John S. Graham

Histopathologic features seen with different animal models following cutaneous sulfur mustard exposure

In an effort to understand the pathophysiology of sulfur mustard (2,2 dichlorodiethyl sulfide, HD)-induced cutaneous lesions, a number of animal models have been used. Animal models have been and will continue to be used in the development of therapeutic strategies to protect against and/or moderate lesions, and to potentiate wound healing after HD exposure. Upon reviewing the histopathologic features seen after HD-exposure, we propose roles for different animal models in HD-research. Hematoxylin and eosin slides from protocols done originally as dose response studies for either liquid or vapor HD-exposures were examined. The animal models reported include the hairless guinea pig (HGP), weanling pig (WP), mouse ear (ME) and hairless mouse (HM). In all these animal models. HD induces subepidermal blister formation as well as epidermal cell death. The HGP appears to be the most sensitive model for epidermal necrosis. The HGP and, to a lesser degree, the HM react with a marked neutrophilic infiltrate. The ME provides a quantitative measure for HD effects and a mild inflammatory infiltrate similar to what is seen in human skin. Doses necessary to produce microblister formation in the WP are usually associated with more significant stromal and vascular changes than in other animal models. In addition to a quantitative measure of the HD effect and a mild inflammatory response, the cost, as well as the availability of specific antibodies, and DNA and RNA probes and primers gives the ME advantages for both drug screening and for the study of the pathophysiology of HD-induced cutaneous lesions. The sensitivity of the HGP and the abundant experience with vapor exposures establishes a place for this animal model in barrier cream and drug screening. The similarity of WP skin to human skin is important in the study of wound healing after HD exposure, as well as in the study of the pathophysiology of the cutaneous lesion and in more definitive therapeutic studies.

Efficacy of laser debridement with autologous split-thickness skin grafting in promoting improved healing of deep cutaneous sulfur mustard burns

The consequences of receiving a cutaneous sulfur mustard (SM) burn are prolonged wound healing and secondary infection. This study was undertaken to find a treatment that promotes quick healing with few complications and minimal disfigurement. Multiple deep SM burns (4 cm diameter) were generated on the ventrum of weanling pigs and treated at 48 h. Four treatments were compared: (1) full-thickness CO(2) laser debridement followed by skin grafting; (2) full-thickness sharp surgical tangential excision followed by skin grafting, the Gold Standard used in deep thermal burns management; (3) partial-thickness laser ablation with no grafting; and (4) partial-thickness sharp excision with no grafting. A computer controlled, raster scanned, high-powered continuous wave (cw) CO(2) laser was utilized. Ulceration, wound geometry, and wound contraction were evaluated during a 36-day healing period. Histopathological evaluations were conducted at the end of the healing period. Engraftment rates were similar between both methods of debridement. Laser debridement followed by skin grafting was as efficacious in improving the wound healing of deep SM burns as the Gold Standard. Full-thickness laser debridement of these small total body surface area (TBSA) burns was time efficient and provided adequate beds for split-thickness skin grafting. Laser debridement offered additional benefits that included hemostatic control during surgery and minimal debridement of normal perilesional skin. Mid-dermal debridement by sharp excision or laser ablation without grafting produced less desirable results but was better than no treatment.

Immunohistochemical studies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs

Sulfur mustard (2,2-dichlorodiethyl sulfide, HD) is a chemical warfare agent that is a threat to both troops and civilians. The focus of HD research has been on intracellular adduct formation leading to apoptosis and/or necrosis in cutaneous lesions. However, there is work which suggests that HD may have a more direct effect on the basement membrane zone. Immunohistochemical staining to desmosomal proteins, cellular fibronectin, laminin 1, laminin 5, collagen IV, collagen VII, p53, Bcl-2, and PCNA was performed on weanling pig skin exposed to vesicating doses of HD, GB3, an antibody to laminin 5, showed a progressive decrease with loss of expression during the time period of clinical vesiculation. The other basement membrane proteins showed no change or inconsistent changes. PCNA, and p53 staining increased in the overlying epidermis in areas of vesiculation without significant necrosis. Bcl-2 positive cells were decreased or absent after exposure. This study implicates laminin 5 as the main basement membrane protein affected acutely by HD exposure. The patterns of staining of PCNA, Bcl-2, and p53 within the epidermis suggest that apoptosis and cellular necrosis both may play a role in cell death secondary to HD.

A cutaneous full-thickness liquid sulfur mustard burn model in weanling swine: clinical pathology and urinary excretion of thiodiglycol.

Sulfur mustard (bis(2‐chloroethyl)sulfide, HD) is a well‐known blistering chemical warfare agent. We have developed a cutaneous full‐thickness HD burn model in weanling pigs for efficacy testing of candidate treatment regimens. This report addresses clinical pathology findings and the urinary excretion profile of a major HD metabolite (thiodiglycol, TDG) in this model. Six female Yorkshire pigs were exposed to HD liquid on the ventral surface for 2 h, generating six 3‐cm diameter full‐thickness dermal lesions per pig. Blood samples were collected throughout a 7‐day observation period for hematology and serum chemistry examinations. Urine was collected in metabolism cages. Routine urinalysis was performed and the urine analyzed for TDG using gas chromatography/mass spectrometry. Examination of clinical pathology parameters revealed subtle HD‐related changes that are suggestive of a mild hemolytic episode. No other signs of clinically significant systemic toxicities were noted, including bone marrow suppression. Thiodiglycol was detected at the earliest time point tested (6–8 h post‐exposure) at levels ranging from 0.66 to 4.98 μg ml−1 with a mean of 2.14 μg ml 2−1. Thiodiglycol concentrations were the highest for half of the animals at this earliest time point and at 24–48 h for the others. By the evening of day 3, the mean level had reached 50 ng ml−1. Mean levels remained 10–40 ng ml−1 for the remainder of the 7‐day observation period, with the highest individual concentration noted during this period of 132 ng ml−1. Our results are in general agreement with the TDG excretion profiles previously described for rodent models and humans. Urinary excretion of absorbed HD in our weanling pig wound healing model appears to follow the same pattern as is seen in other laboratory animals models. In general, urinary excretion of TDG appears to peak within the first 1–4 days following exposure, with detectable levels after 1 week. Relatively high urinary TDG levels may thus indicate agent exposure within the previous 96 h. Low levels significantly above natural background levels may indicate either exposure to low levels of agent or exposure that occurred more than 4 days prior to collection of the sample. Published in 2000 by John Wiley & Sons, Ltd.

Bioengineering methods employed in the study of wound healing of sulphur mustard burns

Background/purpose:u2002 Sulphur mustard (SM) is a potent incapacitating chemical warfare agent that remains a threat to war fighters and civilians worldwide. SM lesions may require weeks or months to heal, depending upon their severity. This study was undertaken to find a treatment regimen that promotes speedier healing of deep cutaneous SM burns in a weanling pig model. The principal objective of the study was to compare four treatment regimens and establish which achieved the shortest healing time.

Depth of morphologic skin damage and viability after one, two, and three passes of a high-energy, short-pulse CO 2 laser (Tru-Pulse) in pig skin

BACKGROUNDnCO2 laser energy is absorbed by water, which is present in all tissue. The depth of penetration of CO2 lasers is narrow with minimal reflection, scatter, or transmission. However, thermal damage has limited the usefulness of conventional, continuous-wave CO2 lasers for debridement as demonstrated by wound healing studies. The development of high-energy CO2 lasers, with pulse durations that are less than the thermal relaxation time of tissue, have made vaporization of skin for resurfacing and wound debridement possible because of the decreased risk of thermal damage.nnnOBJECTIVEnThis study was performed to evaluate thermal damage produced by a CO2 laser.nnnMETHODSnRoutine histopathologic examination and nitroblue-tetrazolium chloride (NBTC) staining were used to evaluate the depth of tissue damage and viability in weanling pig skin after one, two, and three passes of the laser.nnnRESULTSnAt a pulse energy of 300 mJ, with a pulse duration of 60 microseconds, one pass of the laser produced vaporization of the epidermis with minimal thermal damage. Two passes produced areas of denatured collagen with loss of viable cells in the superficial papillary dermis. Three passes extended the damage into the papillary dermis.nnnCONCLUSIONnHyalinization of collagen appears to correspond well with the level of thermal damage as measured by NBTC staining. Our findings suggest that the energy necessary to vaporize the dermis may be greater than that needed to vaporize epidermis.

Medical Management of Cutaneous Sulfur Mustard Injuries

BACKGROUNDnSulfur mustard (2,2-dichlorodiethyl sulfide; HD) is a potent vesicating chemical warfare agent that poses a continuing threat to both military and civilian populations. Significant cutaneous HD injuries can take several months to heal, necessitate lengthy hospitalizations, and result in long-term complications. There are currently no standardized or optimized methods of casualty management. New strategies are needed to provide for optimal and rapid wound healing.nnnOBJECTIVEnThe primary aim of this research was to develop improved clinical strategies (treatment guidelines) for optimal treatment of superficial dermal (second degree) cutaneous HD injuries, with the goal of returning damaged skin to optimal appearance and normal function in the shortest period of time.nnnMETHODSnSuperficial dermal HD injuries were created on the ventral abdominal surface of weanling pigs. At 48h post-exposure, lesions were laser debrided and a treatment adjunct applied. Cultured epithelial allografts and 11 commercial off-the-shelf (COTS) products were examined for their efficacy in improving wound healing of these injuries. Clinical evaluations and a variety of non-invasive bioengineering methods were used at 7 and 14 days post-surgery to follow the progress of wound healing and evaluate various cosmetic and functional properties of the wounds. Measurements included reflectance colorimetry to measure erythema; evaporimetry to examine transepidermal water loss as a method of evaluating barrier function; torsional ballistometry to evaluate the mechanical properties of skin firmness and elasticity; and two-dimensional high frequency ultrasonography (HFU) to monitor skin thickness (e.g., edema, scar tissue). Histopathology and immunohistochemistry were performed 14 days following surgery to examine structural integrity and quality of healing. Logical Decisions((R)) for Windows was used to rank the 12 treatment adjuncts that were studied.nnnRESULTSnThe most efficacious treatment adjuncts included (1) Vacuum Assisted Closure, V.A.C., involving application of topical negative pressure, (2) Amino-Plex Spray (biO(2) Cosmeceuticals International, Inc., Beverly Hills, CA), a nutritive cosmeceutical product that is designed to increase oxygen in cells, stimulate ATP synthesis, improve glucose transportation, stimulate collagen formation, and promote angiogenesis, and (3) ReCell Autologous Cell Harvesting Device (Clinical Cell Culture Americas LLC, Coral Springs, Florida), an innovative medical device that was developed to allow rapid harvesting of autologous cells from a thin split-thickness biopsy followed by spray application of a population of skin cells onto wounds within 30 min of collecting the biopsy, without the need of culturing the keratinocytes in a clinical laboratory.nnnCONCLUSIONSnComplete re-epithelialization of debrided HD injuries in 7 days is possible. In general, shallow laser debridement through the basement membrane zone (100 microm) appears to provide better results than deeper debridement (400 microm) with respect to early re-epithelialization, cosmetic appearance, functional restoration, and structural integrity. Of the 12 treatment adjuncts examined, the most promising included Vacuum Assisted Closure, Amino-Plex Spray, and ReCell Autologous Cell Harvesting Device.

Sulfur mustard-induced skin burns in weanling swine evaluated clinically and histopathologically

Histopathological indicators and clinical observations were used to evaluate wound severity, depth and degree of healing on days 2 and 8 for full‐skin‐thickness sulfur‐mustard (HD)‐induced burns in weanling swine. Six female weanling swine were exposed for 2 h to 400 μl of HD at each of six dose sites on the hairless abdominal skin. Biopsy samples (8 mm) were taken from the periphery and from the center of the wound on day 2, and the wound was excised on day 8. Histopathological indicators evaluated were epidermal necrosis, follicular necrosis, dermal necrosis, vascular necrosis, depth of injury, ulceration (loss of epidermis), granulation tissue response, neovascularization, re‐epithelialization (hyperplasia) and completeness of healing. Wounds were more severe from anterior to posterior. Histopathological assessment of epidermal ulceration and necrosis of epidermis, dermis, basal epithelium, adnexal structures and subcutaneous tissue were useful indicators of wound development on day 2. Granulation tissue response (observed as early as day 8) and re‐epithelialization were good indicators of wound healing. Clinical evaluations were performed on day 2 prior to and after debriding, and on study day 8. Clinical observations on study day 2 were for wound size and for exudation, erythema, edema, necrosis and eschar. Clinical observations on study day 8 were for the previous parameters and for re‐epithelialization, granulation and infection. Wound size and severity increased from anterior to posterior position. Size, exudation and edema were useful indicators of wound development. These histological and clinical observation parameters will be used in future experiments to compare various treatments for HD‐induced burns. Copyright

The skin reservoir of sulphur mustard.

Studies of the percutaneous reservoir of sulphur mustard (HD) formed during absorption carried out during WWI and WWII are inconclusive. More recent studies have indicated that a significant amount of unreacted HD remains in human epidermal membranes during percutaneous penetration studies in vitro. The present study investigated the nature and persistence of the HD reservoir formed during in vitro penetration studies using dermatomed slices of human and pig skin (0.5mm thick). Amounts of (14)C-HD that (a) penetrated, (b) remained on the surface, (c) were extractable from and (d) remained in the skin after extraction were estimated by liquid scintillation counting (confirmed using GC-MS analysis). The results demonstrated that there is a reservoir of HD in human and pig skin for up to 24 h after contamination of the skin surface in vitro with liquid agent. At least some of this reservoir could be extracted with acetonitrile, and the amounts of extracted and unextracted HD exceed the amount required to produce injury in vivo by at least 20 fold. The study demonstrated the presence of a reservoir whether the skin was covered (occluded) or left open to the air (unoccluded). The study concluded that the extractable reservoir was significant in terms of the amount of HD required to induce a vesicant response in human skin. The extractable reservoir was at least 20 times the amount required per cm(2) estimated to cause a response in all of the human population, as defined by studies carried out in human volunteers during the 1940s.

Increased Smooth Muscle Actin, Factor XHIa, and Vimentin-positive Cells in the Papillary Dennis of Carbon Dioxide Laser-debrided Porcine Skin

BACKGROUND. Pulsed carbon dioxide (CO2) laser debridement is now being used as therapy for photodamaged skin. It has been proposed that the long duration of erythema and a tissue scaffold, which results from tightening of the collagen helix induced by the laser heat, may lead to tightening of sagging skin and skin creases of lesser magnitude. METHODS. Weanling pigs exposed to mild and moderate erythema producing doses of sulfur mustard (bis‐2‐chloroethyl sulfide; HD) were treated with the CO2 laser (Tru‐Pulse) at 6, 24, and 48 hours after exposure. In addition to histologic examination of laser‐debrided and nondebrided biopsy specimens obtained at 14 days after exposure, immunohistochemical staining with antibodies to smooth muscle actin, Factor XIIIa, vimentin, and CD3 was performed. RESULTS. CO2 laser debridement of the HD‐exposed skin resulted in clearing of the cytologic atypia induced by this chemical carcinogen and reduced the inflammatory infiltrate. In addition laser debridement resulted in increased numbers of stromal cells within the papillary dermis, which showed immunohistochemical staining for smooth muscle actin, Factor Xllla, and vimen‐tin. CONCLUSIONS. CO2 laser debridement is effective in clearing the epidermis of cytologically damage cells in HD as well as solar‐damaged skin. In addition CO, laser debridemant may result in tightening of sagging skin and produce a decrease in skin creases initially, by inducing increased stromal cells within the papillary dermis, with prominent contractile actin filaments. The collagen produced by these stromal cells may subsequently maintain these improvements in the photoaged skin.