Basic Science Aspects
Systemic Inflammatory Responses and Multiple Organ Dysfunction Syndrome Following Skin Burn Wound and Pseudomonas aeruginosa Infection in Mice
Li, Na; Hu, Xiaolong; Liu, Yang; Wang, Yaojun; Wang, Yunchuan; Liu, Jiaqi; Cai, Weixia; Bai, Xiaozhi; Zhu, Xiongxiang; Han, Juntao; Hu, Dahai
Department of Burns and Cutaneous Surgery, Xijing Hospital, the Fourth Military Medical University, Xi’an, Shaanxi Province, China
Received 15 Mar 2013; first review completed 2 Apr 2013; accepted in final form 8 May 2013
Address reprint requests to Dahai Hu, PhD, MD, Department of Burns and Cutaneous Surgery, Xijing Hospital, the Fourth Military Medical University, Xi’an, Shaanxi Province, China. E-mail: Hudhai@fmmu.edu.cn.
N.L. and X.H. contributed equally to this work.
This study was supported by a grant from the National Natural Science Foundation of China (no. 81101405).
ABSTRACT: Burn wound–related sepsis is associated with the development of systemic inflammatory response syndrome and multiple organ dysfunction syndrome (MODS). This study is aimed at investigating the development and progression of SIS and MODS in a mouse model of skin burn sepsis. C57BL/6J mice were randomly divided into the sham, burn, Pseudomonas, and burn/Pseudomonas groups. The back skin of the sham, burn, and burn/Pseudomonas groups was burned about 10% of total area with using 37°C or 98°C water for 8 s, respectively, followed by inoculating with Pseudomonas aeruginosa. The Pseudomonas group was infected with P. aeruginosa without burn injury. Their body weights, mortality, organ histology, and function as well as systemic inflammation were measured longitudinally. The burn/Pseudomonas mice lost more body weights than did mice from the other groups and had a significantly higher mortality rate (P < 0.05). The burn/Pseudomonas mice exhibited significantly higher levels of bacterial loads in different organs and serum endotoxin, interleukin 1β, interleukin 6, tumor necrosis factor α, and C-reactive protein than those in mice from the other groups (P < 0.05). The burn/Pseudomonas mice also displayed more severe liver, lung, and kidney tissue damage and impaired organ functions, particularly at 72 h after inoculation than did the burn and Pseudomonas groups of mice. Our data indicate that burn and P. aeruginosa infection induced severe sepsis and rapidly progressed into systemic inflammatory response syndrome and MODS in mice.
Patients with severe extensive burn wound are sensitive to bacterial infection, which is associated with the development of sepsis. Without rapid and optimal control, patients with severely extensive burns and sepsis can rapidly develop systemic inflammatory response syndrome (SIRS), which results in the damage of many organs, such as the lung, liver, and kidney, leading to the development of multiple organ dysfunction syndrome (MODS) (1, 2). There are more than 18 million patients with severe sepsis every year, and the incidence of severe sepsis is increasing in the world. Approximately, 30% of patients with severe sepsis are attributed to burn infection. Although the approaches in medial researches have led to the development of therapeutic strategies for the treatment and prevention of burn sepsis in the clinic, the mortality rate of patients with burn sepsis remains high because of the pathogenic complex of sepsis (3). The outcomes of patients with burn sepsis are affected by many factors, including preexisting diseases, organisms, and other variables. However, the pathogenesis of burn sepsis is still not fully understood. Therefore, further understanding the pathogenic process of burn sepsis will be of great significance.
Previous studies have generated several animal models of sepsis. Although the in vivo lipopolysaccharide (LPS) model has been widely used to study the pathogenesis of sepsis (4, 5) endotoxemia-related sepsis does not reflect the same entity and complexity of sepsis in humans (6). Animal models of peritonitis have also been used for studying sepsis (7–9), but these models are not feasible for studying the pathogenic process of burn-related hypovolemic shock and Pseudomonas aeruginosa infection. There are some animal models of burn wound infection (10–14); however, these models are not involved in P. aeruginosa infection or center only on the lung injury. Other models of skin burn sepsis are established by subcutaneous or subeschar inoculation with bacteria or by inoculating with clinical isolates of P. aeruginosa (PA14, PAK, or PAO1) (19, 20). More importantly, many of these studies mainly center on the therapeutic effects of reagents on the mortality and bacterial loads in animals. Accordingly, it is unclear whether inoculation of burn wound with standard P. aeruginosa can induce severe sepsis, which progresses into the development of SIRS and MODS, similar to that in human patients with burn sepsis.
The present study used a mouse model of skin burn–related sepsis by inoculating the burn wound surface with a higher dose of standard P. aeruginosa to characterize the development and progression of burn sepsis longitudinally in C57BL/6J mice. We found that the mice with burn sepsis reproducibly developed severe SIRS and MODS and had a high rate of mortality, fulfilling the sepsis criteria, as defined by the 2001 International Sepsis Definition Conference (21).
MATERIALS AND METHODS
Female C57BL/6J mice 8 to 10 weeks of age were from Experimental Animal Center of the Fourth Military Medical University. Animals were housed in ventilated cages in a specific pathogen-free facility with 12-h light-dark cycles, 23°C (SD, 2°C), 30% to 60% humidity, and all mice were allowed free access to a standard laboratory rodent chow and water. Individual animals were anesthetized intraperitoneally with 0.1% pentobarbital (5 mg/kg body weight) and subjected to surgical procedures or sample collection. The experimental protocols were approved by Experimental Animal Committee of the Fourth Military Medical University.
Preparation of P. aeruginosa
Pseudomonas aeruginosa (ATCC27853) was from the American Type Culture Collection (ATCC, Manassas, Va). Bacterium was cultured in lysogeny broth (LB) medium overnight, and the bacteria were resuspended in 10% glycerin medium, aliquoted, and stored at −80°C. The bacterium suspension was subjected to serial dilutions and plated on LB agar plates for the determination of colony-forming unit (CFU).
Establishment of burn sepsis model in mice
The mouse model of skin burn sepsis was established, as described previously with minor modification (19, 30, 31). Briefly, mice were randomly assigned to the sham burn, burn, Pseudomonas, and burn/Pseudomonas groups (n = 10–20 per group, respectively). The mice were anesthetized and shaved on their back.. The mice in the sham group were burned about 2 cm in diameter (about 10% total body surface area) using 37°C water for 8 s, and the mice in the burn/Pseudomonas group were burned about 2 cm in diameter throughout the full thickness of skin (III°) using 95°C to 98°C water steam for 8 s. When the burned regions were cooled down for about 5 min to normal body temperature, the surface area was inoculated with 50 μL (2.5 × 105 CFU) of P. aeruginosa bacterial suspension using a microinjector. The mice in the burn group were burned using 95°C to 98°C water steam without bacterial inoculation, and the mice in the Pseudomonas group were inoculated with the same dose of bacteria without prior burn injury. Subsequently, the mice were administered intraperitoneally with 1 mL of physiological saline immediately. The mice were monitored daily for their survival up to 1 week after burn injury. Their body weights were measured, and their blood samples were collected daily for the first 3 days, excluding dead mice at each time point. Some mice from each group were anesthetized at 24, 48, and 72 h after burn injury and subjected to perfusion with 15 to 20 mL of saline. Briefly, individual mice were placed on the operating plate with their back down and subjected to a long midline incision to expose the heart carefully. After the right atrium was cut with a hole, the left ventricle was inserted with a butterfly needle that was connected with a plastic tubing filled with perfusion saline. Individual mice were subjected with saline perfusion at 1 mL/min using a micropump for 15 to 20 min until the flow-out was clear from the right atrium hole. Subsequently, their heart, liver, spleen, lung, and kidney tissues were dissected and weighed for further examinations.
The lung, liver, and kidney tissue specimens were fixed in 4% paraformaldehyde overnight and paraffin embedded. The tissue sections (5 μM) were stained with hematoxylin-eosin, followed by examining under a light microscope in a blinded manner.
Real-time polymerase chain reaction
Total RNA was extracted from individual lung, liver, and kidney samples using Trizol reagent, according to the manufacturer’s instructions (Invitrogen, Grand Island, NY) and reversely transcribed into cDNA using the ImProm-II Reverse Transcription kit and poly-dT primers (Promega, Beijing, China). The relative levels of target gene mRNA transcripts were determined by real-time polymerase chain reaction (PCR) using the SYBR Premix Ex Taq kit (Takara Biomedical Technology, Beijing China) and specific primers. The sequences of primers were sense 5′-TCCAGGATGAGGACATGAGCAC-3′ and antisense 5′-GAACGTCACACACCAGCAGGTTA-3′ for interleukin 1β (IL-1β) (305 base pairs [bp]); sense 5′-CCACTTCACAAGTCGGAGGCTTA-3′ and antisense 5′-GCAAGTGCATCATCGTTGTTCATAC-3′ for IL-6 (147 bp); sense 5′-GTTCTATGGCCCAGACCCTCAC-3′ and antisense 5′-GGCACCACTAGTTGGTTGTCTTTG-3′ for tumor necrosis factor α (TNF-α) (209 bp); sense 5′-TGTGTCCGTCGTGGATCTGA-3′ and antisense 5′-TTGCTGTTGAAGTCGCAGGAG-3′ for GAPDH (379 bp), respectively. The amplification was performed in triplicate at 94°C for 2 min and subjected to 35 cycles of 94°C for 30 s and 60°C for 45 s. The relative levels of IL-1β, IL-6, and IL-10 mRNA transcripts to GAPDH were calculated by 2-ΔΔCt.
Biochemistry and enzyme-linked immunosorbent assay
Blood samples were centrifuged at 4,000 revolutions/min for 10 min, and individual plasma samples were prepared and stored at −20°C. The concentrations of plasma total proteins, creatinine (Cr), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and C-reactive protein (CRP) were measured by routine methods. The levels of serum IL-1β, IL-6, and TNF-α were tested by enzyme-linked immunosorbent assay using specific cytokine kits, according to the manufacturer’s instructions (R&D Systems, Beijing, China). The limitation of detection for IL-1β, TNF-α, and IL-6 was 16 to 2,000 pg/mL, respectively. The concentrations of serum endotoxin in individual samples were detected using the HEK-Blue LPS Detection Kit, according to the manufacturers’ instruction (Invitrogen).
Organ bacterial quantification
Individual heart, liver, spleen, kidney, and lung wet tissue samples were weighed and homogenized in phosphate-buffered saline at 0.1 g/mL, followed by centrifuging at 3,000g for 15 min. The supernatants were collected and subjected to serial dilutions. Subsequently, the tissue homogenates were plated in triplicate on LB agar plates and cultured at 37°C overnight. The numbers of bacterial colonies in individual tissue samples were counted in a blinded manner.
Data are expressed as the mean (SD). The difference among the different groups or time points was analyzed by one-way analysis of variance and post hoc Tukey-Kramer analysis, or by Kruskal-Wallis test, as appropriate. The difference between two groups was analyzed by Mann-Whitney U test. The survival curves were estimated by the Kaplan-Meier method and analyzed by the log-rank test. All statistical analyses were performed by SPSS software 16.0 version. P < 0.05 was considered statistically significant.
Mice with burn sepsis lost body weights and had a high mortality
To study the pathogenesis of burn sepsis, female adult C57BL/6 mice were randomized and subjected to skin sham burn, burn injury, Pseudomonas alone, or burn/Pseudomonas groups. Three days after burn injury, whereas there was no obvious damage on the burned skin in the sham group of mice, there were brown dry scab and redness on the burned skin area of the burn group of mice, but obvious skin injury in the infection area of the Pseudomonas group of mice (Fig. 1A). In contrast, the burn region in the burn/Pseudomonas group of mice exhibited swelling and black necrosis. Furthermore, measurement of body weights indicated that there was no significant change in the body weights in the sham group of mice during the 3-day observation period, but the mean body weights in the burn group of mice were reduced by 5% during this period. However, the body weights in the Pseudomonas and burn/Pseudomonas groups of mice significantly decreased by near 10% at 24 h after burn and were further reduced by near 12.5% and 20% at the end of observation, respectively (Fig. 1B). Furthermore, the mice in the burn/Pseudomonas group, but not in other groups, displayed inactive, slow movement, decreased alertness, and hunched postures. In addition, whereas all mice in the sham and burn groups survived through the experimental period, 90% of mice in the Pseudomonas group survived (Fig. 1C). In contrast, 70% of mice in the burn/Pseudomonas group survived at 24 h after inoculation and 40% of mice at 72 h after inoculation. Similar mortality rates were observed in the burn/Pseudomonas group of mice in five independent trials conducted over a period of 5 months (Fig. 1D). Therefore, this mouse model of burn sepsis had a high mortality rate.
Quantification of plasma endotoxin and tissue bacteria
Next, the concentrations of serum endotoxin were measured using an endotoxin-sensitive HEK-Blue-4 cells and specific kit. There was no detectable level of serum endotoxin in the sham and burn groups of mice, and only very low levels of serum endotoxin were detected in the Pseudomonas group of mice (Fig. 2A). In contrast, the concentrations of serum endotoxin in the burn/Pseudomonas group of mice were dramatically elevated at 3 days after inoculation. Further characterization indicated that whereas there were no detectable bacteria in the sham and burn groups of mice, there were moderate numbers of bacteria detected in the liver, lung, spleen, and kidney, but not in the heart, of the Pseudomonas group of mice (Fig. 2B). The numbers of bacteria in the liver, lung, spleen, and kidney of the burn/Pseudomonas group of mice increased by twofold to threefold and also detected in the heart of this group of mice. Collectively, these data provided evidence of P. aeruginosa–related septicemia in the Pseudomonas and burn/Pseudomonas groups of mice.
Changes of inflammatory cytokines
Systemic bacterial infection usually causes potent inflammatory responses. To investigate the inflammatory responses in the different organs, the relative levels of IL-1β, IL-6, and TNF-α mRNA transcripts in the liver and lung of the different groups of mice were determined by quantitative reverse transcriptase–PCR (Fig. 3A). Although very low levels of IL-1β, IL-6, and TNF-α mRNA transcripts in the liver and lung of the sham group of mice slightly increased, levels of IL-1β, IL-6, and TNF-α mRNA transcripts were detected in the liver and lung of the burn group and Pseudomonas group of mice. In contrast, the levels of IL-1β, IL-6, and TNF-α mRNA transcripts in the liver and lung of the burn/Pseudomonas group of mice were significantly elevated, as compared with that in other groups. Similarly, there were very low levels of serum IL-1β, IL-6, and TNF-α in the sham and burn groups of mice throughout the observation period (Fig. 3B) and moderate levels of serum CRP in the burn group, but not in the sham group. Gradually increased levels of serum IL-1β and TNF-α and moderate levels of serum IL-6 and CRP were detected in the Pseudomonas group of mice (Fig. 3B). As expected, significantly higher levels of IL-1β, IL-6, and TNF-α as well as CRP were detected in the burn/Pseudomonas group of mice throughout the observation period. These data clearly demonstrated that mice in the Pseudomonas and burn/Pseudomonas groups developed varying levels of systemic inflammatory responses.
Multiple organ injury and dysfunction
Systemic bacterial infection and inflammation can result in multiple organ injury. Histological analysis revealed that whereas normal lung morphology was observed in the sham and burn groups of mice, obviously pulmonary interstitial edema and moderate degrees of inflammatory infiltrates were detected in the lung sections of the Pseudomonas group of mice at 24 h after inoculation (Fig. 4A). Moderate levels of pulmonary interstitial edema and inflammation were observed in the burn group, but not in the sham group of mice, and severe inflammation was detected in the lung of the Pseudomonas group of mice at later time points. In contrast, severe pulmonary interstitial edema and inflammatory infiltrates around bronchia as well as bloody effusion in the alveolar cavity were observed in the burn/Pseudomonas group of mice at 24 h after inoculation, and worse inflammation was detected at later time points.
Similarly, whereas there were a few inflammatory infiltrates around the liver lobules in the Pseudomonas group, but not in the sham and burn groups of mice at 24 h after inoculation, obvious hepatocyte swelling, degeneration, and vacuolation were observed in the liver sections of the Pseudomonas group of mice at later time points (Fig. 4B). In contrast, the liver sections of the burn/Pseudomonas group of mice displayed abnormal hepatic lobules, hemorrhage, necrosis, hepatocyte swelling, degeneration, and vacuolation, accompanied by inflammatory infiltrates at 24 h after inoculation, and the pathological changes were worse at later time points (Fig. 4B). Furthermore, there were a few inflammatory infiltrates in the glomeruli of the Pseudomonas group, but not in the sham and burn groups of mice at 24 h after inoculation, but obvious renal tubular damages were observed in the kidney sections of the burn and Pseudomonas groups of mice at later time points (Fig. 4C). In contrast, there were many inflammatory infiltrates in the glomeruli and obvious renal tubular damage in the burn/Pseudomonas group of mice at 24 h after inoculation, and the pathological changes were worse at later time points in the burn/Pseudomonas group of mice.
Finally, we found that in comparison with that in the sham group of mice, the concentrations of plasma total proteins in the burn and Pseudomonas groups were significantly reduced, but were further reduced in the burn/Pseudomonas group of mice throughout the observation period (Fig. 5). Furthermore, in comparison with that in the sham group, the levels of plasma BUN, Cr, AST, ALT, and total bilirubin in the burn and Pseudomonas groups of mice were temporally elevated, whereas the levels of plasma BUN, Cr, AST, ALT, and total bilirubin were significantly elevated in the burn/Pseudomonas group of mice throughout the observation period. The histological changes in the liver, lung, and kidney, together with increased values of abnormal liver and kidney functional measures, clearly indicated that the burn/Pseudomonas mice developed MODS.
Burn wound–related sepsis remains serious risk for patients with severe burns. In the present study, we used a mouse model of burn sepsis with a III° burn wound covering about 10% of total body surface area, followed by inoculating with a high dose of P. aeruginosa in female C57BL/6 mice. We found that the mice with burn sepsis developed SIRS and MODS and had a high rate of mortality, which were reproducible. This mouse model of burn sepsis assembled the pathogenesis and clinical characteristics of human with burn sepsis.
In comparison with that of available models (12–20), this mouse model of burn sepsis had several characteristics. First, this mouse model of burn sepsis used P. aeruginosa for the infection of burn wound skin because P. aeruginosa is gram-negative bacterium and a common source of skin burn wound infection and pneumonia in burn patients, whereas Staphylococcus aureus infection tends to rise. The cell wall of P. aeruginosa contains high amount of endotoxin, such as LPS. We detected significantly higher levels of plasma endotoxin in the burn/Pseudomonas mice. Given that the concentrations of blood endotoxin are closely related to the prognosis of patients with gram-negative bacterial infection, the high levels of endotoxin in the burn/Pseudomonas mice suggest that the concentrations of blood endotoxin may be used as a biomarker for the prognosis of individuals with burn sepsis. Second, unlike the burn sepsis model using P. aeruginosa from clinical patients (19, 20), we inoculated the burn skin wound with a standard stain of P. aeruginosa (ATCC27853) with a high virulence to establish this model, which had a better universality, stability, and reproducibility. Furthermore, unlike other models with ovine or BALB/c mice (12, 20), we used C57BL/6 mice, which are prone to proinflammatory responses. There are many genetic mutants of C57BL/6 mice available for further studying the molecular mechanisms underlying the pathogenesis of burn sepsis. Although the burn injury and infection at a short period in this model may be not common in patients, the burn/Pseudomonas group of mice did rapidly develop SIRS and MODS. Evidentially, significantly higher levels of bacterial loads in different organs and serum endotoxin, IL-1β, IL-6, TNF-α, and CRP were detected in the burn/Pseudomonas group of mice, accompanied by more severe liver, lung, and kidney tissue damage and impaired organ functions. Thus, this model had clinical pathogenic characteristics similar to that in patients with severe burn sepsis, and it simulated almost the smallest burn area (∼10%–12%), causing sepsis in the clinic.
An animal model of burn sepsis should meet the criteria for sepsis, established by the 2001 International Sepsis Definitions Conference (21). Our mouse model of burn sepsis met the criteria. Unlike some models using human and rodent stools to induce bacterial infection (6, 24–26), the skin burn wound was infected with P. aeruginosa, one of the best known infection factors (23). Infection with P. aeruginosa, one of the commonest bacteria in the intensive care unit (27), usually stimulates the natural pathogenic process of sepsis and clinical characteristics in patients with severe burn patients. This mouse model of burn sepsis had a mortality of 50% to 80%, just slightly higher than the requirement of ∼30% to 60% (23). Following inoculation, all of the mice in the burn/Pseudomonas group gradually lost body weights nearly 20% and had a reduced level of plasma total proteins, consistent with continuous hypermetabolism (19). All of the mice in the burn/Pseudomonas group displayed significantly increased levels of plasma endotoxin and proinflammatory cytokines, such as IL-1β, TNF-α and IL-6, and CRP, accompanied by a great number of bacterial invasion in the lung, liver, spleen, and kidney, demonstrating septicemia and SIRS (5, 27, 28). In addition, the mice in the burn septic group developed MODS at 24 h after inoculation, evidenced by tissue damage and increased levels of plasma renal and hepatic dysfunctional indicators, consistent with an interval between the onset MODS and bacterial infection (22, 29). The reliable and reproducible burn infection–induced sepsis model met the standard criteria and mimicked the pathogenic characteristics of patients with burn sepsis. Conceivably, this model may be valuable for further studies of molecular mechanisms underlying the pathogenic process and drug screening.
We recognized that our study had limitations, including the lack of therapeutic approaches and systemic immune response, single gender, and artificial infection. Further studies are necessary to validate the importance of this model in investigating the molecular mechanisms underlying the pathogenesis of burn sepsis and evaluating the efficacy of therapeutic strategies.
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Sepsis; inflammation; organ dysfunction; interleukin 6; tumor necrosis factor α
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