Burned Rat Model Research Articles

Immunopotentiating properties of chimeric OprF-OprI-PopB protein against Pseudomonas aeruginosa PAO1 in the infected burned rat model.

Objective(s): Pseudomonas aeruginosa, as an opportunistic pathogen, is known to cause nosocomial infections among patients suffering from burn injuries and also cystic fibrosis patients. The objective of our research was to develop a novel vaccine against P. aeruginosa. Materials and Methods:A recombinant P. aeruginosa subunit vaccine based on the outer membrane proteins, including the OprF-OprI region and its major protein in the type III secretion system, PopB (called FIB protein) was formulated. To induce a robust immune response, our preferred regions were conjugated to a carrier protein, GMCSF (Granulocyte-macrophage colony-stimulating factor). FIB protein’s immunogenicity with and without adjuvant was evaluated in vaccinated rats and also burned rat models, which were subcutaneously challenged by the PAO1 strain of P. aeruginosa.Results:Antibody levels were increased in sera of rats in this study. Assessment of the resident memory CD4+ T cells in splenocytes from vaccinated rats demonstrated that the FIB conjugated with GMCSF could cause higher responses in comparison with other groups. Moreover, immunization with the FIB plus adjuvant protein could improve interferon-gamma (IFN-γ) production, interleukin 17A (IL-17A), and IL-4, contributing to elicit humoral and cellular immune responses and decreased post-infection bacterial loads after PA challenge, pathology, and inflammatory cell infiltration. Conclusion:These observations demonstrated that FIB conjugated with GMCSF can be a putative vaccine candidate against P. aeruginosa in burnt rat models.

Fabrication and Characterization of Chitosan-Polyethylene Glycol (Ch-Peg) Based Hydrogels and Evaluation of Their Potency in Rat Skin Wound Model.

Thermal burns are a major cause of death and suffering around the globe. They can cause debilitating, life-altering injuries as well as lead to significant psychological and financial consequences. Several research works have been conducted in attempt to find a wound healing therapy that is successful. At present, hydrogels have been widely used in cutting-edge research for this purpose because they have suitable properties. This study aimed to see how therapy with chitosan-polyethylene glycol (Ch-Peg) based hydrogels affected the healing of burn wounds in rats. With the concern of public health, xanthan gum (X), boric acid (B), gelatin (Ge), polyethylene glycol (Peg), chitosan (Ch), glutaraldehyde (G), and HPLC-grade water were prepared using X : Ge : G, X : Ge : Peg : G, X : Ge : Ch : G, X : Ge : Peg : Ch : G, X : Ge : B : Ch : G, X : Ge : B : Peg : G, and X : Ge : B : Peg : Ch : G. The produced composite hydrogels were examined for swelling ability, biodegradability, rheological characteristics, and porosity. The 3D structure of the hydrogel was revealed by scanning electron microscopy (SEM). After that, the structural characterization technique named Fourier-transform infrared spectroscopy (FTIR) was used to describe the composites (SEM). Lastly, in a rat skin wound model, the efficacy of the produced hydrogels was studied. Swelling ability, biodegradability, rheological properties, and porosity were all demonstrated in composite hydrogels that contained over 90% water. Hydrogels with good polymeric networks and porosity were observed using SEM. The existence of bound water and free, intra- and intermolecule hydrogen-linked OH and NH in the hydrogels was confirmed using FTIR. In a secondary burned rat model, all hydrogels showed significant wound healing effectiveness when compared to controls. When compared to other composite hydrogels, wounds treated with X : Ge : Peg : Ch : G, X : Ge : B : Peg : G, and X : Ge : B : Peg : Ch:G recovered faster after 28 days. In conclusion, this research suggests that X : Ge : Peg : Ch : G, X : Ge : B : Peg : G, and X : Ge : B : Peg : Ch : G could be used to treat skin injuries in the clinic.

Human Umbilical Cord Mesenchymal Stem Cells Transplantation Promotes Cutaneous Wound Healing of Severe Burned Rats

BackgroundSevere burns are a common and highly lethal trauma. The key step for severe burn therapy is to promote the wound healing as early as possible, and reports indicate that mesenchymal stem cell (MSC) therapy contributes to facilitate wound healing. In this study, we investigated effect of human umbilical cord MSCs (hUC-MSCs) could on wound healing in a rat model of severe burn and its potential mechanism.MethodsAdult male Wistar rats were randomly divided into sham, burn, and burn transplanted hUC-MSCs. GFP labeled hUC-MSCs or PBS was intravenous injected into respective groups. The rate of wound closure was evaluated by Image Pro Plus. GFP-labeled hUC-MSCs were tracked by in vivo bioluminescence imaging (BLI), and human-specific DNA expression in wounds was detected by PCR. Inflammatory cells, neutrophils, macrophages, capillaries and collagen types I/III in wounds were evaluated by histochemical staining. Wound blood flow was evaluated by laser Doppler blood flow meter. The levels of proinflammatory and anti-inflammatory factors, VEGF, collagen types I/III in wounds were analyzed using an ELISA.ResultsWe found that wound healing was significantly accelerated in the hUC-MSC therapy group. The hUC-MSCs migrated into wound and remarkably decreased the quantity of infiltrated inflammatory cells and levels of IL-1, IL-6, TNF-α and increased levels of IL-10 and TSG-6 in wounds. Additionally, the neovascularization and levels of VEGF in wounds in the hUC-MSC therapy group were markedly higher than those in other control groups. The ratio of collagen types I and III in the hUC-MSC therapy group were markedly higher than that in the burn group at indicated time after transplantation.ConclusionThe study suggests that hUC-MSCs transplantation can effectively improve wound healing in severe burned rat model. Moreover, these data might provide the theoretical foundation for the further clinical application of hUC-MSC in burn areas.

Parecoxib Reduces Systemic Inflammation and Acute Lung Injury in Burned Animals with Delayed Fluid Resuscitation

Burn injuries result in the release of proinflammatory mediators causing both local and systemic inflammation. Multiple organ dysfunctions secondary to systemic inflammation after severe burn contribute to adverse outcome, with the lungs being the first organ to fail. In this study, we evaluate the anti-inflammatory effects of Parecoxib, a parenteral COX-2 inhibitor, in a delayed fluid resuscitation burned rat model. Anaesthetized Sprague Dawley rats were inflicted with 45% total body surface area full-thickness scald burns and subsequently subjected to delayed resuscitation with Hartmann's solution. Parecoxib (0.1, 1.0, and 10 mg/kg) was delivered intramuscularly 20 min after injury followed by 12 h interval and the rats were sacrificed at 6 h, 24 h, and 48 h. Burn rats developed elevated blood cytokines, transaminase, creatinine, and increased lung MPO levels. Animals treated with 1 mg/kg Parecoxib showed significantly reduced plasma level of CINC-1, IL-6, PGEM, and lung MPO. Treatment of 1 mg/kg Parecoxib is shown to mitigate systemic and lung inflammation without significantly affecting other organs. At present, no specific therapeutic agent is available to attenuate the systemic inflammatory response secondary to burn injury. The results suggest that Parecoxib may have the potential to be used both as an analgesic and ameliorate the effects of lung injury following burn.

Sustained oxidative stress causes late acute renal failure via duplex regulation on p38 MAPK and Akt phosphorylation in severely burned rats.

BackgroundClinical evidence indicates that late acute renal failure (ARF) predicts high mortality in severely burned patients but the pathophysiology of late ARF remains undefined. This study was designed to test the hypothesis that sustained reactive oxygen species (ROS) induced late ARF in a severely burned rat model and to investigate the signaling mechanisms involved.Materials and MethodsRats were exposed to 100°C bath for 15 s to induce severe burn injury (40% of total body surface area). Renal function, ROS generation, tubular necrosis and apoptosis, and phosphorylation of MAPK and Akt were measured during 72 hours after burn.ResultsRenal function as assessed by serum creatinine and blood urea nitrogen deteriorated significantly at 3 h after burn, alleviated at 6 h but worsened at 48 h and 72 h, indicating a late ARF was induced. Apoptotic cells and cleavage caspase-3 in the kidney went up slowly and turned into significant at 48 h and 72 h. Tubular cell ROS production shot up at 6 h and continuously rose during the 72-h experiment. Scavenging ROS with tempol markedly attenuated tubular apoptosis and renal dysfunction at 72 h after burn. Interestingly, renal p38 MAPK phosphorylation elevated in a time dependent manner whereas Akt phosphorylation increased during the first 24 h but decreased at 48 h after burn. The p38 MAPK specific inhibitor SB203580 alleviated whereas Akt inhibitor exacerbated burn-induced tubular apoptosis and renal dysfunction. Furthermore, tempol treatment exerted a duplex regulation through inhibiting p38 MAPK phosphorylation but further increasing Akt phosphorylation at 72 h postburn.ConclusionsThese results demonstrate that sustained renal ROS overproduction induces continuous tubular cell apoptosis and thus a late ARF at 72 h after burn in severely burned rats, which may result from ROS-mediated activation of p38 MAPK but a late inhibition of Akt phosphorylation.

Melatonin Protection against Burn-Induced Hepatic Injury by Down-Regulation and Nuclear Factor Kappa B Activation

Melatonin exhibits a wide variety of biological activity including antioxidant and anti-inflammatory effects. We have previously reported its protective effect on hepatic oxidative hepatic injury in burns. In this study, we investigated the role of nuclear factor kappa B (NF-κB) in melatonin-mediated protection against liver injury by using the burned-rat model. Melatonin (N-acetyl-5-methoxytriptamin, 10mg/kg (-1), i.p.) was administered immediately and 12 hours after thermal skin injury. Hepatic NF-κB expression was determined by Western blotting. TNF-α level in liver homogenate was quantified using enzyme-linked immunosorbent assay (ELISA) kit. Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined to assess liver injury at the 24th hour after burns. Thermal skin injury caused significant elevation of hepatic NF-κB expression by 48 percent, TNF-α level by 55 percent and plasma AST and ALT activities by 2- and 3-fold, respectively, in comparison with normal control rats. Treatment with melatonin decreased significantly elevated hepatic NF-κB activity and TNF-α, maintaining the levels close to the control values Melatonin suppressed the elevation of plasma AST and ALT activities (p less than 0.001), which remained significantly increased compared to controls. In conclusion, thermal skin injury causes hepatic NF-κB activation that may mediate the release of hepatic TNF-α and contribute to liver damage. Melatonin protects against burn-induced hepatic injury as to a certain extent this effect may result from the suppression of NF-κB-mediated inflammatory response.

Effective melatonin protection of burn-induced hepatic disorders in rats

Abstract We studied the effect of melatonin on morphological and functional disorders using serum markers of liver dysfunction such as cholinesterase and gamma glutamyl transpeptidase, hepatic protein content and malondialdehyde in a burned-rat model. Melatonin (10 mg/kg (−1), i.p) was administered immediately and then 12 h after 30% of total body surface area burns of male Wistar rats. The burns induced an increase of hepatic malondialdehyde levels by 166% (p<0.001), and also vascular congestion, leukocyte infiltration around the central veins, intracellular vacuolization, hepatic cell degeneration and apoptotic bodies (Councilman’s bodies). These changes were associated with significantly reduced serum cholinesterase (36%), gamma glutamyl transpeptidase (76%), hepatic proteins (52%) and serum albumin (37%) (p<0.001–0.0001). Treatment with melatonin reduced elevated hepatic malondialdehyde values by 50% (p<0.01). Melatonin restricted degenerative alteration in the hepatocytes: it protected the burninduced decrease of serum gamma glutamyl transpeptidase activity by 48% (p<0.01), hepatic proteins by 64% (p<0.01), and serum activity of cholinesterase as the only marker of liver damaged synthetic function by 57% (p<0.0001) but did not exert any significant influence on serum albumin concentration. Melatonin repaired the pathomorphological lesions and functional disorders. It could restore liver damage following thermal injury in humans.

Escherichia coli endotoxin enhances acute renal failure in rats after thermal injury

This study was designed to evaluate the burned rat model to determine whether there are any differences in endotoxin-sensitive kidney functions between an infant rat (10-day-old pup) and an adult rat (10-week-old rat). Renal failure was observed in the infant burned rat and histological changes showed the adhesion of inflammatory cells in the glomerular capillaries and vacuolar changes in the renal proximal tubular cell. A horseradish peroxidase (HRP) tracer experiment suggested that the intestinal barrier damage of the infant burned rat was more severe than that of the adult burned rat. Therefore, more bacterial translocation of the intestinal flora, rich in endotoxin, might be expected in the infant versus the adult rats. Renal failure was not observed in the adult burned rat, so we investigated to determine the effects of endotoxin on the kidney function of the adult burned rat with low lethal lipopolysaccharide (LPS) or carrageenan (CAR). CAR is known to increase sensitivity to the lethal effects of endotoxin in rodents. Our present data demonstrated that renal failure was observed in the LPS- or CAR-treated adult burned rat and LPS- and CAR-treated adult rat (non-burned). These results show the possibility that endotoxin enhances renal failure in a burned rat model and provide additional support for the hypothesis that postburn renal failure is mediated, in part, by endotoxin associated with bacterial translocation.

Oxidative stress and metallothionein expression in the liver of rats with severe thermal injury

The aim of this work was to study oxidative stress and the compensating mechanisms implicated in severe thermal injury using the burned rat model. Results showed that after thermal injury glutathione (GSH) level was decreased, oxidized glutathione (GSSG) and the ratio of GSSG/GSH increased both at 24 and 48 h in the liver. The activities of GSH-reductase (GSH-Rx) in the liver and GSH-peroxidase (GSH-Px) both in the liver and erythrocytes increased at 24 h and then decreased at 48 h. The level of α-tocopherol in plasma was reduced at 24 h. Lipid peroxide levels increased both at 24 and 48 h in the liver. The serum zinc level decreased, reaching a minimum at 12 h, whereas liver zinc level was elevated and reached the maximum at 12 h. After severe thermal injury enhancement of metallothionein (MT) expression has been discovered for the first time. MT content in the liver increased both at 24 and 48 h. Expression of MT-I mRNA was activated at 3 h and reached the top at 24 h postburn. The conclusion is that severe thermal injury gives rise to oxidative stress and dramatic enhancement of MT expression could be one of the important compensative mechanisms of natural defense system postburn.

Changes in the tissue and plasma superoxide dismutase (SOD) levels in a burned rat model.

To examine whether thermal injury alters the superoxide dismutase (SOD) concentrations in various types of tissue or plasma, we studied the plasma and tissue Mn- and Cu/Zn-SOD levels in a rodent burn model. The animals were resuscitated with saline (50 mg/kg, i.p.) immediately following thermal injury and thereafter were sacrificed at either 6 or 24 hours post-burn. The Mn- and Cu/Zn-SOD levels were measured using an enzyme-linked immunosorbent assay (ELISA). The plasma Mn- and Cu/Zn-SOD concentrations significantly increased 6 hours after the injury and positively correlated with the burn size. The kidney Mn-SOD concentrations were significantly higher 24 hours after the injury in the animals with 30% burns than in those with either sham or 50% burn injuries. The lung Cu/Zn-SOD concentrations were also significantly higher 6 hours after the injury in animals with 30% burns than in the other two types above. These findings suggest that the changes in the SOD concentrations after burn injury vary according to the type of SOD and also the type of tissue. As a result, the SOD concentrations may play some role in the early response to thermal trauma.

Modulating the functions of neutrophils and lipid peroxidation by FK506 in a rat model of thermal injury

Neutrophils diffusely invade lung, liver, kidney, intestine, muscle and burned skin following burn injury. To ameliorate this invasion and minimize its effects, neutrophils can be modulated by giving neutrophil inhibitors and modulators. In this study, FK506 was used to decrease neutrophil infiltration and lipid peroxidation in remote organs (lung, liver, kidney and intestine) in a burned rat model. FK506 is a new major immunosuppressive agent that is known to modulate neutrophils during inflammation. Neutrophil infiltration was assessed indirectly by measuring myeloperoxidase (MPO) activity biochemically in remote organs following 30% full thickness burn injury. Malondialdehyde (MDA), the end product of lipid peroxidation, was measured biochemically in remote organs and plasma to determine if there is a relationship between neutrophil infiltration and lipid peroxidation after burn injury. FK506 was given intramuscularly at the dose of 0.5 and 1.0 mg/kg for three days before burn injury. Thermal trauma to the skin caused a statistically significant increase in MPO activity and MDA content in remote organs. FK506 was effective in reducing lipid peroxidation and neutrophil infiltration especially at 24 h postinjury in lung, liver and kidney. FK506 may have some benefit (prophylactic) in reducing systemic neutrophilic injury and related lipid peroxidation in burns.

Chronic Indomethacin Administration Blocks Increased Body Temperature After Burn Injury in Rats

This study investigates the hypothesis that continuous administration of indomethacin (a cyclo-oxygenase inhibitor) will chronically reduce body temperature (TB) in burned rats (i.e., modulate "true" fever). Male Sprague-Dawley rats had radio transmitters and osmotic pumps (containing indomethacin) placed in the peritoneal cavity, and 7 days later large full-thickness scald burns were produced. Activity and TB were continually recorded through the 14th postburn day (PBD). There were 4 experimental groups: burn + indomethacin (B-In), n = 9; burn + polyethylene glycol (B-Peg), n = 6; control + indomethacin (C-In), n = 9; and control + polyethylene glycol (C-Peg), n = 6. From PBD 5 through PBD 10, the B-Peg group had consistently and significantly higher TB during light hours than the B-In, C-In, and C-Peg groups. From PBD 7 through PBD 12, the B-In group had an average TB during light hours significantly lower or not different than the C-In and C-Peg groups. These results support the hypothesis that in this burned-rat model chronically increased TB during the light hours may be "fever" (rather than hyperthermia) and that the final effector link could be a prostanoid because changes in activity do not explain the changes in body temperature.

Activity and protein synthesis of manganese superoxide dismutase in a burned rat model

Activity and protein synthesis of manganese superoxide dismutase in a burned rat model

The effect of an SOD derivative (SM-SOD) administration in a burned rat model.

To determine whether superoxide radicals may influence the pathogenesis of burn injury, we investigated the effects of a long-acting, site-directed superoxide dismutase derivative (SM-SOD) on the rats subjected to burn shock. Anesthesized animals were injected intravenously with either 2 ml/kg of saline or SM-SOD (10 mg/kg; dissolved in saline). After 30 min they were subjected to full-thickness burns of about 40% of total body surface area. The 7-day survival rate was significantly higher in the SM-SOD-treated animals than in the untreated controls. Also, administration of SM-SOD markedly inhibited the increase in the levels of thiobarbituric acid reactive metabolites, such as lipid peroxides in the plasma, lung and kidney, and the decrease in plasma protein levels particularly at the early stage of burn injury. These findings suggested that superoxide radicals may play a critical role in the pathogenesis following thermal injury.

Effect of Early Feeding on the Postburn Hypermetabolic Response in Rats

The effect of early enteral feeding on the hypermetabolic response following burn injury in a rat burn model has been investigated. The rates of heat production and partitioned heat loss were determined on the fourteenth postburn day for five treatment groups: I) burn, fed rat chow ad libitum starting 2 hours postburn; II) burn, fed by gastrostomy beginning 2 hours postburn; III) burn, fed by gastrostomy, delayed until 72 hours postburn; IV) controls, fed by gastrostomy 2 hours post anesthesia; and V) controls, fed rat chow ad libitum 2 hours post anesthesia. Gastrostomy feedings delivered 175 kcal/kg.day. The mean rates of heat production and heat loss for the three burn groups did not differ significantly whether rats were fed chow ad libitum, or by gastrostomy early or late. Contrary to previous studies using a guinea pig model, method and timing of feeding in this burned rat model had no significant effect on the postburn increment in the rate of heat loss and the corresponding increment in the rate of heat production.

The effect of ibuprofen on postburn metabolic and immunologic function

Patients suffering severe trauma, including thermal injuries, demonstrate both a hypermetabolic response and an immunosuppressed state following the injury. Biochemically, these patients produce extremely large amounts of cyclooxygenase products, including prostaglandin E. We have investigated the effect of a drug, ibuprofen, which blocks the synthesis of prostaglandins in a burned rat model. Ibuprofen at high doses was found to significantly diminish the hypermetabolic response to burn injury and sepsis. The same dosage of ibuprofen increased the mortality rate in the same burn sepsis model. Prostaglandin E may therefore exert some beneficial effects in traumatized patients by altering their metabolism.

Plasma proteolytic activity following burns.

Because a number of metabolic events which are triggered by proteolysis in the bloodstream are activated following trauma, net proteolytic activity (P.A.) in the plasma of 37 pediatric burned patients was measured. The P.A. assay involved incubating plasma with a radioiodinated protein substrate and counting the isotopic activity of the hydrolyzed fragments in the acid-soluble fraction. In patients, plasma P.A. increased in direct proportion to the extent of burn injury. To examine additional trends in plasma P.A. suggested by the patient P.A. data, we measured plasma P.A. in a burned rat model: circulating P.A. was significantly elevated at 6 hours and until at least 2 weeks postburn; infection with Pseudomonas and use of the proteolytic debriding agent, Travase, each further elevated this activity; the plasma P.A. was not directly derived from the burn site. We postulate that this elevated circulating P.A. triggers some of the pathologic as well as some physiologic sequelae which follow burn trauma.

Active and passive immunization with Pseudomonas aeruginosa ribosomal vaccines and antisera in the burned rat model

Pseudomonas aeruginosa ribosomal vaccines were tested for their ability to protect rats subjected to a 20% total body surface burn against the lethal effects of infection with homologous organisms. When administered prior to burning, the vaccines provided 100% protection. When administered postburning, the vaccine from one strain also provided 100% protection when the time interval between vaccination and infection was 3 days. When this time interval was reduced to 1 or 2 days, approximately 50% protection was obtained with the same vaccine. The vaccine from a second strain tested provided about 50% protection with a 3-day time interval. In addition, passive immunization using antiserum to a ribosomal vaccine was also demonstrated to be effective in protecting burned and infected rats, especially when multiple doses of antiserum were used. In this case, 80% protection was obtained (with no protection observed using multiple doses of normal serum). Finally, a comparison of ribosomal and lipopolysaccharide vaccines and antisera was also performed.

Effect of transfusion on immune function in a traumatized animal model.

Blood transfusions repeatedly have been shown to prolong allograft survival, probably by stimulating suppressor T lymphocytes. The effects of transfusions on immune function in traumatized patients has not previously been investigated. We investigated the effects of transfusions on the immune system using a burned rat model. The transfusions were found to have no effect on the white blood cell counts, differential cell count, or neutrophil migration and bactericidal index. Those animals that received transfusion did exhibit impaired cell-mediated immunity and macrophage migration. Blood transfusions seem to increase further the immunosuppression seen with trauma and surgery.

Characterization of mouse monoclonal antibodies directed against Pseudomonas aeruginosa lipopolysaccharides.

Mouse monoclonal antibodies that react with O-side chain specific, O-side chain cross-reactive, and core P. aeruginosa lipopolysaccharide determinants have been isolated. The monoclonals directed at O-side chain determinants are generally opsonophagocytic with human neutrophils and human complement. They also protect mice from intraperitoneal and intravenous challenge and protect in the burned rat model of infection.