TY - JOUR
AU - MD, James C. Jeng,
AB - Abstract Burn wound progression refers to the phenomenon of continued tissue necrosis in the zone of stasis after abatement of the initial thermal insult. A multitude of chemical and mechanical factors contribute to the local pathophysiologic process of burn wound progression. Prolonged inflammation results in an accumulation of cytotoxic cytokines and free radicals, along with neutrophil plugging of dermal venules. Increased vascular permeability and augmentations of interstitial hydrostatic pressure lead to edema with vascular congestion. Hypercoagulability with thrombosis further impairs blood flow, while oxidative stress damages endothelial cells and compromises vascular patency. A number of studies have investigated the utility of various agents in modulating these mechanisms of burn wound progression. However, as many of studies have used animal models of burn injury, often with administration of therapy preburn, obscuring the clinical applicability of the results to burn patients is of questionable benefit. An understanding of the complex, interrelated mediators of burn wound progression and their ultimate point of convergence in effecting tissue necrosis—cell apoptosis or oncosis—will allow for the future development of therapeutic interventions. In 1953, Jackson1 described three concentric zones of a burn wound: the central zone of coagulation, the intermediate zone of stasis, and the outer zone of hyperemia (Figure 1). The white tissue in the zone of coagulation sustains the greatest direct damage from a thermal trauma and is characterized by irreversible necrosis. Microscopically, the zone of coagulation demonstrates complete destruction of the subpapillary vasculature.2 Conversely, the edematous, red tissue in the zone of hyperemia invariably recovers. In his 1953 article, Jackson proposed that the natural history of the zone of stasis involves complete cessation of blood flow within 24 hours and tissue necrosis, so that the zones of stasis and coagulation become indistinguishable and “inevitably separate as slough.”1,3 In 1969, after observing the ability of the zone of stasis to accept an autograft and bleed from a few arterioles on excision, he realized that the zone of stasis is not avascular and may not be “dead.”3 In noting that the same dermal layer capable of receiving and supporting a graft becomes a “brown dry slough” in the absence of grafting, Jackson raised a question whose answer, even today, continues to be elusive: what factors contribute to or ameliorate the progression of tissue destruction in the zone of stasis? Figure 1. View largeDownload slide Zones of thermal injury. Three-dimensional illustration of the three zones of burn injury. Figure 1. View largeDownload slide Zones of thermal injury. Three-dimensional illustration of the three zones of burn injury. The answer to this question is of paramount clinical significance. The ability to halt tissue destruction in the zone of stasis may significantly influence the overall depth and surface area extent of a burn wound, which often continue to progress for 2 weeks after the initial injurious event.4,5 The extent of surface area involvement and depth of a burn wound are key determinants of both mortality and morbidity, correlating with rates of delayed wound healing, hypertrophic scarring, dyspigmentation, contractures, infection, and shock.6,–8 Indeed, when a burn involves 20% or more of the TBSA, there is an increased risk of massive fluid shifts, sepsis, and multiorgan failure.9 Burns are commonly classified according to the depth of injury; that is, superficial, superficial partial-thickness, deep partial-thickness, full-thickness, and subdermal burns (Figure 2A). Superficial burns affect only the epidermis. Clinically, they are characterized by erythema and pain that resolves within 3 to 5 days without scarring. Superficial partial-thickness burns extend through the epidermis into the papillary dermis, manifesting as blisters, erythema, and edema. These burns blanch on pressure and retain a brisk capillary refill. Deep partial-thickness burns involve the reticular dermis and exhibit a sluggish capillary refill. The wound is very moist and edematous with diminished sensation to pinprick and possibly complete analgesia. The tissue injury of full-thickness burns extends into the subcutaneous tissue, whereas that of subdermal burns extends into the fascia, muscles, and bone.1,6 Figure 2. View largeDownload slide Anatomy and histology of the skin. A, Three-dimensional illustration of the skin. B, Important histological zones and location of different cell types. Figure 2. View largeDownload slide Anatomy and histology of the skin. A, Three-dimensional illustration of the skin. B, Important histological zones and location of different cell types. The depth of a burn influences the potential for successful wound healing, in part, because reservoirs of keratinocytes that have the potential to repopulate the epidermis are located in the appendages of the reticular dermis (Figure 2B).7 In addition, growth factors such as platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-β1 and the extracellular matrix produced by dermal macrophages and fibroblasts are important in wound healing.8,10 Thus, it follows that a superficial or superficial partial-thickness burn will heal more readily and satisfactorily than a deep partial-thickness, full-thickness, or subdermal burn in which the majority of keratinocyte-containing hair follicles, sweat glands, and sebaceous glands are destroyed. In the years following Jackson's re-examination of the fate of the zone of stasis in 1969, a number of mechanisms for burn wound progression were proposed. Some, including Jackson, argued that epithelial cell death was a direct result of heat injury.3,11 Others gave more weight to microthrombosis or wound dehydration.11 No doubt, all of these, among other more recently implicated factors, contribute to burn wound progression. The aim of this study is to review the literature regarding the molecular and cellular biology of burn wounds, thereby elucidating the phenomenon of burn wound progression. An understanding of the mediators of burn wound progression may allow for the development of novel therapies capable of limiting two dominant contributors——Mburn wound depth and surface area extent——Mto burn injury morbidity and mortality. This review begins with a general overview of cell death. Next is an analysis of the mechanisms of cell death in the zone of stasis. Then, an integrated summary of the scientific research on inflammatory cell infiltrates, local blood flow changes, and oxygen-derived free radicals as applicable to burn wound progression is provided. Figure 3 has been provided to assist in navigation of this article, and a glossary of abbreviations has been created as a reference (Table 1). Figure 3. View largeDownload slide Table of contents. Figure 3. View largeDownload slide Table of contents. Table 1. Frequently used abbreviations in this review View Large Table 1. Frequently used abbreviations in this review View Large Table. No caption available. View Large Table. No caption available. View Large GENERAL OVERVIEW OF CELL DEATH Cell death has typically been conceptualized as occurring through one of the two biochemical mechanisms: apoptosis or necrosis. Apoptosis is regarded as a programmed, active, and organized process, whereas necrosis is believed to be accidental, passive, and disorganized,12 although recent evidence suggests that necrosis can also be the result of death by design.13 In addition, autophagy has been alternately described as either a mechanism of survival or a nonapoptotic form of programmed cell death. All three forms of cell death seem to play a role in normal physiological processes and in disease.14,15 However, because there are as yet no studies to suggest a role for autophagy in burn wound conversion, this review will focus on the roles of apoptosis and necrosis. Both forms of cell death have been implicated in burn wound progression, although the relative contribution of each depends on the burn model used, the cell type examined, and the time course studied (see later). The term apoptosis was originally used by Kerr in 1972 to describe the morphological pattern of cell death seen during embryonic development and healthy adult cell turnover.12 The Greek translation of apoptosis suggests the image of leaves “falling off” a tree.12,16 Apoptosis is characterized by shrinkage and condensation of cell contents, DNA fragmentation, and budding. There is a lack of cellular organelle swelling and a minimal inflammatory response.16 Necrosis is the term commonly used to refer to nonapoptotic cell death characterized by cellular swelling, depletion of intracellular energy stores, and massive release of inflammatory mediators.12 However, pathologists also use the term necrosis in reference to the histological appearance of dead tissue, irrespective of whether the process culminating in death occurred through apoptosis or necrosis. To avoid such confusion, it has been suggested that the term oncosis rather than the term necrosis be used when referring to the process of cell death accompanied by cellular and organelle swelling, membrane permeability, and depletion of energy stores.12,16 For purposes of clarity and consistency, the remainder of this article will use the term oncosis to refer to the earlier-described pathway of cell death and the term necrosis to indicate the pathological finding of dead tissue (Figure 4). Figure 4. View largeDownload slide Oncosis vs necrosis. Figure 4. View largeDownload slide Oncosis vs necrosis. Apoptosis The process of apoptosis can proceed through either the “intrinsic” or the “extrinsic” pathway. Both pathways lead to the activation of intracellular caspases or cysteine proteases.17 Caspases are categorized as initiator caspases (caspases-2, -8, -9, and -10) or effector caspases (caspases-3, -6, and -7).12,17 Activation of initiator caspases begins sequelae wherein downstream effector caspases are activated through cleavage at internal aspartic acid residues. The effector caspases then alter the activities of various target proteins within the cell, through proteolytic cleavage, resulting within minutes in the morphologic and biochemical changes characteristic of apoptosis within.17,18 Apoptosis: Intrinsic Pathway In the intrinsic pathway, cellular stress causes the mitochondria to release cytochrome C, among other death-promoting molecules. Cytochrome C acts in concert with another protein, apoptosis activating protease-1, to activate caspase-9 by recruiting it to the heptameric “apoptosome” complex. The Bcl-2 family of proteins is implicated in effecting the release of cytochrome C and other mitochondrial constituents. The Bcl-2 family is divided into three groups. Group 1 comprises the antiapoptotic Bcl-2 subfamily that includes Bcl-xL and Bcl-w (among others). Group 2 is the proapoptotic Bax subfamily (Bax, Bak, and Bok), and group 3 is the proapoptotic BH3 subfamily comprising Bad, Bim, and Bik among others. A cell's balance of anti- and proapoptotic Bcl-2 family members determines its overall sensitivity to death.18 When the balance is shifted to proapoptotic Bcl-2 family members, it is postulated that Bax subfamily members may form a pore in the outer mitochondrial membrane through which cytochrome C can pass. Alternatively, the Bax/Bak/Bok proteins may alter mitochondrial homeostasis, such that the organelle swells and ruptures, releasing cytochrome C and other mitochondrial constituents from the intermembrane space.18 Apoptosis: Extrinsic Pathway In the extrinsic pathway, the binding of a ligand to its “death receptor” results in the formation of a membrane-bound death-inducing signaling complex and the mobilization of several molecules of procaspase-8 through homotypic interaction with adapter molecules, including Fas-associated protein with death domain. The relatively high local concentration of the zymogen form of procaspase-8 provides a milieu in which the weak, intrinsic protease activity of the procaspase is sufficient for reciprocal cleavage and activation.18 In skin, constitutive expression of various death receptors is restricted to certain cells: Fas to basal keratinocytes, tumor necrosis factor (TNF)- related apoptosis-inducing ligand (TRAIL) receptor 1 (DR4) to suprabasal keratinocytes, TRAIL receptor 2 (DR5) to granular keratinocytes, TNF receptor 1 to keratinocytes in all layers, and TNF receptor 2 to eccrine sweat ducts and to dermal dendritic cells.19 In healthy skin, the ligands for these death receptors, Fas ligand (FasL), TNF-α, and TRAIL, are found, although usually at low levels.19 It should be noted that TNF-α is not only capable of inhibiting apoptosis through nuclear factor (NF)-κB activation but also involved with a variety of biological and signaling processes in addition to apoptosis, such as inflammation.19 Oncosis The cellular mechanisms underlying the process of oncosis include inhibition of ATP production, acceleration of ATP consumption, and alteration of intracellular calcium concentrations. An example of accelerated ATP consumption is as follows: DNA damage (strand breaks) activates the DNA repair enzyme poly (ADP-ribose) polymerase (PARP), which uses nuclear nicotinamide adenine dinucleotide (NAD; 95% of the total cellular NAD) to covalently attach polymers of ADP-ribose to its substrates. In instances of massive DNA damage, the resulting high intracellular activity of PARP depletes the supply of its substrate, NAD. To replenish the supply of NAD, large amounts of ATP are consumed, exhausting the cell's energy reserve with consequent failure of membrane ionic pumps, cellular swelling, and lysis. This hyperconsumption of ATP is prevented in apoptotic cells by the action of caspases. Caspases-3 and -7 cleave and inhibit PARP, thereby retaining the cell's energy reserve in the presence of DNA damage. In addition to ATP, altered calcium homeostasis is associated with plasma membrane integrity characteristic of oncosis. High levels of intracellular calcium activate the calpain family of cysteine proteases, which degrade cytoskeletal and plasma membrane proteins. In addition, high intracellular calcium levels initiate the translocation of PLA2s from the cytosol to the plasma membrane. In the plasma membrane, PLA2s hydrolyzes phospholipids, compromising the membrane's strength.12 APOPTOSIS AND ONCOSIS IN BURN WOUNDS To better understand the cellular basis of burn wound progression, a number of studies have examined the mechanisms of cell death in the zone of stasis. In 2006, intrigued by the idea that the zone of stasis seen in deep partial-thickness burns bears similarity to the “stunned” zones of myocardial infarctions, Gravante et al examined human deep-partial thickness burns for evidence of apoptosis. In their studies, a cell was considered apoptotic if it was positive for both terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) and FasL. They found that 44% of dermal cells in the burns were apoptotic, and that the presence of apoptotic cells persisted up to 13 days after the burn injury.4 These data were in agreement with earlier studies demonstrating the ability of thermal injury to induce apoptosis.20,21 To confirm the significance of the earlier finding, they conducted a study comparing the apoptotic rate in deep partial-thickness burns with that in normal skin. Again, apoptotic cells were identified by co- localization of TUNEL and FasL. Results showed that 48.7% of dermal cells were apoptotic in deep partial-thickness burns, whereas only 18.6% of dermal cells were apoptotic in healthy skin—a statistically significant difference.4 Gravante et al5 also reported that apoptotic dermal cells were found at higher numbers in deep partial-thickness burns (44.5%) when compared with superficial partial-thickness burns (5.6%) or full-thickness burns (0%). In 2008, they published the results of an investigation, looking at the relationship between the apoptotic rate of dermal cells in deep partial-thickness burns and the time elapsed from burn injury. They discovered that the apoptotic rate was highest early after the burn injury but persisted for 23 days. They also found that the apoptotic rate did not correlate with mortality, as the burn wounds of nonsurvivors demonstrated a lower apoptotic rate (30.1%) than survivors (44.0%).22 Finally, another descriptive study by the same group investigated the impact of a patient's general health quantified by a prognostic mortality index on apoptotic rate. They found that a patient's prognostic mortality index did not correlate with apoptotic rate.23 Overall, these studies by Gravante et al allow three conclusions to be drawn: 1) apoptotic dermal cells are found at a higher rate in deep partial-thickness burns than in normal skin, superficial partial-thickness burns, and full-thickness burns, suggesting but not confirming that apoptosis plays a role in the burn wound progression of deep partial-thickness wounds; 2) apoptosis continues for at least 23 days after the initial burn injury, providing a large window of opportunity for pharmacologic intervention of the inciting factor; and 3) apoptotic rates are not influenced by a patient's prognostic mortality index, implying that local processes may be more important than systemic conditions in effecting apoptosis in the zone of stasis. However, one limitation of these studies is the failure to definitively demonstrate a relationship between apoptotic rate and evolution of deep partial-thickness burns to full-thickness burns. Although the aforementioned studies of Gravante et al suggest a prominent role of apoptosis in burn wound conversion, a study by Singer et al reported on the contributions of both apoptosis and oncosis to cell death in the zone of stasis. In their study, they used a rat comb thermal injury model.24 Biopsies were taken from the interspaces of comb-induced full-thickness burns at 30 minutes, 24 hours, and 48 hours postthermal injury. Apoptotic cells were identified by the presence of cleaved caspase-3 (CC3a), and “necrotic” (oncotic) cells were identified by the redistribution of high-mobility group box-1 from the nucleus to the cytoplasm. At 30 minutes postinjury, a small number of basal keratinocytes and those surrounding hair follicles stained positive for CC3a, indicating apoptosis. Minimal CC3a staining was found in hair follicle cells at 24 and 48 hours postinjury. Evidence of oncosis as indicated by diffuse cytoplasmic high-mobility group box-1 staining was present in the interspace sebaceous glands, hair follicles, keratinocytes, and dermal fibroblasts at 24 hours postinjury, with diminished staining at 48 hours postinjury. The authors concluded that the roles of apoptosis and oncosis in burn wound conversion may be time dependent with apoptosis and oncosis playing greater roles in initial and later stages, respectively.24 Although this study does confirm the concept of oncosis in burn wound progression, it is difficult to compare the findings of Singer et al regarding apoptosis with those of Gravante et al because different burn models (and species), biomarkers, and time periods were used by the two groups. Assuming that apoptosis in the zone of stasis is causally linked to the progression of deep partial-thickness to full-thickness burns, it would be advantageous to develop a pharmaceutical agent to block this mechanism of death before irreversible loss of cell viability. Giles et al25 investigated inhibition of c-Jun, a transcription factor involved in multiple cell processes, including inflammation, proliferation, and apoptosis. Specifically, the phosphorylation of c-Jun plays an important role in the “extrinsic” apoptotic pathway initiated by the TNF death receptors, which activate c-Jun N-terminal kinase.26,27 Giles et al25 found that direct application of the c-Jun inhibitor to full-thickness burn wounds in mice resulted in improved reepithelialization and a reduction in apoptotic cells, as assessed by TUNEL, at 24 hours after burn injury. The positive results of this study reinforce the idea that preventing cell death, particularly apoptosis, in the zone of stasis may enhance wound healing. It should be noted, however, that other consequences of c-Jun inhibition such as decreased vascular permeability and inflammation may be responsible for the observed outcome. Furthermore, it is possible that the topically applied peptide acted by a distinct mechanism unrelated to c-Jun inhibition to effect the aforementioned results.25 In discussing the time course of the apoptosis of skin cells after photodamage, Godar28,–30 used the terms immediate, intermediate, and delayed. Immediate apoptosis occurs within 30 minutes and does not require protein synthesis postinjury. Intermediate apoptosis occurs between 30 minutes and 4 hours and is prompted by death receptor cross-linking. Delayed apoptosis occurs after 4 hours and requires protein synthesis postinjury.31 It seems appropriate to apply this general concept to the apoptosis that results from burns, whether they are thermal, electrical, radiation, or chemical insults. Immediate apoptosis can be thought of as resulting from the direct effects of the burn injury, whereas delayed apoptosis can be thought of as likely to be resulting from a combination of direct effects of the burn injury and secondary damage that occurs after the burn. A logical strategy for limiting burn wound progression would be to block this delayed apoptosis, either by targeting the actual proteins and transcription factors involved in the apoptotic process or by ameliorating secondary damages. The following sections will analyze local sources of secondary damage that mediate delayed apoptosis, as well as oncosis, and their involvement in burn wound progression. These include inflammatory cell infiltrates, blood flow changes, and oxygen-derived free radicals. BURN WOUNDS AND INFLAMMATORY CELL INFILTRATES Although inflammation is an important factor in the promotion of wound healing, it can also hinder the healing process. Beneficial effects of local inflammation include clearance of cellular debris and protection from microbial agents.32 However, a prolonged acute inflammatory reaction, dominated by neutrophils and macrophages, may lead to persistently increased levels of proinflammatory cytokines, resulting in collagen degradation and keratinocyte apoptosis; adherence of neutrophils to the venular endothelium, resulting in microvascular compromise; and production of oxygen-derived free radicals, resulting in disruption of plasma membranes, DNA cross-links and strand breaks, and peptide fragmentation.33,–35 Mediators of inflammatory cell activation and infiltration after burn injury include oxygen-free radicals (specifically the superoxide anion released by capillary endothelial cells in the dermal papillae), complement, cytokines, and bacterial endotoxin33,36 (Figure 5). Figure 5. View largeDownload slide Inflammatory cell infiltration. IL, interleukin; TNF, tumor necrosis factor; ICAM-1, intercellular adhesion molecule-1; ROS, reactive oxygen species; MMP, matrix metalloproteinase. Figure 5. View largeDownload slide Inflammatory cell infiltration. IL, interleukin; TNF, tumor necrosis factor; ICAM-1, intercellular adhesion molecule-1; ROS, reactive oxygen species; MMP, matrix metalloproteinase. Delayed Neutrophil Apoptosis A number of studies have shown that apoptosis of neutrophils, which is necessary for the resolution of inflammation, is inhibited in burn patients.33,37 These studies compared the percentage of apoptotic cells in the peripheral blood of burn patients with that of healthy controls and found that the control patients had a statistically higher percentage of apoptotic neutrophils than the burn patients. Factors generally known to prolong neutrophil survival include glucocorticoids, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α.38,–40 Chitnis et al32 purport that in burn patients the inhibition of neutrophil apoptosis may be related to a heat-labile factor that induces GM-CSF production. In their experiments, they observed decreased inhibition of neutrophil apoptosis when cells were cultured in burn-derived plasma that was either first heated to 56°C for 30 minutes or mixed with neutralizing antibodies to GM-CSF. However, these researchers were unable to demonstrate increased levels of GM-CSF in the plasma of burn patients. Treatment of burn-derived plasma with antibodies to interleukin (IL)-1, TNF-α transforming growth factor-β, IL-6, and lipopolysaccharide failed to reverse the delay in neutrophil apoptosis. Although Chitnis et al have begun to elucidate mediators of the delayed apoptotic response in neutrophils after burn injury, it is unclear whether the finding that GM-CSF inhibits neutrophil apoptosis is applicable to burn patients, because the described study, as well as another study of 12 burn patients, failed to demonstrate detectable plasma levels of GM-CSF.32 To delineate the cellular and molecular mechanisms of inhibited neutrophil apoptosis, Hu et al studied the mitochondrial morphology changes and the balance of pro apoptotic and antiapoptotic Bcl-2 family members in peripheral neutrophils in a rat scald burn injury model. When compared with sham rats, the neutrophils of the burned rats showed increased expression of antiapoptotic Bcl-xL at 2 hours postinjury, decreased expression of proapoptotic Bax and Bad at 8 hours postinjury, decreased mitochondria aggregation (an indicator of nonsurviving neutrophils) at 2 and 8 hours postinjury, decreased mitochondrial release of cytochrome C, and decreased levels of active caspase-3 and -9.42 This study suggests that the inhibited apoptotic response of neutrophils after burn injury is due, in part, to suppression of the intrinsic mitochondrial apoptotic pathway. Hu et al further investigated the role of protein kinase B (PKB) signaling in inhibited neutrophil apoptosis. PKB mediates survival signals downstream to phosphoinositide-3 kinase. For example, PKB phosphorylates and sequesters the proapoptotic Bcl-2 family member Bad. Using a rat burn injury model, the authors found that incubating neutrophils from burned rats with an inhibitor of PI-3 kinase (Wortmannin or LY-294002) significantly increased the rate of neutrophil apoptosis. In addition, they demonstrated that NF-κB, a downstream target of phosphoinositide-3 kinase that is involved with survival signaling, was activated in neutrophils from burned rats after 2 hours of culture. After 8 hours of neutrophil culture, phosphorylation of proapoptotic Bad was increased.37 Similar to their earlier study, this work reaffirms the importance of the intrinsic apoptotic pathway in inhibited neutrophil apoptosis after burn injury and suggests several time-dependent targets for pharmacologic intervention. Ogura et al used flow cytometry to analyze the expression of heat shock proteins (HSPs) in neutrophils in eight severely burned patients. They found that neutrophils exhibited decreased apoptosis in comparison with controls, expressed higher levels of HSP27, HSP60, and HSP70 during the first 28 days postburn, and had increased oxidative activity.40 This study was not able to define a causal relationship, if any, between increased HSP expression and the oxidative and apoptotic activity of neutrophils. If HSPs contribute to enhanced neutrophil oxidative activity and decreased neutrophil apoptosis, they may prove to be another target for intervening in the prolonged postburn inflammatory response. It is important to note that studies demonstrating a parallel inhibition of neutrophil apoptosis locally in the burn wound are lacking. However, it is feasible that inhibited neutrophil apoptosis in the burn wound could contribute to tissue injury and burn wound progression. Future investigations aimed at correlating the apoptotic rate of neutrophils in deep partial-thickness burn wounds with wound healing or progression will help to elucidate this matter. Neutrophil Aggregation, Venule Occlusion, and Cytokine Production Irrespective of the status of local neutrophil apoptosis in the burn wound, the fact that inflammation can negatively impact the burn wound is well established.33,38,39,41,43,–45 Recent evidence from a clinical study points to the complement product C3d and C-reactive protein as having roles in skin leukocyte sequestration after burn injury.46 A number of studies explore the utility of various inhibitors of postburn inflammatory cell infiltration (Table 2). Table 2. Interventions aimed at modulating postburn inflammatory infiltrates View Large Table 2. Interventions aimed at modulating postburn inflammatory infiltrates View Large As noted earlier, products of the complement system attract neutrophils to the cutaneous site of burn injury. Henze et al studied the ability of a C1 inhibitor (Berinert) to reduce the progression of burn wounds in a porcine deep partial-thickness burn model. C1 inhibitors are known to decrease bradykinin levels and to inhibit the release of C3a and C5a. This study demonstrated that C1 inhibitor maintained the patency of the lower dermal vascular network, which, in pigs not treated with the C1 inhibitor, demonstrated leukocyte adherence to the endothelium, hyaline thrombus, denuded basement membranes, and necrosis of portions of the vascular wall. These vascular alterations were only seen in the middermal and subepidermal vessels in C1 inhibitor-treated pigs. In addition, Henze et al examined progression of burn wound depth in C1 inhibitor-treated pigs by assessing tissue staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. In viable cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is converted in the mitochondria to formazan dye. They found that from 28 hours onward, the depth of burn wound of C1 inhibitor-treated pigs was significantly less than that of nontreated pigs. The authors concluded that burn wound progression in C1 inhibitor-treated pigs was only secondary to decreased neutrophil activation and endothelial cell damage.47 The findings of Suber et al similarly support the role of complement-induced inflammation in burn wound progression. In a mouse burn model, they found that C4−/− complement-deficient mice healed without contracture, hair loss, or neutrophil infiltration, and that complement-sufficient mice pretreated with a complement inhibitor sCR1 that blocks cleavage of C3 likewise demonstrated reduced injury postburn. Moreover, their experiments revealed that IgM is responsible for stimulating the complement-induced injury in this mouse burn model. Thus, they found that pretreatment with systemic N2 peptide (a peptide that binds IgM) or posttreatment with topical N2 peptide blocks inflammatory exacerbation of burn wound injury.50 Once leukocytes are attracted to a site of burn injury, whether by complement breakdown products or other mediators, they adhere to the vascular endothelium through interactions between leukocyte surface receptors and specialized ligands on the endothelial surface. In a rabbit burn model, the effect of an antibody to CD18 (MAb 60.3) on wound healing was tested. The burn wounds of treated rabbits demonstrated less edema, thinner eschar, and reduced surface area at 24 hours postinjury compared with control rabbits. Histologically, burn wounds of the control rabbits were characterized by occlusion of venules secondary to aggregated neutrophils and thrombus at the dermis-subcutaneous border and destruction of hair follicles. (This histologic finding is similar to an earlier study by Cotran and Majno51 in which leukocyte margination and emigration was appreciated in subcutaneous venules.) The wounds of antibody-treated rabbits were spared from these pathologic changes. At 8 days postinjury, antibody-treated rabbits had 96.6% reepithelialization of burn wounds, whereas control rabbits had only 55.4% reepithelialization. There was no significant difference in wound healing or histologic appearance of the 10-second (full thickness) burn wounds of antibody-treated and control rabbits. In a similar experiment, monoclonal antibodies to CD18 or its endothelial cell ligand, intercellular adhesion molecule 1, were used in a rabbit burn model to investigate the effects of neutrophils on postburn microvascular injury as assessed by laser Doppler imaging (LDI). Blood flow in the “zone of stasis” between areas of full-thickness burns was higher in antibody-treated rabbits than in control rabbits. Pretreatment with the antibodies or treatment 30 minutes postburn was equally beneficial.39 These two studies underscore the contribution of neutrophils to the progression of deep partial-thickness burn wounds through microvascular plugging and illuminate one potential intervention through a blockade of neutrophil adhesion receptors. Indeed, the success with targeting neutrophil adhesion in animal models of burn injury has translated into effective therapy for patients. In 2003, Mileski et al published the results of a phase II clinical trial of the murine monoclonal antibody against intercellular adhesion molecule 1, enlimomab. At 21 days postburn, 7% more of the burn wounds from patients treated with enlimomab had healed when compared with the placebo group. Although this endpoint failed to reach statistical significance (as one would expect with a phase II trial), when analyzing only the “high risk” burn wound sites as characterized by low blood flow on LDI, the odds of healing by day 21 were 3.1 times higher in patients receiving enlimomab than in patients receiving placebo, a statistically significant difference. In addition, there was a decrease in grafting requirements in the enlimomab arm. Notably, there was no increased risk of infection in the enlimomab group. This study again reiterates the central role of leukocyte adherence to cutaneous vascular endothelium in burn wound progression and suggests that the inhibition of this step in the inflammation process may prove to be a viable treatment option for patients.48 It has been postulated by Cetinkale et al that neutrophil aggregation in postcapillary venules in the zone of stasis can result in vascular damage directly through increased pressure gradients and indirectly through the release of granule contents and oxygen-derived radicals. In their study, using a rat full-thickness comb burn model, pretreatment with cyclosporine A (believed to modulate neutrophil accumulation during acute inflammation) resulted in increased perfusion of the interspace panniculus carnosus muscle and a reduction in the interspace water content, an indicator of edema.41 This study demonstrated that neutrophils are an important cause of ischemia in the zone of stasis that is associated with progression to tissue necrosis. Ipaktchi et al target the potential damage from excessive inflammation in a burn wound from a different angle. They looked at the ability of a topical p38 mitogen-activated protein kinase inhibitor (SB202190) to reduce postburn inflammation in a rat partial-thickness burn model. In response to external stress, p38 mitogen-activated protein kinase activates a number of proapoptotic factors and induces keratinocyte production of the proinflammatory cytokines IL-6, IL-1β, and TNF-α. Analyzing tissue homogenates taken from burn sites with ELISA and reverse-transcriptase polymerase chain reaction, they found that rats treated with the inhibitor produced less IL-6, TNF-α, and IL-1β. Treatment also resulted in decreased levels of macrophage inflammatory protein 2 and decreased skin myeloperoxidase activity, indicating reduced neutrophil migration to and sequestration in the burn wound. Finally, SB202190 attenuated the apoptotic response of hair follicle cells to burn injury and inhibited microvascular injury as assessed by vascular albumin leakage. The observed reduction in microvascular injury may be due to decreased neutrophil adhesion to the vascular endothelium, as neutrophil sequestration to the burn wound was diminished and TNF-α was reduced. The ultimate effect of decreased hair follicle cell apoptosis was likely mediated both directly by SB202190 in its ability to inhibit apoptotic genes and indirectly by its antiinflammatory properties. This study bolsters the hypothesis that prolonged inflammation after burn injury contributes to apoptosis of hair follicle cells and thus burn wound progression.44 Yet another approach to reduce the prolonged inflammatory response that follows burn injury is through local liposomal gene transfer of insulin-like growth factor (IGF)-1 to keratinocytes and fibroblasts. Although systemic administration of IGF-1 has been shown to promote wound healing, it is also associated with adverse side effects such as hypoglycemia and electrolyte imbalances. A study by Spies et al demonstrated that liposomal IGF-1 cDNA gene transfer ameliorates local increases in proinflammatory IL-1β and TNF-α mRNA levels and stimulates local increases in antiinflammatory IL-4 mRNA levels in a rat burn model. TNF-α induces keratinocyte apoptosis through interaction with the death receptors of the TNF receptor superfamily and activation of the caspase cascade. IL-1β is a neutrophil chemoattractant and may also contribute to keratinocyte apoptosis. Both cytokines promote collagen degradation through matrix metalloproteinases 1 and 3 activation.45 In a follow-up study, Dasu et al examined the alterations in transcription factors induced by liposomal IGF-1 gene transfer in a rat burn model. The authors found that burn wounds of IGF-1-treated rats showed decreased expression of the proapoptotic proteins Bax and caspase-3 and increased activation of antiapoptotic NF-κB transcription factors and AP1 DNA binding activity.38 These studies highlight the contribution to delayed burn wound healing by prolonged neutrophil and macrophage production of proinflammatory cytokines.52 The decrease in proapoptotic protein expression demonstrated by treatment with IGF-1 in the study by Dasu et al is concordant with the expected outcome of decreasing TNF-α and IL-1β levels. However, the direct effects of IGF-1 vs those induced by decreased levels of proinflammatory cytokines cannot be separated in this study. Wound Infection and Inflammation Bacterial infection and endotoxin production further compound and contribute to the deleterious effects of burn wound inflammatory cell infiltrates. Microbial organisms not only produce toxins and proteases that destroy tissue but also perpetuate the prolonged inflammatory response. As resistance to antibiotics has increased, the use of topical silver in preventing and treating burn wound infections has gained ground. Silver has broad antimicrobial activity and is effective against yeast, mold, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococcus, and other bacteria.53 Indeed, Sawhney et al54 demonstrated that 1% silver sulfadiazine decreased the rate of burn wound progression from deep dermal burns to full-thickness burns and increased the rate of wound healing in burn patients.8 Although the positive impact in terms of burn wound progression attributable to treatment with topical silver is likely due to diminished direct microbial tissue damage, moderation of inflammatory cell infiltration is possibly an additional mechanism of action. Of note the concert of responses exerted by silver species have yet to be fully elucidated. Recent studies conducted with silver nanoparticles suggest that a proliferative advantage may exist compared with silver sulfadiazene.55 In vitro and in vivo models have shown a relative toxicity of silver in dressing products (on examination of keratinocyte and fibroblast function); however, silver species, concentration, wound type, and dressing preparation all seem to contribute to varying conclusions. BURN WOUNDS AND BLOOD FLOW As the name implies, the zone of stasis or the zone of ischemia is an area of decreased circulation. It is believed that the reduced blood flow observed in the reticular dermis of this zone results from a combination of hypercoagulability, fluid shifts and edema, vasoconstriction, neutrophil occlusion, erythrocyte plugging, and oxygen radical-mediated injury to the vascular endothelium.56 Insufficient blood supply in the reticular dermis compromises the viability of the reservoirs of keratinocytes in the skin adnexa, among other cells, leading to delayed wound healing and possibly burn wound progression.57 This section of the article will focus on the first three causes of decreased blood flow because neutrophil plugging of the microcirculation was discussed previously, and the role of oxygen radicals in vascular injury will be elaborated in the next section. Although this article discusses the roles of inflammatory cell infiltrates, blood flow, and oxygen-free radicals in separate sections, it should be appreciated that each of these is very much interrelated. Edema One of the most serious events that occurs after burn injury is local and systemic edema.58 The massive shift of fluid from the intravascular space to the interstitial and intracellular spaces results from alterations in any one of the variables in Starling's equation:  Where Jv is the net capillary filtration rate, Lp is the hydraulic conductivity of the capillary wall, A is the capillary surface area, Pc is the capillary hydrostatic pressure, Pif is the interstitial hydrostatic pressure, ς is the capillary reflection coefficient for plasma proteins, πc is the capillary colloid osmotic pressure, and πif is the interstitial colloid osmotic pressure.59 A positive value of Jv indicates that fluid is moving out of the vascular space, whereas a negative value of Jv indicates that fluid is moving into the vascular space. When Jv is positive and fluid is moving out of the vasculature into the interstitium, the effective blood volume may be reduced and the tissue pressure may be increased, impairing circulation (Figure 6). In the zone of stasis, such impaired circulation may lead to ischemia, tissue necrosis, and progression of a deep partial-thickness burn to a full-thickness burn.43 Figure 6. View largeDownload slide Starling's forces and edema. Figure 6. View largeDownload slide Starling's forces and edema. Vascular Permeability. Alterations in ς have been implicated in the development of postburn edema.60 For reference, when ς = 1, the capillary wall is impermeable to plasma proteins, and when ς = 0, the capillary wall is freely permeable to plasma proteins.59 In normal skin, ς ranges from 0.85 to 0.95. Reductions in ς after burn injury have been demonstrated in canine scald burn models. Pitt et al61 reported a decrease in ς from 0.87 to 0.45 postburn, and Ferrara et al60 reported an immediate and sustained decrease in ς postburn. The consequence of lowering ς is a reduction in the ordinarily resorptive colloid osmotic pressure gradient (πc − πif).59 Increased capillary permeability, corresponding to a decreased ς, may be due to a number of factors. Contraction of venule endothelial cells (with an increase in interendothelial spaces) may be mediated by a variety of agents, including histamine, bradykinin, leukotrienes, substance P, and NO.62,63 Direct endothelial injury, such as that of a burn, may impair an endothelial cell's ability to maintain its transmembrane potential with resulting influx of sodium ions and water, swelling, oncosis, and detachment.56 Direct injury affects arterioles, capillaries, and venules.63 In burns, adherent neutrophils exacerbate the endothelial damage from the direct burn injury with their release of cytotoxic phospholipase, granule contents, and free oxygen radicals.33 Finally, some chemical modulators, such as vascular endothelial growth factor (VEGF), have been shown to increase transcytosis of venule endothelial cells, which is the process whereby proteins and fluids are transported through endothelial cell pores.58,63 A number of investigators have sought to explore the mechanisms and mediators of increased vascular permeability specific to burn injury (Table 3). Table 3. Interventions aimed at modulating postburn edema View Large Table 3. Interventions aimed at modulating postburn edema View Large In 1965, Cotran used carbon labeling and electron microscopy in the study of a rat burn model to elucidate the intricacies of postburn vascular permeability. In mild burn injuries (54°C for 20 seconds), an immediate increased permeability of venules was followed by a delayed, prolonged increased permeability of capillaries. In severe burn injuries (60°C for 20 seconds), all types of vessels—arterioles, capillaries, and venules—showed increased vascular permeability.62 Examination of subepidermal capillaries, dermal vessels, and subcutaneous venules with electron microscopy 1 to 3 hours after mild burn injury (54°C for 20 seconds) showed accumulation of tracer material between endothelial cells and in the basement membrane and 1- to 2-μm gaps between endothelial cells. Of note, a majority of the endothelial cells were normal in appearance with occasional cytoplasmic or organelle swelling. Some intact vessels contained large aggregations of platelets, and a number of intact and leaky venules contained emigrating and marginating leukocytes. Within 1 hour of severe burn injury (60°C for 20 seconds), many endothelial cells exhibited prominent mitochondrial and endoplasmic reticulum swelling, nuclear condensation, and cellular fragmentation. However, some leaky vessels retained normal appearing endothelium. A few vessels were occluded with platelets, fibrin, and endothelial cell debris.62 Cotran62 hypothesized that in mild burn injury, delayed vascular permeability is the result of mediator-induced gaps in the vascular endothelium, and that increased permeability after severe burn injury results partly from direct damage (leaky vessels with disrupted, sloughing endothelial cells) and partly from mediators (leaky vessels with normal appearing endothelial cells). One potential mediator of burn-induced increases in vascular permeability is histamine. Burn trauma stimulates the release of histamine from mast cells, which acts to increase the production of oxygen-free radicals through the induction of xanthine oxidase.75 Substance P, neurokinins, and complement may play a role in triggering histamine release.65 In a rat burn model, it was demonstrated that cromolyn sodium, a mast cell stabilizer, is able to attenuate the increases in histamine and xanthine oxidase postburn and effect a 92.8% decrease in vascular permeability, as assessed by 125I-bovine serum albumin leakage.64 The utility of histamine antagonists in reducing postburn edema is controversial. In the study by Friedl et al, cimetidine, but not diphenhydramine (H1 antagonist), reduced postburn edema as measured by water content and 125I-bovine serum albumin leakage.64 However, the positive results with the H2 antagonist were attributed to the ability of cimetidine to scavenge hydroxyl radicals. In a more recent study in 2003, the contribution of H1, H2, and H3 receptors to postburn vasculature permeability and blood flow was explored.65 To assess vascular permeability they quantified accumulation of Evans blue albumin in burned skin, and to assess blood flow they used LDI. They found that that the H1 receptor antagonist did not effect vascular permeability or blood flow. The H2 receptor antagonist ranitidine actually reduced blood flow in full-thickness burns, most likely due to impeded vasodilation. H3 receptors did not contribute to vascular permeability, but the H3 receptor agonist significantly increased blood flow in partial-thickness and full-thickness burn wounds with a concomitant increase in mean arterial pressure.65 The contribution of histamine to postburn vascular permeability and overall blood flow remains to be fully studied and understood. VEGF is stimulated by ischemia and hypoxia and ultimately results in the neovascularization of a damaged vascular bed.76 VEGF is released by mast cells, macrophages, neutrophils, platelets, and keratinocytes. Potential mechanisms for the vascular permeability induced by VEGF include alterations in intracellular calcium, release of NO with activation of guanylyl cyclase and protein kinase, endothelial fenestration, increased synthesis of platelet-activating factor, and activation of the cyclooxygenase (COX) pathway.58,76 A study by Infanger et al revealed that levels of VEGF in serum are increased in burn patients when compared with control. The increase in VEGF serum levels is seen as early as 1 day postburn and persists until day 35 postburn. Local and general tissue edema, measured by weighing patients, was present in burn patients during the period of increased VEGF. These results indicate that VEGF is important in generating edema after burn injury, and that inhibition of VEGF may potentially ameliorate tissue edema and progression of burn wound depth.58 Another hypothesized method to reduce capillary leak at the site of burn injury is through the use of α-trinositol. α-Trinositol is produced by yeast through the hydrolysis of phytic acid. It was originally studied as an antiinflammatory agent but has also been shown to antagonize the vasoconstrictor neuropeptide-Y and to modulate reactive oxygen species (ROS) production. In burns, α-trinositol acts on the arachidonic acid cascade to reduce inflammation. In a canine burn model, Ferrara et al studied that ability of α-trinositol to reduce capillary leak and found that pretreatment with α-trinositol slightly stabilized the decrease in ς seen after burn injury but that α-trinositol had no effect on ς when it was administered postburn. The authors concluded that from a clinical standpoint, α-trinositol has no utility in preventing capillary leak. However, treatment with α-trinositol postburn did result in an overall decrease in local edema, as measured by weight loss. As postburn increases in the capillary filtration coefficient Kf (equivalent to LpA in the preceding Starling equation) were blunted by α-trinositol, the authors attribute the overall edema reduction to the agent's ability to reduce transvascular fluid flux through stabilizing the transmembrane fluid conductance (permeability of endothelial cells to water).60 These results stand in contrast to those of earlier studies by Lund and Reed66 and Tarnow et al.67 Both groups used a rat model and found that administration of α-trinositol after a burn injury significantly reduced capillary leak as measured by tissue extravasation of albumin.66,67 Perhaps, as suggested, these divergent results can be explained by species difference and different methods of measuring capillary leak (ς vs albumin extravasation).60 Regardless, it is apparent that further research is needed to define the therapeutic role of α-trinositol in reducing postburn edema and progression of burn wounds. Bradykinin, a potent proinflammatory mediator, has been implicated in the development of postburn edema. Bradykinin stimulates the release of NO through interaction with B2 receptors on endothelial cells with resulting increases in vasodilation and vascular permeability.69 A role for B1 receptors in postburn edema has been proposed.77 Nwariaku et al used a rabbit burn model to assess the impact of a B2 antagonist (NPC 17731) on skin blood flow as measured by LDI and edema as measured by weight in the zone of stasis. They found that the bradykinin antagonist improved blood flow in partial-thickness burns and in the zone of stasis between full-thickness burns. The antagonist also resulted in moderate improvement in edema in the zone of stasis but no improvement in edema of partial-thickness burns.68 The mechanism by which bradykinin inhibition may improve postburn edema was explored in a recent study using a sheep. These data outlined a reduction in transvascular fluid flux attributed to the B2 antagonist Icatibant. The treatment with the agent also was associated with a decrease in net transvascular protein flux.69 These studies suggest that inhibition of bradykinin may have a role in the prevention of postburn edema and reduced blood flow through stabilization of transvascular fluid and protein flux. Arachidonic acid metabolites (ie, prostaglandin H, LTA4-F4, prostacyclin [PGI], thromboxane A2 [TXA2], and thromboxane B2) in progressive destruction of the zone of stasis has been studied intensely. Alexander et al reported that ibuprofen (COX inhibitor), FPL 55712 (LTA4 receptor blocker), and ketoconazole (thromboxane synthetase inhibitor) were able to reduce extravasation of Evans blue dye after burn injury regardless of whether the drug was administered preburn or postburn. However, the only agent able to impart a decrease in edema when given after burn injury was FPL 55712. This finding may have resulted from the inhibition of LTA4-induced increases in vascular permeability and vasoconstriction of postcapillary venules.70 Barrow et al71 demonstrated that ibuprofen administered preburn in a rabbit model, through its inhibition of TXA2 production, is able to preserve local tissue perfusion and decrease local edema. The clinical applicability of the finding of Barrow et al is limited, because ibuprofen was given before the burn injury. Yonehara and Yoshimura analyzed the contribution of substance P and NO to postburn alterations in vascular permeability and edema in a rat scald burn model. They found that NO metabolites were locally increased after injury. Chronic pretreatment with a nitric oxide synthase (NOS) inhibitor decreased extravasation of Evans blue. Local administration of substance P increased levels of NO, whereas RP67580 (NK-1R antagonist) inhibited postburn increases in NO and Evans blue leakage.77,72 The authors concluded that burn injury induces the release of substance P from small-diameter afferent fibers, leading to NO production through NK-1R, with ultimate increases in vascular permeability and edema.72 Another group also found that administration of a NOS inhibitor is able to decrease postburn tissue accumulation of Evans blue. However, in their study, both pretreatment and therapeutic administration of the NOS inhibitor was effective.78 Through the earlier discussion it is clear that the control of vascular permeability after a burn injury is complex. A variety of chemical mediators and growth factors—histamine, VEGF, α-trinositol, bradykinin, NO, substance P, and arachidonic acid metabolites—among others have been demonstrated to modulate postburn vascular permeability. Interstitial Hydrostatic Pressure. To account for the extent of tissue edema observed postburn, there must be another mechanism, in addition to increased vascular permeability, contributing to fluid imbalances.59 A number of studies have demonstrated that intradermal Pif transiently decreases immediately after burn injury.79,80 The normal Pif of skin ranges between −2 and +2.81 In a rat burn model, Shimizu et al82 found that dermal Pif significantly decreased in full-thickness burns but not in superficial dermal burns. This suggests that the contribution of a negative Pif to postburn edema may depend on burn depth. The mechanism of negative Pif after burn injury is not well defined, but a possible mechanism involves degradation of collagen.59,83 Thermal injury is able to induce collagen denaturation, transforming the protein into a water-soluble gelatin.59 Rodt et al proposed one model of increased negativity of Pif. According to this model, dermal fibroblasts loosen their attachment to a collagen network, which releases the constraining forces on matrix hyaluronan gel. Hyaluronan gel has an inherent tendency to expand, and as the tissue attempts to swell, it is inhibited by a lack of fluid. Thus, Pif becomes negative until the forces of tissue expansion and negative Pif are balanced.84 Multiple investigators have examined potential ways to mitigate this postburn drop in Pif. Shi and Li proposed the use of HBO to attenuate the drop in Pif observed postburn. In a rat burn model, they observed that rats placed in a high air pressure environment (30 cm H2O) after scalding exhibited less tissue edema than controls.73 Lund and Reed66 demonstrated that when the antiinflammatory agent α-trinositol was given to rats after burn injury, it was able to significantly mitigate the decrease in Pif. Tanaka et al74 found that treating rats after burn injury with vitamin C resulted in a significant attenuation of the increased negativity of Pif, a moderate reduction in total tissue water content, and no effect on albumin extravasation. The beneficial effect of Vitamin C on Pif may be a result of preservation of matrix elements.81 Vasodilation. Yet another mechanism contributing to a net movement of fluid out of the vasculature and into the interstitial space, manifest as edema, is an increase in capillary hydrostatic pressure, Pc. After a burn injury, assuming hemodynamic stability, the initial vasoconstriction mediated by sympathetic discharge is followed by local vasodilation with a concomitant increase in Pc.81 The increase in Pc, as the Starling equation makes evident, contributes to fluid efflux and edema.81 Mediators of this local vasodilation may include leukotrienes, prostaglandins, histamine, serotonin, nitric oxide, and lactate, among others.77,85 The effect of this vasodilation on transvascular fluid exchange and overall blood flow may be nominal when compared with the effects of increased capillary permeability and decreased Pif. Vasoconstriction Although vasodilation may contribute to progressive tissue damage in the zone of stasis through the formation of edema and increased tissue pressure, vasoconstriction also likely plays a role. By using a rabbit burn model, Knabl et al86 found that systemic administration of the vasoconstrictor epinephrine resulted in an increase in burn wound depth (28.6% unburned dermis) when compared with controls (43.5% unburned dermis). One endogenous molecule with vasoconstrictive properties that is increased after burn injury is TXA2. TXA2 stimulates vasoconstriction, platelet aggregation, neutrophil activation, and vascular permeability.71 As discussed earlier, Barrow et al have reported that pretreatment of rabbits with ibuprofen provides for maintenance of local tissue perfusion as well as decreased tissue edema. They assert that the effect of ibuprofen is likely mediated through the inhibition of the arachidonic acid cascade at the level of COX71 with resultant decreases in vasoconstriction, platelet aggregation, and neutrophil activation. Again, the clinical applicability of this study is questionable given the fact that ibuprofen was administered preburn. However, it does highlight the complexity of burn wound progression in that a single pathway is implicated in numerous mechanisms of tissue injury. Furthermore, different products of the same pathway may have redundant or opposite activity. In contrast to the work of Barrow et al, Tan et al found that ibuprofen administered to guinea pigs did not decrease the depth of dermal ischemia after burn injury, as measured by an India ink perfusion technique. They reasoned that the lack of effect of ibuprofen to prevent progressive dermal ischemia may be due to its inhibition of protective PGI2 and collagenase activity.87 Perhaps, the somewhat conflicting results between these two studies are related to the timing of the treatment. Hypercoagulability Platelet aggregation with thrombus formation is another mechanism, which may account for the compromised blood flow in the zone of stasis. Indeed, vessel occlusion by thrombus has been documented by histologic examination of the postburn dermal vasculature.33,62 Compounding the problem of thrombus formation in the postburn vasculature is a reduction in plasmin-mediated fibrinolysis.88,89 The prothrombotic, antifibrinolytic nature of the vascular endothelium after burn injury is due, in part, to the shedding of thrombomodulin. Thrombomodulin is an endothelial cell surface glycoprotein that has important antithrombotic and fibrinolytic properties through its inhibition of thrombin and activation of protein S. It also reduces endothelial cell production of inflammatory cytokines and decreases leukocyte adhesion. Thrombomodulin can be transcriptionally down-regulated by shear stress and hypoxia, among other factors. In burn injury, thrombomodulin is decreased secondary to direct thermal injury and subsequent inflammation. Uygur et al studied the ability of simvastatin to decrease progressive tissue necrosis in the zone of stasis in a rat comb burn model. They found that postburn treatment with simvastatin yielded increased blood flow within the zone of stasis and decreased histologic tissue damage. Because thrombomodulin expression was maintained in simvastatin-treated rats, it was concluded that simvastatin protects the zone of stasis from necrosis through its stimulation of thrombomodulin expression.90 Instead of targeting the restoration of the body's intrinsic antithrombotic, profibrinolytic systems, such as thrombomodulin expression, Isik et al have focused on the administration of an exogenous fibrinolytic agent. They studied the ability of recombinant tissue-type plasminogen activator (r-tPA) to maintain the patency of vessels in the zone of stasis and prevent progressive tissue necrosis in a rat comb burn model. On a macroscopic level, they observed that the r-tPA group interspaces (zones of stasis) appeared alive, whereas those of the control group appeared necrotic. Blood flow in the interspaces, as measured by LDI, was significantly greater on day 7 in the treatment group. By using a radioactive marker and a gamma camera, they found that the mean percentage of surviving interspace and vertical space area was higher in the treatment group at 24 hours and 7 days postburn. The authors postulate that thrombus formation is the main mechanism accounting for the restriction of blood flow in the zone of stasis, and that the administration of a fibrinolytic agent would likely help to salvage the zone of stasis.88 Mahajan et al present another strategy for combating postburn thrombus formation. They investigated rNAPc2, which binds to factor X and inhibits fibrin formation, in a rat comb burn model. The rats treated with rNAPc2 (before and after thermal injury) showed decreased macroscopic tissue destruction in the interspace areas (zones of stasis), increased wound perfusion as assessed by LDI, and decreased cutaneous vascular fibrin content.91 Although this study does show the beneficial effects of anticoagulation on limiting the extension of a burn wound, the administration of rNAPc2 before burn injury limits its clinical applicability. Battal et al explored the utility of a PGI2 analogue (Beraprost) in limiting tissue necrosis in the zone of stasis of a rat comb burn model. In contrast to its TXA2 counterpart, PGI2 acts as a vasodilator as well as a platelet aggregation inhibitor. Results of the study indicated that the PGI2 analogue increased blood flow in the zone of stasis as measured by LDI and decreased edema and necrosis. Histologically, at 24 hours postburn, Beraprost prevented accumulation of neutrophils, parenchymal hemorrhage, and thrombus formation, and at 7 days postburn, it prevented full-thickness tissue necrosis and destruction of hair follicles in the zone of stasis. These researches concluded that Beraprost serves as a potential therapeutic intervention for limiting tissue destruction in the zone of stasis through its effects on platelet aggregation, thrombus formation, neutrophil chemotaxis, and ROS production.92 The positive results from the aforementioned three studies in terms of correlating treatment with reduction in tissue necrosis in the zone of stasis support the idea that vascular occlusion is responsible for a large part of the circulatory deficiency observed in the zone of stasis. The agents targeted different steps of thrombosis and thrombolysis. Simvastatin targeted the expression of thrombomodulin, r-tPA targeted the degradation of fibrin, rNAPc2 targeted the coagulation cascade, and sodium Beraprost targeted platelet aggregation. However, the potential clinical applicability of some of these agents to patients remains questionable, especially in light of the bleeding risks associated with the multiple grafting and debridement surgeries a burn patient may require. There have been attempts to use heparin sulfate as a treatment for burn wounds. A recent analysis of these studies concluded that because of poor experimental design there was no strong evidence to prove the benefit of heparin treatment.93 BURN WOUNDS AND FREE RADICALS A free radical is a molecule with an unpaired outer orbit electron that possesses powerful oxidizing or reducing capacity. ROS refers to both free radicals and other molecules involved in the production of free radicals. Notable ROS are superoxide radical (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH)94 (Figure 7). Under normal conditions, cellular metabolism generates ROS as a byproduct of oxidative phosphorylation in the mitochondria, oxidation of purines by xanthine oxidase, neutrophil activation of the NADPH-dependent oxidase system, and the division of arachidonic acid to prostaglandins and leukotrienes.95 Antioxidants are compounds or enzyme systems that combat the potential cellular damage resulting from ROS. Examples of antioxidants include superoxide dismutase (SOD), catalase, glutathione peroxidase, α-tocopherol (vitamin E), ascorbic acid (vitamin C), and glutathione. When there is an imbalance of ROS compared with antioxidants, a biologic system is under oxidative stress. Mechanisms by which oxidative stress may contribute to progressive tissue destruction in the zone of stasis include lipid peroxidation of cell membranes and disruption of proteins and nucleic acids, culminating in cell death.35,94,96 Figure 7. View largeDownload slide Reactive oxygen species. Figure 7. View largeDownload slide Reactive oxygen species. In general, ROS may mediate apoptosis through the cleavage of PARP, increases in mitochondrial permeability with the release of cytochrome C, and activation of caspases (see General Overview of Cell Death section).97 ROS-triggered release of lysosomal contents can also lead to apoptosis. Hydrogen peroxide can freely diffuse across cell membranes, including the lysosomal membrane. Within the lysosome, hydrogen peroxide can react with iron to form the potent hydroxide radical, ultimately resulting in damage to the lysosomal membrane and leakage of contents. The translocation of the protease cathepsin D from the lysosome to the cytosol results in the induction of apoptosis.98 Source of Oxidative Stress The cause of oxidative stress after burn injury is multifactorial. First, thermal energy is capable of directly producing free radicals by homolytic bond fission.99 Second, there is increased activity of xanthine oxidase and phagocyte-related NADPH oxidase. Recently, attention has been given to the role of increased NO postburn. Inoue et al78 found that inducible NOS-like immunoreactive proteins were detected in the ear tissue of mice after burn injury. Paulsen et al100 demonstrated increased levels of NOS in proliferating keratinocytes, capillary endothelial cells, and arterial smooth muscle cells from human burn wounds. In addition to altering blood flow and initiating the production of inflammatory cytokines through up-regulation of NF-κB, NO interacts with superoxide radical to produce the highly reactive peroxynitrite compound. The increased ROS level is further compounded by reductions in α-tocopherol, ascorbic acid, SOD, catalase, and glutathione antioxidant defenses.101 A number of studies have been conducted to assess the source of ROS within the burn wound and to gauge the utility of inhibitors of free radical production, inhibitors of free radical-mediated injury, and antioxidant replacement in limiting burn wound progression. The relative contribution of xanthine oxidase and phagocyte-related NADPH oxidase to the local increase in ROS postburn is controversial. The mechanism by which xanthine oxidase activity is amplified postburn may involve complement-dependent increases in histamine, which then acts to stimulate the catalytic activity of xanthine oxidase.76 Ischemia-reperfusion injury also contributes to increases in xanthine oxidase activity. Specifically, under conditions of ischemia, hypoxanthine is formed as a by-product of AMP metabolism. The increased levels of hypoxanthine then serve as a substrate for xanthine oxidase when the tissue is reperfused.101 Jeng and coworkers actually demonstrated that fluctuations in burn wound blood flow, as assessed by LDI, mirror cyclic changes in base deficit, reinforcing the idea that ischemia-reperfusion injury leads to free radical production in burn wounds.102 Enhancement of phagocyte-related NADPH oxidase is brought about by phagocyte activation by inflammatory cytokines such as TNF-α, leading to the “respiratory burst” with production of microbicidal ROS.94,103 Till et al report that depleting a rat burn model of neutrophils to less than 250/mm3 did not ameliorate postburn wound edema. They hypothesized that xanthine oxidase is the primary source of ROS because pretreatment with the xanthine oxidase inhibitors, allopurinol and lodoxamide tromethamine, significantly attenuated postburn edema.104 Conversely, a number of other studies have demonstrated the beneficial impact of reducing local neutrophil aggregation on postburn edema, blood flow, and tissue viability in the zone of stasis, presumably due, in part, to decreased levels of neutrophil-derived ROS.33,39,41 Xanthine Oxidase The study by Cetinkale et al demonstrated that pretreatment of rats with the xanthine-oxidase inhibitor, allopurinol, increased perfusion during the first 24 hours postburn in the interspace panniculus carnosus, as assessed by radiopharmaceutical uptake, and decreased interspace edema. It is likely that these results are the consequence of the ability of allopurinol to limit the lipid peroxidation of endothelial cell membranes.41 Another study by different investigators yielded more disappointing results regarding the utility of allopurinol to salvage the zone of stasis. These experiments focused on the effects of allopurinol, dimethyl sulfoxide, and SOD on epithelization and hair follicle retention in a guinea pig burn model. The antioxidants were administered for 5 days postburn. Histologic examination at 1 and 3 weeks showed no difference between animals treated with any of the three antioxidants and controls.105 NADPH Oxidase The work by Fazal described a means by which ROS can be decreased through manipulation of a second important enzyme, NADPH oxidase, that is increased after burn injury. Examination was focused on the role of platelet-activating factor (PAF) in priming neutrophils for subsequent enhanced ROS production in a rat burn model. They found that a PAF receptor antagonist is able to prevent the PAF-potentiated neutrophil ROS response, possibly through modulating the PKC signaling pathway.106 It is believed that postburn increases in phagocyte-related NADPH oxidase play a role in the generation of damaging ROS locally in the burn wound. Paradoxically, some studies suggest that neutrophils in the peripheral blood are actually deficient in NADPH activity, leaving the burn patient susceptible to infection.107,108 It is clear that local and systemic neutrophil NADPH activity needs further definition. Lipid Peroxidation The polyunsaturated fatty acids in cell membranes are a main target of free radicals. Lipid peroxidation results in decreased membrane fluidity, thereby altering membrane-bound proteins.35 Indeed, in animal models of burn injury, malondialdehyde (MDA) and conjugated dienes, end products of lipid peroxidation, are increased locally in the burn wound, plasma, and peripheral tissues.109,110 Choi et al used a rat comb burn model to study the role that lipid peroxidation plays in progressive tissue destruction in the zone of stasis. Specifically, they compared blood flow, vascular patency, tissue necrosis, and lipid peroxidation in the zones of stasis in rats treated with U75412E, a lipid peroxidation inhibitor, and control rats. They found that at 16 and 24 hours postburn, blood flow in the interspaces of U75412E-treated rats was significantly higher than that of control rats. At 24 hours postburn, 4 of 30 interspaces in U75412E-treated rats became necrotic, whereas 60 of 60 interspaces in control rats became necrotic. At 5 days postburn, 26 of U75412E-treated rat interspaces were viable with hair growth, whereas control rat interspaces were necrotic with eschars. Also, at 24 hours postburn, latex casting of vessels revealed that vasculature in the interspaces of U75412E-treated rats remained patent, whereas vasculature in the interspaces of control rats was occluded secondary to thrombosis. The conjugated diene level in the interspaces of U75412E-treated rats never increased above baseline, whereas that in the interspaces of control rats increased three times from baseline. This study by Choi et al demonstrates that U75412E is able to reverse tissue loss in the zone of stasis by preserving skin appendages. They conclude that U75412E was capable of maintaining vascular patency and blood flow through the inhibition of lipid peroxidation, as demonstrated by the absence of an increase in conjugated dienes in the interspaces of U75412E-treated rats, thereby protecting endothelial cell membranes.111 Rosiglitazone is a peroxisome proliferator-activated receptor-δ agonist. Rosiglitazone is reported to modulate inflammatory responses by decreasing IL-6, IL-1B, and TNF-α levels, inhibiting inflammatory gene expression, and reducing levels of NO, inducible NOS, and neutrophil-derived free radicals.109,112,113 Taira et al recently looked at the ability of rosiglitazone to beneficially impact tissue levels of ROS, macroscopic interspace necrosis, and microscopic reepithelialization, neutrophil infiltration, and thrombosis. Tissue levels of ROS, as assessed by quantification of the lipid peroxidation by-product MDA, were lower in treated rats compared with control rats, although the difference failed to reach statistical significance. On postburn days 3 to 7, the rosiglitazone-treated animals had a significantly lower rate of interspace necrosis. Although the rosiglitazone-treated rats did demonstrate histologic evidence of decreased neutrophil infiltration and thrombosis and increased reepithelialization, these findings were not statistically significant. In showing a parallel decrease in interspace necrosis and MDA levels, this study supports the involvement of ROS and lipid peroxidation in burn wound progression and suggests that a peroxisome proliferator-activated receptor-δ agonist may be able to modulate such progression.109 Antioxidants and Free Radical Scavengers One component of the cellular antioxidant system is esterified glutathione (GSH). This molecule acts as an electron acceptor for hydrogen peroxide by forming an oxidized thiol and then undergoing degradation by glutathione reductase. GSH levels have been reported to be decreased in lung, liver, and kidney of rats after burn injury.114 Zor et al found that on postburn day 10, the interspaces of glutathione-treated rats were macroscopically viable, whereas those of control rats were necrotic. Nuclear imaging and autoradiography confirmed that the percentage of surviving interspace tissue was higher in the treated rats (87.8%) compared with controls (26.9%).115 This study suggests that antioxidant therapy, specifically glutathione replacement, may be a practical therapy for limiting progressive destruction in the zone of stasis. Additional important antioxidants that are decreased after burn injury include vitamins C and E. A number of studies have indicated that postburn, vitamin C supplementation in animal models leads to positive systemic and local effects. For example, Matsuda et al116 reported that resuscitation with high-dose vitamin C led to increased cardiac output and decreased burn wound edema in a guinea pig burn model. Tanaka et al74 later reported that vitamin C supplementation in a rat burn model results in attenuation of the postburn drop in Pif and total tissue water content, possibly through its matrix stabilization and antioxidant properties. With regard to levels of vitamin E after burn injury, Shimoda et al117 demonstrated that in a sheep model of inhalation and burn injury, vitamin E levels in the plasma, liver, and lung are decreased. Naziro˘glu et al found that intraperitoneal administration of α-tocopherol led to decreased MDA levels in plasma, liver, and skin and increased superoxide dismutase activity in skin and red blood cells in a guinea pig burn model. The authors proposed that the protective effect of α-tocopherol may be the result of upregulation of the cellular enzymatic antioxidant machinery.118 Yet another agent that has been studied to combat postburn oxidative stress is the tryptophan derivation melatonin. Melatonin acts both directly as a free radical scavenger and indirectly to stimulate a cell's antioxidant enzymes, such as SOD and glutathione reductase. In burn patients, melatonin levels may be low secondary to inflammatory cytokine inhibition of pineal gland serotonin, a precursor of melatonin.119 Sener et al120 used a rat burn model to demonstrate that intraperitoneal administration of melatonin results in increased GSH and decreased MDA levels in the lung, liver, and intestines. Tunali et al121 also used a rat burn model and showed that intraperitoneal melatonin administration normalized prothrombin time and fibrin degradation product levels, decreased skin lipid peroxidation, and increased skin GSH levels. Thus, because melatonin is able to modify oxidative stress both peripherally and locally in the skin, it may serve as a therapeutic intervention for limiting free radical-induced burn wound progression. Table 4 outlines interventions that have attempted to modulate free radical damage. Table 4. Interventions aimed at modulating free radical damage View Large Table 4. Interventions aimed at modulating free radical damage View Large SUMMARY AND CONCLUSION Despite significant technological advances since Jackson's 1969 rethinking of the viability of the zone of stasis, a single unifying cause of progressive tissue necrosis in the zone of stasis has failed to be revealed. This article has explored a selection of the prominent local mediators of burn wound progression; namely, inflammatory cell infiltration, compromised perfusion, and oxidative stress. After burn injury, the burn wound experiences a prolonged inflammatory response in which neutrophils release cytotoxic cytokines and ROS. Moreover, persistent neutrophil aggregation in postcapillary venules contributes to vascular occlusion and edema. Interstitial edema, and the resulting increased tissue pressure and vascular congestion, is exacerbated by increases in vascular permeability and a drop in the interstitial hydrostatic pressure. Perfusion to the burn wound is further reduced secondary to hypercoagulability and thrombus formation. Oxidative stress, because of increased xanthine oxidase and NADPH oxidase activity as well as reductions in antioxidant mechanisms, alters vital cellular molecules, including lipids, proteins, and nucleic acids. Many of the described studies have attempted to address the problem of burn wound progression by targeting one of the aforementioned mediators. For example, investigators pursued neutrophil infiltration by using an antibody to a cell surface adhesion receptor, whereas others focused on replenishing a cell's antioxidant stores with glutathione replacement. The success of both of these research teams in terms of demonstrating reduced necrosis in the zone of stasis reflects both the varied sources of burn wound progression (eg, neutrophil infiltration and oxidative stress) and the interrelatedness of these sources (eg, ROS released by aggregated neutrophils). The ultimate point of convergence of the multiple mediators of burn wound progression is cell death. It has been demonstrated that cell death in the vicinity of the burn wound occurs through apoptosis and oncosis.4,5,22,–24,123,49 Perhaps, future research regarding the progression of burn wounds will be centered on the development of small molecules capable of blocking steps in these pathways, which are common to multiple mediators. Indeed, the promise of such intervention is highlighted by the work of Giles et al,25 who have already successfully used topical administration of c-Jun in burned mice. REFERENCES 1. Jackson DM [The diagnosis of the depth of burning.]. Br J Surg  1953; 40: 588– 96. Google Scholar CrossRef Search ADS PubMed  2. Koenig PA Diagnosis of depth of burning. Br Med J  1965; 1: 1622– 3. Google Scholar CrossRef Search ADS PubMed  3. Jackson DM Second thoughts on the burn wound. J Trauma  1969; 9: 839– 62. Google Scholar CrossRef Search ADS PubMed  4. Gravante G, Filingeri V, Delogu D, et al.   Apoptotic cell death in deep partial thickness burns by coexpression analysis of TUNEL and Fas. Surgery  2006; 139: 854– 5. Google Scholar CrossRef Search ADS PubMed  5. Gravante G, Palmieri MB, Esposito G, et al.   Apoptotic death in deep partial thickness burns vs. normal skin of burned patients. J Surg Res  2007; 141: 141– 5. Google Scholar CrossRef Search ADS PubMed  6. Johnson RM, Richard R. Partial-thickness burns: identification and management. Adv Skin Wound Care 2003;16:178–87; quiz 188–9. 7. Shakespeare P Burn wound healing and skin substitutes. Burns  2001; 27: 517– 22. Google Scholar CrossRef Search ADS PubMed  8. Singh V, Devgan L, Bhat S, Milner SM The pathogenesis of burn wound conversion. Ann Plast Surg  2007; 59: 109– 15. Google Scholar CrossRef Search ADS PubMed  9. Grunwald TB, Garner WL Acute burns. Plast Reconstr Surg  2008; 121: 311e– 9e. Google Scholar CrossRef Search ADS PubMed  10. Wolff K, Goldsmith LA, Katz SI, Gilchrest BA, Paller AS, Leffell DJ. Fitzpatrick's Dermatology in General Medicine. 7th ed. Chapter 249: Wound repair: mechanisms and practical considerations. McGraw-Hill; 2008. Available at http://www.accessmedicine.com/resourceTOC.aspx?resourceID=505. 11. Zawacki BE Reversal of capillary stasis and prevention of necrosis in burns. Ann Surg  1974; 180: 98– 102. Google Scholar CrossRef Search ADS PubMed  12. Fink SL, Cookson BT Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun  2005; 73: 1907– 16. Google Scholar CrossRef Search ADS PubMed  13. Golstein P, Kroemer G Cell death by necrosis: towards a molecular definition. Trends Biochem Sci  2007; 32: 37– 43. Google Scholar CrossRef Search ADS PubMed  14. Edinger AL, Thompson CB Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol  2004; 16: 663– 9. Google Scholar CrossRef Search ADS PubMed  15. Hotchkiss RS, Strasser A, McDunn JE, Swanson PE Cell death. N Engl J Med  2009; 361: 1570– 83. Google Scholar CrossRef Search ADS PubMed  16. Majno G, Joris I Apoptosis, oncosis, and necrosis. An overview of cell death. Am J Pathol  1995; 146: 3– 15. Google Scholar PubMed  17. Beurel E, Jope RS The paradoxical pro- and anti-apoptotic actions of GSK3 in the intrinsic and extrinsic apoptosis signaling pathways. Prog Neurobiol  2006; 79: 173– 89. Google Scholar CrossRef Search ADS PubMed  18. Hengartner MO The biochemistry of apoptosis. Nature  2000; 407: 770– 6. Google Scholar CrossRef Search ADS PubMed  19. Contassot E, Gaide O, French LE Death receptors and apoptosis. Dermatol Clin  2007; 25: 487– 501; vii. Google Scholar CrossRef Search ADS PubMed  20. Iwasaki K, Izawa M, Mihara M Thermal injury induces both necrosis and apoptosis in rat skin. Br J Dermatol  1997; 137: 647– 8. Google Scholar CrossRef Search ADS PubMed  21. Matylevitch NP, Schuschereba ST, Mata JR, et al.   Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury. Am J Pathol  1998; 153: 567– 77. Google Scholar CrossRef Search ADS PubMed  22. Gravante G, Delogu D, Palmieri MB, Santeusanio G, Montone A, Esposito G Inverse relationship between the apoptotic rate and the time elapsed from thermal injuries in deep partial thickness burns. Burns  2008; 34: 228– 33. Google Scholar CrossRef Search ADS PubMed  23. Gravante G, Palmieri MB, Esposito G, et al.   Dermal apoptosis is not influenced by the severity of clinical conditions in burned patients. Eur Rev Med Pharmacol Sci  2006; 10: 207– 9. Google Scholar PubMed  24. Singer AJ, McClain SA, Taira BR, Guerriero JL, Zong W Apoptosis and necrosis in the ischemic zone adjacent to third degree burns. Acad Emerg Med  2008; 15: 549– 54. Google Scholar CrossRef Search ADS PubMed  25. Giles N, Rea S, Beer T, Wood FM, Fear MW A peptide inhibitor of c-Jun promotes wound healing in a mouse full-thickness burn model. Wound Repair Regen  2008; 16: 58– 64. Google Scholar CrossRef Search ADS PubMed  26. Brach MA, Gruss HJ, Sott C, Herrmann F The mitogenic response to tumor necrosis factor alpha requires c-Jun/AP-1. Mol Cell Biol  1993; 13: 4284– 90. Google Scholar CrossRef Search ADS PubMed  27. Lee HY, Youn SW, Kim JY, et al.   FOXO3a turns the tumor necrosis factor receptor signaling towards apoptosis through reciprocal regulation of c-Jun N-terminal kinase and NF-kappaB. Arterioscler Thromb Vasc Biol  2008; 28: 112– 20. Google Scholar CrossRef Search ADS PubMed  28. Godar DE Preprogrammed and programmed cell death mechanisms of apoptosis: UV-induced immediate and delayed apoptosis. Photochem Photobiol  1996; 63: 825– 30. Google Scholar CrossRef Search ADS PubMed  29. Godar DE UVA1 radiation triggers two different final apoptotic pathways. J Invest Dermatol  1999; 112: 3– 12. Google Scholar CrossRef Search ADS PubMed  30. Godar DE Singlet oxygen-triggered immediate preprogrammed apoptosis. Methods Enzymol  2000; 319: 309– 30. Google Scholar CrossRef Search ADS PubMed  31. Sheehan JM, Young AR The sunburn cell revisited: an update on mechanistic aspects. Photochem Photobiol Sci  2002; 1: 365– 77. Google Scholar CrossRef Search ADS PubMed  32. Chitnis D, Dickerson C, Munster AM, Winchurch RA Inhibition of apoptosis in polymorphonuclear neutrophils from burn patients. J Leukoc Biol  1996; 59: 835– 9. Google Scholar PubMed  33. Bucky LP, Vedder NB, Hong HZ, et al.   Reduction of burn injury by inhibiting CD18-mediated leukocyte adherence in rabbits. Plast Reconstr Surg  1994; 93: 1473– 80. Google Scholar CrossRef Search ADS PubMed  34. Han YP, Tuan TL, Wu H, Hughes M, Garner WL TNF-alpha stimulates activation of pro-MMP2 in human skin through NF-(kappa)B mediated induction of MT1-MMP. J Cell Sci  2001; 114: 131– 9. Google Scholar PubMed  35. Parihar A, Parihar MS, Milner S, Bhat S Oxidative stress and anti-oxidative mobilization in burn injury. Burns  2008; 34: 6– 17. Google Scholar CrossRef Search ADS PubMed  36. Moore FD Jr, Davis C, Rodrick M, Mannick JA, Fearon DT Neutrophil activation in thermal injury as assessed by increased expression of complement receptors. N Engl J Med  1986; 314: 948– 53. Google Scholar CrossRef Search ADS PubMed  37. Hu Z, Sayeed MM Activation of PI3-kinase/PKB contributes to delay in neutrophil apoptosis after thermal injury. Am J Physiol Cell Physiol  2005; 288: C1171– 8. Google Scholar CrossRef Search ADS PubMed  38. Dasu MR, Herndon DN, Nesic O, Perez-Polo JR IGF-I gene transfer effects on inflammatory elements present after thermal trauma. Am J Physiol Regul Integr Comp Physiol  2003; 285: R741– 6. Google Scholar CrossRef Search ADS PubMed  39. Mileski W, Borgstrom D, Lightfoot E, et al.   Inhibition of leukocyte-endothelial adherence following thermal injury. J Surg Res  1992; 52: 334– 9. Google Scholar CrossRef Search ADS PubMed  40. Ogura H, Hashiguchi N, Tanaka H, et al.   Long-term enhanced expression of heat shock proteins and decelerated apoptosis in polymorphonuclear leukocytes from major burn patients. J Burn Care Rehabil  2002; 23: 103– 9. Google Scholar CrossRef Search ADS PubMed  41. Cetinkale O, Demir M, Sayman HB, Ayan F, Onsel C Effects of allopurinol, ibuprofen and cyclosporin A on local microcirculatory disturbance due to burn injuries. Burns  1997; 23: 43– 9. Google Scholar CrossRef Search ADS PubMed  42. Hu Z, Sayeed MM Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury. Am J Physiol Cell Physiol  2004; 286: C170– 8. Google Scholar CrossRef Search ADS PubMed  43. Baskaran H, Yarmush ML, Berthiaume F Dynamics of tissue neutrophil sequestration after cutaneous burns in rats. J Surg Res  2000; 93: 88– 96. Google Scholar CrossRef Search ADS PubMed  44. Ipaktchi K, Mattar A, Niederbichler AD, et al.   Topical p38MAPK inhibition reduces dermal inflammation and epithelial apoptosis in burn wounds. Shock  2006; 26: 201– 9. Google Scholar CrossRef Search ADS PubMed  45. Spies M, Nesic O, Barrow RE, Perez-Polo JR, Herndon DN Liposomal IGF-1 gene transfer modulates pro- and anti-inflammatory cytokine mRNA expression in the burn wound. Gene Ther  2001; 8: 1409– 15. Google Scholar CrossRef Search ADS PubMed  46. van de Goot F, Krijnen PA, Begieneman MP, Ulrich MM, Middelkoop E, Niessen HW Acute inflammation is persistent locally in burn wounds: a pivotal role for complement and C-reactive protein. J Burn Care Res  2009; 30: 274– 80. Google Scholar CrossRef Search ADS PubMed  47. Henze U, Lennartz A, Hafemann B, Goldmann C, Kirkpatrick CJ, Klosterhalfen B The influence of the C1-inhibitor BERINERT and the protein-free haemodialysate ACTIHAEMYL20% on the evolution of the depth of scald burns in a porcine model. Burns  1997; 23: 473– 7. Google Scholar CrossRef Search ADS PubMed  48. Mileski WJ, Burkhart D, Hunt JL, et al.   Clinical effects of inhibiting leukocyte adhesion with monoclonal antibody to intercellular adhesion molecule-1 (enlimomab) in the treatment of partial-thickness burn injury. J Trauma  2003; 54: 950– 8. Google Scholar CrossRef Search ADS PubMed  49. Singer AJ, McClain SA, Hacht G, Batchkina G, Simon M Semapimod reduces the depth of injury resulting in enhanced re-epithelialization of partial-thickness burns in swine. J Burn Care Res  2006; 27: 40– 9. Google Scholar CrossRef Search ADS PubMed  50. Suber F, Carroll MC, Moore FD Innate response to self-antigen significantly exacerbates burn wound depth. Proc Natl Acad Sci USA  2007; 104: 3973– 7. Google Scholar CrossRef Search ADS PubMed  51. Cotran RS, Majno G The delayed and prolonged vascular leakage in inflammation. I. Topography of the leaking vessels after thermal Injury. Am J Pathol  1964; 45: 261– 81. Google Scholar PubMed  52. Hubner G, Brauchle M, Smola H, Madlener M, Fassler R, Werner S Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice. Cytokine  1996; 8: 548– 56. Google Scholar CrossRef Search ADS PubMed  53. Atiyeh BS, Costagliola M, Hayek SN, Dibo SA Effect of silver on burn wound infection control and healing: review of the literature. Burns  2007; 33: 139– 48. Google Scholar CrossRef Search ADS PubMed  54. Sawhney CP, Sharma RK, Rao KR, Kaushish R Long-term experience with 1 per cent topical silver sulphadiazine cream in the management of burn wounds. Burns  1989; 15: 403– 6. Google Scholar CrossRef Search ADS PubMed  55. Liu X, Lee PY, Ho CM, et al. Silver nanoparticles mediate differential responses in keratinocytes and fibroblasts during skin wound healing. ChemMedChem 5:468–75. 56. Baskaran H, Toner M, Yarmush ML, Berthiaume F Poloxamer-188 improves capillary blood flow and tissue viability in a cutaneous burn wound. J Surg Res  2001; 101: 56– 61. Google Scholar CrossRef Search ADS PubMed  57. Papp A, Romppanen E, Lahtinen T, Uusaro A, Harma M, Alhava E Red blood cell and tissue water content in experimental thermal injury. Burns  2005; 31: 1003– 6. Google Scholar CrossRef Search ADS PubMed  58. Infanger M, Schmidt O, Kossmehl P, Grad S, Ertel W, Grimm D Vascular endothelial growth factor serum level is strongly enhanced after burn injury and correlated with local and general tissue edema. Burns  2004; 30: 305– 11. Google Scholar CrossRef Search ADS PubMed  59. Lund T, Onarheim H, Reed RK Pathogenesis of edema formation in burn injuries. World J Surg  1992; 16: 2– 9. Google Scholar CrossRef Search ADS PubMed  60. Ferrara JJ, Kukuy EL, Gilman DA, Choe EU, Franklin EW, Flint LM Alpha-trinositol reduces edema formation at the site of scald injury. Surgery  1998; 123: 36– 45. Google Scholar CrossRef Search ADS PubMed  61. Pitt RM, Parker JC, Jurkovich GJ, Taylor AE, Curreri PW Analysis of altered capillary pressure and permeability after thermal injury. J Surg Res  1987; 42: 693– 702. Google Scholar CrossRef Search ADS PubMed  62. Cotran RS The delayed and prolonged vascular leakage in inflammation. II. An Electron microscopic study of the vascular response after thermal injury. Am J Pathol  1965; 46: 589– 620. Google Scholar PubMed  63. Kumar V, Abbas AK, Fausto N, Aster JC. Robbins and Cotran Pathologic Basis of Disease, Professional Edition. 8th ed. Chapter 2: Acute and chronic inflammation. Philadelphia: Saunders. Available at http://www.mdconsult.com/das/book/body/221411575-2/0/2060/28.html. 64. Friedl HP, Till GO, Trentz O, Ward PA Roles of histamine, complement and xanthine oxidase in thermal injury of skin. Am J Pathol  1989; 135: 203– 17. Google Scholar PubMed  65. Räntfors J, Cassuto J Role of histamine receptors in the regulation of edema and circulation postburn. Burns  2003; 29: 769– 77. Google Scholar CrossRef Search ADS PubMed  66. Lund T, Reed RK Alpha-trinositol inhibits edema generation and albumin extravasation in thermally injured skin. J Trauma  1994; 36: 761– 5. Google Scholar CrossRef Search ADS PubMed  67. Tarnow P, Jonsson A, Nellgard P, Cassuto J Reduced albumin extravasation in experimental rat skin and muscle burn injury by D-myo-inositol-1,2,6-trisphosphate treatment. J Burn Care Rehabil  1996; 17: 207– 12. Google Scholar CrossRef Search ADS PubMed  68. Nwariaku FE, Sikes PJ, Lightfoot E, Mileski WJ, Baxter C Effect of a bradykinin antagonist on the local inflammatory response following thermal injury. Burns  1996; 22: 324– 7. Google Scholar CrossRef Search ADS PubMed  69. Jonkam CC, Enkhbaatar P, Nakano Y, et al.   Effects of the bradykinin B2 receptor antagonist icatibant on microvascular permeability after thermal injury in sheep. Shock  2007; 28: 704– 9. Google Scholar PubMed  70. Alexander F, Mathieson M, Teoh KH, et al.   Arachidonic acid metabolites mediate early burn edema. J Trauma  1984; 24: 709– 12. Google Scholar CrossRef Search ADS PubMed  71. Barrow RE, Ramirez RJ, Zhang XJ Ibuprofen modulates tissue perfusion in partial-thickness burns. Burns  2000; 26: 341– 6. Google Scholar CrossRef Search ADS PubMed  72. Yonehara N, Yoshimura M Interaction between nitric oxide and substance P on heat-induced inflammation in rat paw. Neurosci Res  2000; 36: 35– 43. Google Scholar CrossRef Search ADS PubMed  73. Shi Z, Li C The effect of air pressure on edema and healing of scalded tissue of rats. J Burn Care Res  2007; 28: 286– 90. Google Scholar CrossRef Search ADS PubMed  74. Tanaka H, Lund T, Wiig H, et al.   High dose vitamin C counteracts the negative interstitial fluid hydrostatic pressure and early edema generation in thermally injured rats. Burns  1999; 25: 569– 74. Google Scholar CrossRef Search ADS PubMed  75. Santos FX, Arroyo C, Garcia I, et al.   Role of mast cells in the pathogenesis of postburn inflammatory response: reactive oxygen species as mast cell stimulators. Burns  2000; 26: 145– 7. Google Scholar CrossRef Search ADS PubMed  76. Hippenstiel S, Krull M, Ikemann A, Risau W, Clauss M, Suttorp N VEGF induces hyperpermeability by a direct action on endothelial cells. Am J Physiol  1998; 274: L678– 84. Google Scholar PubMed  77. Rawlingson A Nitric oxide, inflammation and acute burn injury. Burns  2003; 29: 631– 40. Google Scholar CrossRef Search ADS PubMed  78. Inoue H, Ando K, Wakisaka N, Matsuzaki K, Aihara M, Kumagai N Effects of nitric oxide synthase inhibitors on vascular hyperpermeability with thermal injury in mice. Nitric Oxide  2001; 5: 334– 42. Google Scholar CrossRef Search ADS PubMed  79. Lund T, Wiig H, Reed RK Acute postburn edema: role of strongly negative interstitial fluid pressure. Am J Physiol  1988; 255: H1069– 74. Google Scholar PubMed  80. Onarheim H, Reed RK Thermal skin injury: effect of fluid therapy on the transcapillary colloid osmotic gradient. J Surg Res  1991; 50: 272– 8. Google Scholar CrossRef Search ADS PubMed  81. Demling RH The burn edema process: current concepts. J Burn Care Rehabil  2005; 26: 207– 27. Google Scholar CrossRef Search ADS PubMed  82. Shimizu S, Tanaka H, Sakaki S, Yukioka T, Matsuda H, Shimazaki S Burn depth affects dermal interstitial fluid pressure, free radical production, and serum histamine levels in rats. J Trauma  2002; 52: 683– 7. Google Scholar PubMed  83. Lund T, Onarheim H, Wiig H, Reed RK Mechanisms behind increased dermal imbibition pressure in acute burn edema. Am J Physiol  1989; 256: H940– 8. Google Scholar PubMed  84. Rodt SA, Reed RK, Ljungstrom M, Gustafsson TO, Rubin K The anti-inflammatory agent alpha-trinositol exerts its edema-preventing effects through modulation of beta 1 integrin function. Circ Res  1994; 75: 942– 8. Google Scholar CrossRef Search ADS PubMed  85. Taheri PA, Lippton HL, Force SD, et al.   Analysis of regional hemodynamic regulation in response to scald injury. J Clin Invest  1994; 93: 147– 54. Google Scholar CrossRef Search ADS PubMed  86. Knabl JS, Bauer W, Andel H, et al.   Progression of burn wound depth by systemical application of a vasoconstrictor: an experimental study with a new rabbit model. Burns  1999; 25: 715– 21. Google Scholar CrossRef Search ADS PubMed  87. Tan Q, Lin Z, Ma W, et al.   Failure of Ibuprofen to prevent progressive dermal ischemia after burning in guinea pigs. Burns  2002; 28: 443– 8. Google Scholar CrossRef Search ADS PubMed  88. Isik S, Sahin U, Ilgan S, Guler M, Gunalp B, Selmanpakoglu N Saving the zone of stasis in burns with recombinant tissue-type plasminogen activator (r-tPA): an experimental study in rats. Burns  1998; 24: 217– 23. Google Scholar CrossRef Search ADS PubMed  89. Rockwell WB, Ehrlich HP Fibrinolysis inhibition in human burn blister fluid. J Burn Care Rehabil  1990; 11: 1– 6. Google Scholar CrossRef Search ADS PubMed  90. Uygur F, Evinc R, Urhan M, Celikoz B, Haholu A Salvaging the zone of stasis by simvastatin: an experimental study in rats. J Burn Care Res  2009; 30: 872– 9. Google Scholar CrossRef Search ADS PubMed  91. Mahajan AL, Tenorio X, Pepper MS, et al.   Progressive tissue injury in burns is reduced by rNAPc2. Burns  2006; 32: 957– 63. Google Scholar CrossRef Search ADS PubMed  92. Battal MN, Hata Y, Matsuka K, et al.   Reduction of progressive burn injury by using a new nonselective endothelin-A and endothelin-B receptor antagonist, TAK-044: an experimental study in rats. Plast Reconstr Surg  1997; 99: 1610– 9. Google Scholar CrossRef Search ADS PubMed  93. Oremus M, Hanson MD, Whitlock R, et al.   A systematic review of heparin to treat burn injury. J Burn Care Res  2007; 28: 794– 804. Google Scholar CrossRef Search ADS PubMed  94. Latha B, Babu M The involvement of free radicals in burn injury: a review. Burns  2001; 27: 309– 17. Google Scholar CrossRef Search ADS PubMed  95. Horton JW Free radicals and lipid peroxidation mediated injury in burn trauma: the role of antioxidant therapy. Toxicology  2003; 189: 75– 88. Google Scholar CrossRef Search ADS PubMed  96. Haycock JW, Ralston DR, Morris B, Freedlander E, MacNeil S Oxidative damage to protein and alterations to antioxidant levels in human cutaneous thermal injury. Burns  1997; 23: 533– 40. Google Scholar CrossRef Search ADS PubMed  97. Li C, Jackson RM Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol  2002; 282: C227– 41. Google Scholar CrossRef Search ADS PubMed  98. Roberg K, Ollinger K Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes. Am J Pathol  1998; 152: 1151– 6. Google Scholar PubMed  99. Slater TF Free-radical mechanisms in tissue injury. Biochem J  1984; 222: 1– 15. Google Scholar CrossRef Search ADS PubMed  100. Paulsen SM, Wurster SH, Nanney LB Expression of inducible nitric oxide synthase in human burn wounds. Wound Repair Regen  1998; 6: 142– 8. Google Scholar CrossRef Search ADS PubMed  101. Horton JW, White DJ, Maass DL, Hybki DP, Haudek S, Giroir B. Antioxidant vitamin therapy alters burn trauma-mediated cardiac NF-kappaB activation and cardiomyocyte cytokine secretion. J Trauma 2001;50:397–406; discussion 407–8. 102. Jaskille AD, Jeng JC, Sokolich JC, Lunsford P, Jordan MH Repetitive ischemia-reperfusion injury: a plausible mechanism for documented clinical burn-depth progression after thermal injury. J Burn Care Res  2007; 28: 13– 20. Google Scholar CrossRef Search ADS PubMed  103. Dang PM, Elbim C, Marie JC, Chiandotto M, Gougerot-Pocidalo MA, El-Benna J Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity. FASEB J  2006; 20: 1504– 6. Google Scholar CrossRef Search ADS PubMed  104. Till GO, Guilds LS, Mahrougui M, Friedl HP, Trentz O, Ward PA Role of xanthine oxidase in thermal injury of skin. Am J Pathol  1989; 135: 195– 202. Google Scholar PubMed  105. Melikian V, Laverson S, Zawacki B Oxygen-derived free radical inhibition in the healing of experimental zone-of-stasis burns. J Trauma  1987; 27: 151– 4. Google Scholar CrossRef Search ADS PubMed  106. Fazal N, Al-Ghoul WM, Choudhry MA, Sayeed MM PAF receptor antagonist modulates neutrophil responses with thermal injury in vivo. Am J Physiol Cell Physiol  2001; 281: C1310– 7. Google Scholar PubMed  107. Parment K, Zetterberg A, Ernerudh J, Bakteman K, Steinwall I, Sjoberg F Long-term immunosuppression in burned patients assessed by in vitro neutrophil oxidative burst (Phagoburst). Burns  2007; 33: 865– 71. Google Scholar CrossRef Search ADS PubMed  108. Rosenthal J, Thurman GW, Cusack N, Peterson VM, Malech HL, Ambruso DR Neutrophils from patients after burn injury express a deficiency of the oxidase components p47-phox and p67-phox. Blood  1996; 88: 4321– 9. Google Scholar PubMed  109. Taira BR, Singer AJ, McClain SA, et al.   Rosiglitazone, a PPAR-gamma ligand, reduces burn progression in rats. J Burn Care Res  2009; 30: 499– 504. Google Scholar CrossRef Search ADS PubMed  110. Till GO, Hatherill JR, Tourtellotte WW, Lutz MJ, Ward PA Lipid peroxidation and acute lung injury after thermal trauma to skin. Evidence of a role for hydroxyl radical. Am J Pathol  1985; 119: 376– 84. Google Scholar PubMed  111. Choi M, Ehrlich HP U75412E, a lazaroid, prevents progressive burn ischemia in a rat burn model. Am J Pathol  1993; 142: 519– 28. Google Scholar PubMed  112. Cuzzocrea S, Pisano B, Dugo L, et al.   Rosiglitazone, a ligand of the peroxisome proliferator-activated receptor-gamma, reduces acute inflammation. Eur J Pharmacol  2004; 483: 79– 93. Google Scholar CrossRef Search ADS PubMed  113. Rizzo G, Fiorucci S PPARs and other nuclear receptors in inflammation. Curr Opin Pharmacol  2006; 6: 421– 7. Google Scholar CrossRef Search ADS PubMed  114. Sener G, Sehirli AO, Gedik N, Dulger GA Rosiglitazone, a PPAR-gamma ligand, protects against burn-induced oxidative injury of remote organs. Burns  2007; 33: 587– 93. Google Scholar CrossRef Search ADS PubMed  115. Zor F, Ozturk S, Deveci M, Karacalioglu O, Sengezer M Saving the zone of stasis: is glutathione effective? Burns  2005; 31: 972– 6. Google Scholar CrossRef Search ADS PubMed  116. Matsuda T, Tanaka H, Shimazaki S, et al.   High-dose vitamin C therapy for extensive deep dermal burns. Burns  1992; 18: 127– 31. Google Scholar CrossRef Search ADS PubMed  117. Shimoda K, Nakazawa H, Traber MG, Traber DL, Nozaki M Plasma and tissue vitamin E depletion in sheep with burn and smoke inhalation injury. Burns  2008; 34: 1137– 41. Google Scholar CrossRef Search ADS PubMed  118. Naziro˘glu M, Kökçam I, Yilmaz S Beneficial effects of intraperitoneally administered alpha-tocopheryl acetate on the levels of lipid peroxide and activity of glutathione peroxidase and superoxide dismutase in skin, blood and liver of thermally injured guinea pigs. Skin Pharmacol Appl Skin Physiol  2003; 16: 36– 45. Google Scholar CrossRef Search ADS PubMed  119. Maldonado MD, Murillo-Cabezas F, Calvo JR, et al.   Melatonin as pharmacologic support in burn patients: a proposed solution to thermal injury-related lymphocytopenia and oxidative damage. Crit Care Med  2007; 35: 1177– 85. Google Scholar CrossRef Search ADS PubMed  120. Sener G, Sehirli AO, Satiroglu H, et al.   Melatonin improves oxidative organ damage in a rat model of thermal injury. Burns  2002; 28: 419– 25. Google Scholar CrossRef Search ADS PubMed  121. Tunali T, Sener G, Yarat A, Emekli N Melatonin reduces oxidative damage to skin and normalizes blood coagulation in a rat model of thermal injury. Life Sci  2005; 76: 1259– 65. Google Scholar CrossRef Search ADS PubMed  122. Singer AJ, McClain SA, Romanov A, Rooney J, Zimmerman T Curcumin reduces burn progression in rats. Acad Emerg Med  2007; 14: 1125– 9. Google Scholar CrossRef Search ADS PubMed  123. Gravante G, Palmieri MB, Esposito G, et al.   Apoptotic cells are present in ischemic zones of deep partial-thickness burns. J Burn Care Res  2006; 27: 688– 93. Google Scholar CrossRef Search ADS PubMed  Copyright © 2010 by the American Burn Association
TI - A Review of the Local Pathophysiologic Bases of Burn Wound Progression
JF - Journal of Burn Care & Research
DO - 10.1097/BCR.0b013e3181f93571
DA - 2010-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-review-of-the-local-pathophysiologic-bases-of-burn-wound-progression-Awt1O5oR5R
SP - 849
EP - 873
VL - 31
IS - 6
DP - DeepDyve
ER -