Full-thickness Contact Burns Research Articles

A natural history study of coagulopathy in a porcine 40% total body surface area burn model reveals the time-dependent significance of functional assays

Various studies have reported discordant results on the magnitude and direction of burn-induced coagulopathy (BIC), which has recently been associated with multiple organ dysfunction syndrome (MODS) and death. The increased mechanistic understanding of BIC is due, in part, to novel assays that have expanded the armamentarium beyond traditional tests like PT and aPTT. Still, BIC is a dynamic process, and the progression is difficult to define in the thermally-injured. To this end, we aimed to enhance the understanding of burn-induced coagulation abnormalities by employing functional assessments of platelet aggregation, viscoelastic kinetics, and thrombin generation in an extensive burn model in swine. Anesthetized Yorkshire pigs sustained 40% total body surface area (TBSA) full-thickness contact burns and recovered in metabolic cages. Blood was collected at baseline (BL), as well as 6, 24, and 48 h after injury. A significant effect of burn (P < 0.0001) was seen on platelets, with mild thrombocytopenia apparent at 24 h. While slight decreases in aPTT were not significant, rotational thromboelastometry (ROTEM) analysis revealed hypercoagulation 6 and 24 h after burn by a decreased clotting time. Maximum clot firmness increased after burn, but was not statistically significant until 48 h. Hypercoagulation was not supported by platelet aggregation, as the response to ADP was greatly and persistently diminished, and the response to collagen was unchanged. Endogenous thrombin potential was significantly reduced at 6 and 24 h after burn (P < 0.0001), and also correlated with a number of ROTEM parameters and collagen-induced platelet aggregation. In contrast, PT was not correlated with other measured parameters. Taken together, novel coagulation parameters may be more sensitive than PT in characterizing coagulopathy in the setting of burns. The data presented herein makes initial strides to report the natural history of several of these variables over time in a large animal model of extensive burns, indicating early hypercoagulability followed by hypocoagulation. Future work will elucidate the effects of standard of care.

T5 Tracking Cardiac Output During Burn Resuscitation via Pulse Wave Analysis

IntroductionUrine output (UOP) still remains the primary endpoint utilized as a surrogate for cardiac output (CO) and adequacy of perfusion during burn resuscitation. The role of arterial blood pressure (BP) waveform as a guiding tool for burn resuscitation has not been rigorously explored. As we move toward developing and validating novel endpoint to complement UOP, we compared the potential of pulse wave analysis (PWA) of arterial BP waveforms for estimating cardiac output (CO) and stroke volume (SV) in a large animal model of 40% total body surface area (TBSA) burns with varying resuscitation paradigms.MethodsAnesthetized swine were instrumented and hemorrhaged 15% of their blood volume, and sustained a 40% TBSA full-thickness contact burn with aluminum billets. Animals were kept in a surgical ICU setting overnight, during which anesthesia was maintained with a combination of propofol, ketamine, and fentanyl. Animals were randomized to 3 different intravenous fluid (lactated Ringer’s, LR) levels: under resuscitation with no IV fluids, adequate resuscitation protocolized with a clinical decision support tool, or over resuscitation with a starting rate of 500mL/hour. We computed 20 surrogate measures of CO and SV (10 each) via PWA of arterial BP, and calibrated them to reference CO via thermo-dilution and SV on a subject-by-subject basis. Surrogate performance was quantified in terms of correlation coefficient and root-mean-squared error (RMSE) relative to reference CO and SV.ResultsAnimals received 0±0, 4.4±1.6, and 9.1±1.3 mL/kg/% TBSA in the under, adequate, and over resuscitation groups (p=0.0036), respectively. Among the ten surrogate measures of CO and SV, 4 surrogates had positive proportionality to reference CO and SV consistently. The best surrogate measure of CO and SV was [mean BP-diastolic BP]/[heart period] and [mean BP-diastolic BP], respectively, which yielded r value of 0.82+/-0.19 and RMSE of 0.23+/-0.14 lpm for CO and r value of 0.85+/-0.17 and RMSE of 2.7+/-2.0 ml for SV (Fig. 1).ConclusionsThe initial results suggest that PWA-based surrogates of CO and SV have the potential to track reference CO and SV in extremes of resuscitation post-burn. The current model produces under-resuscitated, adequately resuscitated, and over-resuscitated animals, making it possible to rigorously examine the efficacy of PWA-based tracking of CO and SV in a wide spectrum of burn resuscitation scenarios. Hence, PWA may provide metrics useful for developing surrogates of CO and SV as novel treatment endpoints of burn resuscitation that indicate perfusion and volume status of burn injury patients.

Increased oxidative phosphorylation in lymphocytes does not atone for decreased cell numbers after burn injury.

The acute systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction (MOD) that occur in large burn injuries may be attributed, in part, to immunosuppressive responses such as decreased lymphocytes. However, the mitochondrial bioenergetics of lymphocytes after severe burn injury are poorly understood. The purpose of this study was to examine mitochondrial function of lymphocytes following severe burns in a swine model. Anesthetized Yorkshire swine (n = 17) sustained 40% total body surface area full-thickness contact burns. Blood was collected at pre-injury (Baseline; BL) and at 24 and 48 h after injury for complete blood cell analysis, flow cytometry, cytokine analysis, and ficoll separation of intact lymphocytes for high-resolution mitochondrial respirometry analysis. While neutrophil numbers increased, a concomitant decrease was found in lymphocytes (P < 0.001) after burn injury, which was not specific to CD4+ or CD8+ lymphocytes. No changes in immune cell population were observed from 24 h to 48 h post-injury. IL 12-23 decreased while a transient increase in IL 4 was found from BL to 24h (P < 0.05). CRP progressively increased from BL to 24h (P < 0.05) and 48h (P < 0.001) post-injury. Routine and maximal mitochondrial respiration progressively increased from BL to 24h (P < 0.05) and 48 h post-injury (P < 0.001). No changes were found in leak respiration or residual oxygen consumption. When considering the reduction in lymphocyte number, the total peripheral lymphocyte bioenergetics per volume of blood significantly decreased from BL to 24h and 48h (P < 0.05). For the first time, we were able to measure mitochondrial activity in intact lymphocyte mitochondria through high-resolution respirometry in a severely burned swine model. Our data showed that the non-specific reduction in peripheral T cells after injury was larger than the increased mitochondrial activity in those cells, which may be a compensatory mechanism for the total reduction in lymphocytes. Additional studies in the metabolic activation of T cell subpopulations may provide diagnostic or therapeutic targets after severe burn injury.

Plasma and Urinary Glycosaminoglycans as Evidence for Endotheliopathy in a Swine Burn Model

BackgroundThe endothelial glycocalyx controls vascular permeability, cellular signaling, blood-endothelial cell adhesion, extravasation, and transmission of shear stress signals. Burn injury compromises integrity of this layer increasing vascular permeability, which is further exacerbated by large volumes of (intravenous) crystalloids. We have shown that enteral resuscitation is able to reverse burn-induced acute kidney injury (AKI), and herein, we present a follow-up examination of the integrity of the glycocalyx layer and its relationship with renal dysfunction after burn injury. Materials and methodsAnesthetized Yorkshire pigs sustained 40% total body surface area full-thickness contact burns and recovered in metabolic cages for one of three treatments: no fluids (oral or intravenous); (n = 6), ad libitum water (n = 6), or volume-matched oral rehydration solution (ORS; n = 6) for 48 h. Urine and blood were collected at baseline (BL), 6, 12, 24, 32, and 48 h after burn at which point kidneys were harvested. ResultsIn no fluid and water groups (but not ORS), plasma levels of glycosaminoglycans (GAGs) were elevated after burn (P ≤ 0.031). Syndecan-1 was elevated by 6 h after burn in all animals, but levels declined by 24 h with enteral fluids. Urinary GAGs in the no-fluid group were elevated after burn. No differences among treatments were detected in syndecan-1 levels, or glomerular lectin within the kidney. ConclusionsCollectively, these data demonstrate that ORS prevented increases in circulating GAGs. Furthermore, an inexpensive and simple method for detecting GAGs provides a sensitive measure of endotheliopathy after burn.

SAT-006 Dihydrotestosterone (DHT) Enhances Major Burn Injury Wound Healing by Regulating the Inflammatory Response in Mice

Androgen analogy (Oxandrolone) have been reported to better maintain lean body mass, with improved hypermetabolic responses and shortened healing time for major burn injured patients. This is contradictory to that androgens inhibit local wound repair in men and male mice. The aim of this study therefore is to identify the role of pure androgen dihydrotesterone (DHT) in complex major burn injury, in particularly whether androgen targets local healing process or systemic burn induced hypermetabolism. A DHT silastic tube was subcutaneously implanted to male Balb/c mice prior to surgery as the treatment group. A 2 X 2 cm2 full thickness contact burn wound was created on the dorsal skin of wild type littermates (control) or DHT treated mice. Wound healing rate and body weight changes were measured and compared between treatment and non-treatment group. The serum level of inflammatory cytokine/chemokine was measured using a Multiplex Immunoassay System. Spleen immune cells enumeration was analysis by flow cytometry. Inflammation, re-epithelialization, cell proliferation and collagen deposition was analysed using histology, immunohistochemistry and RT-PCR. In the present study, we found DHT treatment better maintained the body weight in mice and significantly promoted wound healing over 14 days, whereas DHT treatment had no effect on burn-induced hypermetabolism. In control group, major burn injury triggered an acute systematic inflammation response, resulting in significant increased weight of spleen, excess infiltration of nucleated erythroid cells in red pulps of spleen and a significant increase in number of splenic monocytes over 21 days. DHT treatment shortened the systemic inflammation response, evidenced via reduced splenic weight and the number of monocytes in spleen and circulation at day 14 and 21. This finding is further confirmed by less infiltration of macrophages in wound area at day 14 and 21 compared to control group. Taken together, our results suggesting the DHT treatment significantly improve wound healing by accelerated turnover of inflammation response but not through the metabolism. Further studies are necessary to define the exact mechanisms and DHT treatment could be a new therapeutic approach to improve the survivability of major burn injured patient.

One-hit wonder: Late after burn injury, granulocytes can clear one bacterial infection but cannot control a subsequent infection

ObjectiveBurn injury induces an acute hyperactive immune response followed by a chronic immune dysregulation that leaves those afflicted susceptible to multiple secondary infections. Many murine models are able to recapitulate the acute immune response to burn injury, yet few models are able to recapitulate long-term immune suppression and thus chronic susceptibility to bacterial infections seen in burn patients. This has hindered the field, making evaluation of the mechanisms responsible for these susceptibilities difficult to study. Herein we describe a novel mouse model of burn injury that promotes chronic immune suppression allowing for susceptibility to primary and secondary infections and thus allows for the evaluation of associated mechanisms. MethodsC57Bl/6 mice receiving a full-thickness contact burn were infected with Pseudomonas aeruginosa 14 days (primary infection) and/or 17 days (secondary infection) after burn or sham injury. The survival, pulmonary and systemic bacterial load as well as frequency and function of innate immune cells (neutrophils and macrophages) were evaluated. ResultsFollowing secondary infection, burn mice were less effective in clearance of bacteria compared to sham injured or burn mice following a primary infection. Following secondary infection both neutrophils and macrophages recruited to the airways exhibited reduced production of anti-bacterial reactive oxygen and nitrogen species and the pro-inflammatory cytokineIL-12 while macrophages demonstrated increased expression of the anti-inflammatory cytokine interleukin-10 compared to those from sham burned mice and/or burn mice receiving a primary infection. In addition the BALF from these mice contained significantly higher level so of the anti-inflammatory cytokine IL-4 compared to those from sham burned mice and/or burn mice receiving a primary infection. ConclusionsBurn-mediated protection from infection is transient, with a secondary infection inducing immune protection to collapse. Repeated infection leads to increased neutrophil and macrophage numbers in the lungs late after burn injury, with diminished innate immune cell function and an increased anti-inflammatory cytokine environment.

Enteral resuscitation with oral rehydration solution to reduce acute kidney injury in burn victims: Evidence from a porcine model.

Intravenous (IV) resuscitation of burn patients has greatly improved outcomes and become a cornerstone of modern burn care. However, the heavy fluids and vascular access required may not be feasible in austere environments, mass casualty, or delayed transport scenarios. Enteral resuscitation has been proposed for these situations; we sought to examine the effectiveness of this strategy on improving burn-induced kidney injury. Anesthetized Yorkshire swine sustaining 40% TBSA full-thickness contact burns were randomized to three groups (n = 6/group): fluid deprivation, ad libitum water access, or 70 mL/kg/d Oral Rehydration Salt solution (ORS). Urine and blood were collected at baseline (BL), 6, 12, 24, 32, and 48h post-burn, at which point tissue was harvested and CT angiography performed. Although fluid consumption by ad libitum and ORS groups were matched (132±54mL/kg versus 120±24mL/kg, respectively), ORS intake increased urine output compared with water and no water (47.3±9.0 mL/kg versus 16.1±2.5 mL/kg, and 24.5±1.7 mL/kg respectively). Plasma creatinine peaked 6h following burn (1.67±0.07mg/dL) in all animals, but at 48h was comparable to BL in animals receiving water (1.23±0.06mg/dL) and ORS (1.30±0.09mg/dL), but not fluid deprived animals (1.56±0.05mg/dL) (P<0.05). Circulating levels of blood urea nitrogen steadily increased, but also decreased by 48h in animals receiving enteral fluids (P<0.05). Water deprivation reduced renal artery diameter (-1.4±0.17mm), whereas resuscitation with water (-0.44±0.14 mm) or ORS maintained it (-0.63±0.20 mm;P< 0.02). Circulating cytokines IL-1β, IL-6, IFNγ, and GM-CSF were moderately elevated in the fluid-deprived group. Taken together, the data suggest that enteral resuscitation with ORS rescues kidney function following burn injury. Incorporating enteral fluids may improve outcomes in resource-poor environments and possibly reduce IV fluid requirements to prevent co-morbidities associated with over-resuscitation. Studies into different volumes/types of enteral fluids are warranted. While ORS has saved many lives in cholera-associated dehydration, it should be investigated further for use in burn patients.

Antecedent thermal injury worsens split-thickness skin graft quality: A clinically relevant porcine model of full-thickness burn, excision and grafting

Current standard of care for full-thickness burn is excision followed by autologous split-thickness skin graft placement. Skin grafts are also frequently used to cover surgical wounds not amenable to linear closure. While all grafts have potential to contract, clinical observation suggests that antecedent thermal injury worsens contraction and impairs functional and aesthetic outcomes. This study evaluates the impact of antecedent full-thickness burn on split-thickness skin graft scar outcomes and the potential mediating factors. Full-thickness contact burns (100°C, 30s) were created on the backs of anesthetized female Yorkshire Pigs. After seven days, burn eschar was tangentially excised and covered with 12/1000th inch (300μm) split-thickness skin graft. For comparison, unburned wounds were created by sharp excision to fat before graft application. From 7 to 120days post-grafting, planimetric measurements, digital imaging and biopsies for histology, immunohistochemistry and gene expression were obtained. At 120days post-grafting, the Observer Scar Assessment Scale, colorimetry, contour analysis and optical graft height assessments were performed. Twenty-nine porcine wounds were analyzed. All measured metrics of clinical skin quality were significantly worse (p<0.05) in burn injured wounds. Histological analysis supported objective clinical findings with marked scar-like collagen proliferation within the dermis, increased vascular density, and prolonged and increased cellular infiltration. Observed differences in contracture also correlated with earlier and more prominent myofibroblast differentiation as demonstrated by α-SMA staining. Antecedent thermal injury worsens split-thickness skin graft quality, likely by multiple mechanisms including burn-related inflammation, microscopically inadequate excision, and dysregulation of tissue remodeling. A valid, reliable, clinically relevant model of full-thickness burn, excision and skin replacement therapy has been demonstrated. Future research to enhance quality of skin replacement therapies should be directed toward modulation of inflammation and assessments for complete excision.

NLRP12 limits TNFα-induced apoptosis of monocyte progenitor cells during emergency hematopoiesis

Abstract The NLR family member NLRP12, expressed in peripheral leukocytes and bone marrow progenitor cells, is implicated in controlling the response of the innate immune system by suppressing non-canonical NfκB signaling. Given the hematopoietic system’s ability to respond to insults by rapidly increasing myeloid cell production, and the importance of tightly controlled cytokine regulation during these processes, we hypothesized NLRP12 to be a key regulator of the innate immune system’s reconstitution following a hematopoietic emergency. To test this, wild type and Nlrp12−/− C57BL/6 female mice underwent a radiation-thermal combined injury (RCI) consisting of a 20% total body surface area, full thickness contact burn in addition to receiving a single whole-body dose of 5 Gy γ-radiation. Following injury, Nrlp12−/− mice showed a three-fold increase in specific apoptosis of granulocyte-monocyte progenitor cells leading to a reduction in the total number of bone marrow cells and thus systemic leukopenia. We also observed that the lack of NLRP12 led to elevated levels of TNFα systemically following injury. Utilization of a neutralizing antibody against TNFα following injury abrogated the granulocyte-monocyte progenitor cell apoptosis and leukopenia seen in vehicle-treated mice. These data lead us to conclude that NLRP12 acts as a suppressor of TNFα signaling during emergency hematopoiesis; a novel finding, since no member of the NLR family has been previously found to interact with TNFα signaling.

Dendritic cells modulate burn wound healing by enhancing early proliferation.

Adequate wound healing is vital for burn patients to reduce the risk of infections and prolonged hospitalization. Dendritic cells (DCs) are antigen presenting cells that release cytokines and are central for the activation of innate and acquired immune responses. Studies have showed their presence in human burn wounds; however, their role in burn wound healing remains to be determined. This study investigated the role of DCs in modulating healing responses within the burn wound. A murine model of full-thickness contact burns was used to study wound healing in the absence of DCs (CD11c promoter-driven diphtheria toxin receptor transgenic mice) and in a DC-rich environment (using fms-like tyrosine kinase-3 ligand, FL- a DC growth factor). Wound closure was significantly delayed in DC-deficient mice and was associated with significant suppression of early cellular proliferation, granulation tissue formation, wound levels of TGFβ1 and formation of CD31+ vessels in healing wounds. In contrast, DC enhancement significantly accelerated early wound closure, associated with increased and accelerated cellular proliferation, granulation tissue formation, and increased TGFβ1 levels and CD31+ vessels in healing wounds. We conclude that DCs play an important role in the acceleration of early wound healing events, likely by secreting factors that trigger the proliferation of cells that mediate wound healing. Therefore, pharmacological enhancement of DCs may provide a therapeutic intervention to facilitate healing of burn wounds.

NLRP12 suppresses host innate immune signaling to limit inflammation during emergency hematopoiesis (INM6P.404)

Abstract The hematopoietic system is capable of rapidly increasing myeloid cell production in response to tissue damage. NLRP12 is expressed in peripheral leukocytes and by osteoclasts and marrow progenitor cells. This NLR family member has been controversial but has been implicated in regulation of innate immune responses. We hypothesized that NLRP12 is a central regulator of innate immune reconstitution following an emergency hematopoietic event. To test this, wild type and Nlrp12-/- C57BL/6 female mice underwent a radiation-thermal combined injury (RCI) consisting of a 20% total body surface area, full thickness contact burn in addition to receiving a single whole body dose of 5 Gy γ-radiation. Following RCI, NLRP12-deficient mice display a lack of peripheral innate immune cell reconstitution as seen in wild type mice; specifically a ten-fold decrease in total numbers of splenic and pulmonary neutrophils and immature monocytes compared to wild type, and a three-fold increase in apoptosis of bone marrow granulocyte/monocyte progenitor cells resulting in a three-fold reduction of total bone marrow cells. Lack of NLRP12 results in a sustained increase in inflammation after injury, with significant increase in serum levels of TNFα, IL-6, IL-12, and type I interferons compared to injured, wild type mice that is inflammasome-independent. These data suggest that NLRP12 acts as an important regulator of inflammation during events that trigger immune hematopoeisis and reconstitution.

Combined radiation and thermal injury induces a non-suppressive myeloid CD11b+ Gr1+ cell population (P4025)

Abstract Increasing sophistication of terrorist attacks and recent nuclear energy crises have amplified the interest in the immunobiology of combined radiation and burn injury (RCI). Burn is associated with immunosuppression and increased mortality with lower doses of ionizing radiation. Following trauma, putative myeloid derived suppressor cells (MDSCs; CD11b+Gr1+) have been described. We hypothesized that RCI-MDSCs display a suppressive phenotype and RCI will result in increased susceptibility to bacterial infection. C57BL/6 female mice underwent either a 20% total body surface area full thickness contact burn or sham procedure. All mice then received a single whole body dose of 5Gy γ-radiation. Mice were infected 14 days post injury with Pseudomonas aeruginosa intratracheally. Immune cells underwent apoptosis following RCI and infection, with CD11b+Gr1(Ly6C)+ cells being the predominant surviving cell type, which we isolated and cocultured 1:1 with untouched T cells. Surprisingly, T cells incubated with CD11b+Gr1(Ly6C)+ cells from RCI mice displayed greater proliferation, increased IFN-γ and decreased IL-10 production compared to MDSC from radiation-only mice. RCI mice were able to clear the P. aeruginosa infection significantly better than radiation-only mice. These data suggest that while RCI depletes immune cells, resistant CD11b+Gr1(Ly6C)+ cells represent a target population for modulation of bacterial clearance after injury.

Radiation Combined With Thermal Injury Induces Immature Myeloid Cells

The continued development of nuclear weapons and the potential for thermonuclear injury necessitates the further understanding of the immune consequences after radiation combined with injury (RCI). We hypothesized that sublethal ionization radiation exposure combined with a full-thickness thermal injury would result in the production of immature myeloid cells. Mice underwent either a full-thickness contact burn of 20% total body surface area or sham procedure followed by a single whole-body dose of 5-Gy radiation. Serum, spleen, and peripheral lymph nodes were harvested at 3 and 14 days after injury. Flow cytometry was performed to identify and characterize adaptive and innate cell compartments. Elevated proinflammatory and anti-inflammatory serum cytokines and profound leukopenia were observed after RCI. A population of cells with dual expression of the cell surface markers Gr-1 and CD11b were identified in all experimental groups, but were significantly elevated after burn alone and RCI at 14 days after injury. In contrast to the T-cell-suppressive nature of myeloid-derived suppressor cells found after trauma and sepsis, myeloid cells after RCI augmented T-cell proliferation and were associated with a weak but significant increase in interferon γ and a decrease in interleukin 10. This is consistent with previous work in burn injury indicating that a myeloid-derived suppressor cell-like population increases innate immunity. Radiation combined injury results in the increase in distinct populations of Gr-1CD11b cells within the secondary lymphoid organs, and we propose these immature inflammatory myeloid cells provide innate immunity to the severely injured and immunocompromised host.

Radiation combined with thermal injury: induction of a radio-resistant immature myeloid cell. (118.10)

Abstract Increasing sophistication of terrorist attacks and recent nuclear energy crises have amplified the interest in the immunobiology of radiation combined with burn injury (RCI). Burn is associated with immunosuppression and increased mortality with lower doses of ionizing radiation. We hypothesize that RCI results in reduction of immune cell populations with a decrease in serum cytokine levels. C57BL/6 female mice underwent either a 20% TBSA full thickness contact burn or sham procedure. One burn group and one sham group received a single whole body dose of 5 Gy gamma radiation. Spleens and burn draining lymph nodes (dLN) were harvested 3 and 14 days later. Flow cytometry was performed to identify adaptive and innate cells. Serum was analyzed by multiplex bead array assay. IL-1β (day3), MCP-1 and IP-10 (days 3 and 14) were significantly elevated in RCI mice when compared to all other groups. At day 14, splenic and dLN T-cells, B-cells and neutrophils were significantly decreased after irradiation and RCI when compared to sham and burn alone. In contrast, numbers and percentages of Gr.1+CD11b+ cells were significantly increased. We confirmed these were not myeloid-derived suppressor cells as they did not suppress T cell proliferation in vitro. These data suggest that while RCI depletes immune cells, there are specific cytokine alterations and induction of a population of immature myeloid-derived cells which may act as a potential target for immune countermeasures.

Pseudomonas aeruginosa infection after burn injury polarizes innate immune cells towards an alternatively activated phenotype (55.19)

Abstract Severe burn injury causes profound immunosuppression resulting in prevalent bacterial infections. We hypothesized that bacterial infection after burn injury polarizes macrophage (Mo) and neutrophil (No) responses into an alternatively activated (M2/N2) phenotype. To test this, we used a murine model of burn injury and bacterial challenge. Female C57BL/6 mice underwent either a 20% TBSA full thickness contact burn or control sham procedure. Twenty-four hours later mice were either given a subcutaneous injection of Pseudomonas aeruginosa or control saline and sacrificed two days later to determine bacterial burden in skin, liver and lung. Serum cytokine levels were measured by cytometric bead array. Splenocytes were cultured in the presence of Brefeldin-A for intracellular cytokine analysis, and Mo and No were identified by surface staining. Burn mice had bacterial dissemination indicated by positive cultures from all tissue samples. No bacteria were recovered from sham mice. Burn injury, but not infection, induced a significant increase in serum IFN-γ and TNF-α. IL-6 was also significantly increased after burn injury alone but was significantly higher in infected burn mice. IL-10 was elevated in infected burn mice only. Flow cytometry confirmed expression of IL-10 protein within Mo and No. These data suggest that after burn injury, bacterial infection polarizes serum cytokine and innate cell phenotype into an alternatively activated phenotype allowing systemic infection.

Fibrin sealant improves graft adherence in a porcine full-thickness burn wound model

Introduction Autograft take and rapid wound closure is essential for the survival of severely burned patients. Loss of skin grafts typically occurs during the first few days after coverage, mainly due to shear forces and inadequate contact with the wound bed. Slow-clotting fibrin sealant, applied with a spray-on device, has been shown to improve healing of skin grafts in large wounds. However, its use in burn wounds has not been studied so far. Study aim To evaluate the effectiveness of sprayed fibrin sealant in excised and grafted full-thickness burns. Material and methods Ten female Yorkshire pigs (30–45 kg) received a full-thickness contact burn of approximately 15% total body surface area. The burns were excised to the level of the muscular fascia after 24 h and covered with meshed skin autograft (mesh ratio 1:3). Wounds were randomized to either fibrin sealant ( n = 20) or standard skin staples ( n = 16) for graft fixation. Fibrin sealant was used as a slow-clotting spray (4 IU thrombin/ml). Outcome measurements included clinical scoring at days 2, 5, 9 and 14 postoperatively, planimetric analysis of wound closure, and histological examination of epidermal and dermal thickness 14 days after autografting. Results In the fibrin sealant group, graft adherence scores were significantly increased ( p < 0.02) and graft dislocation scores significantly decreased ( p < 0.01) at days 2 and 5 postoperatively, when compared to controls. Planimetric analysis of remaining open mesh interstices showed acceleration of wound closure in the fibrin sealant group but did not reach statistical significance (day 14 p = 0.04 at significance level p < 0.025). Wound contraction, occurrence of hematoma, and dermal as well as epidermal thickness were not different between the groups at 14 days postoperatively. Conclusion The results indicate that the use of slow-clotting fibrin sealant spray for autograft fixation is advantageous over skin staples. Easy handling and reduced graft dislocation at early time points are key qualities of this method.

Increased neutrophil migration rescues impaired function and susceptibility to Pseudomonas pneumonia after burn injury (56.25)

Abstract Bacterial lung infection is a major cause of mortality late after burn injury. Neutrophil dysfunction is implicated in the susceptibility of wound infection with Pseudomonas aeruginosa. We hypothesized that burn-induced impaired neutrophil migration and function within the lungs would increase susceptibility to intratracheal P. aeruginosa infection. Female C57BL/6 mice were anesthetized and subjected to a 20% full thickness contact burn or sham injury. Fourteen days later, mice were inoculated with 1x106 CFU of P. aeruginosa. After 24 hours, pulmonary bacterial load was evaluated. Flow cytometric analysis was used to quantify neutrophils and evaluate their function by dihydrorhodamine (DHR) oxidation. Contrary to our hypothesis, burn mice had a significant increase in bacterial clearance compared to sham. This correlated with an increased percentage and absolute number of lung neutrophils in burn mice (mean±SEM, burn: 2.5±1.4x108, sham: 2.7±0.8x107cells, p=0.007). However, these neutrophils had reduced reactive-oxygen production compared to sham (burn: 1044±21, sham: 51431±182 MFI of oxidized DHR, p=0.0131). As predicted, lung neutrophil function was significantly impaired after burn injury. However, burn mice exhibited a larger neutrophil influx than sham. The net result was that burn mice cleared the infection quicker than sham mice. This implies a fine balance exists between neutrophil migration and function after burn injury.

Full-Thickness Contact Burn from a Warming Bottle in a Newborn

Major full-thickness burn injuries in newborns are rare but have been reported following the use of medical and warming devices during the early neonatal period. In this report, we present a newborn infant with major full-thickness contact burn injuries of 15% of the total body surface area (TBSA) caused by a water-filled warming bottle applied to the naked skin, and describe the sequential progress of epidermal necrosis and wound healing with autografting.

Severe burn injury leads to spontaneous generation of Th17 CD4+ T cells in wound draining lymph nodes (42.18)

Abstract Severe burn injury leads to early and prolonged immune dysfunction resulting in a reduced resistance to bacterial infection. In a model of burn injury, we have previously shown that burn mice challenged with Pseudomonas aeruginosa develop systemic infection and have high mortality rates whereas sham mice do not. Wound tissue TGF-β levels and serum IL-6 levels are elevated in the infected burn (IL-6: 32.68 +/- 9.53 pg/mL, n=4) compared to infected sham (2.87 +/- 1.43, n=3) mice. IFN-γ levels are similar between the groups. Since TGF-β and IL-6 are key for differentiation of naïve CD4+ T cells into the Th17 phenotype, we hypothesized that Th17 T cells are generated following burn injury. Female C57BL/6 mice were anesthetized and subjected to 20% full thickness contact burn or sham procedure. After 3, 7 or 14 days, CD4+ T cells selected from wound-draining lymph nodes were in vitro stimulated with mitogen. Flow cytometry was used to identify intracellular IL-17 in CD3+ CD4+ T cells. At all time points there was a statistically significant burn-dependent increase in both percentage of CD4+ T cells that were Th17 cells and absolute Th17 cell number (e.g. Day 3, 7384 +/- 1370 cells, n=3) compared to sham (1604 +/- 499, n=5). While burn injury spontaneously induces Th17 cells, known to recruit neutrophils, this does not protect against subsequent P. aeruginosa challenge. This may provide a novel mechanism for post-burn immunosuppression.

Burn injury induces a myeloid suppressor phenotype and susceptibility to Pseudomonas infection (129.24)

Abstract Following severe burn injury, the immune system is dramatically altered resulting in a susceptibility to bacterial infections. Using a mouse model of burn injury and subsequent Pseudomonas aeruginosa infection, we hypothesized that 1) clearance will be decreased after burn and 2) expression of TLR2, important for P. aeruginosa clearance, and/or Gr1.1, a marker of myeloid suppressive ability, is altered by burn. Female C57BL/6 mice were subjected to a 20% full thickness contact burn or sham procedure. After one day, mice were inoculated s/c at the wound with 2 x104 CFU of P. aeruginosa. CFU were calculated for various organs after two days. Splenocytes were analyzed by FACS for CD11b, Gr1.1 and TLR2. Burn mice had significant morbidity (weight loss) and mortality versus sham. Burn mice developed a systemic infection (e.g. mean±SEM 4.4±0.6 x104 CFU/lung, n=6) while no CFU were recovered from sham (n=6). Infected burn mice had a significantly higher number and percent of CD11b+ TLR2hi splenocytes (22.6±3.5%) than the infected sham (3.4±0.4%). A higher number and percent of burn CD11b+ cells were Gr1.1+ (75.5±5.7%) compared to sham mice (43.3±1.2%). Therefore, although we observed an increase in TLR2+ myeloid cells following burn injury, the increased percentage of GR1.1+ CD11b+ myeloid suppressor cells could contribute to the development of a systemic P. aeruginosa infection after burn. Supported by NIH K08 067147.