Burned Mouse Model Research Articles

Burn mouse models.

Severe thermal injury induces immunosuppression, involving all parts of the immune system, especially when large fractions of the total body surface area are affected. An animal model was established to characterize the burn-induced immunosuppression. In our novel mouse model a 6 % third-degree burn injury was induced with a hot-air blower. The third-degree burn was confirmed histologically. At 48 h, a decline in the concentration of peripheral blood leucocytes was observed in the group of mice with burn wound. The reduction was ascribed to the decline in concentration of polymorphonuclear neutrophil leucocytes and monocytes. When infecting the skin with Pseudomonas aeruginosa, a dissemination of bacteria was observed only in the burn wound group. Histological characterization of the skin showed an increased polymorphonuclear neutrophil granulocytes dominated inflammation in the group of mice with infected burn wound compared with the burn wound only group. The burn mouse model resembles the clinical situation and provides an opportunity to examine or develop new strategies like new antibiotics and immune therapy, in handling burn wound victims much.

Antimicrobial Blue Light Therapy for Multidrug-Resistant Acinetobacter baumannii Infection in a Mouse Burn Model: Implications for Prophylaxis and Treatment of Combat-related Wound Infections

In this study, we investigated the utility of antimicrobial blue light therapy for multidrug-resistant Acinetobacter baumannii infection in a mouse burn model. A bioluminescent clinical isolate of multidrug-resistant A. baumannii was obtained. The susceptibility of A. baumannii to blue light (415 nm)-inactivation was compared in vitro to that of human keratinocytes. Repeated cycles of sublethal inactivation of bacterial by blue light were performed to investigate the potential resistance development of A. baumannii to blue light. A mouse model of third degree burn infected with A. baumannii was developed. A single exposure of blue light was initiated 30 minutes after bacterial inoculation to inactivate A. baumannii in mouse burns. It was found that the multidrug-resistant A. baumannii strain was significantly more susceptible than keratinocytes to blue light inactivation. Transmission electron microscopy revealed blue light-induced ultrastructural damage in A. baumannii cells. Fluorescence spectroscopy suggested that endogenous porphyrins exist in A. baumannii cells. Blue light at an exposure of 55.8 J/cm(2) significantly reduced the bacterial burden in mouse burns. No resistance development to blue light inactivation was observed in A. baumannii after 10 cycles of sublethal inactivation of bacteria. No significant DNA damage was detected in mouse skin by means of a skin TUNEL assay after a blue light exposure of 195 J/cm(2).

Evaluation of skin regeneration after burns in vivo and rescue of cells after thermal stress in vitro following treatment with a keratin biomaterial

Thermal burns typically display an injury pattern dictated by the transfer of the thermal energy into the skin and underlying tissues and creation of three zones of injury represented by a necrotic zone of disrupted cells and tissue, an intermediate zone of injured and dying cells, and a distant zone of stressed cells that will recover with proper treatment. The wound healing capabilities of a keratin biomaterial hydrogel were studied in two pilot studies, one using a chemical burn model in mice and the other a thermal burn model in swine. In both studies, keratin was shown to prevent enlargement of the initial wound area and promote faster wound closure. Interestingly, treating thermally stressed dermal fibroblast in culture demonstrated that soluble keratin was able to maintain cell viability and promote proliferation. Separation of so-called alpha and gamma fractions of the keratin biomaterial had differential effects, with the gamma fraction producing more pronounced cell survival and recovery. These results suggest that the gamma fraction, composed essentially of degraded alpha keratin proteins, may facilitate cell rescue after thermal injury. Treatment of burns with gamma keratin may therefore represent a potential therapy for wounds with an intermediate zone of damaged tissue that has the potential to contribute to spontaneous healing.

Mitochondria‐targeted antioxidant promotes recovery of skeletal muscle mitochondrial function after burn trauma assessed byin vivo31P nuclear magnetic resonance and electron paramagnetic resonance spectroscopy

Burn injury causes a major systemic catabolic response that is associated with mitochondrial dysfunction in skeletal muscle. We investigated the effects of the mitochondria-targeted peptide antioxidant Szeto-Schiller 31 (SS-31) on skeletal muscle in a mouse burn model using in vivo phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy to noninvasively measure high-energy phosphate levels; mitochondrial aconitase activity measurements that directly correlate with TCA cycle flux, as measured by gas chromatography mass spectrometry (GC-MS); and electron paramagnetic resonance (EPR) to assess oxidative stress. At 6 h postburn, the oxidative ATP synthesis rate was increased 5-fold in burned mice given a single dose of SS-31 relative to untreated burned mice (P=0.002). Furthermore, SS-31 administration in burned animals decreased mitochondrial aconitase activity back to control levels. EPR revealed a recovery in redox status of the SS-31-treated burn group compared to the untreated burn group (P<0.05). Our multidisciplinary convergent results suggest that SS-31 promotes recovery of mitochondrial function after burn injury by increasing ATP synthesis rate, improving mitochondrial redox status, and restoring mitochondrial coupling. These findings suggest use of noninvasive in vivo NMR and complementary EPR offers an approach to monitor the effectiveness of mitochondrial protective agents in alleviating burn injury symptoms.

Pseudomonas aeruginosa PA1006, Which Plays a Role in Molybdenum Homeostasis, Is Required for Nitrate Utilization, Biofilm Formation, and Virulence

Pseudomonas aeruginosa (Pae) is a clinically important opportunistic pathogen. Herein, we demonstrate that the PA1006 protein is critical for all nitrate reductase activities, growth as a biofilm in a continuous flow system, as well as virulence in mouse burn and rat lung model systems. Microarray analysis revealed that ΔPA1006 cells displayed extensive alterations in gene expression including nitrate-responsive, quorum sensing (including PQS production), and iron-regulated genes, as well as molybdenum cofactor and Fe-S cluster biosynthesis factors, members of the TCA cycle, and Type VI Secretion System components. Phenotype Microarray™ profiles of ΔPA1006 aerobic cultures using Biolog plates also revealed a reduced ability to utilize a number of TCA cycle intermediates as well as a failure to utilize xanthine as a sole source of nitrogen. As a whole, these data indicate that the loss of PA1006 confers extensive changes in Pae metabolism. Based upon homology of PA1006 to the E. coli YhhP protein and data from the accompanying study, loss of PA1006 persulfuration and/or molybdenum homeostasis are likely the cause of extensive metabolic alterations that impact biofilm development and virulence in the ΔPA1006 mutant.

Effects of Umbilical Cord Serum Eye Drops in a Mouse Model of Ocular Chemical Burn

Purpose: To evaluate the effect of umbilical cord serum (UCS) eye drops on corneal wound healing and haze in a mouse model of ocular chemical burn and compare with that of peripheral blood serum (PBS) eye drops or artificial tears (AT).Methods: Chemical burn of the ocular surface was induced by 1N NaOH in C57BL/6 mice. Injured mice were topically treated with 20% UCS, 20% PBS, or AT four times daily. The changes of corneal epithelial defects and degree of haze were analyzed at 6 h, 1, 2, 3, 5, and 7 days, and histological examination was performed at 3 and 7 days. The concentration of IL-1β in the cornea was measured by enzyme-linked immunosorbent assay at 7 days after treatment.Results: The UCS group showed lower epithelial defect parameters compared with the PBS group at 1 and 2 days (p < 0.05), and with the AT group from 1–5 days (p < 0.05). The haze scores were significantly lower in the UCS group than in the PBS group at 2 and 3 days (p < 0.05), and in the AT group from 2–7 days (p < 0.05). Histological examination showed better epithelial integrity and lower stromal inflammation and edema in the UCS group than the other groups. IL-1β levels were 99.71 ± 85.22 and 230.76 ± 102.67 pg/ml in the UCS and PBS groups, respectively (p = 0.03).Conclusions: UCS eye drops are more effective in improving corneal wound healing and reducing corneal haze compared with PBS eye drops and AT in experimental chemical burns.

Chitosan dressing promotes healing in third degree burns in mice: Gene expression analysis shows biphasic effects for rapid tissue regeneration and decreased fibrotic signaling

Burns are a significant health challenge and healing can result in scar formation. Chitosan, a derivative of chitin, has been used to promote wound healing. In this study we used gene expression profiling in a mouse model of full thickness cutaneous burn to assess the benefits of treating with a chitosan lactate dressing. Three days after wounding mice treated with chitosan showed increased expression of genes associated with formation of granulation tissue. At a later time point, seven days after wounding, genes that initially showed increased expression were now down-regulated, and there was increased expression of genes involved in remodeling suggesting that the chitosan treatment results in accelerated healing. Quantitative RT-PCR showed modulated mRNA levels for TGFβ1 by the chitosan dressing. TGFβ1 initially promotes healing but extended activity can result in scarring. Importantly we found that expression was elevated at day three, but decreased at day seven suggesting that chitosan treatment will not result in scar formation, and may even be beneficial in preventing scar formation. Additionally, the biphasic regulation of expression of TGFβ1 could be a powerful biomarker for future studies of the wound-healing potential of chitosan based and other treatments for burn wounds.

In Vivo Molecular Imaging of Murine Embryonic Stem Cells Delivered to a Burn Wound Surface via Integra® Scaffolding

It has been demonstrated that restoration of function to compromised tissue can be accomplished by transplantation of bone marrow stem cells and/or embryonic stem cells (ESCs). One limitation to this approach has been the lack of noninvasive techniques to longitudinally monitor stem cell attachment and proliferation. Recently, murine ESC lines that express green fluorescent protein (GFP), luciferase (LV), and herpes simplex thymidine kinase (HVTK) were developed for detection of actively growing cells in vivo by imaging. In this study, the authors investigated the use of these ESC lines in a burned mouse model using Integra® as a delivery scaffolding/matrix. Two different cell lines were used: one expressing GFP and LV and the other expressing GFP, LV, and HVTK. Burn wounds were produced by application of a brass block (2 × 2 cm kept in boiling water before application) to the dorsal surface of SV129 mice for 10 seconds. Twenty-four hours after injury, Integra® with adherent stem cells was engrafted onto a burn wound immediately after excision of eschar. The stem cells were monitored in vivo by measuring bioluminescence with a charge-coupled device camera and immunocytochemistry of excised tissue. Bioluminescence progressively increased in intensity over the time course of the study, and GFP-positive cells growing into the Integra® were detected. These studies demonstrate the feasibility of using Integra® as a scaffolding, or matrix, for the delivery of stem cells to burn wounds as well as the utility of bioluminescence for monitoring in vivo cellular tracking of stably transfected ESC cells.

Passive immunisation against Pseudomonas aeruginosa recombinant flagellin in an experimental model of burn wound sepsis

Background This study was aimed to investigate whether anti-recombinant flagellin type A (anti r-fla-A) immunoglobulin G (IgG) provides protection in a mouse burn model of infection, and to determine the role of anti r-fla-A IgG as an opsonin and motility inhibitor in local and systemic infections. Methods Following the preparation of r-flagellin type A, rabbit polyclonal IgG was prepared. Specificity of anti r-flagellin for r-flagellin was evaluated by immunoblot analysis. After burn and challenge, mortality rate was screened in the mice treated with anti r-fla-A IgG. The ability of antiserum to promote phagocytosis of bacteria was assessed by the opsonophagocytosis testing. Functional activity of anti r-fla-A IgG was assessed in vitro by motility inhibition assay. Bacterial quantity in skin and internal organs was evaluated to study systemic infection. Results In vivo administration of anti r-fla-A IgG resulted in a significant improvement in survival in mice infected by a homologous strain of Pseudomonas aeruginosa from 16.6% to 75% compared with the control IgG. By contrast, this rate was 33.3% in the mice infected by the heterologous strain, PAO1 (type B flagellated strain). Protection was improved by giving a second treatment of r-flagellin antisera at 24-h post-burn and infection. Furthermore, anti r-fla-A IgG enhanced considerably the phagocytosis of the homologous strain but it was slight in the heterologous strain. The antiserum against r-flagellin type A was able to inhibit the motility of the PAK strain (type A flagellated strain), but slight inhibition was observed against PAO1. Meanwhile, anti r-fla-A IgG inhibited the systemic spread of PAK strain from the site of infection to internal organs. Conclusion In this study, passive immunisation with anti r-fla-A IgG was active against a homologous strain of infecting P. aeruginosa, but lost most of its efficiency against a heterologous strain. Therefore, passive treatment with anti r-fla-A IgG might protect burned mice against local and systemic infection of P. aeruginosa.

Active immunization using exotoxin A confers protection against Pseudomonas aeruginosainfection in a mouse burn model

BackgroundPseudomonas aeruginosa is an important cause of nosocomial infection and may lead to septicemia and death. We evaluated the immunogenicity of semi-purified exotoxin A from the bacterium in a mouse burn model.MethodsThe toxoid was prepared from exotoxin A taken from toxigenic strains of P. aeruginosa (PA 103). 50 mice were immunized with the toxoid, burned with hot metal and infected with 1 × 108 CFU of toxigenic strains of P. aeruginosa (experimental group); 25 non-immunized mice were also burned and infected (control group). The mortality rate and presence of any exotoxin and P. aeruginosa in the sera, liver and spleen were determined.ResultsIn the experimental group, 2 mice died before the burns were administered and were excluded from the study. The remainder (48 mice) were challenged with a lethal dose of P. aeruginosa and followed for 70 days. 3 of these mice died. Neither P. aeruginosa nor exotoxin A was not detected in the liver, spleen or sera of the surviving mice. The protective efficacy of toxoid vaccination was therefore 93.8%. In the control group, all mice died from bacteremia and septicemia, most (80%) within 6 days, and P. aeruginosa and exotoxin A were isolated from sera, spleen and liver.ConclusionActive immunization of mice using a semi-purified exotoxin A derived from P. aeruginosa was 93.8% effective at protecting mice from subsequent P. aeruginosa infections in a mouse burn model.

Differences between Pseudomonas aeruginosa in a clinical sample and in a colony isolated from it: Comparison of virulence capacity and susceptibility of biofilm to inhibitors

We study the differences between Pseudomonas aeruginosa from an infected wound (clinical strain) and a colony isolated from it. We assessed the in vitro inhibition of these P. aeruginosa biofilms by DNase and filtrate of Lactobacillus plantarum cultures (acid=AF and neutralize=NF) with crystal violet technique. Inhibition by AF was greatest than DNase for clinical and isolated strain (p<0.001) and greatest than NF for clinical (p<0.05) and isolated strain (p<0.001). Using a burn model in mice, we compared the infection producing by clinical and isolated strains in planktonic and biofilm form. Deaths were quantified and the infection was assessed by determining CFU/g of tissue in the lesion, spleen and liver. The infections with planktonic bacteria tended to become systemic and more deadly than biofilm infections. All infected wounds required the same healing period (30 days). These findings were independent of the origin of the bacteria (clinical or colony isolated strain).

Cytokine expression profile over time in burned mice

The persistent inflammatory response induced by a severe burn increases patient susceptibility to infections and sepsis, potentially leading to multi-organ failure and death. In order to use murine models to develop interventions that modulate the post-burn inflammatory response, the response in mice and the similarities to the human response must first be determined. Here, we present the temporal serum cytokine expression profiles in burned mice in comparison to sham mice and human burn patients. Male C57BL/6 mice were randomized to control ( n = 47) or subjected to a 35% TBSA scald burn ( n = 89). Mice were sacrificed 3, 6, 9, 12, 24, and 48 h and 7, 10, and 14 days post-burn; cytokines were measured by multi-plex array. Following the burn injury, IL-6, IL-1β, KC, G-CSF, TNF, IL-17, MIP-1α, RANTES, and GM-CSF were increased, p < 0.05. IL-2, IL-3, and IL-5 were decreased, p < 0.05. IL-10, IFN-γ, and IL-12p70 were expressed in a biphasic manner, p < 0.05. This temporal cytokine expression pattern elucidates the pathogenesis of the inflammatory response in burned mice. Expression of 11 cytokines were similar in mice and children, returning to lowest levels by post-burn day 14, confirming the utility of the burned mouse model for development of therapeutic interventions to attenuate the post-burn inflammatory response.

Therapy with anti-flagellin A monoclonal antibody limits Pseudomonas aeruginosa invasiveness in a mouse burn wound sepsis model

Background The aim of this study was to evaluate the effect of an anti-flagellin sub-type monoclonal antibody (anti-fla-a) on Pseudomonas aeruginosa infection in a mouse burn model and to assay bacterial dissemination and invasiveness. Methods After immediate post-burn infection with P. aeruginosa, mortality and morbidity (daily weight changes) were monitored in mice treated with anti-fla-a as compared to untreated mice. Bacterial dissemination and invasiveness were monitored by bacterial counts at the burn site and spleen. Three different timing regimens for anti-fla-a treatment were studied: (a) prophylaxis (pre-infection), (b) therapeutic (post-infection), and (c) combined mode. Results Combined regimen of anti-fla-a markedly improved survival of mice infected with P. aeruginosa from 6% to 96% ( p < 0.0001), similar to treatment with Imipenem. Furthermore, a significant improvement in survival was obtained when anti-fla-a was given prior to (75% survival) or post-infection (50% survival). It reduced bacterial load in the spleen ( p = 0.01), preventing bacterial sepsis. Conclusion Anti-fla-a is effective in reducing mortality and morbidity in murine P. aeruginosa-infected burn model.

The effect of CAG repeat length polymorphism in the murine glucocorticoid receptor on transactivation potential

Severe sepsis remains an important cause of death, particularly among trauma and burn patients. The severity of the systemic inflammatory response after trauma or burns and susceptibility to sepsis vary among individuals. One possible mechanism is through differential effects on glucocorticoid receptor (GR) expression by pro-inflammatory mediators (e.g. lipopolysaccharide signaling). In a mouse burn model, we demonstrated differential GR levels, size, and transcriptional activity in CD14 knockout (KO) mice when compared to wild-type (C57BL/6J) after injury. Sequence analysis revealed only 8 CAG repeats in the GR transactivation domain in the CD14 KO; the wild-type contained seventeen. A survey of genomic DNA from 51 mouse strains demonstrated CAG repeat length range from 7 - 17. We then studied the effect of polymorphism in CAG repeat length on GR activity. Fragments with CAG repeats varying from 8-40 (8, 17, 30, 38, and 40) were engineered and shuttled into the wild-type GR expression vector. The resulting plasmids were then co-transfected with a glucocorticoid response element linked to a luciferase in order to compare their transactivation potentials. Transactivation potential was highest in the 17-CAG GR. The effect of GR polymorphisms on GR activity warrants more research as this data suggests a mechanism for the individual differences in response to steroid treatment and the response to injury.

Pulmonary changes in a mouse model of combined burn and smoke inhalation-induced injury

The morbidity and mortality of burn victims increase when burn injury is combined with smoke inhalation. The goal of the present study was to develop a murine model of burn and smoke inhalation injury to more precisely reveal the mechanistic aspects of these pathological changes. The burn injury mouse group received a 40% total body surface area third-degree burn alone, the smoke inhalation injury mouse group received two 30-s exposures of cotton smoke alone, and the combined burn and smoke inhalation injury mouse group received both the burn and the smoke inhalation injury. Animal survival was monitored for 120 h. Survival rates in the burn injury group, the smoke inhalation injury group, and the combined injury group were 70%, 60%, and 30%, respectively. Mice that received combined burn and smoke injury developed greater lung damage as evidenced by histological changes (septal thickening and interstitial edema) and higher lung water content. These mice also displayed more severely impaired pulmonary gas exchange [arterial PO2 (PaO2)/inspired O2 fraction (FiO2)<200]. Lung myeloperoxidase activity was significantly higher in burn and smoke-injured animals compared with the other three experimental groups. Plasma NO2-/NO3-, lung inducible nitric oxide synthase (iNOS) activity, and iNOS mRNA increased with injury; however, the burn and smoke injury group exhibited a higher response. Severity of burn and smoke inhalation injury was associated with more pronounced production of nitric oxide and accumulation of activated leukocytes in lung tissue. The murine model of burn and smoke inhalation injury allows us to better understand pathophysiological mechanisms underlying cardiopulmonary morbidity secondary to burn and smoke inhalation injury.

Towards understanding Pseudomonas aeruginosa burn wound infections by profiling gene expression

Pseudomonas aeruginosa is a key opportunistic pathogen causing severe acute and chronic nosocomial infections in immunocompromised or catheterized patients. It is prevalent in burn wound infections and it is generally multi-drug resistant. Understanding the genetic programs underlying infection is essential to develop highly needed new strategies for prevention and therapy. This work reviews expression profiling efforts conducted worldwide towards gaining insights into pathogenesis by P. aeruginosa, in particular in burn wounds. Work on various infection models, including the burned mouse model, has identified several direct virulence factors and elucidated their mode of action. In vivo gene expression experiments using In vivo Expression Technology (IVET) ascertained distinct regulatory circuits and traits that have helped explain P. aeruginosa s success as a general pathogen. The sequencing of the whole genome from a number of P. aeruginosa strains and the construction of genome-wide microarrays have paved the road to the several insightful studies on the (interacting) traits underlying infection. A series of in vitro and initial in vivo gene expression studies revealed specific traits pivotal for infection, such as quorum sensing systems, iron acquisition and oxidative stress responses, and toxin production among others. The data sets obtained from global transcriptional profiling provide insights that will be essential for the development of new targets and options for prevention and intervention.

Control of Pseudomonas aeruginosa Skin Infections in Mice Is Mast Cell-Dependent

Mast cells (MCs) have recently been shown to be essential for the elicitation of efficient immune responses in murine sepsis. To explore whether MCs also contribute to the control of bacterial skin infections, we studied skin lesions induced by Pseudomonas aeruginosa (PA) in genetically MC-deficient Kit(W)/Kit(W-v) mice, normal Kit(+/+) mice, and MC-reconstituted Kit(W)/Kit(W-v) mice. PA injections resulted in strikingly (>2-fold) larger skin lesions in Kit(W)/Kit(W-v) mice than in Kit(+/+) mice, which exhibited pronounced MC degranulation at infection sites. In addition, neutrophil recruitment following PA injections and bacterial clearance from sites of infection was significantly impaired in Kit(W)/Kit(W-v) mice compared with Kit(+/+) mice. Notably, the adoptive transfer of MCs to the skin of Kit(W)/Kit(W-v) mice before PA infection resulted in normal neutrophil accumulation as well as skin lesions comparable with those in Kit(+/+) mice in both bacterial burden and size. These findings demonstrate for the first time that activated MCs are crucial for the induction of protective innate immune responses to bacterial skin infections.

Innate response to self-antigen significantly exacerbates burn wound depth

A major component of burn injury is caused by additional local damage from acute inflammation. Using a scald burn model in mice, we find that this part of the injury is dependent on recognition of self-antigen by specific natural IgM, leading to activation of the complement system. We propose that the depth of a burn wound is a sum of the thermal energy applied and of the degree of host inflammatory response.

The Establishment of a Pseudomonas aeruginosa-Infected Burn-Wound Sepsis Model and the Effect of Imipenem Treatment

We present a standardized Pseudomonas aeruginosa (PA) infected burn-wound model in mice for evaluating new antimicrobials and therapy strategies for PA infections and demonstrate the effect of the antibiotic imipenem in this model. A 6%-8% total body surface area, full-thickness, scald-burn wound was induced in anesthetized mice. Two study groups (PA-infected burn) were compared with 1 treatment group (systemic imipenem) and 3 control groups (noninfected burn, infected nonburned, and burn with distant infection). Seven-day mortality, quantitative culture from eschars and from spleens, weight loss, and time to healing were compared. The 25%-100% mortality rate in the nontreated PA-infected burn group was directly related to the infecting inoculum. Imipenem treatment reduced the mortality rate to 0-17%. No control animal died. Systemic bacterial dissemination at 48 hours was significantly higher in the study group. Morbidity paralleled survival results. Wound healing was quicker in the imipenem-treated group and control groups compared with the infected nontreated group. The mice model is a useful tool for evaluating new antibacterial agents and strategies for treating PA-infected burn injuries. Imipenem was found to be efficacious in the treatment of severe PA sepsis.

Cytokine expression profile over time in a mouse burn model

Introduction: Severe burns remain a serious clinical problem with few therapeutic options to improve outcome. Cytokines are thought to contribute to the underlying inflammatory pathogenesis. The aim of the present study was to determine the cytokine expression profile over time in a mice burn model in order to determine a time frame that appears to be ideal for intervention. Methods: Male C57/BL/6 mice (n=8 per group and time point) were subjected to either a scald burn injury (35% total body surface area burn) or sham procedure.