Comb Burn Research Articles

Rapid and Selective Enzymatic Debridement of Porcine Comb Burns With Bromelain-Derived Debrase®: Acute-Phase Preservation of Noninjured Tissue and Zone of Stasis

Deep burns are associated with the formation of an eschar, which delays healing and increases the risk of infection. Surgical debridement of the eschar is, at present, the fastest means to achieve an eschar-free bed, but the process can not differentiate between the viable tissue and the eschar and follow the minute irregularities of the interface between the two. We evaluated the efficacy and selectivity of a novel enzymatic bromelain-based debriding agent, Debrase Gel Dressing (Debrase), in a porcine comb burn model. We hypothesized that Debrase would result in rapid debridement of the eschar without adverse effects on the surrounding uninjured skin. This is a prospective, controlled, animal experiment. Five domestic pigs (20-25 kg) were used in this study. Sixteen burns were created on each animal's dorsum using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds, resulting in four rectangular 10 x 20 mm full-thickness burns and separated by three 5 x 20 mm unburned interspaces representing the zone of stasis. The burned keratin layer (blisters) was removed, and the burns were treated with a single, topical, Debrase or control vehicle application for 4 hours. The Debrase/control was then wiped off using a metal forceps handle, and the burns were treated with a topical silver sulfadiazine (SSD). The wounds were observed, and full-thickness biopsies were obtained at 4 and 48 hours for evidence of dermal thickness, vascular thrombosis, and burn depth, both within the comb burns and the unburned interspaces in between them. Chi-square and t tests are used for data analysis. A single 4-hour topical application of Debrase resulted in rapid and complete eschar dissolution of all the burns in which the keratin layer was removed. The remaining dermis was thinner (1.1 +/- 0.7 mm) than in the control burns (2.1 +/- 0.3 mm; difference 0.9 mm [95% confidence interval: 0.3-1.4]) and was viable with no injury to the normal surrounding skin or to the unburned interspaces between the burns, which represents the zone of stasis. In control burns, the entire thickness of the dermis was necrotic. At 48 hours, Debrase-treated areas were found partially desiccated under SSD treatment. The unburned interspaces demonstrated partial-thickness necrosis in two third and full-thickness necrosis in one third of wounds. In contrast, full-thickness necrosis was noted in all control interspaces (P = .05). In a porcine comb burn model, a single, 4-hour topical application of Debrase resulted in rapid removal of the necrotic layer of the dermis with preservation of unburned tissues. At 48 hours, SSD treatment resulted in superficial tissue damage and partial preservation of the unburned interspaces.

Saving the zone of stasis in burns with activated protein C: An experimental study in rats

Salvage of the zone of stasis is a major subject of focus in burn research. Use of various antithrombotic, anti-inflammatory and antioxidant drugs have been studied experimentally, with reports of favourable results; however, none became popular in clinical practice. Activated protein C (APC) is a well-known physiologic anticoagulant. Recent studies revealed that APC contributes not only to systemic anticoagulant activities but also to anti-inflammatory activities by inhibiting leucocyte activation associated with TNF production. The likely favourable effects of APC on salvage of the zone of stasis were investigated on a well-described experimental rat burn model representing the zone of stasis according to the ‘burn comb model’. Twenty Sprague-Dawley rats were used and randomly separated into experimental and control groups. Two hours after inducing injury, 100 μg kg −1 APC (Sigma, Boehringer Ingelheim, Germany) was administered to the experimental group through the caudal vein while 0.9% saline was injected through the same route in the control group. Laser Doppler flowmetry measurements and autoradiography were used for evaluation of perfusion and viability in the zone of stasis. At day 3, the differences between the results obtained from the treatment and the control groups were found to be statistically significant ( p < 0.05). Our experimental study revealed that APC improved tissue perfusion and decreased the area of skin necrosis in the zone of stasis in rats. The dual effect of APC, each of which has been shown to be favourable in saving the zone of stasis, may make this agent effective with a single effect in treatment of burn injury.

Is HBOT treatment effective in recovering zone of stasis? An experimental immunohistochemical study

Studies aimed at recovering the zone of stasis are one of the major issues of experimental burn studies. Hypoxia and oedema at that zone may cause irreversible changes. Due to anti-oedematous and antihypoxic effects of hyperbaric oxygen treatment (HBOT), it may be beneficial in recovering the zone of stasis. We performed an experimental study using 20 Sprague–Dawley rats, each weighing 350–450 g. The rats were first divided into two groups as 24 h and 5 days. Subsequently, control and treatment groups (five rats in each group) were formed. For burn wounds, we used the burn comb model, which was described by Regas and Ehrlich. In the treatment group, 2.5 ATA HBOT was applied for 90 min twice daily. A 0.8-cm punch biopsy was performed and samples for histological examination were taken from the centre of burn area. The rats were sacrificed by administering ‘3 mci technetium-99m methoxy butyl nitrite’ ( 99Tc m MIBI) through the femoral vein. Biopsy materials were evaluated by histological and immunohistochemical methods. Dorsal skin fragment, excised to 1 cm margin, was imaged by scintigraphic measurements with a gamma camera. Wet and dry weight measurements of excised skin fragments were taken. As a result, HBOT showed a positive effect at the cellular level in the first 24 h. It increased recovery potential by augmenting neovascularisation and decreasing oedema in the 5-day group.

Rosiglitazone, a PPAR-γ Ligand, Reduces Burn Progression in Rats

Burns induce the activation of an inflammatory cascade that generates reactive oxygen radicals and lipid peroxidation leading to burn wound progression and extension. Peroxisome proliferation-activated receptor-gamma is a nuclear hormone receptor that is activated by transcription factors and plays an important role in the regulation of cellular proliferation and inflammation. We hypothesized that treatment of burns with rosiglitazone, a peroxisome proliferation-activated receptor-gamma ligand, would reduce burn wound progression. This is a randomized controlled study of 20 Sprague-Dawley rats. Two burns were created on each animal's dorsum using a brass comb with four rectangular prongs preheated in boiling water and applied for 30 seconds resulting in four rectangular 10 x 20 mm full thickness burns separated by three 5 x 20 mm unburned interspaces (zone of ischemia). Animals were randomized to rosiglitazone 4 mg/kg or vehicle by oral gavage 30 minutes after injury and at 24 and 48 hours after injury. Wounds were observed at 1, 2, 3, and 4 days after injury for visual evidence of necrosis in the unburned interspaces. Full thickness biopsies from the interspaces were evaluated with hematoxylin and eosin staining 7 days after injury for evidence of necrosis. The percentage of interspaces that progressed to necrosis was compared with chi tests. Forty comb burns with 120 unburned interspaces were evenly distributed between rosiglitazone and vehicle. The number of interspaces that progressed to full thickness necrosis at 1, 2, 3, 4, and 7 days after injury in the rosiglitazone and vehicle groups were 9/60 (15%) versus 13/60 (21%) (P = .48), 16/60 (27%) versus 15/60 (20%) (P = 1.00), 24/60 (40%) versus 46/60 (77%) (P = .001), 35/60 (58%) versus 53/60 (88%) (P = .001), and 43/60 (72%) versus 54/60 (90%) (P = .02), respectively. Treatment with oral rosiglitazone reduces the percentage of unburned skin interspaces that progress to full necrosis in a rat comb burn model.

Validation of a porcine comb burn model

Objective A brass comb burn model that creates 3 full-thickness burns separated by 3 interspaces of unburned skin representing the zone of ischemia has been described in rats. We evaluated this model in pigs. Methods Design—observational. Subjects—6 pigs (20-25 kg). Interventions—4 burns created on each animal on the dorsum using a brass comb with 4 rectangular prongs preheated in boiling water and applied for 30 seconds resulting in 4 rectangular 10 × 20–mm full-thickness burns separated by three 5 × 20–mm unburned interspaces. Outcomes—wounds observed at 1, 2, 3, and 7 days for evidence of necrosis in unburned interspaces. Full-thickness biopsies from interspaces were evaluated with hematoxylin-eosin staining 7 days after injury for evidence of necrosis. Data analysis—the percentages of interspaces progressing to necrosis were analyzed with descriptive statistics. Results Twenty-four comb burns with 72 unburned interspaces were created evenly distributed between the animals. The percentages of interspaces that progressed to full-thickness necrosis at 1, 2, 3, and 7 days after injury were 88.9% (64/72; 95% confidence interval [CI], 79.6%-94.3%), 88.9% (64/72; 95% CI, 79.6%-94.3%), 88.9% (64/72; 95% CI, 79.6%-94.3%), and 97.7% (70/72; 95% CI, 90.4%-99.2%), respectively. There was perfect agreement between gross inspection and histomorphology. Conclusions The comb burn model in swine results in the progression of most unburned ischemic interspaces to full-thickness necrosis within 1 to 7 days.

Apoptosis and Necrosis in the Ischemic Zone Adjacent to Third Degree Burns

Burns are characterized by a central zone of necrosis surrounded by a zone of potentially reversible ischemia. The authors explored the contribution of necrosis and apoptosis to cell death in the zone of ischemia. A previously established rat contact thermal injury model that utilizes a brass comb to produce four distinctive burns sites separated by three "interspaces" of unburned skin was used. The interspaces represent the zone of stasis or ischemia while the burn sites represent the zone of coagulation. With this model, most unburned interspaces progress to necrosis over 2 to 3 days. Full-thickness 3-mm biopsies were obtained from the interspaces, burns, and normal skin controls at 30 minutes, 24 hours, and 48 hours after injury. Slides were stained with hematoxylin and eosin as well as activated cleaved caspase-3 (CC3a) for evidence of apoptosis and high-mobility group box 1 (HMGB1) for evidence of necrosis. Necrosis was not seen at 30 minutes, but was found in a large number of cells within the epidermis, sebaceous glands, and follicles at 24 and 48 hours. Faint nuclear CC3a staining indicative of apoptosis was present in a minority of cells within the epidermis, dermal fibroblasts, dermal follicles, and dermal sebaceous glands at 30 minutes and to a lesser degree at 24 and 48 hours. Both early apoptosis and delayed necrosis are present in the zone of ischemia, contributing to injury progression. Necrosis appears to play a larger role than apoptosis in injury progression in the comb burn model.

Validation of an Ischemic Comb Burn Model in Swine

Validation of an Ischemic Comb Burn Model in Swine

Saving the zone of stasis in burns with recombinant tissue-type plasminogen activator (r-tPA): an experimental study in rats

Hypercoagulability 2–3 h following scald injury and vessel thrombosis are well-known complications of burn injury. The present study was designed to determine the effect of recombinant tissue-type plasminogen activator (r-tPA) on vessels thrombosis in zones of stasis postburn. Twenty rats were assigned to experimental and control groups (N:10). After shaving the backs, a ‘comb burn’ was given bilaterally on the back of the rats 0.5 cm lateral and parallel to the midline by using a brass probe consisting of four rows (10 × 20 mm) and three interspaces (5 × 20 mm). Standardized full-thickness burns of the experimental group were treated with r-tPA via femoral veins 2 h after the burn, while the control group rats were infused the same volume of saline. Interspaces between the rows and vertical space area were evaluated as zone of stasis. The skin blood flow measurements of interspaces revealed a decreased level immediately and 2 h after the burn injury, averaging 11.3 and 9.8 perfusion units, respectively. On day 7, blood flow measurements of interspace and vertical space areas significantly differed between the groups, averaging 11.9 PU in the experimental and 1.8 PU in the control groups ( P < 0.05). To evaluate perfusion, each rat received 3 mCi of technetium-99m methoxyisobutylisonitrile on the first or seventh day postburn. After 30 min, the entire back skins including the panniculus carnosus muscle layer were dissected away. Gamma camera images revealed that in the experimental group the interspace areas took up 60–90 per cent more radioactivity than the burned areas while in the control group 20–50 per cent more activity was taken up by the interspace area when compared to burned areas. Autoradiography gave the exact borders of the necrotic and survived tissues. The percentage of live interspace and vertical space areas in the experimental group was 87.8 per cent on day 7, while it was 31.8 per cent in the control group ( P < 0.05). Dry/wet ratios did not reveal any significant difference at 24 h postburn. These results confirm that treatment with this selective fibrinolytic agent (r-tPA) after burn injury would have some benefits on saving the zone of stasis in burns.

Preventing the Infiltration of Leukocytes by Monoclonal Antibody Blocks the Development of Progressive Ischemia in Rat Burns

Tissue loss as a consequence of thermal trauma occurs in two stages. There is immediate necrosis in tissues directly killed by the thermal energy, followed by a delayed secondary necrosis in neighboring tissues. The infiltration of neutrophils into traumatized tissues is a hallmark of the inflammatory response. Neutrophils have the machinery to kill invading microorganisms, but these same weapons have the capacity to destroy the host's viable tissues as well. Leukocyte infiltration requires their adherence to the vascular endothelial cell surface. Masking these adhesion sites on neutrophils will block the adhesion of neutrophils to the endothelium. A monoclonal antibody (mAb) was developed to guinea pig leukocyte adhesion sites CD11b/ CD18, and this mAb cross-reacts with rat leukocytes, blocking their adherence. Rats received a "comb burn" composed of four rectangular full-thickness burns placed in a row and separated by three areas left unburned. The four individual burns convert into a single large wound because the blood flow to the interspaces was terminated, blood vessels were occluded, and leukocytes were present in the extravascular space. The systemic administration of the mAb (50 to 150 microliters) immediately following a comb burn promoted the survival of the interspace, demonstrated by the prevention of loss of blood flow by laser Doppler monitoring, maintained patent vessels by latex vascular casts, blocked extravascular migration of neutrophils histologically at 2 hours, and limited the tissue loss to the original four burns.