box was applied to the skin and made airtight with surgical tape. Continuous suction, applied for 135 min at a pressure of 200mmHg below atmospheric, produced a blister, which was excised at the base, thus exposing the blister wound. Excess fluid was absorbed with a gauze compress. Some of the wounds were then selectively irradiated from a distance of 10 cm for 30 min, the lower arm was covered by a tin-foil with a hole corresponding to the wound being irradiated. All the wounds were dressed either with a hydrocolloid dressing (Metoderm) or with adhesive polyurethane film (Tegaderm). Comparison of non-irradiated and irradiated blister wounds Series 1: video microscopy Two blister wounds were made; one was irradiated (n = 6). The lesions were studied 60 min after infliction (day 0) and then again on days 1 and 2. Series 2 Six blister wounds were made, four were irradiated (n = 8). Two of the irradiated wounds and one non-irradiated wound were dressed with adhesive polyurethane film, the remaining three with hydrocolloid. If not otherwise indicated the following assessments were made daily for 6 days concerning the wounds dressed with the adhesive film: 1. Maximum
length of lesions: three lesions, two irradiated and one non-irradiated, were measured. 2. ER. 3. LDF: these measurements were made also on day 10. 4. Exudation: the assessments were made for the wounds dressed with hydrocolloid. The appearance of the lesions was assessed after the experiment.
6 months
Calculations and statistical analysis The ER values decreased from day to day, the end-point of epithelialization being defined as the first day when the ER value was not followed by a further decrease on the
43
Svedman et al.: Blister wound exposed to U.V. irradiation
Figure 1. Representative videoprints from the surface of blister wounds on day I. a, Non-UV-irradiated. b, UV-irradiated ( x 120).
following day. When the lowest value was recorded on day 6, the sixth day was designated as the end-point. The sample units for determining the end-points of epithelialization in the two groups were based on the values from one irradiated and one non-irradiated wound situated adjacent to each other. The sample units for the irradiated wound maximum length, the flowmetry and the exudation rate values, were defined as the mean of values measured on the same site(s) in two wounds, while the non-irradiated wound sample unit was based on the value(s) from one wound. The LDF findings were presented either as a linear scan or as a ‘centre’ and a ‘periphery’ value. The ‘centre’ value was calculated from the mean of the five central values in each scan, and the ‘periphery’ value based on the six peripheral values. Statistical differences were assessed using Wilcoxon’s signed rank test. A P level of 0.05 or less was considered significant.
Results The wounds were inflicted painlessly, but slight and transient itching was experienced, particularly at the time when the blisters were beginning to form. The edges of the u.v.-irradiated wounds showed some swelling and erythema during the first days, a reaction which was distinctly less obvious in the non-irradiated wounds. Soft, thin fibrin clots developed daily in the irradiated wounds, but were less regularly observed in the others. These clots were easily removed (series 1). There were no signs of infection. In the non-irradiated wounds in series I, the red blood cells were found to delineate clearly the microvascular network in the wound bed. This network appeared to consist of both evenly distributed papillary loops and vessels in the superficial horizontal plexus. The blood cells were seen to be passing through most of the vessels on day 0 as well as on days 1 and 2. However, already on day 0, blood cell stagnation and extravasation was observed, corresponding to some papillary loops, particularly peripherally in the wound. The irradiated wound structures, on the other hand, could not be so perfectly focused, and the papillary loops were typically less evenly distributed, some containing larger volumes of stagnant red blood cells than in the non-irradiated group. There were signs of extravasation in excess of those observed in the non-irradiated lesions. In some vessels the movement of red cells was uneven, and at
times the cells passed through these vessels extremely quickly. By day I the focusing was even less perfect, there was no movement in the papillary loops, and stagnant red cells had often aggregated into microthrombi. Extravasation was more prominent and widespread. However, movement of red blood cells was still observed in a few vessels in the underlying horizontal plexus. Flow in the papillary loops had ceased also on day 2, at which point, however, the superficial horizontal plexus could not be sufficiently focused to ascertain whether the red cells were moving or not. Video prints taken on day I from representative non-irradiated and irradiated wounds are shown in Figttre 7. In series 2 the maximum lengths reached their peak on day I in both groups, when the value in the non-irradiated lesions was 4.5 f 0.5 mm (mean f s.d.) and in the irradiated ones 10.8 f 1.1 mm (PC 0.01). (Figttre2). Differences between the two groups were observed also on days 2,3,4, 5 and 6 (P < 0.05). On day 6 the value for the non-irradiated lesions was 2.8 f 0.9 mm while it was 5.5 f 0.5 mm in the irradiated ones. The endpoint of the epithelialization was reached after 5.1 f 0.6 days in the non-irradiated wounds and after 5.5 f 0.5 days in the irradiated ones (n.s.). The LDF scans, which were approximately bell shaped in both groups, are shown for day 3 in Figure 3. All scan values were lower within the irradiated wound (n.s.). On day 1 the ‘centre’ LDF values in the non-irradiated and the irradiated wounds were 95 f 39 PU vs. 93 f 25 PU, and the corresponding ‘periphery’ values were 56 f 17 PU vs. 56 f 5 PU. For comparison the value on the adjacent skin was 7 f 4 PU. As observed in Figttre 4 both the ‘centre’ and the ‘periphery’ values in non-irradiated as well as in irradiated wounds reached their maximum on day 3, and then decreased until day 6 or 10. The ‘periphery’ values were found to be slightly higher in the irradiated than in the non-irradiated wounds from day 2, and in comparisons made on a daily basis these differences were significant (PC 0.05) on day 4, when the values were 56 f 16 PU and 31f 11PU, and on day 5, when they were 50 f 19 PU and 32 f 18PU. The ‘centre’ values, on the other hand, were higher in the non-irradiated than in the irradiated wounds from day 3, but these differences were not significant. The rates of exudation were 146f 22 pg/24 h in the non-irradiated wounds and 180f 75 pg/24 h in the irradiated ones on day I (n.s.). On day 2 the respective values were 97 f 19 pg/24 h vs. 108 f 37 pg/24 h (n.s.), and
Bums (1991) Vol. 17/No. 1
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Length (mm)
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Figure 2. Maximum length (including oedema adjacent to the wound) (mean, s.d.) of non-UV-irradiated (0) and UV-irradiated (0) blister wounds, as a function of time. The value on day 0 is approximated to the diameter of the suction cup used for creating the blister.
Figure 3. Laser Doppler flowmetry (LDF) linear scans (mean, s.d.) made over non-UV-irradiated (0) and IN-irradiated (0) blister wounds on day 3. The scans were made also on the skin adjacent to the wounds. 160
160 t
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III 10
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Figure 4. a, LDFvalues (mean, s.d.) from the centre of non-UV-irradiated (0) and UV-irradiated (0) blister wounds, as a function of tie. b, LDF values (mean, s.d.) from the periphery of non-W-irradiated (0) and UV-irradiated (0) blister wounds, as a function of time. The values from a and b are not directly comparable. on day 3 they were 63 f 21 vs. 82 f 21 (n.s.).The decreases in the non-irradiated wounds was 33 per cent between days 1 and 2, and 23 per cent between days 2 and 3. In the irradiated wounds the corresponding values were 40 per cent (Pc0.05) and 14 per cent (PCO.05). The complete time-courses are shown in Figure 5. While the values were higher after the irradiation for the duration of the experiment, significant differences (PC 0.05) were observed only on day 5, when the values were 1Of 5 pg in the nonirradiated wounds and 21 f 10 pg in the irradiated ones, and on day 6 when the values were 2zt2l.tg and 14f6pg respectively. Table I summarizes some of the findings. Six months after the experiments slight discolouring was noted on some wound sites. There were no palpable scars.
Discussion The irradiated suction blister wound model is benign and standardized and should thus be acceptable for experimental use in humans. The suction injury results in oedema, exudation and a pronounced dermal reactive hyperaemia of long duration. The U.V. irradiation of the exposed dermis causes a severe microvascular derangement in the papillary dermis, including abolition of flow in the papillary loops,
creating a superficial, partial skin thickness radiation bum. The reaction is completely different from the hyperaemic response seen after U.V. irradiating skin with intact epidermis. The re-epithelialization occurs rapidly even after irradiation. The more pronounced inflammatory reaction after irradiation is the expected consequence of the increased injury. The present findings indicate that the microckulatory changes, and the end-point of the epithelialization, can be determined with the use of the relatively simple, non-invasive methods of measurement that were applied. The blister separation occurs in the basal membrane zone through the lamina lucida of the basal lamina, and the basal cell plasma membrane appears initially to be intact (Saksela et al., 1981). Epidermal appendages are tom off near their
entrance points into the dermis (Saksela et al., 1981), and papillary loop vessels are destroyed (Saksela et al., 1981; Svedman and Kutlu, 1990). Functional aspects of the suction injury, and the histological changes in the dermal wound bed after suctioning have been described elsewhere (Kutlu et al., 1990). Functionally, laser Doppler flowmetry shows that there are two phases of reactive hyperaemia during suctioning, one occurs at the beginning and the other at the end. The latter develops when the blister is starting to form, and continues after cessation of the stimulus. In between these phases the LDF values are lower than on normal skin,
Svedman et al.: Blister wound exposed to U.V. irradiation
45
Table I. Summary findings: relative values are given
Length LD F centre LDFperiphery Exudation rate Time to epithelialization end-point
Day
Blister wound
W-irradiated blister wound
1 3 4
100 100 100
240 85 171
PC 0.005 ns. PC 0.005
5
100
212
PC 0.005
100
107
ns.
Significance
but they never reach the low levels observed during complete arterial occlusion. A superficial dermal inflammatory infiltrate increases in extent and magnitude to a maximum by day 2 after injury, and then subsides rapidly. There are signs of angiectasis and perivascular inflammation in the mid-dermis by day 3. The dermal hyperaemia continues to be present for several days (Svedman et al., 1990). Exposure of intact human skin to U.V. light results in an acute inflammatory reaction characterized by oedema, erythema, and an inflammatory cell response (Logan and Wilhelm, 1966; Eaglstein et al., 1979; Grange and Parish, 1983; Greaves, 1986), and may lead to epithelial hyperplasia (Blum and Soffer, 1961; Epstein et al., 1970). U.V. exposure may also cause diverse immunological effects (Daynes et al., 1986). The effects of irradiating expcrsed dermis have not been reported in the literature. However, since energy is lost during passage of the radiation through the tissue, the dermal response to a certain U.V.dose is expected to be more pronounced in the absence of the epidermis. Video microscopy has previously been used by us for study of the superficial dermal microcirculation of nonirradiated dermal blister wounds (Svedman and Kutlu, 1990). Removal of soft fibrin clots from the wound surface has not been shown to result in any certain additional
damage to the superficial microvascular network. The problem with focusing in irradiated wounds, which is obviously caused by oedema in the superficial dermis, has not been an obstacle when examining non-irradiated lesions. The ER values relate to the water vapour pressure measured in a small volume of air over the surface of the wound, and simply show when the skin water barrier in the stratum comeum has been re-established. The technique has been described in more detail elsewhere (Svedman et al., IWO). The exudation rate values indicate leakage from the microvascular tree, and are a measure of the volume of fluid that reaches the surface of the wound during 24 h. These rates are, of course, influenced by the size of the wound. In the following we do not attempt to equalize the day-to-day values, but assume that the differences in wound area between the two groups on days I,2 and 3 are so small that they do not significantly affect the exudation rates. The laser Doppler measuring volume includes also the deeper dermal microcirculation, and the Doppler values are thus related to the ‘total’ blood flow in the dermis as opposed to the flow through the superficial parts of the dermal microvascular network -which can be studied by video microscopy. In the statistical evaluation the sample units of the different parameters that were measured in the irradiated wound were usually determined based on values from two wounds in each volunteer, increasing the accuracy of measurement for the individual unit. The results suggest that while this may affect the variability of the LDF values in the irradiated
1
1
3
4
5
6
Time (days)
Exudation (mean, s.d.) in non-U/-irradiated (0) and U/-irradiated (0 ) blister wounds, as a function of time.
Figure
5.
group slightly, the interindividual variation remains as the major factor in determining the total variability. In the wounds exposed to suction only the early microvascular damage in the superfical wound vessels was limited to a lesser portion of the papillary loops. A sustained hyperaemia of the full thickness of the dermal wound bed was demonstrated by flowmetry. The exudation was maximal during the initial 24 h, and then rapidly decreased. The oedema, in and adjacent to the wound, was found to be small. The epithelialization proceeded rapidly. These findings correspond to previous ones (Svedman et al., 1990). In the irradiated wounds, on the other hand, a progressive, superficial microcirculatory disturbance was observed, and microvascular thrombosis with complete absence of flow was observed by microscopy on day 1. Separate experiments indicate that flow cessation may occur as early as 6 h after irradiation (N. Kutlu and P. Svedman, personal communication, 1990). This reaction is clearly an effect of U.V. irradiating the exposed dermis. A superficial radiation burn, induced in a way that has not previously been described, is thus formed. The lesion may have an interesting parallel in the frequently discussed clinical partial skin thickness bum which progressively develops into a full skin
thickness lesion by gradual cessation of flow in microvessels that remain functional directly after the original injury. The oedema within the irradiated wounds, as observed by microscopy, was present to a lesser degree already on day 0 and was maximal on day 2 (when the experiment was ended). This oedema, which is a consequence of the increased capillary injury with additional leakage, involves also the skin adjacent to the wound, as shown by the increased maximal length of the lesions (Figure 2). The increased capillary leakage also explains the higher exudation rates in the irradiated wounds. The findings that the maximal length values, as well as the exudation rate values, reach their peak on day I, and thereafter quickly decrease, suggest that the overall capillary leakage may be maximal during the initial 24 h. From a methodological point of view, the components of the exudate may be collected for further analysis using a chamber technique (Dubertret et al., 1982). Both the ‘centre’ and the ‘periphery’ LDF values were maximal on day 3 in both groups. Thus the dermal blood flow may reach its peak when the oedema and exudation
46
Bums (1991) Vol. 17/No. 1
rates are subsiding. The increase in the ‘periphery’ LDF values of the irradiated wounds suggests an increased hyperaemia in response to the irradiation injury (Logan and Wilhelm, 1966; Grange and Parish, 1983; Greaves, 1986). This hyperaemia constitutes a lesser fraction of the total peripheral hyperaemia, and the difference was statistically significant only on days 4 and 5 after the injury. At this time after the injury a part of the response may be a consequence of new vessel formation (Svedman and Kutlu, 1990). Even in the presence of abolished papillary loop flow there were no significant differences between the ‘centre’ LDF values of the groups. One explanation for this may be that the papillary flow constitutes such a small fraction of the total dermal flow that it cannot be detected by the flowmetry. Another explanation may be that while the blood flow is abolished or severely compromised in the papillary dermis, there may have been a relative flow increase in the deeper dermis of the wound. The previously discussed data show that by irradiating a blister wound a model is formed that may allow better insight into the early course of a more pronounced microvascular injury, as may be seen in a burn. From a clinician’s point of view the model may be of particular interest for studying the effects of early treatment on bum microcirculation in humans. One may also envisage applying, for instance, a thermistor needle through the whole thickness of the dermis at the centre of a blister wound, creating a localized full skin thickness lesion with an adjacent, visible microvascular network. There was no significant delay in reaching the end-point of the epithelialization in spite of the severe microvascular derangement observed after irradiation. The epithelialization process is thus forceful in healthy, young adults, and assessing stimulation of epithelialization in a small number of such individuals may be of limited value even after increasing the degree of tissue damage by U.V. irradiation. The model should, however, be suitable for assessing inhibitory stimuli.
by biosynthetic epidennal growth factor. J. Erp. Med. 163, 1319. Daynes R. A., Samlowski W. E., Bumham D. K. et al. (1986) Immunobiological consequences of acute and chronic UV exposure. Cut-r. Pro&l.Den&ol. 15, 176.
Dubertret L., Lebreton C. and Touraine R. (1982). Neutrophil studies in psoriatics: in vivo migration, phagocytosis and bacterial killing. J. Invest. Dermatol., 79, 681. Eaglstein W. H., Sakai M. and Mizuno N. (1979) Ultraviolet radiation induced inflammation and leucocytes. 1, Invest. Dermatol. 72, 59. Epstein J. H., Fukyjama K. and Fye K. (1970) Effects of ultraviolet radiation on the mitotic cycle and DNA, RNA and protein synthesis in mammalian epidermis in vivo. Pkofockem. Pkotobiol. 12,57.
Grange R. W. and Parrish J. (1983) Acute effects of ultraviolet radiation upon the skin. In: Parrish J. (ed.), Photoimmunology. New York: Plenum, p. 77. Greaves M. W. (1986). Ultraviolet erythema: causes and consequences. Curr. Pro&l.Dermatol. 15, 18.
Hammarlund C., Svedman C. and Svedman P. (1990) Hyperbaric oxygen treatment of healthy volunteers with UV-irradiated blister wounds. Abstract, XVI European Conference on Miaocirculation. Inf. 1. Microcirc. Clin. Exp. 9, Suppl. I, 185. Hunt T. K. and Pai M. P. (1972) The effects of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg. Gynecol. Obstef. 135, 561. Kolari P.J. (1985) Penetration of unfocused laser light into the skin. Arch. Dermafol. Res. 277,342.
Kutlu, N., Rausing A. and Svedman P. (1990) Suction blister wound, morphological and functional aspects. Abstract, XVI European Conference Microcirculation Inf. 1. Micro&c. Clin. 4. 9, Suppl. 1, 160. Logan G. and Wilhelm D. L. (1966) The inflammatory reaction in ultraviolet injury. Br. 1. &p. Pat/&. 47,286. Niinikoski J., Kivisari J. and Viljanto J. (1977) Local hyperalimentation of experimental granulation tissue. Acfa Ckir. Stand. 143, 201.
Conclusions In this report, a benign and reproducible wound model in humans - based on a combination of a mechanical suction injury and a superficial radiation burn -has been described. In wounds exposed to suction only, the early microvascular damage was limited to the most superficial capillaries. A pronounced hyperaemia of the ‘total dermal wound bed was present for some days. The exudation was maximal during the initial 24 h, and then rapidly decreased. The oedema adjacent to the wound was found to be small. The epithelialization proceeded rapidly. U.V. irradiation of blister wounds resulted in cessation of the blood flow in the papillary loops, marked oedema and increased exudation, and increased hyperaemia at the wound edges. The epithelialization was not significantly retarded by the irradiation injury. The model may be of interest, for instance, for studying effects of bums treatment (Hammarlund et al., 1990).
Acknowledgement This study was supported Foundation.
ultraviolet light. J Ceil. Camp. Physiol 58, 97. Brown G. (1986) Enhancement of epidermal regeneration
by a grant from the Bo Rydin
References Blum H. F. and Soffen B. A. (1961) Quantitative analysis of epidermal hyperplasia in mouse skin following single doses of
Nilsson G. E. (1977) Measurement of water exchange through skin. Med. Biol. Eng. Compuf. 15, 209. Nilsson G. E. Tenland T. and Oberg P. A. (198Oa) A new instrument for continuous measurement of tissue blood flow by light beating spectroscopy. IEEE Trans. Biomed. Eng. BME, 27,
1.2. Nilsson G. E., Tenland T. and &erg P. A. (198Ob) Evaluation of a laser Doppler flowmeter for measurement of blood flow. IEEE Trans. Biowred.Eng. BME 27,597. Saksela O., AIiraIo K., Kiistala U. et al. (1981) Basal lamina components in experimentally induced skin blisters. J. Invest. Dermafol. 77, 283. Svedn-an P. and Kutlu N. (1990) Videomicroscopy of the superficial microcirculation in suction blister wounds on healthy volunteers. Abstract, XVI European Conference on Microcirculation. Microcirc. C/in. Exp. 9, Suppl 1, 160. Svedman P., Svedman C., Hammarlund C. et al (1989) Iatrogenic wounds on humans for studying wound healing. Proceedings, Swedish Society for Plasfic Surgery Stockholm. Svedman P., Svedman C. and Njalsson T. (1990) Epithelialization and blood flow in suction blister wounds on healthy volunteers. J. Invest. Surg. (accepted for publication). Paper accepted
8 October
1990.
Correspondence should be addressed to: C. Svedman, Department of Plastic and Reconstructive Surgery, Allmiinna Sjukhuset, !%1401 Malmii, Sweden.
Prinkd in Great Britain
41
Skin suction blister wound exposed to U.V. irradiation: a burn wound model for use in humans C. Svedmanl, C. Hammarlundz, N. Kutlu’ and P. Svedman’ ‘Department Helsingborg
of Plastic and Reconstructive Surgery, Allmba Sjukhuset, MalmG and 2Department Hospital, Helsingborg, University of Lund, Lund, Sweden
The effects of exposing blister wounds to U.V.irradiation were assessed in 14 male voluntem, on whose foream blister wounds (diameter 5mm, suctionset at 200 mm@ below atmospheric for2 h 15 min, blister roof cuf at base), and irradiated blister wounds (as above, in addition xv. irradiation given selectively for 30 min from a distance of 10 MI), were produced. In non-xv.-irradiated wounds, flow cessation, asxssed by video microscopy (n = 6), was obsemti in a small proportion of the papillary loop ve5sels. The oedema adjacent to the wound was poorly &eloped. Laser Doppler linear scans (n = 8) demonstrated a pronounced hyperaemia in the wound bed and also in the adjacent skin, the reaction subsiding over a few days. The exudation rate, deknined by weighing the hydrocolloid dressingsapplied to the wound, was makmal on day I ana’then rapidly decreased. Epithelialization, assessed evapoketkally as the time taken for reinstatement of the epidetmal water barn& was complete in 5.1 hys. In the xv.-irradiated wounds Hz blood flow had ceased in all the papilkzry hyperaemia at loop vessels by day I, and increased oedema, exudation ana’ the wound edges were observed Epithelializalion was not significantly retarded by the irradiation injury. After 6 months, slight discolouration of the skin was occasionally observed, but no cosmetically disturbing scars. This benign and standardized wound model in humans - based on a combination of a mechanical strctioninjury ana’a supe$cial radiation burn - may prove to be useful, for instance when studykg the effects of bums treatment.
Introduction Wound models in animals are indispensable for studying wound healing since human wounds are complex and variable and are difficult to sample and measure. At present, findings from research in experimental animals, such as the stimulating effects of oxygen (Hunt and Pai, 1972), and of nutritional (Niinikoski et al., 1977) and growth factors (Brown, 1986) on healing need to be corroborated in human patients. It is also important to study the way diabetes and peripheral vascular diseases affect the healing processes in humans, as well as how the same processes are influenced by the common drugs in clinical use. Furthermore, many therapy modes are advocated for treating wounds and burns without adequate clinical testing. Thus measurement of wound healing in patients is a necessity. This challenge can be met by developing standardized, benign and miniaturized wound models by which healing can be measured quantitatively, preferably with non0 1991 Butterworth-Heineman Ltd 0305/4179/91/010041-06
of Anaesthesiology,
invasive techniques (Svedman et al., 1989). By means of evaporimetry and laser Doppler flowmetry, we have studied the blister wound, formed painlessly after sucking loose and excising the epidermis within a small area of skin (Svedman et al., 1990). Initially, wound fluid evaporated quickly in the absence of the epidermal barrier, but within a week the rate normalized as the barrier reformed. A simple model for determining the end-point of epithelialization in humans was thus devised. The flowmetry revealed a strong hyperaemic reaction to the dermal injury caused by the suction. In this investigation suction blister wounds in healthy volunteers were exposed to U.V.irradiation, creating an irradiation injury in addition to the original mechanical one. The damage to the superficial wound bed of both non-irradiated and irradiated lesions was assessed by video microscopy. During the initial days after the injury the total diameter including the oedema zone - was measured, and the epithelialization was assessed evaporimetrically. The microcirculatory events of the healing process were studied by laser Doppler flowrnetry, and by determining the rate of exudation from the wound.
Materials and methods Fourteen healthy, non-smoking male volunteers were studied, their ages ranging from 24 to 29 (median = 26.5) years. The volunteers wore normal, indoor clothing, and were sitting comfortably, resting their lower arm with its exposed measuring sites at heart level. The measurements were begun after 10 min of rest. Conversation was avoided. The room temperature was controlled with a thermostat and maintained at 22-23°C. Video microscopy A stereomicroscope (M3 C Kombistereoscope, Wild Leitz, Switzerland) equipped with a TV camera (Panasonic WV 1850/G, Japan), and connected to a video monitor (Panasonic WV 5360/G), a recorder (Goldstar GI-IV 1323 P, South Korea) and a video printer (Sony, UP 850, Japan) was used. The wound was illuminated with cold light (Intralux 6000, Volpi AG, Switzerland) that had been passed through a blue filter (Volpi AC). The microscope had a three-stage magnification changer. The total magnifications obtained on the monitor were 40 x , 120 x and 450 x . The forearm
Bums (1991) Vol. 17/No. 1
42
with the blister wound was laid in a rigid, lightly padded rest which could be moved in a controlled way in the horizontal plane (Svedman and Kutlu, 1990). When present, fibrin clots on the wounds surface were removed under magnification (2.5 x ) using ophthalmological tweezers. During the examination the wound was covered with a transparent polyester film (Mylar, thickness 6 pm; BioRad, UK). The appearance of the superficial dermal microvascular network was described from video images, and the presence or absence of moving blood cells in the vessels was noted. Video-prints were made from the filmed material.
Evaporimetry The evaporation rate (ER) was measured using an evaporimeter (EPI, Servomed AB, Stockholm, Sweden) with a digital output (Nilsson, 1977). The open cylinder probe contains two sets of sensors, each consisting of a capacitive film and a thermistor. The water vapour pressure gradient in the volume of air between the two sets of sensors is estimated, and from this estimate the ER value, determined as the approximate mean of values displayed during a minimum of 2 min after stabilization, was determined in g/m2/h.
Measurement of blood flow A laser Doppler flowmeter (Periflux PF 3, Perimed KB, Sweden) was used (Nilsson et al., 1980a,b). Light (632.8 nm) from the flowmeter’s laser (He-Ne, 2mW) is brought to transilluminate the measuring volume by an optic fibre within a conductor. The backscattered light is transmitted through a separate pair of optical fibres to a photodetector system. Photons scattered in moving blood cells change wavelength according to the Doppler principle, and the laser Doppler flowmetry (LDF) output is related to the number of Doppler-shifted beams and the average Doppler frequency shift generated by movement of the blood cells. The measuring volume is thought to be hemispherical, with a radius of about 1 mm (Nilsson et al., 1980b; Kolani, 1985). The cut-off frequency of the flowmeter filter was set at 12 kHz, except for measurement on intact skin sites when it was set at 4 kHz. The time constant of the output amplifier was set at 3 s. The LDF values were displayed digitally and read in perfusion units (PU) which were defined by calibration against a motility standard (Perimed) giving a reading of 250PU (2.5 V, gain x 1) at 12 kHz. Each LDF reading was determined as the approximate mean of values displayed during a minimum of 2 min after stabilization. The measurements were made through a freshly applied transparent film of polyurethane (Tegaderm, 3M, USA). The conductor tip was secured in a holder within a rigid frame which in turn was taped to the skin adjacent to the wound (Svedman et al., 1990). The holder could be moved on the frame in a controlled way by turning a screw. This allowed linear advancement of the conductor tip along a line passing through the centre of the wound onto the skin on either side. The conductor tip was kept at roughly the same minimal distance from the underlying polyurethane film. Each LDF scan included I I separate measurements made at sites l.Omm apart. The sixth site was positioned approximately over the centre of the wound. At the end of each experiment the LDF value was measured randomly on the forearm during arterial occlusion produced by a bra&al cuff (2OOmmHg). These values were in the range 0.8-2.5 PU.
Maximum length of the lesions The maximum distance between the outer edges of the lesion was measured under magnification (2.5 X) using a ruler (scale unit = 0.1 mm). Oedema adjacent to the wound was included in the lesion. Measurement of exudation Hydrocolloid dressings (2 x 2 cm, Metoderm, Molnlycke Health Care AB, Sweden) were used; for each assessment one dressing was applied to the wound, and another on adjacent, intact skin. The dressings were weighed (Mettler P 163N, Mettler Instrument AC, Switzerland) before and after use. The rate of exudation &g/24 h) from the wound was determined by calculating the difference in weight increase between the two. Infliction of the lesions Rigid, transparent rectangular boxes, with either two or six circular perforations (diameter = 5 mm, spaced 15 mm apart) on one side, were connected by pliable tubing to a suction pump (Junior DK, Fricat AG, Switzerland). A mineral-light lamp (Mineral-light lamp, model S-52-T, Ultra Violet Products Inc., USA) was also used, its radiation wavelengths ranging between 200 and 400 nm with a peak at 254 nm. The volunteer’s trunk and extremities were covered with a blanket. The forearm was exposed, and the skin on the volar part was cleansed with ethyl alcohol (70 per cent). Five minutes later the perforated side of the box was applied to the skin and made airtight with surgical tape. Continuous suction, applied for 135 min at a pressure of 200mmHg below atmospheric, produced a blister, which was excised at the base, thus exposing the blister wound. Excess fluid was absorbed with a gauze compress. Some of the wounds were then selectively irradiated from a distance of 10 cm for 30 min, the lower arm was covered by a tin-foil with a hole corresponding to the wound being irradiated. All the wounds were dressed either with a hydrocolloid dressing (Metoderm) or with adhesive polyurethane film (Tegaderm). Comparison of non-irradiated and irradiated blister wounds Series 1: video microscopy Two blister wounds were made; one was irradiated (n = 6). The lesions were studied 60 min after infliction (day 0) and then again on days 1 and 2. Series 2 Six blister wounds were made, four were irradiated (n = 8). Two of the irradiated wounds and one non-irradiated wound were dressed with adhesive polyurethane film, the remaining three with hydrocolloid. If not otherwise indicated the following assessments were made daily for 6 days concerning the wounds dressed with the adhesive film: 1. Maximum
length of lesions: three lesions, two irradiated and one non-irradiated, were measured. 2. ER. 3. LDF: these measurements were made also on day 10. 4. Exudation: the assessments were made for the wounds dressed with hydrocolloid. The appearance of the lesions was assessed after the experiment.
6 months
Calculations and statistical analysis The ER values decreased from day to day, the end-point of epithelialization being defined as the first day when the ER value was not followed by a further decrease on the
43
Svedman et al.: Blister wound exposed to U.V. irradiation
Figure 1. Representative videoprints from the surface of blister wounds on day I. a, Non-UV-irradiated. b, UV-irradiated ( x 120).
following day. When the lowest value was recorded on day 6, the sixth day was designated as the end-point. The sample units for determining the end-points of epithelialization in the two groups were based on the values from one irradiated and one non-irradiated wound situated adjacent to each other. The sample units for the irradiated wound maximum length, the flowmetry and the exudation rate values, were defined as the mean of values measured on the same site(s) in two wounds, while the non-irradiated wound sample unit was based on the value(s) from one wound. The LDF findings were presented either as a linear scan or as a ‘centre’ and a ‘periphery’ value. The ‘centre’ value was calculated from the mean of the five central values in each scan, and the ‘periphery’ value based on the six peripheral values. Statistical differences were assessed using Wilcoxon’s signed rank test. A P level of 0.05 or less was considered significant.
Results The wounds were inflicted painlessly, but slight and transient itching was experienced, particularly at the time when the blisters were beginning to form. The edges of the u.v.-irradiated wounds showed some swelling and erythema during the first days, a reaction which was distinctly less obvious in the non-irradiated wounds. Soft, thin fibrin clots developed daily in the irradiated wounds, but were less regularly observed in the others. These clots were easily removed (series 1). There were no signs of infection. In the non-irradiated wounds in series I, the red blood cells were found to delineate clearly the microvascular network in the wound bed. This network appeared to consist of both evenly distributed papillary loops and vessels in the superficial horizontal plexus. The blood cells were seen to be passing through most of the vessels on day 0 as well as on days 1 and 2. However, already on day 0, blood cell stagnation and extravasation was observed, corresponding to some papillary loops, particularly peripherally in the wound. The irradiated wound structures, on the other hand, could not be so perfectly focused, and the papillary loops were typically less evenly distributed, some containing larger volumes of stagnant red blood cells than in the non-irradiated group. There were signs of extravasation in excess of those observed in the non-irradiated lesions. In some vessels the movement of red cells was uneven, and at
times the cells passed through these vessels extremely quickly. By day I the focusing was even less perfect, there was no movement in the papillary loops, and stagnant red cells had often aggregated into microthrombi. Extravasation was more prominent and widespread. However, movement of red blood cells was still observed in a few vessels in the underlying horizontal plexus. Flow in the papillary loops had ceased also on day 2, at which point, however, the superficial horizontal plexus could not be sufficiently focused to ascertain whether the red cells were moving or not. Video prints taken on day I from representative non-irradiated and irradiated wounds are shown in Figttre 7. In series 2 the maximum lengths reached their peak on day I in both groups, when the value in the non-irradiated lesions was 4.5 f 0.5 mm (mean f s.d.) and in the irradiated ones 10.8 f 1.1 mm (PC 0.01). (Figttre2). Differences between the two groups were observed also on days 2,3,4, 5 and 6 (P < 0.05). On day 6 the value for the non-irradiated lesions was 2.8 f 0.9 mm while it was 5.5 f 0.5 mm in the irradiated ones. The endpoint of the epithelialization was reached after 5.1 f 0.6 days in the non-irradiated wounds and after 5.5 f 0.5 days in the irradiated ones (n.s.). The LDF scans, which were approximately bell shaped in both groups, are shown for day 3 in Figure 3. All scan values were lower within the irradiated wound (n.s.). On day 1 the ‘centre’ LDF values in the non-irradiated and the irradiated wounds were 95 f 39 PU vs. 93 f 25 PU, and the corresponding ‘periphery’ values were 56 f 17 PU vs. 56 f 5 PU. For comparison the value on the adjacent skin was 7 f 4 PU. As observed in Figttre 4 both the ‘centre’ and the ‘periphery’ values in non-irradiated as well as in irradiated wounds reached their maximum on day 3, and then decreased until day 6 or 10. The ‘periphery’ values were found to be slightly higher in the irradiated than in the non-irradiated wounds from day 2, and in comparisons made on a daily basis these differences were significant (PC 0.05) on day 4, when the values were 56 f 16 PU and 31f 11PU, and on day 5, when they were 50 f 19 PU and 32 f 18PU. The ‘centre’ values, on the other hand, were higher in the non-irradiated than in the irradiated wounds from day 3, but these differences were not significant. The rates of exudation were 146f 22 pg/24 h in the non-irradiated wounds and 180f 75 pg/24 h in the irradiated ones on day I (n.s.). On day 2 the respective values were 97 f 19 pg/24 h vs. 108 f 37 pg/24 h (n.s.), and
Bums (1991) Vol. 17/No. 1
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Length (mm)
TimeCdays)
Figure 2. Maximum length (including oedema adjacent to the wound) (mean, s.d.) of non-UV-irradiated (0) and UV-irradiated (0) blister wounds, as a function of time. The value on day 0 is approximated to the diameter of the suction cup used for creating the blister.
Figure 3. Laser Doppler flowmetry (LDF) linear scans (mean, s.d.) made over non-UV-irradiated (0) and IN-irradiated (0) blister wounds on day 3. The scans were made also on the skin adjacent to the wounds. 160
160 t
OL
III 10
0
a
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Figure 4. a, LDFvalues (mean, s.d.) from the centre of non-UV-irradiated (0) and UV-irradiated (0) blister wounds, as a function of tie. b, LDF values (mean, s.d.) from the periphery of non-W-irradiated (0) and UV-irradiated (0) blister wounds, as a function of time. The values from a and b are not directly comparable. on day 3 they were 63 f 21 vs. 82 f 21 (n.s.).The decreases in the non-irradiated wounds was 33 per cent between days 1 and 2, and 23 per cent between days 2 and 3. In the irradiated wounds the corresponding values were 40 per cent (Pc0.05) and 14 per cent (PCO.05). The complete time-courses are shown in Figure 5. While the values were higher after the irradiation for the duration of the experiment, significant differences (PC 0.05) were observed only on day 5, when the values were 1Of 5 pg in the nonirradiated wounds and 21 f 10 pg in the irradiated ones, and on day 6 when the values were 2zt2l.tg and 14f6pg respectively. Table I summarizes some of the findings. Six months after the experiments slight discolouring was noted on some wound sites. There were no palpable scars.
Discussion The irradiated suction blister wound model is benign and standardized and should thus be acceptable for experimental use in humans. The suction injury results in oedema, exudation and a pronounced dermal reactive hyperaemia of long duration. The U.V. irradiation of the exposed dermis causes a severe microvascular derangement in the papillary dermis, including abolition of flow in the papillary loops,
creating a superficial, partial skin thickness radiation bum. The reaction is completely different from the hyperaemic response seen after U.V. irradiating skin with intact epidermis. The re-epithelialization occurs rapidly even after irradiation. The more pronounced inflammatory reaction after irradiation is the expected consequence of the increased injury. The present findings indicate that the microckulatory changes, and the end-point of the epithelialization, can be determined with the use of the relatively simple, non-invasive methods of measurement that were applied. The blister separation occurs in the basal membrane zone through the lamina lucida of the basal lamina, and the basal cell plasma membrane appears initially to be intact (Saksela et al., 1981). Epidermal appendages are tom off near their
entrance points into the dermis (Saksela et al., 1981), and papillary loop vessels are destroyed (Saksela et al., 1981; Svedman and Kutlu, 1990). Functional aspects of the suction injury, and the histological changes in the dermal wound bed after suctioning have been described elsewhere (Kutlu et al., 1990). Functionally, laser Doppler flowmetry shows that there are two phases of reactive hyperaemia during suctioning, one occurs at the beginning and the other at the end. The latter develops when the blister is starting to form, and continues after cessation of the stimulus. In between these phases the LDF values are lower than on normal skin,
Svedman et al.: Blister wound exposed to U.V. irradiation
45
Table I. Summary findings: relative values are given
Length LD F centre LDFperiphery Exudation rate Time to epithelialization end-point
Day
Blister wound
W-irradiated blister wound
1 3 4
100 100 100
240 85 171
PC 0.005 ns. PC 0.005
5
100
212
PC 0.005
100
107
ns.
Significance
but they never reach the low levels observed during complete arterial occlusion. A superficial dermal inflammatory infiltrate increases in extent and magnitude to a maximum by day 2 after injury, and then subsides rapidly. There are signs of angiectasis and perivascular inflammation in the mid-dermis by day 3. The dermal hyperaemia continues to be present for several days (Svedman et al., 1990). Exposure of intact human skin to U.V. light results in an acute inflammatory reaction characterized by oedema, erythema, and an inflammatory cell response (Logan and Wilhelm, 1966; Eaglstein et al., 1979; Grange and Parish, 1983; Greaves, 1986), and may lead to epithelial hyperplasia (Blum and Soffer, 1961; Epstein et al., 1970). U.V. exposure may also cause diverse immunological effects (Daynes et al., 1986). The effects of irradiating expcrsed dermis have not been reported in the literature. However, since energy is lost during passage of the radiation through the tissue, the dermal response to a certain U.V.dose is expected to be more pronounced in the absence of the epidermis. Video microscopy has previously been used by us for study of the superficial dermal microcirculation of nonirradiated dermal blister wounds (Svedman and Kutlu, 1990). Removal of soft fibrin clots from the wound surface has not been shown to result in any certain additional
damage to the superficial microvascular network. The problem with focusing in irradiated wounds, which is obviously caused by oedema in the superficial dermis, has not been an obstacle when examining non-irradiated lesions. The ER values relate to the water vapour pressure measured in a small volume of air over the surface of the wound, and simply show when the skin water barrier in the stratum comeum has been re-established. The technique has been described in more detail elsewhere (Svedman et al., IWO). The exudation rate values indicate leakage from the microvascular tree, and are a measure of the volume of fluid that reaches the surface of the wound during 24 h. These rates are, of course, influenced by the size of the wound. In the following we do not attempt to equalize the day-to-day values, but assume that the differences in wound area between the two groups on days I,2 and 3 are so small that they do not significantly affect the exudation rates. The laser Doppler measuring volume includes also the deeper dermal microcirculation, and the Doppler values are thus related to the ‘total’ blood flow in the dermis as opposed to the flow through the superficial parts of the dermal microvascular network -which can be studied by video microscopy. In the statistical evaluation the sample units of the different parameters that were measured in the irradiated wound were usually determined based on values from two wounds in each volunteer, increasing the accuracy of measurement for the individual unit. The results suggest that while this may affect the variability of the LDF values in the irradiated
1
1
3
4
5
6
Time (days)
Exudation (mean, s.d.) in non-U/-irradiated (0) and U/-irradiated (0 ) blister wounds, as a function of time.
Figure
5.
group slightly, the interindividual variation remains as the major factor in determining the total variability. In the wounds exposed to suction only the early microvascular damage in the superfical wound vessels was limited to a lesser portion of the papillary loops. A sustained hyperaemia of the full thickness of the dermal wound bed was demonstrated by flowmetry. The exudation was maximal during the initial 24 h, and then rapidly decreased. The oedema, in and adjacent to the wound, was found to be small. The epithelialization proceeded rapidly. These findings correspond to previous ones (Svedman et al., 1990). In the irradiated wounds, on the other hand, a progressive, superficial microcirculatory disturbance was observed, and microvascular thrombosis with complete absence of flow was observed by microscopy on day 1. Separate experiments indicate that flow cessation may occur as early as 6 h after irradiation (N. Kutlu and P. Svedman, personal communication, 1990). This reaction is clearly an effect of U.V. irradiating the exposed dermis. A superficial radiation burn, induced in a way that has not previously been described, is thus formed. The lesion may have an interesting parallel in the frequently discussed clinical partial skin thickness bum which progressively develops into a full skin
thickness lesion by gradual cessation of flow in microvessels that remain functional directly after the original injury. The oedema within the irradiated wounds, as observed by microscopy, was present to a lesser degree already on day 0 and was maximal on day 2 (when the experiment was ended). This oedema, which is a consequence of the increased capillary injury with additional leakage, involves also the skin adjacent to the wound, as shown by the increased maximal length of the lesions (Figure 2). The increased capillary leakage also explains the higher exudation rates in the irradiated wounds. The findings that the maximal length values, as well as the exudation rate values, reach their peak on day I, and thereafter quickly decrease, suggest that the overall capillary leakage may be maximal during the initial 24 h. From a methodological point of view, the components of the exudate may be collected for further analysis using a chamber technique (Dubertret et al., 1982). Both the ‘centre’ and the ‘periphery’ LDF values were maximal on day 3 in both groups. Thus the dermal blood flow may reach its peak when the oedema and exudation
46
Bums (1991) Vol. 17/No. 1
rates are subsiding. The increase in the ‘periphery’ LDF values of the irradiated wounds suggests an increased hyperaemia in response to the irradiation injury (Logan and Wilhelm, 1966; Grange and Parish, 1983; Greaves, 1986). This hyperaemia constitutes a lesser fraction of the total peripheral hyperaemia, and the difference was statistically significant only on days 4 and 5 after the injury. At this time after the injury a part of the response may be a consequence of new vessel formation (Svedman and Kutlu, 1990). Even in the presence of abolished papillary loop flow there were no significant differences between the ‘centre’ LDF values of the groups. One explanation for this may be that the papillary flow constitutes such a small fraction of the total dermal flow that it cannot be detected by the flowmetry. Another explanation may be that while the blood flow is abolished or severely compromised in the papillary dermis, there may have been a relative flow increase in the deeper dermis of the wound. The previously discussed data show that by irradiating a blister wound a model is formed that may allow better insight into the early course of a more pronounced microvascular injury, as may be seen in a burn. From a clinician’s point of view the model may be of particular interest for studying the effects of early treatment on bum microcirculation in humans. One may also envisage applying, for instance, a thermistor needle through the whole thickness of the dermis at the centre of a blister wound, creating a localized full skin thickness lesion with an adjacent, visible microvascular network. There was no significant delay in reaching the end-point of the epithelialization in spite of the severe microvascular derangement observed after irradiation. The epithelialization process is thus forceful in healthy, young adults, and assessing stimulation of epithelialization in a small number of such individuals may be of limited value even after increasing the degree of tissue damage by U.V. irradiation. The model should, however, be suitable for assessing inhibitory stimuli.
by biosynthetic epidennal growth factor. J. Erp. Med. 163, 1319. Daynes R. A., Samlowski W. E., Bumham D. K. et al. (1986) Immunobiological consequences of acute and chronic UV exposure. Cut-r. Pro&l.Den&ol. 15, 176.
Dubertret L., Lebreton C. and Touraine R. (1982). Neutrophil studies in psoriatics: in vivo migration, phagocytosis and bacterial killing. J. Invest. Dermatol., 79, 681. Eaglstein W. H., Sakai M. and Mizuno N. (1979) Ultraviolet radiation induced inflammation and leucocytes. 1, Invest. Dermatol. 72, 59. Epstein J. H., Fukyjama K. and Fye K. (1970) Effects of ultraviolet radiation on the mitotic cycle and DNA, RNA and protein synthesis in mammalian epidermis in vivo. Pkofockem. Pkotobiol. 12,57.
Grange R. W. and Parrish J. (1983) Acute effects of ultraviolet radiation upon the skin. In: Parrish J. (ed.), Photoimmunology. New York: Plenum, p. 77. Greaves M. W. (1986). Ultraviolet erythema: causes and consequences. Curr. Pro&l.Dermatol. 15, 18.
Hammarlund C., Svedman C. and Svedman P. (1990) Hyperbaric oxygen treatment of healthy volunteers with UV-irradiated blister wounds. Abstract, XVI European Conference on Miaocirculation. Inf. 1. Microcirc. Clin. Exp. 9, Suppl. I, 185. Hunt T. K. and Pai M. P. (1972) The effects of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg. Gynecol. Obstef. 135, 561. Kolari P.J. (1985) Penetration of unfocused laser light into the skin. Arch. Dermafol. Res. 277,342.
Kutlu, N., Rausing A. and Svedman P. (1990) Suction blister wound, morphological and functional aspects. Abstract, XVI European Conference Microcirculation Inf. 1. Micro&c. Clin. 4. 9, Suppl. 1, 160. Logan G. and Wilhelm D. L. (1966) The inflammatory reaction in ultraviolet injury. Br. 1. &p. Pat/&. 47,286. Niinikoski J., Kivisari J. and Viljanto J. (1977) Local hyperalimentation of experimental granulation tissue. Acfa Ckir. Stand. 143, 201.
Conclusions In this report, a benign and reproducible wound model in humans - based on a combination of a mechanical suction injury and a superficial radiation burn -has been described. In wounds exposed to suction only, the early microvascular damage was limited to the most superficial capillaries. A pronounced hyperaemia of the ‘total dermal wound bed was present for some days. The exudation was maximal during the initial 24 h, and then rapidly decreased. The oedema adjacent to the wound was found to be small. The epithelialization proceeded rapidly. U.V. irradiation of blister wounds resulted in cessation of the blood flow in the papillary loops, marked oedema and increased exudation, and increased hyperaemia at the wound edges. The epithelialization was not significantly retarded by the irradiation injury. The model may be of interest, for instance, for studying effects of bums treatment (Hammarlund et al., 1990).
Acknowledgement This study was supported Foundation.
ultraviolet light. J Ceil. Camp. Physiol 58, 97. Brown G. (1986) Enhancement of epidermal regeneration
by a grant from the Bo Rydin
References Blum H. F. and Soffen B. A. (1961) Quantitative analysis of epidermal hyperplasia in mouse skin following single doses of
Nilsson G. E. (1977) Measurement of water exchange through skin. Med. Biol. Eng. Compuf. 15, 209. Nilsson G. E. Tenland T. and Oberg P. A. (198Oa) A new instrument for continuous measurement of tissue blood flow by light beating spectroscopy. IEEE Trans. Biomed. Eng. BME, 27,
1.2. Nilsson G. E., Tenland T. and &erg P. A. (198Ob) Evaluation of a laser Doppler flowmeter for measurement of blood flow. IEEE Trans. Biowred.Eng. BME 27,597. Saksela O., AIiraIo K., Kiistala U. et al. (1981) Basal lamina components in experimentally induced skin blisters. J. Invest. Dermafol. 77, 283. Svedn-an P. and Kutlu N. (1990) Videomicroscopy of the superficial microcirculation in suction blister wounds on healthy volunteers. Abstract, XVI European Conference on Microcirculation. Microcirc. C/in. Exp. 9, Suppl 1, 160. Svedman P., Svedman C., Hammarlund C. et al (1989) Iatrogenic wounds on humans for studying wound healing. Proceedings, Swedish Society for Plasfic Surgery Stockholm. Svedman P., Svedman C. and Njalsson T. (1990) Epithelialization and blood flow in suction blister wounds on healthy volunteers. J. Invest. Surg. (accepted for publication). Paper accepted
8 October
1990.
Correspondence should be addressed to: C. Svedman, Department of Plastic and Reconstructive Surgery, Allmiinna Sjukhuset, !%1401 Malmii, Sweden.