Apoptosis is differentially regulated by burn severity and dermal location.

BACKGROUND: The cellular processes that contribute to cell death in burns are poorly understood. This study evaluated the distribution and extent of apoptosis in an established rat model of acute dermal burn injury. MATERIALS AND METHODS: A branding iron (100 degrees C) was applied to the depilated dorsum of seven rats, creating burn contact times of 1-8, 10, 12, and 14 s. Biopsies were collected and immunohistochemistry performed for apoptosis and cell injury/necrosis by detection of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and high-mobility group box 1 (HMGB1), respectively. The slides were scored by evaluating staining in superficial, middle, and deep dermal fields. Within these, basal keratinocytes of the epidermis, mesenchymal cells, adnexal epithelia, and vasculature wall cells were morphometrically analyzed for stain detection of selected markers. RESULTS: TUNEL staining had an inverse relationship with contact time in most fields except in deep dermal mesenchymal cells where it was increased. HMGB1 nuclear staining was significantly decreased with progressive contact time consistent with transition to cell injury/necrosis. CONCLUSIONS: This study is the first to demonstrate that apoptosis rate is dependent on dermal location, cell type, and severity of thermal injury. Furthermore, this work suggests that for most dermal locations increased thermal injury corresponds with decreased apoptosis and increased cell injury/necrosis. Together, these findings indicate that many parameters can regulate apoptosis in burn wounds, and these results will be critical to understanding burn pathogenesis and assessing future therapies.

Pharmacologic modification to resuscitation fluid after thermal injury--is drotrecogin alfa the answer to arrest burn depth progression?

HYPOTHESIS: The addition of drotrecogin alfa (DA), an anti-inflammatory useful in septic shock, to standard burn shock resuscitation fluids will protect burned, injured skin from further injury. METHODS: Anesthetized animals were subjected to a standardized burn pattern by applying a branding iron to 10 different locations on the back of the rat for 1 seconds to 14 seconds, creating a range of burn depths and severities. DESIGN: Animal burn shock and resuscitation model. PARTICIPANTS: Thirty-one male adult Sprague-Dawley rats. INTERVENTIONS: Control animals were resuscitated with lactated Ringer's solution (LRS) at 2 mL/kg/percent total body surface area/24 h; experimental animals received LRS plus DA 24 microg/kg/h (LRS + DA). OUTCOME MEASURES: Perfusion to each burned area was assessed using a laser Doppler imaging technology. Punch biopsies at each burned area were stained with hematoxylin and eosin and assessed for burn depth and for inflammation using previously reported measures. Samples from 14 animals were stained for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and caspase-3 (apoptosis markers). RESULTS: Increasing branding iron contact times worsened perfusion, burn depth, and apoptotic ratios. There was no correlation between inflammatory markers and burn contact time. The addition of DA leads to worse perfusion, deeper burns, worse inflammation, and decreased apoptotic ratios. CONCLUSIONS: Laser Doppler imaging is a useful technology to assess burn depth. The addition of DA to traditional resuscitation fluids for burn shock is deleterious to the injured, burned skin. Modifying the traditional burn shock resuscitation fluids, although intellectually attractive, needs to be rigorously studied.

Morphological parameters for assessment of burn severity in an acute burn injury rat model.

Determination of burn severity (i.e. burn depth) is important for effective medical management and treatment. Using a recently described acute burn model, we studied various morphological parameters to detect burn severity. Anaesthetized Sprague-Dawley rats received burns of various severity (0- to 14-s contact time) followed by standard resuscitation using intravenous fluids. Biopsies were taken from each site after 5 h, tissues fixed in 10% neutral-buffered formalin, processed and stained with haematoxylin and eosin. Superficial burn changes in the epidermis included early keratinocyte swelling progressing to epidermal thinning and nuclear elongation in deeper burns. Subepidermal vesicle formation generally decreased with deeper burns and typically contained grey foamy fluid. Dermal burns were typified by hyalinized collagen and a lack of detectable individual collagen fibres on a background of grey to pale eosinophilic seroproteinaceous fluid. Intact vascular structures were identified principally deep to the burn area in the collagen. Follicle cell injury was identified by cytoplasmic clearing/swelling and nuclear pyknosis, and these follicular changes were often the deepest evidence of burn injury seen for each time point. Histological scores (epidermal changes) or dermal parameter depths (dermal changes) were regressed on burn contact time. Collagen alteration (r(2) = 0.91) correlated best to burn severity followed by vascular patency (r(2) = 0.82), epidermal changes (r(2) = 0.76), subepidermal vesicle formation (r(2) = 0.74) and follicular cell injury was useful in all but deep burns. This study confirms key morphological parameters can be an important tool for the detection of burn severity in this acute burn model.

Repetitive ischemia-reperfusion injury: a plausible mechanism for documented clinical burn-depth progression after thermal injury.

Our previous studies confirmed the phenomenon of burn depth progression despite adequate Parkland formula resuscitation [Kim et al. J Burn Care Rehabil 2001;22960:406-6]. Repetitive ischemia-reperfusion injury (I-R) is a plausible explanation and is suggested by the concomitant swings we have observed in serum base deficit (BD) during resuscitation from burn shock. We chose to explore whether laser Doppler imaging (LDI) evidence of burn depth progression mirrored cycles of I-R (episodic swings in continuously measured BD). Positive findings would support the hypothesis that repetitive episodes of I-R is a factor in burn depth progression despite apparently adequate resuscitation. A total of 14 patients with severe life-threatening burns (median 51% TBSA) underwent continuous BD monitoring using a Paratrend 7 (Malvern PA) during 48 hours of resuscitation. Fluid needs were estimated using the Parkland formula, then were titrated to urine output. The slopes of BD changes were then analyzed. Worsening of BD greater than 0.2 mmol/l/min was noted, and a proportion derived relative to pooled data on 5-minute intervals. In four of the patients, LDI scans were performed on six representative areas sequentially every 4 hours. The analysis of median flux in these LDI images provided real-time determination of burn depth progression. Eight patients eventually died. Only four patients achieved a normal BD within 12 hours of monitoring despite exceeding the Parkland formula estimate and meeting urinary output parameters. Our analysis also showed cyclical peaks and valleys in the BD curve (P < .001), suggesting repetitive I-R insults. All increases in BD preceded changes that could be detected in vital signs or urine output. Finally, LDI confirmed that the burn depths continued to progress despite apparently adequate resuscitation, and also showed that there are similar peaks and valleys in the perfusion of the wounds (P < .0001), which mimic the changes in the BD curve. Responses to fluid resuscitation do not follow a linear pattern in the case of massive burns. These results in repetitive periods of tissue hypoperfusion evidenced by BD alterations and may contribute to progressive deepening of the burn wound.

Seven years' experience with Integra as a reconstructive tool.

The bilayered dermal substitute Integra (Integra Life Sciences Corp., Plainsboro, NJ) was developed and has been widely used as primary coverage for excised acute burns. Our take has been slightly different, finding it most useful in the management of complex soft-tissue loss and threatened extremities as the result of tendon, joint, or bone exposure. Often tasked to fill significant volume loss, we have become adept at stacked multiple-layer applications. Creative use of this material has resulted in unexpected successes with distal limb salvage; the technique takes its place beside adjacent tissue transfer, composite flaps, and vascular pedicle flaps in our burn reconstructive practice. A prospective registry (44 patients) has been kept during the past 7 years that catalogs wounds with complex soft-tissue loss treated with Integra grafts. Many of these patients were at risk of extremity loss because of exposed tendons, joints, or bone. Integra was applied after 1:1 meshing. With profound soft-tissue defects, multiple layers of Integra were serially applied 1 to 2 weeks apart for reconstitution of soft-tissue contours. Local Integra graft infections were managed by silicone unroofing followed by topical sulfamylon liquid dressings. Wounds addressed included fourth-degree burns, necrotizing fasciitis, pit-viper envenomations, and total abdominal wall avulsion in one patient after being run over by a bus. Patients generally were free of pain from their wounds during the maturation phase of the Integra neodermis. Restoration of tissue contour was significantly better when using multiple layers for deep defects. Second and third layers of Integra were successfully applied after an abbreviated first graft maturation period of 7 days. Epithelial autografts on multilayer Integra applications frequently "ghosted"; they would auto-digest to dispersed cells followed subsequently by the reappearance of a confluent epithelial layer. Final grafted skin morphology over palmar and plantar surfaces assumed the type and fingerprint pattern of the original tissues. Infections were readily visible. Early recognition kept them to easily treated circumscribed areas, which did not jeopardize the entire wound. Lengths of stay were long (range, 2-246 days) but not significantly greater than with traditional techniques. The specific reconstructive use of Integra permitted unexpected salvage of several threatened extremities by protecting exposed tendons, bones and joints. Long-term histologic examination revealed unexpected persistence of Integra collagen. Large volume loss wounds benefited from the ability to fill voids with multilayered applications.