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Non-invasive quantification of skin injury resulting from exposure to sulphur mustard and Lewisite vapours
Burns 26 (2000) 245±250
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Non-invasive quanti®cation of skin injury resulting from exposure to sulphur mustard and Lewisite vapours R.P. Chilcott*, R.F.R. Brown, P. Rice Biomedical Sciences Department, CBD Sector, Defence Evaluation and Research Agency, Porton Down, Salisbury SP4 0JQ, UK. Accepted 13 August 1999
Abstract The severity and progression of skin lesions resulting from exposure to the chemical warfare agents Lewisite (L) and sulphur mustard (SM) have been investigated using the non-invasive biophysical methods of evaporimetry and re¯ectance spectroscopy in large white pigs in vivo. Erythema (redness) expressed immediately after exposure to L or SM vapours appeared to be related to the lesion severity as demonstrated by histopathological analysis. Skin brightness correlated well with scab formation whereas blueness (cyanosis) did not appreciably alter throughout the study. Rates of transepidermal water loss (TEWL) changed both with occlusion (during vapour exposure) and also mirrored the progression of macroscopic skin injury after 12 h. Whilst no single parameter could be used in isolation to ascertain the severity and subsequent progression of the skin lesions, measurement of erythema, skin brightness and TEWL could provide quantitative, non-invasive methods for determining the ecacy of antidotes or therapies to prevent the toxic eects of chemical warfare agents. However, neither colourimetry or TEWL provided a clinical evaluation of such lesions that were comparable with the prognostic capabilities of laser Doppler imaging. Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Burn; Transepidermal water loss (TEWL); Skin colour; Pig; Skin; In vivo; Sulphur mustard; Lewisite
1. Introduction Vesicating warfare agents are chemicals that can produce deep skin burns [1] and include the classic agents Lewisite (L) and sulphur mustard (SM). Skin lesions resulting from exposure to SM dier from thermal burns in that signs and symptoms may be delayed for up to 24 h after exposure depending on dose, damage tends to be partial thickness and spontaneous healing rates are signi®cantly slower [2]. In contrast the development, depth and healing rates of L skin injuries resemble those of thermal burns. Skin lesions resulting from exposure to L or SM vapour may progress to form large bullae that may require intensive medical management and surgical intervention. Previous studies have reported a laser Doppler imaging * Corresponding author.
(LDI) technique for the clinical evaluation of the severity of L and SM skin injuries [3]. Whilst being an invaluable tool for determining vesicant burn depth and subsequent prognosis, the LDI system is currently a bulky instrument which is unsuitable for battle-®eld use. Furthermore, LDI evaluation of burns in experimental models requires the animals to be anaesthetised. This places a restriction on the number of times laser Doppler imaging can be performed. This constraint is particularly relevant when assessing pretreatments or therapies, as skin lesions resulting from exposure to L or SM may take up to 3 months to resolve [4], the duration of which an animal may have to be anaesthetised many times. A variety of alternative techniques are commercially available for the rapid measurement of skin damage that are non-invasive and relatively inexpensive. For example, experimentally induced skin lesions have previously been quanti®ed by measurement of transepi-
0305-4179/00/$20.00+0.00 Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 9 ) 0 0 1 2 9 - 1
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dermal water loss [5], skin colourimetry [6] and electrical capacitance [7]. The purpose of this current investigation was to determine whether transepidermal water loss (TEWL) and skin re¯ectance spectrophotometry (SRS) could be used as an adjunct or alternative to LDI for quanti®cation of severe skin lesions resulting from exposure of pig skin to L and SM vapours under occluded conditions in vivo. The potential bene®ts would be that a limited number of animals could be used in future studies to evaluate treatment strategies against vesicating chemical warfare agents and that measurements could be taken without the additional trauma of general anaesthesia. 2. Materials and methods 2.1. Chemicals Sulphur mustard and Lewisite were obtained from CB Systems Department, CBD, DERA Porton Down and were reported to be 98 and 96% pure by nuclear magnetic resonance (NMR) respectively. All other chemicals were obtained from Merck, Ltd., Poole. 2.2. Animals Eight female large white pigs (weight 22±28kg) were obtained from commercial sources by Animal Services, CBD, DERA Porton Down, and were housed in pairs and allowed access to food and water ad lib. 2.3. Biophysical measurements Transepidermal water loss (TEWL) was measured with a ServoMed EP-2 evaporimeter (ServoMed, Sweden). The instrument was kept running for the whole of the study and was set to zero at the start of each day. The evaporimeter probe was placed under a 1 L plastic beaker during measurements to prevent draught artifacts. Skin colour was measured using a Minolta CM-503i hand-held re¯ectance spectrophotometer (Minolta UK Ltd., England) reporting in CIELAB colour space mode (standard d/8 geometry) after white calibration before each set of measurements. 2.4. Vesicant exposure All procedures in this study were subject to scrutiny by the CBD, DERA Porton Down Animal Ethics Committee and had been licensed by the Home Oce Inspectorate. Following induction of general anaesthesia (1±3% Halothane in an oxygen/nitrous oxide mixture), an area (35 25 cm) of dorsal skin was wet shaved and dried in preparation for application of dos-
ing chambers as previously described [3]. Brie¯y, the animals were exposed over four 10 cm2 sites to vapour produced from a glass ®bre ®lter paper saturated with vesicant solution contained within the roof of a glass chamber that was bonded to the skin with Bostik impact adhesive. Each chamber was retained on the skin until the animal was humanely killed, or a maximum period of 6 h before being carefully removed. Animals were subsequently allowed to recover from anaesthesia and returned to their home pen. Skin lesions were not subsequently dressed and no signs of infection were observed. A total of four animals were exposed to Lewisite and four to sulphur mustard. The calculated dose of vapour absorbed at each site was 1.91 and 0.3 mg cmÿ2 for HD and L respectively, assuming 100% skin absorption over the exposure period. The quantities of agent used represent the minimum amount of vesicant required to cause a reproducible skin injury in this animal model, as previously described [3]. The resulting burns may be considered comparable in severity to human skin lesions following exposure to L or SM vapours under battle®eld conditions. Measurements of TEWL and skin colour were taken in triplicate at 1, 2, 4, 6, 8, 9, 12 and 24 h, and at 3 and 7 days. In addition, histopathology specimens were excised at the same time points in nonrecovery animals after intravenous administration of 15 ml (200 mg mlÿ1) sodium pentobarbitone (Euthatal). Techniques for tissue processing and histopathological assessment have been described elsewhere [3,8]. 3. Results 3.1. Histopathology 3.1.1. Lewisite After 1 h exposure, histology showed focal basal cell vacuolation associated with early acute in¯ammatory cell in®ltration. By 4 h, there were focal areas of epidermal degeneration involving the super®cial portions of adnexal structures. In addition, the upper papillary dermis showed mild in¯ammatory in®ltration with focal thrombosis of the subepidermal capillary network. After 6 h exposure, there was focal dermo-epidermal separation with extensive dermal oedema and evidence of an acute in¯ammation with complete thrombosis of the super®cial capillary network. Seven days postexposure, the lesions were covered by a well developed eschar composed of necrotic debris and coagulated ®brin. At the lesion edge, the eschar was undermined by tongues of regenerative epidermis showing features of recent proliferation including acanthosis, loss of rete ridge pattern, hyperkeratosis and parakeratosis.
R.P. Chilcott et al. / Burns 26 (2000) 245±250
247
3.1.2. Sulphur mustard After 4 h, there were no signi®cant changes in the histopathology of exposed regions. After 6 h exposure, the epidermis was still intact, with early focal perinuclear vacuolation in the basal layers. Within the upper papillary dermis there was marked dilatation and congestion of the subepidermal capillaries with slight perivascular oedema and leucocyte diapedsis. After one day, there was complete structural necrosis of the surface epithelium involving the majority of the super®cial adnexal structures. Within the dermis, there was necrosis of the papillary dermal capillaries with extensive dermal oedema and focal haemorrhage. At 7 days post exposure, histological analysis revealed eschar formation. 3.2. Gross clinical manifestations Mild erythema was apparent on both L and SM exposed sites after 1 h. This became more marked over the next 12 h in L-exposed skin, but did not appreciably alter at SM-exposed sites. Vesicles (>0.5 cm diameter) were discernable on L-exposed skin within 6±8 h that coalesced into larger (>1 cm diameter) blisters after 24 h. In contrast, vesicles were not observed on SM-exposed skin until 24±48 h. Palpable induration was noted on both L and SM sites between 12 and 24 h and 7 days post exposure, respectively. 3.3. Skin colour 3.3.1. Brightness (L) Skin brightness transiently decreased at SM exposed skin sites but returned to the same value as controls after 8 h (2 h after removal of exposure chambers). There was no signi®cant dierence in skin brightness between SM exposed and unexposed skin until 3±7 days post exposure (Fig. 1); this period coincided with eschar formation at SM exposed sites. Brightness of skin at L exposed sites gradually decreased throughout the study period, attaining a minimum value at 24 h (beginning of eschar formation) with no further signi®cant decrease (Fig. 1) thereafter. 3.3.2. Redness (a) After 1 h, both sulphur mustard (SM) and Lewisite (L) exposed sites were signi®cantly redder than controls (Fig. 2). Progressive reddening of exposed skin continued for 4 h (SM) or 8 h (L), with L exposed skin attaining a signi®cantly higher a value, indicative of a more severe erythematous response. After 8 h, the erythema (redness) of SM exposed skin was signi®cantly less than at 4 h but was still signi®cantly higher than control. In comparison, the redness of L exposed sites did not signi®cantly alter for up to 24 h. Control
Fig. 1. Skin brightness (expressed as the CIELAB colour scale parameter, L) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) exposed animals. All values expressed as mean 2 standard deviation.
(unexposed) skin did not signi®cantly change, except at 24 h when a small, transient increase was measured. Seven days post exposure, both L and SM exposed skin areas remained signi®cantly redder than controls but were less red than at 1 h post exposure. 3.3.3. Blueness (b) There were no remarkable changes in the b parameter measured for the duration of the study, although exposed sites had consistently higher b
Fig. 2. Skin erythema (expressed as the CIELAB colour scale parameter, a) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) animals. All values expressed as mean2standard deviation.
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Fig. 3. Skin blueness (expressed as the CIELAB colour scale parameter, b) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) exposed animals. All values expressed as mean2standard deviation.
values than control sites, indicative of slight cyanosis (Fig. 3). 3.4. Transepidermal water loss During the 6 h exposure period, there was a gradual increase in transepidermal water loss (TEWL, Fig. 4) at vesicant exposed sites that reached a maximum at 4±6 h ( p < 0.05). After the exposure chambers were removed, TEWL decreased and did not signi®cantly
dier from control until 24 or 72 h (L and SM, respectively). A second large, signi®cant increase in TEWL was measured after 24 h at L exposed skin sites. This correlated with the histopathological features of a complete loss of stratum corneum from the aected areas. In contrast, TEWL measured at SM exposed sites did not dier from controls until 72 h and reached a maximum value signi®cantly lower than that of L exposed skin. After 1 week, TEWL had substantially decreased at L exposed skin, but was still signi®cantly higher than control. In comparison, TEWL increased between 72 h and 1 week at SM exposed sites.
4. Discussion 4.1. Colour measurements
Fig. 4. Transepidermal water loss (TEWL) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) exposed animals. All values are mean2standard deviation.
Instruments used to measure skin colour can provide quantitative measurements of erythema [9], blanching [10] and pigmentation [11], and are arguably superior to subjective, visual scoring indices. Skin colour is commonly measured using a tristimulus device that measures absorption at ®xed wavelengths under standard illuminating conditions. Tristimulus colourimeters are relatively inexpensive and ideal for measuring colour dierences. In contrast, hand-held re¯ectance spectrophotometers analyse a wide band of frequencies [12] (typically between 400 and 700 nm) and can be used to measure changes in individual skin chromophores [13]
R.P. Chilcott et al. / Burns 26 (2000) 245±250
such as melanin and haemoglobin in addition to providing the same colorimetric data as a tristimulus instrument. In this present study, use of re¯ectance spectroscopy was limited to measuring skin colour changes using the CIELAB colour space model [14]. Basically, this comprises of the three components of brightness (L), redness (a) and blueness (b) and were used to quantitate eschar formation, erythema and cyanosis, respectively. Measurement of redness (a) indicated that skin lesions resulting from exposure to Lewisite (L) were quantitatively more erythematous than those of sulphur mustard (SM). This coincided with histopathological analysis that demonstrated more severe cellular damage in L exposed animals and also corresponded with increased blood ¯ow in the area as measured previously by laser Doppler imaging [3]. A transient, but signi®cant increase in redness was measured 24 h post exposure at control (unexposed) skin sites and was more profound in L exposed animals. This corresponded with the animals scratching their backs within the pens and was fully resolved within 3 days. Changes in brightness (L) could be associated with the process of scab formation (eschari®cation) and mirrored the known temporal dierence in the progression of L and SM lesions [1]: eschar formation on L exposed sites plateau after 24 h, whereas SM exposed skin do not show eschari®cation until 72 h. The blueness (b) of skin did not appear to correlate with any main histopathological features. It is conceivable that the small elevation in the b parameter may have been due to a marginal increase in the back-scattering of red and green light wavelengths due to extravasation, a consequence of the in¯ammatory response. Back-scattering would also decrease the L and a parameters, but the magnitude of this eect would be small in relation to the large changes in L and a measured. 4.2. Transepidermal water loss Measurement of TEWL is generally conducted with an evaporimeter, an instrument that measures the water vapour gradient above the skin surface [15]. The value of TEWL is though to be a re¯ection of the structural integrity [16,17] or the hydration status [18] of the stratum corneum. At 24 h, transepidermal water loss (TEWL) increased dramatically for L, but remained at baseline levels for SM exposed pigs. Histology of L skin lesions at this time showed a complete loss of stratum corneum. The fact that the stratum corneum remained in situ over SM exposed skin again illustrates the temporal dierence in lesion development between the two agents: Maximal TEWL rates of skin exposed to SM
249
occurred at 1 week compared to 24 h for L exposed skin. It should be noted that the steady decrease in TEWL rates measured over L-exposed skin after 24 h may not be completely attributable to epidermal regeneration (and thus restoration of barrier function). Although some repair processes were evident after 3 days, it is likely that the sudden loss of stratum corneum at 24 h contributed to the rapid dehydration of the epidermis and consequently a decrease in TEWL (which could be misinterpreted as barrier repair). The more gradual degeneration of the epidermis at SM exposed sites may have allowed dehydration to lower TEWL rates before complete loss of the stratum corneum was evident. Thus, the fact that L produced higher maximum TEWL rates than SM does not necessarily imply that the L skin lesions were more severe, even though this would correspond with the erythema (a) measurements. Furthermore, when there is near or total loss of stratum corneum barrier function, TEWL rates will attain a maximum value that can not be surpassed irrespective of the burn depth. Thus, unless there is relatively limited damage, measurement of TEWL cannot determine the severity of the lesion but can be used to follow the gross temporal progression of skin injury. 4.3. Conclusions Quanti®cation of the erythema (a) expressed after exposure to L or SM vapours appeared to correspond to histopathological changes during early lesion development. Skin brightness (L) correlated well with the subsequent formation of a scab whereas blueness (b) did not appreciably alter but may have been indicative of extravasation. Rates of TEWL changed both with occlusion (during vapour exposure) and also mirrored the progression of macroscopic skin injury after 12 h. Whilst no single parameter could be used in isolation to ascertain the severity and subsequent progression of the skin lesions, measurement of erythema, skin brightness and TEWL could be useful, non-invasive techniques for determining the ecacy of antidotes or therapies designed to prevent or ameliorate the toxic eects of the agents. However, neither colourimetry or TEWL could provide a clinical evaluation of such lesions that are comparable with the prognostic ability of laser Doppler imaging. Acknowledgements The authors would like to thank Mr. C.E. Kenward, Mrs. J. Platt, Mrs. L. Ba-Tin and Mr. M. Hand of the Biomedical Sciences Department, CBD Sector Porton Down for their invaluable help and technical assistance
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in this study and, express their gratitude to the Defence Evaluation and Research Agency (DERA) for ®nancial assistance.
References [1] Mellor SG, Rice P, Cooper GJ. Vesicant Burns. Br J Plastic Surg 1991;44:434±7. [2] Rice P. Mustard Gas Injuries: Skin. In: Cooper GJ, Dudley DS, Gann DS, Little RA, Maynard RL, editors. Scienti®c Foundations of Trauma. Oxford: Reed Educational and Professional Publishing LTD, 1997. p. 427±47 (Section 6). [3] Brown RFR, Rice P, Bennet NJ. The use of laser Doppler imaging as an aid in clinical management decision making in the treatment of vesicant burns. Burns 1998;24:692±8. [4] Goldman L, Cullen GE. The vesicant chemical warfare agents. Arch Dermatol Syphilol 1944;42:123±36. [5] Freeman S, Maibach HI. Study of irritant contact dermatitis produced by repeat patch test with sodium lauryl sulfate and assessed by visual methods, transepidermal water loss and laser Doppler velocimetry. Irritant Contact Dermatitis 1988;19:496± 502. [6] Grunewald AM, Gloor M, Gehring W, Kleesz P. Damage to the skin by repetitive washing. Contact Dermatitis 1995;32:225± 32. [7] Gon V, PieÂrard GE, Henry F, Letawe C, Maibach HI. Sodium hypochlorite, bleaching agents and the stratum corneum. Ecotoxicol Environ Safety 1997;37:199±202. [8] Brown RFR, Rice P. Histopathological changes in Yucatan minipig skin following challenge with sulphur mustard a sequen-
[9]
[10]
[11] [12] [13] [14]
[15] [16] [17] [18]
tial study of the ®rst 24 h following challenge. Int J Path 1997;78:9±20. Lahti A, Kopola H, Harila A, MyllylaÈ R, Hannuksela M. Assessment of skin erythema by eye laser Doppler ¯owmetry, spectroradiometer, two-channel erythema meter and Minolta chroma-meter. Arch Dermatol Res 1993;285:278±82. Queille-Roussel C, Poncet M, Schaefer H. Quanti®cation of skin-colour changes induced by topical corticosteroid preparations using the Minolta chroma meter. Br J Dermatol 1991;124:264±70. Andersen PH, Bjerring P. Spectral re¯ectance of human skin. Photodermatol Photoimmunol Photomed 1990;7:5±12. Bjerring P, Andersen PH. Skin re¯ectance spectrophotometry. Photodermatology 1985;4:167±71. Andersen PH, Bjerring P. Noninvasive computerised analysis of skin chromophores in vivo by re¯ectance spectroscopy. Photodermatol Photoimmunol Photomed 1990;7:249±57. Serup J, Agner T. Colorimetric quanti®cation of erythema Ð a comparison of two colorimeters with a clinical scoring scheme and laser Doppler ¯owmetry. Clin Exp Dermatol 1990;15:267± 72. Lamke L-O, Nilsson GE, Reithner HL. Insensible perspiration from the skin under standardised environmental conditions. Scand J Clin Lab Invest 1977;37:325±31. Kalia YN, Pirot F, Guy RH. Homogenous transport in a heterogeneous membrane: water diusion across human stratum corneum in vivo. Biophys J 1996;71:2692±700. Jelenko C, Wheeler ML, Anderson AP, Callaway D, Scott RA. Studies in burns. XIII. Eects of a topical lipid on burned subjects and their wounds. Am Surg 1975;41:466±82. Maw-Sheng W. Water diusivity and water concentration pro®le in human stratum corneum from transepidermal water loss measurements. J Soc Cosmet Chem 1983;34:191±6.
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Non-invasive quanti®cation of skin injury resulting from exposure to sulphur mustard and Lewisite vapours R.P. Chilcott*, R.F.R. Brown, P. Rice Biomedical Sciences Department, CBD Sector, Defence Evaluation and Research Agency, Porton Down, Salisbury SP4 0JQ, UK. Accepted 13 August 1999
Abstract The severity and progression of skin lesions resulting from exposure to the chemical warfare agents Lewisite (L) and sulphur mustard (SM) have been investigated using the non-invasive biophysical methods of evaporimetry and re¯ectance spectroscopy in large white pigs in vivo. Erythema (redness) expressed immediately after exposure to L or SM vapours appeared to be related to the lesion severity as demonstrated by histopathological analysis. Skin brightness correlated well with scab formation whereas blueness (cyanosis) did not appreciably alter throughout the study. Rates of transepidermal water loss (TEWL) changed both with occlusion (during vapour exposure) and also mirrored the progression of macroscopic skin injury after 12 h. Whilst no single parameter could be used in isolation to ascertain the severity and subsequent progression of the skin lesions, measurement of erythema, skin brightness and TEWL could provide quantitative, non-invasive methods for determining the ecacy of antidotes or therapies to prevent the toxic eects of chemical warfare agents. However, neither colourimetry or TEWL provided a clinical evaluation of such lesions that were comparable with the prognostic capabilities of laser Doppler imaging. Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. Keywords: Burn; Transepidermal water loss (TEWL); Skin colour; Pig; Skin; In vivo; Sulphur mustard; Lewisite
1. Introduction Vesicating warfare agents are chemicals that can produce deep skin burns [1] and include the classic agents Lewisite (L) and sulphur mustard (SM). Skin lesions resulting from exposure to SM dier from thermal burns in that signs and symptoms may be delayed for up to 24 h after exposure depending on dose, damage tends to be partial thickness and spontaneous healing rates are signi®cantly slower [2]. In contrast the development, depth and healing rates of L skin injuries resemble those of thermal burns. Skin lesions resulting from exposure to L or SM vapour may progress to form large bullae that may require intensive medical management and surgical intervention. Previous studies have reported a laser Doppler imaging * Corresponding author.
(LDI) technique for the clinical evaluation of the severity of L and SM skin injuries [3]. Whilst being an invaluable tool for determining vesicant burn depth and subsequent prognosis, the LDI system is currently a bulky instrument which is unsuitable for battle-®eld use. Furthermore, LDI evaluation of burns in experimental models requires the animals to be anaesthetised. This places a restriction on the number of times laser Doppler imaging can be performed. This constraint is particularly relevant when assessing pretreatments or therapies, as skin lesions resulting from exposure to L or SM may take up to 3 months to resolve [4], the duration of which an animal may have to be anaesthetised many times. A variety of alternative techniques are commercially available for the rapid measurement of skin damage that are non-invasive and relatively inexpensive. For example, experimentally induced skin lesions have previously been quanti®ed by measurement of transepi-
0305-4179/00/$20.00+0.00 Crown Copyright # 2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 5 - 4 1 7 9 ( 9 9 ) 0 0 1 2 9 - 1
246
R.P. Chilcott et al. / Burns 26 (2000) 245±250
dermal water loss [5], skin colourimetry [6] and electrical capacitance [7]. The purpose of this current investigation was to determine whether transepidermal water loss (TEWL) and skin re¯ectance spectrophotometry (SRS) could be used as an adjunct or alternative to LDI for quanti®cation of severe skin lesions resulting from exposure of pig skin to L and SM vapours under occluded conditions in vivo. The potential bene®ts would be that a limited number of animals could be used in future studies to evaluate treatment strategies against vesicating chemical warfare agents and that measurements could be taken without the additional trauma of general anaesthesia. 2. Materials and methods 2.1. Chemicals Sulphur mustard and Lewisite were obtained from CB Systems Department, CBD, DERA Porton Down and were reported to be 98 and 96% pure by nuclear magnetic resonance (NMR) respectively. All other chemicals were obtained from Merck, Ltd., Poole. 2.2. Animals Eight female large white pigs (weight 22±28kg) were obtained from commercial sources by Animal Services, CBD, DERA Porton Down, and were housed in pairs and allowed access to food and water ad lib. 2.3. Biophysical measurements Transepidermal water loss (TEWL) was measured with a ServoMed EP-2 evaporimeter (ServoMed, Sweden). The instrument was kept running for the whole of the study and was set to zero at the start of each day. The evaporimeter probe was placed under a 1 L plastic beaker during measurements to prevent draught artifacts. Skin colour was measured using a Minolta CM-503i hand-held re¯ectance spectrophotometer (Minolta UK Ltd., England) reporting in CIELAB colour space mode (standard d/8 geometry) after white calibration before each set of measurements. 2.4. Vesicant exposure All procedures in this study were subject to scrutiny by the CBD, DERA Porton Down Animal Ethics Committee and had been licensed by the Home Oce Inspectorate. Following induction of general anaesthesia (1±3% Halothane in an oxygen/nitrous oxide mixture), an area (35 25 cm) of dorsal skin was wet shaved and dried in preparation for application of dos-
ing chambers as previously described [3]. Brie¯y, the animals were exposed over four 10 cm2 sites to vapour produced from a glass ®bre ®lter paper saturated with vesicant solution contained within the roof of a glass chamber that was bonded to the skin with Bostik impact adhesive. Each chamber was retained on the skin until the animal was humanely killed, or a maximum period of 6 h before being carefully removed. Animals were subsequently allowed to recover from anaesthesia and returned to their home pen. Skin lesions were not subsequently dressed and no signs of infection were observed. A total of four animals were exposed to Lewisite and four to sulphur mustard. The calculated dose of vapour absorbed at each site was 1.91 and 0.3 mg cmÿ2 for HD and L respectively, assuming 100% skin absorption over the exposure period. The quantities of agent used represent the minimum amount of vesicant required to cause a reproducible skin injury in this animal model, as previously described [3]. The resulting burns may be considered comparable in severity to human skin lesions following exposure to L or SM vapours under battle®eld conditions. Measurements of TEWL and skin colour were taken in triplicate at 1, 2, 4, 6, 8, 9, 12 and 24 h, and at 3 and 7 days. In addition, histopathology specimens were excised at the same time points in nonrecovery animals after intravenous administration of 15 ml (200 mg mlÿ1) sodium pentobarbitone (Euthatal). Techniques for tissue processing and histopathological assessment have been described elsewhere [3,8]. 3. Results 3.1. Histopathology 3.1.1. Lewisite After 1 h exposure, histology showed focal basal cell vacuolation associated with early acute in¯ammatory cell in®ltration. By 4 h, there were focal areas of epidermal degeneration involving the super®cial portions of adnexal structures. In addition, the upper papillary dermis showed mild in¯ammatory in®ltration with focal thrombosis of the subepidermal capillary network. After 6 h exposure, there was focal dermo-epidermal separation with extensive dermal oedema and evidence of an acute in¯ammation with complete thrombosis of the super®cial capillary network. Seven days postexposure, the lesions were covered by a well developed eschar composed of necrotic debris and coagulated ®brin. At the lesion edge, the eschar was undermined by tongues of regenerative epidermis showing features of recent proliferation including acanthosis, loss of rete ridge pattern, hyperkeratosis and parakeratosis.
R.P. Chilcott et al. / Burns 26 (2000) 245±250
247
3.1.2. Sulphur mustard After 4 h, there were no signi®cant changes in the histopathology of exposed regions. After 6 h exposure, the epidermis was still intact, with early focal perinuclear vacuolation in the basal layers. Within the upper papillary dermis there was marked dilatation and congestion of the subepidermal capillaries with slight perivascular oedema and leucocyte diapedsis. After one day, there was complete structural necrosis of the surface epithelium involving the majority of the super®cial adnexal structures. Within the dermis, there was necrosis of the papillary dermal capillaries with extensive dermal oedema and focal haemorrhage. At 7 days post exposure, histological analysis revealed eschar formation. 3.2. Gross clinical manifestations Mild erythema was apparent on both L and SM exposed sites after 1 h. This became more marked over the next 12 h in L-exposed skin, but did not appreciably alter at SM-exposed sites. Vesicles (>0.5 cm diameter) were discernable on L-exposed skin within 6±8 h that coalesced into larger (>1 cm diameter) blisters after 24 h. In contrast, vesicles were not observed on SM-exposed skin until 24±48 h. Palpable induration was noted on both L and SM sites between 12 and 24 h and 7 days post exposure, respectively. 3.3. Skin colour 3.3.1. Brightness (L) Skin brightness transiently decreased at SM exposed skin sites but returned to the same value as controls after 8 h (2 h after removal of exposure chambers). There was no signi®cant dierence in skin brightness between SM exposed and unexposed skin until 3±7 days post exposure (Fig. 1); this period coincided with eschar formation at SM exposed sites. Brightness of skin at L exposed sites gradually decreased throughout the study period, attaining a minimum value at 24 h (beginning of eschar formation) with no further signi®cant decrease (Fig. 1) thereafter. 3.3.2. Redness (a) After 1 h, both sulphur mustard (SM) and Lewisite (L) exposed sites were signi®cantly redder than controls (Fig. 2). Progressive reddening of exposed skin continued for 4 h (SM) or 8 h (L), with L exposed skin attaining a signi®cantly higher a value, indicative of a more severe erythematous response. After 8 h, the erythema (redness) of SM exposed skin was signi®cantly less than at 4 h but was still signi®cantly higher than control. In comparison, the redness of L exposed sites did not signi®cantly alter for up to 24 h. Control
Fig. 1. Skin brightness (expressed as the CIELAB colour scale parameter, L) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) exposed animals. All values expressed as mean 2 standard deviation.
(unexposed) skin did not signi®cantly change, except at 24 h when a small, transient increase was measured. Seven days post exposure, both L and SM exposed skin areas remained signi®cantly redder than controls but were less red than at 1 h post exposure. 3.3.3. Blueness (b) There were no remarkable changes in the b parameter measured for the duration of the study, although exposed sites had consistently higher b
Fig. 2. Skin erythema (expressed as the CIELAB colour scale parameter, a) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) animals. All values expressed as mean2standard deviation.
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R.P. Chilcott et al. / Burns 26 (2000) 245±250
Fig. 3. Skin blueness (expressed as the CIELAB colour scale parameter, b) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) exposed animals. All values expressed as mean2standard deviation.
values than control sites, indicative of slight cyanosis (Fig. 3). 3.4. Transepidermal water loss During the 6 h exposure period, there was a gradual increase in transepidermal water loss (TEWL, Fig. 4) at vesicant exposed sites that reached a maximum at 4±6 h ( p < 0.05). After the exposure chambers were removed, TEWL decreased and did not signi®cantly
dier from control until 24 or 72 h (L and SM, respectively). A second large, signi®cant increase in TEWL was measured after 24 h at L exposed skin sites. This correlated with the histopathological features of a complete loss of stratum corneum from the aected areas. In contrast, TEWL measured at SM exposed sites did not dier from controls until 72 h and reached a maximum value signi®cantly lower than that of L exposed skin. After 1 week, TEWL had substantially decreased at L exposed skin, but was still signi®cantly higher than control. In comparison, TEWL increased between 72 h and 1 week at SM exposed sites.
4. Discussion 4.1. Colour measurements
Fig. 4. Transepidermal water loss (TEWL) following exposure to saturated sulphur mustard (*) and Lewisite vapours (R) in comparison to control areas of SM (w) or L (r) exposed animals. All values are mean2standard deviation.
Instruments used to measure skin colour can provide quantitative measurements of erythema [9], blanching [10] and pigmentation [11], and are arguably superior to subjective, visual scoring indices. Skin colour is commonly measured using a tristimulus device that measures absorption at ®xed wavelengths under standard illuminating conditions. Tristimulus colourimeters are relatively inexpensive and ideal for measuring colour dierences. In contrast, hand-held re¯ectance spectrophotometers analyse a wide band of frequencies [12] (typically between 400 and 700 nm) and can be used to measure changes in individual skin chromophores [13]
R.P. Chilcott et al. / Burns 26 (2000) 245±250
such as melanin and haemoglobin in addition to providing the same colorimetric data as a tristimulus instrument. In this present study, use of re¯ectance spectroscopy was limited to measuring skin colour changes using the CIELAB colour space model [14]. Basically, this comprises of the three components of brightness (L), redness (a) and blueness (b) and were used to quantitate eschar formation, erythema and cyanosis, respectively. Measurement of redness (a) indicated that skin lesions resulting from exposure to Lewisite (L) were quantitatively more erythematous than those of sulphur mustard (SM). This coincided with histopathological analysis that demonstrated more severe cellular damage in L exposed animals and also corresponded with increased blood ¯ow in the area as measured previously by laser Doppler imaging [3]. A transient, but signi®cant increase in redness was measured 24 h post exposure at control (unexposed) skin sites and was more profound in L exposed animals. This corresponded with the animals scratching their backs within the pens and was fully resolved within 3 days. Changes in brightness (L) could be associated with the process of scab formation (eschari®cation) and mirrored the known temporal dierence in the progression of L and SM lesions [1]: eschar formation on L exposed sites plateau after 24 h, whereas SM exposed skin do not show eschari®cation until 72 h. The blueness (b) of skin did not appear to correlate with any main histopathological features. It is conceivable that the small elevation in the b parameter may have been due to a marginal increase in the back-scattering of red and green light wavelengths due to extravasation, a consequence of the in¯ammatory response. Back-scattering would also decrease the L and a parameters, but the magnitude of this eect would be small in relation to the large changes in L and a measured. 4.2. Transepidermal water loss Measurement of TEWL is generally conducted with an evaporimeter, an instrument that measures the water vapour gradient above the skin surface [15]. The value of TEWL is though to be a re¯ection of the structural integrity [16,17] or the hydration status [18] of the stratum corneum. At 24 h, transepidermal water loss (TEWL) increased dramatically for L, but remained at baseline levels for SM exposed pigs. Histology of L skin lesions at this time showed a complete loss of stratum corneum. The fact that the stratum corneum remained in situ over SM exposed skin again illustrates the temporal dierence in lesion development between the two agents: Maximal TEWL rates of skin exposed to SM
249
occurred at 1 week compared to 24 h for L exposed skin. It should be noted that the steady decrease in TEWL rates measured over L-exposed skin after 24 h may not be completely attributable to epidermal regeneration (and thus restoration of barrier function). Although some repair processes were evident after 3 days, it is likely that the sudden loss of stratum corneum at 24 h contributed to the rapid dehydration of the epidermis and consequently a decrease in TEWL (which could be misinterpreted as barrier repair). The more gradual degeneration of the epidermis at SM exposed sites may have allowed dehydration to lower TEWL rates before complete loss of the stratum corneum was evident. Thus, the fact that L produced higher maximum TEWL rates than SM does not necessarily imply that the L skin lesions were more severe, even though this would correspond with the erythema (a) measurements. Furthermore, when there is near or total loss of stratum corneum barrier function, TEWL rates will attain a maximum value that can not be surpassed irrespective of the burn depth. Thus, unless there is relatively limited damage, measurement of TEWL cannot determine the severity of the lesion but can be used to follow the gross temporal progression of skin injury. 4.3. Conclusions Quanti®cation of the erythema (a) expressed after exposure to L or SM vapours appeared to correspond to histopathological changes during early lesion development. Skin brightness (L) correlated well with the subsequent formation of a scab whereas blueness (b) did not appreciably alter but may have been indicative of extravasation. Rates of TEWL changed both with occlusion (during vapour exposure) and also mirrored the progression of macroscopic skin injury after 12 h. Whilst no single parameter could be used in isolation to ascertain the severity and subsequent progression of the skin lesions, measurement of erythema, skin brightness and TEWL could be useful, non-invasive techniques for determining the ecacy of antidotes or therapies designed to prevent or ameliorate the toxic eects of the agents. However, neither colourimetry or TEWL could provide a clinical evaluation of such lesions that are comparable with the prognostic ability of laser Doppler imaging. Acknowledgements The authors would like to thank Mr. C.E. Kenward, Mrs. J. Platt, Mrs. L. Ba-Tin and Mr. M. Hand of the Biomedical Sciences Department, CBD Sector Porton Down for their invaluable help and technical assistance
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in this study and, express their gratitude to the Defence Evaluation and Research Agency (DERA) for ®nancial assistance.
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