- Email: [email protected]
Simvastatin treatment improves survival in a murine model of burn sepsis: Role of interleukin 6
burns 37 (2011) 222–226
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Simvastatin treatment improves survival in a murine model of burn sepsis: Role of interleukin 6 David C. Beffa, Alan J. Fischman, Shawn P. Fagan, Victoria F. Hamrahi, Kasie W. Paul, Masao Kaneki, Yong-Ming Yu, Ronald G. Tompkins, Edward A. Carter * Surgical Service, Massachusetts General Hospital, Shriners Hospitals for Children, and Harvard Medical School, Boston, MA 02114, USA
article info
abstract
Article history:
Infection is the most common and most serious complication of a major burn related to burn
Accepted 16 October 2010
size. Recent studies have demonstrated that statin treatment can decrease mortality in murine or human sepsis. In the current study mice were anesthetized and subjected to a
Keywords:
dorsal 30% TBSA scald burn. Simvastatin or placebo were administered by intraperitoneal
Sepsis
injection once daily or every 12 h. On post burn day 7 cecal ligation and puncture with a 21-
Cecal ligation
gauge needle (CLP) was performed under ketamine/xylazine anesthesia, the two different
Simvastatin
dosing schedules were continued and survival was monitored. In other groups of mice,
Burn
interleukin-6 (IL-6) levels in blood were measured in mice at 7 days after injury. A simva-
Sepsis
statin dependent improvement in survival was observed in the burn sepsis model. This
Survival
protection was found to be dose and time dependent. In addition, statin treatment reduced
Interleukin-6 (IL-6)
the elevation in IL-6 levels of mice burned 7 days previously. However, IL-6 levels in burned mice with or without statin treatment were elevated by CLP to the same degree. The results of these studies suggest that statin treatment reduces mortality in mice with burns and CLP and that this effect may not be mediated via IL-6 levels. # 2010 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Sepsis and septic shock are the tenth most common causes of death in the United States [1,2]. Infection is the most common and most serious complication of a major burn related to burn size [1,3]. Despite improvements in antimicrobial therapies, sepsis still accounts for 50–60% of deaths in burn patients. Sepsis in burn patients is commonly due to bronchopneumonia, pyelonephritis, thrombophlebitis, or invasive wound infection. The burn wound is an ideal media for bacterial growth and provides a wide portal for invasion. Microbial colonization of open burn wounds, primarily from endoge-
nous sources, is usually established by the end of the first week after injury. Infection is further promoted by loss of the epithelial barrier, malnutrition induced by the hypermetabolic response to burns, and by a generalized post-burn immunosuppression. Burn leads to suppression of nearly all aspects of the immune response [4,5]. Granulocytopenia is commonly seen in burn patients. Blood levels of immunoglobulins, fibronectin, and complement are reduced, in combination with a diminished ability for opsonization, chemotaxis and phagocytosis and reduced killing function of neutrophils, monocytes and macrophages. The cellular immune response is also
* Corresponding author at: Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114, USA. Tel.: +1 617 371 4941; fax: +1 617 3714951. E-mail address: [email protected] (E.A. Carter). 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.10.010
burns 37 (2011) 222–226
impaired, as evidenced by anergy to common antigens, altered lymphocyte mitogenesis, and mixed lymphocyte responsiveness [4,5]. Burn also results in reductions of interleukin-2 (Il-2) production, T-cell and NK cell cytotoxicity, and helper to suppressor T-cell ratio (HSR) [4,5]. Given the acute onset and unpredictable nature of sepsis, primary prevention is rarely attempted in its management. However, recent studies have demonstrated that statin treatment can decrease mortality is a murine model of sepsis by preservation of cardiac function and reversal of inflammatory alterations. In addition, it has been shown that treatment with statins is associated with reduced incidence of sepsis in patients [6–11]. In the present study, we developed a murine model of sepsis in burned mice and demonstrated that Simvastatin treatment reduces mortality. We also examined IL-6 serum levels in burned mice with and without statin treatment, before and after CLP since it has been suggested that the level of IL-6 relates to the survival of burned patients with sepsis [21].
2.
Materials and methods
2.1.
Animals models
2.1.1.
Burns
Male CD-1 mice weighing 25–28 g were purchased from Charles River Breeding Laboratories, Boston MA. Full-thickness, non-lethal thermal injury (30% total body surface area [TBSA]) was produced, as described previously [12] using a protocol approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital. Briefly, the mice were anesthetized with ether and their backs were shaved with animal hair clippers. Under ether anesthesia, they were placed in molds exposing 30% TBSA followed by emersion of the exposed area in a water bath at 90 8C for 9 s. The animals were immediately resuscitated with saline (50 ml/kg) by intraperitoneal injection. After the procedure, the animals were caged individually for the duration of the study and food and water were provided ab libitum. There were 11–12 mice in each group. Sham animals were anesthetized, shaven, resuscitated with saline (50 ml/kg) by intraperitoneal injection and subsequently caged individually.
2.2.
Induction of sepsis
To produce cecal ligation puncture (CLP) on day 7 after burn, the animals were anesthetized (IP injection of ketamine [60 mg/g BW] plus xylazine [10 mg/g BW]), a 1 cm midline abdominal incision was made and the cecum was ligated with 3-0 silk below the ileocecal valve (with careful attention to avoiding obstruction of the ileum or colon) [14]. The cecum was subjected to a single ‘‘through-and-through’’ perforation with a 21-gauge needle and a small amount of feces was expressed. The incision was closed in layers. After the procedure, food and water were provided ab libitum. Pain medication (Tramadol, 20 mg/g BW, s.c.) and volume support (normal saline, 50 ml/kg) were administered immediately after CLP.
2.3.
223
Preparation of simvastatin and placebo solutions
Simvastatin (Merck Biosciences, Inc.) was dissolved in ethanol at a concentration of 10 mg/ml and diluted with normal saline to yield a final concentration of 10 mg/ml. The placebo was prepared to contain ethanol and saline in the ratio 1:1000.
2.4.
Simvastatin treatment protocol
In preliminary experiments, groups of unburned mice (n = 11) were subjected to CLP. Half of the mice were injected with saline (0.02 ml/g BW) while others were injected IP with Simvastatin (0.2 mg/g BW). In the primary experiments, at 2 h after burn, mice were divided into four groups of 12 each and were treated as follows:
Group Group Group Group
I: IP injection of placebo (0.02 ml/g BW), q12h. II: IP injection of placebo (0.02 ml/g BW), q 24h. III: IP injection of Simvastatin (0.2 mg/g), q12h. IV: IP injection of Simvastatin (0.2 mg/g), q24h.
In these studies we decided to use the dose of simvastatin (0.2 mg/g) described in previous investigations by Merx et al. [13] and to perform CLP 7 days after burn. In another series of experiments the dose of statin administered to the burned mice was reduced or increased by a factor of 10 (n = 12/group). In a final series of experiments statin treatment was not started until 4 or 6 days after burn (n = 12) or given 2 or 4 h after CLP. Placebo animals received only the vehicle at these time points. The animals were maintained according to federal regulations and all studies were approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital.
2.5.
Interleukin-6 serum analysis
IL-6 serum levels were assayed using the R + D Systems kit (R&D Systems, Inc., Minneapolis, MN). Seven days after burn with or without statin treatment (Statin (0.2 mg/g), q12h 7 days prior to CLP) or sham treatment, mice were subjected to CLP. Eighteen hours after CLP the mice (6 in each group) were anesthetized (IP injection of ketamine [60 mg/g BW] plus xylazine [10 mg/g BW]) and blood was collected by cardiac puncture. The blood samples were allowed to clot for 1 h at room temperature before centrifuging for 20 min at 2000 g. The serum was removed, aliquoted and frozen at 80 8C. Analysis was performed on freshly thawed aliquots. Where necessary, the samples were diluted using the manufacture’s reagents and instructions.
[()TD$FIG]
Fig. 1 – Schematic diagram of the study protocol.
224
burns 37 (2011) 222–226
[()TD$FIG]
Fig. 2 – Survival curves for mice treated at 24 h (panel A) and 12 h (panel B) intervals with Simvastatin or placebo. As these results illustrate, there was a clear survival advantage of Simvastatin treatment and statistical analysis by the Log-Rank (Mantel-Cox) method demonstrated a significant effect ( p < 0.01). There was greater survival in the Simvastatin treated animals at 24 and 72 h compared with animals that received placebo ( p < 0.01). In contrast, survival was not significantly improved in animals treated with a daily dose of Simvastatin vs. placebo.
2.6.
Data analysis
The animals were inspected several times each day and number of survivors in each group was recorded. The LogRank (Mantel-Cox) method was used for statistical analysis. Pvalues of <0.05 were considered to be statistically significant. The IL-6 data was analyzed by ANOVA.
3.
Results
We then proceeded to examine the effect of burn and CLP. A schematic diagram of the study protocol is shown in Fig. 1. Fig. 2 illustrates, the effect of Simvastatin and placebo treatment on the survival of mice with full-thickness (30% TBSA) burn and sepsis produced by CLP at 7 days after burn; treatment with simvastatin and placebo was initiated 2 h after burn. In the animals treated at 12-h intervals, statistical analysis by the Log-Rank (Mantel-Cox) method demonstrated
significantly greater survival in the Simvastatin treated animals at 24 and 72 h compared with animals that received placebo ( p < 0.01). At 48 h there was tendency for greater survival in the Simvastatin treated animals, however, the difference was not statistically significant ( p = 0.15). In contrast, survival was not significantly improved in animals treated with a daily dose of Simvastatin vs. placebo (24 h, p > 0.2; 48 h, p > 0.5; 72 h, p > 0.2). As can be seen in Table 1, a 10-fold reduction in the dose of statin administered q12h resulted in the loss of the protective effect of statin pretreatment ( p < 0.01). In contrast, a 10-fold increase in statin dose seemed to provide even better protection than the standard statin dose ( p < 0.001). Administration of a single dose of statin at 2 or 4 h after CLP did not provide a protective effect compared to the 7 days of statin pretreatment ( p < 0.005). Reduction of the statin pretreatment schedule to 4 days of statin after burn continued to demonstrate a protective effect against CLP induced sepsis carried out on day 7; similar to 7 days of statin treatment.
Table 1 – Effect of statin dose and time of administration on burn induced increase in CLP induced mortality. Group (n = 12) Standard Burn+ CLP + standard statin dosea Statinb 1/10 dose Statinc 10 dose Statind single dose 2 h after CLP Statind single dose 4 h after CLP Statin standard dose begun 4 days after burne Statin standard dose begun 6 days after burne a b c d e
Survival after CLP 24 h
Survival after CLP 48 h
Survival after CLP 72 h
12/12 9/12 12/12 6/12 6/12 12/12 7/12
12/12 6/12 12/12 5/12 5/12 12/12 5/12
7/12 3/12 12/12 3/12 3/12 7/12 2/12
Statin (0.2 mg/g), q12h 7 days prior to CLP. Statin (0.02 mg/g), q12h 7 days prior to CLP. Statin (2.0 mg/g), q12h 7 days prior to CLP. Statin (0.2 mg/g) given as single dose to mice burned 7 days prior to CLP. Statin (0.2 mg/g), q12h 4 days prior to CLP.
Survival after CLP 96 h 6/12 0/12 12/12 1/12 1/12 6/12 0/12
burns 37 (2011) 222–226
However, statin pretreatment started at 6 days after burn did not show any protective effect against CLP induced sepsis carried out on day 7. In contrast the effect of statin, injection of placebo had not effect on survival (data not shown). Seven days following burn, there was a significant increase in IL-6 in the serum of burned mice compared to sham controls (93 7 pg/mL vs. 13 2 pg/mL, mean SEM, p < 0.01, n = 6 each group). Statin treatment significantly reduced the burn induced increase in IL-6 (21 4 pg/mL vs. 93 7 pg/mL, p < 0.01, n = 6 each group). At 18 hrs after CLP induced sepsis, IL-6 serum levels were elevated in both the burned and burned plus statin treated groups (6324 1462 vs. 8841 3032 pg/mL, n = 6 each group) and the difference was not statistically significant.
4.
Discussion
Sepsis can be broadly defined as the presence of pus-forming bacteria or their toxins in the blood or tissues. Sepsis remains the leading cause of death in patients treated in intensive care units with mortality ranging from 30 to 70% [1,2]. The present study was designed to address two questions: (1) Does statin treatment, which has been shown to ameliorate the increased mortality produced by CLP protect in the burn plus CLP murine model? (2) Does statin treatment effect the elevation of serum IL-6 levels produced by burn or burn combined with CLP? Ward et al. has recently reviewed the use of CLP in rodents [14]. These authors stated that cecal ligation and puncture in rodents has become the most widely used model for experimental sepsis and is currently considered as the ‘‘gold standard’’ in sepsis research [14]. These authors also stated that despite its clinical relevance and widespread use in sepsis research, one of the major concerns of the CLP model is consistency [14]. We used the 7-day burn model since it is associated with increased infection related mortality of burned mice and also produces elevations in serum Il-6 (18, this laboratory, and unpublished observations). It should be noted that we used statin initially dissolved in ethanol and then diluted in saline. We did not measure blood ethanol levels in any of the mice since we did not want subject the burned mice with CLP to any further stress. However, based on calculation of the amount of ethanol that might be in the blood of the mice, assuming 7% total blood volume, there should be less than 1 mg% ethanol in the blood of the treated mice, a level that should not alter the inflammatory response. This is supported by the work of Arbabi et al. [15] which demonstrated that ethanol did not inhibit LPS stimulation of TNF production at concentrations less than 100 mg%. We first examined whether we could confirm the increased mortality associated with sepsis in our murine model, and if so, would statin treatment ameliorate this increased mortality. We chose the dosing route/regimen of statin to replicate previous work by Merx et al. [13] which demonstrated that statins are protective in the CLP model. Our studies confirmed that statin treatment improved survival in un-burned CLP mice. This was done to establish the validity of our sepsis model. We then demonstrated that the mortality of the mice burned 7 days previously and then subjected to CLP sepsis was greater than that of the mice with CLP sepsis only. Similar
225
results of increased susceptiablity to CLP sepsis in mice burned 7 days previously has been reported by Schwacha et al. [5] and by Shelley et al. [16] in mice burned 10 days previously. We next examined the effect of the statin treatment on survival of burned mice with sepsis. These studies demonstrated that statin treatment has a dose dependent protective effect against CLP induced sepsis. Hence, a lower dose of statin was less protective while a higher dose was more protective. The statin effect appeared to be temporally related since initiation of statin pretreatment 4 days after burn prior to CLP treatment on day 7 still appeared to be protective. However, pretreatment with statin at 6 days after burn prior to CLP treatment on day 7 after burn did not have a protective effect. The present data suggest the q12 and q24 simvastatin group have basically the same survival curve with about 30– 40% survival after 80 h. This is not significantly different than the q24 placebo. However it appeared that q12 placebo administration had significantly worse outcome. The reasons for this observation is unclear, but may be related to the pharmacokinetics of statins in burned mice. Since statins are cleared by extensive first pass effects at the intestinal and/or hepatic level [17] and burn associated changes at the gut/liver level have been demonstrated in previous work in this laboratory [18], there may be differences in the clearance and/or metabolism of statin which need to be investigated. Diagnosis of sepsis in burn patients can be difficult to distinguish from the usual hyper dynamic, hyperthermia, hypermetabolic post-burn state. In addition, blood cultures are commonly negative in burn patients who go on to develop sepsis, and elevations of temperature may not be proportional to the degree of infection. Since it has been suggested that blood levels of IL-6 relate to the survival of burned patients with sepsis [19], we measured IL-6 levels in the burned mice with statin treatment before and after CLP. These studies confirmed that IL-6 is elevated in mice at 7 days after burn. Statin treatment significantly reduced this elevation in the burned mice, consistent with the hypothesis that statins exert anti-inflammatory effect(s) in the burned mice. There is considerable evidence for a statin-mediated regulation of the inflammatory pathways in atherogenesis [20]. CLP treatment of burned mice produced significant elevations in Il-6 levels. However, the Il-6 levels in statin treated burned mice with CLP and burned mice with CLP without statin treatment were not significantly different. It should be noted that these samples were collected prior to any mortality in either group, suggesting that statin pretreatment associated increased survival of burned mice may not be related to sepsis induced changes in IL-6 levels alone. In support this position that the absolute serum level of IL-6 may not be a predictor of survival in the CLP model, there is literature demonstrating that survival of IL-6 knockout mice (that cannot produce Il-6) subjected to CLP, is the same as wild type mice that mount robust elevations of IL-6 [21]. It is also important to note that Dobesh et al. [11] demonstrated that statin treatment in patients prior to the development of severe sepsis appears to produce a significant reduction in mortality compared with patients that were not treated with statins. In addition, it has been reported by Fogerty et al. that treatment with statins prior to injury
226
burns 37 (2011) 222–226
significantly decreased mortality and showed a trend toward reduced septic shock in a retrospective study of 223 elderly burn patients [22]. The model described in this study may play an important role in determining the mechanism(s) by which statins prevent burn induced sepsis.
5.
Conclusions
The results of these studies suggest that statin treatment reduces mortality in mice with burn and CLP and that this effect may not be mediated via modulation of IL-6 levels. Future studies of the levels of other cytokines after burn and CLP are warranted.
Conflict of interest statement None of the authors have financial and personal relationships with other people or organizations that could inappropriately influence (bias) this work.
Acknowledgments This work was supported in part by grants from the National Institutes of Health (NIGMS P50 GM21000) and Shriners Hospitals for Children.
references
[1] Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348:1546–54. [2] Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003;348(2):138–50. [3] Pruitt Jr BA. Infection and the burn patient. Br J Surg 1990;77:1081. [4] O’Sullivan ST, O’Conner TPF. Immunosuppression following thermal injury: the pathogenesis of immunocysfunction. Br J Plastic Surg 1997;50:615–23. [5] Schwacha MG, Schneider CP, Chaudry IH. Differential expression and tissue compartmentalization of the inflammatory responses following thermal injury. Cytokine 2002;17:266–74. [6] Mortensen EM, Restrepo MI, Anzueto A, Pugh J. The effect of prior statin use on 30-day mortality for patients hospitalized with community-acquired pneumonia. Respir Res 2005;6:82.
[7] Kruger P, Fitzsimmons K, Cook D, Jones M, Nimmo G. Statin therapy is associated with fewer deaths in patients with bacteraemia. Intensive Care Med 2006;32(1):75–9. [8] Almog Y, Shefer A, Novack V, Maimon N, Barski L, Eizinger M, et al. Prior statin therapy is associated with a decreased rate of severe sepsis. Circulation 2004;110(7):880–5. [9] Almog Y, Novack V, Eisinger M, Porath A, Novack L, Gilutz H. The effect of statin therapy on infection-related mortality in patients with atherosclerotic diseases. Crit Care Med 2007;35(2):372–8. [10] Hackam DG, Mamdani M, Li P, Redelmeier DA. Statins and sepsis in patients with cardiovascular disease: a population-based cohort analysis. Lancet 2006;367(9508):413–8. [11] Dobesh PP, Klepser DG, McGuire TR, Morgan CW, Olsen KM. Reduction in mortality associated with statin therapy in patients with severe sepsis. Pharmacotherapy 2009;29:621– 30. [12] Pawley TM, Carter EA, Bode BP, Hirschman A, Tompkins RAG. Central role of interleukin-6 in burned induced stimulation of hepatic amino acid transport. Int J Mol Med 2003;12:541–54. [13] Merx MW, Liehn EA, Janssens U, Lutticken R, Schrader J, Hanrath P, et al. HMG-CoA reductase inhibitor simvastatin profoundly improves survival in a murine model of sepsis. Circulation 2004;109(21):2560–5. [14] Rittirsch D, Huber-Lang MS, Flier MA, Ward PA. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protocol 2009;4(1):31–6. [15] Arbabi S, Garcia I, Bauer GJ, Maier R. Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway. J Immunol 1999;162:7441–5. [16] Shelley O, Murphay T, Paterson H, Mannick J, Lederer J. Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury. Shock 2003;20:123–9. [17] Garcia MJ, Reinoso RF, Navarrok AS, Prous JR. Clincal pharmacokinetics of statins. Methods Find Exp Clin Pharmacol 2003;25:457–60. [18] Carter EA, Hatz RA, Walker WA, Tompkins RG. Injuryinduced inhibition of small intestinal protein and nucleic acid synthesis. Gastroenterology 1990;98:1445–51. [19] Schluter B, Konig B, Bergmann U, Muller FE, Kong W. Interleukin 6—a potential marker of lethal sepsis after major thermal injury: evidence for increased IL-6 production by peripheral blood mononuclear cells. J Trauma 1991;31:1663–70. [20] Montecucco F, Mach F. Update on statin-meidated antiinflammatory activities in atherosclerosis. Semin Immunopathol 2009;31:127–42. [21] Remick DG, Bolgos G, Copeland S, Siddiqui J. Role of interleukin-6 in mortality from and physiologica response to sepsis. Infect Immun 2005;73:2751–7. [22] Fogerty MD, Efron D, Morandi A, Guy J, Abumrad N, Barbul A. The effect of preinjury statin use on mortality and septic shock in elderly burn patients. J Trauma 2010;69(1):99–103.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/burns
Simvastatin treatment improves survival in a murine model of burn sepsis: Role of interleukin 6 David C. Beffa, Alan J. Fischman, Shawn P. Fagan, Victoria F. Hamrahi, Kasie W. Paul, Masao Kaneki, Yong-Ming Yu, Ronald G. Tompkins, Edward A. Carter * Surgical Service, Massachusetts General Hospital, Shriners Hospitals for Children, and Harvard Medical School, Boston, MA 02114, USA
article info
abstract
Article history:
Infection is the most common and most serious complication of a major burn related to burn
Accepted 16 October 2010
size. Recent studies have demonstrated that statin treatment can decrease mortality in murine or human sepsis. In the current study mice were anesthetized and subjected to a
Keywords:
dorsal 30% TBSA scald burn. Simvastatin or placebo were administered by intraperitoneal
Sepsis
injection once daily or every 12 h. On post burn day 7 cecal ligation and puncture with a 21-
Cecal ligation
gauge needle (CLP) was performed under ketamine/xylazine anesthesia, the two different
Simvastatin
dosing schedules were continued and survival was monitored. In other groups of mice,
Burn
interleukin-6 (IL-6) levels in blood were measured in mice at 7 days after injury. A simva-
Sepsis
statin dependent improvement in survival was observed in the burn sepsis model. This
Survival
protection was found to be dose and time dependent. In addition, statin treatment reduced
Interleukin-6 (IL-6)
the elevation in IL-6 levels of mice burned 7 days previously. However, IL-6 levels in burned mice with or without statin treatment were elevated by CLP to the same degree. The results of these studies suggest that statin treatment reduces mortality in mice with burns and CLP and that this effect may not be mediated via IL-6 levels. # 2010 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Sepsis and septic shock are the tenth most common causes of death in the United States [1,2]. Infection is the most common and most serious complication of a major burn related to burn size [1,3]. Despite improvements in antimicrobial therapies, sepsis still accounts for 50–60% of deaths in burn patients. Sepsis in burn patients is commonly due to bronchopneumonia, pyelonephritis, thrombophlebitis, or invasive wound infection. The burn wound is an ideal media for bacterial growth and provides a wide portal for invasion. Microbial colonization of open burn wounds, primarily from endoge-
nous sources, is usually established by the end of the first week after injury. Infection is further promoted by loss of the epithelial barrier, malnutrition induced by the hypermetabolic response to burns, and by a generalized post-burn immunosuppression. Burn leads to suppression of nearly all aspects of the immune response [4,5]. Granulocytopenia is commonly seen in burn patients. Blood levels of immunoglobulins, fibronectin, and complement are reduced, in combination with a diminished ability for opsonization, chemotaxis and phagocytosis and reduced killing function of neutrophils, monocytes and macrophages. The cellular immune response is also
* Corresponding author at: Shriners Hospitals for Children, 51 Blossom Street, Boston, MA 02114, USA. Tel.: +1 617 371 4941; fax: +1 617 3714951. E-mail address: [email protected] (E.A. Carter). 0305-4179/$36.00 # 2010 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2010.10.010
burns 37 (2011) 222–226
impaired, as evidenced by anergy to common antigens, altered lymphocyte mitogenesis, and mixed lymphocyte responsiveness [4,5]. Burn also results in reductions of interleukin-2 (Il-2) production, T-cell and NK cell cytotoxicity, and helper to suppressor T-cell ratio (HSR) [4,5]. Given the acute onset and unpredictable nature of sepsis, primary prevention is rarely attempted in its management. However, recent studies have demonstrated that statin treatment can decrease mortality is a murine model of sepsis by preservation of cardiac function and reversal of inflammatory alterations. In addition, it has been shown that treatment with statins is associated with reduced incidence of sepsis in patients [6–11]. In the present study, we developed a murine model of sepsis in burned mice and demonstrated that Simvastatin treatment reduces mortality. We also examined IL-6 serum levels in burned mice with and without statin treatment, before and after CLP since it has been suggested that the level of IL-6 relates to the survival of burned patients with sepsis [21].
2.
Materials and methods
2.1.
Animals models
2.1.1.
Burns
Male CD-1 mice weighing 25–28 g were purchased from Charles River Breeding Laboratories, Boston MA. Full-thickness, non-lethal thermal injury (30% total body surface area [TBSA]) was produced, as described previously [12] using a protocol approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital. Briefly, the mice were anesthetized with ether and their backs were shaved with animal hair clippers. Under ether anesthesia, they were placed in molds exposing 30% TBSA followed by emersion of the exposed area in a water bath at 90 8C for 9 s. The animals were immediately resuscitated with saline (50 ml/kg) by intraperitoneal injection. After the procedure, the animals were caged individually for the duration of the study and food and water were provided ab libitum. There were 11–12 mice in each group. Sham animals were anesthetized, shaven, resuscitated with saline (50 ml/kg) by intraperitoneal injection and subsequently caged individually.
2.2.
Induction of sepsis
To produce cecal ligation puncture (CLP) on day 7 after burn, the animals were anesthetized (IP injection of ketamine [60 mg/g BW] plus xylazine [10 mg/g BW]), a 1 cm midline abdominal incision was made and the cecum was ligated with 3-0 silk below the ileocecal valve (with careful attention to avoiding obstruction of the ileum or colon) [14]. The cecum was subjected to a single ‘‘through-and-through’’ perforation with a 21-gauge needle and a small amount of feces was expressed. The incision was closed in layers. After the procedure, food and water were provided ab libitum. Pain medication (Tramadol, 20 mg/g BW, s.c.) and volume support (normal saline, 50 ml/kg) were administered immediately after CLP.
2.3.
223
Preparation of simvastatin and placebo solutions
Simvastatin (Merck Biosciences, Inc.) was dissolved in ethanol at a concentration of 10 mg/ml and diluted with normal saline to yield a final concentration of 10 mg/ml. The placebo was prepared to contain ethanol and saline in the ratio 1:1000.
2.4.
Simvastatin treatment protocol
In preliminary experiments, groups of unburned mice (n = 11) were subjected to CLP. Half of the mice were injected with saline (0.02 ml/g BW) while others were injected IP with Simvastatin (0.2 mg/g BW). In the primary experiments, at 2 h after burn, mice were divided into four groups of 12 each and were treated as follows:
Group Group Group Group
I: IP injection of placebo (0.02 ml/g BW), q12h. II: IP injection of placebo (0.02 ml/g BW), q 24h. III: IP injection of Simvastatin (0.2 mg/g), q12h. IV: IP injection of Simvastatin (0.2 mg/g), q24h.
In these studies we decided to use the dose of simvastatin (0.2 mg/g) described in previous investigations by Merx et al. [13] and to perform CLP 7 days after burn. In another series of experiments the dose of statin administered to the burned mice was reduced or increased by a factor of 10 (n = 12/group). In a final series of experiments statin treatment was not started until 4 or 6 days after burn (n = 12) or given 2 or 4 h after CLP. Placebo animals received only the vehicle at these time points. The animals were maintained according to federal regulations and all studies were approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital.
2.5.
Interleukin-6 serum analysis
IL-6 serum levels were assayed using the R + D Systems kit (R&D Systems, Inc., Minneapolis, MN). Seven days after burn with or without statin treatment (Statin (0.2 mg/g), q12h 7 days prior to CLP) or sham treatment, mice were subjected to CLP. Eighteen hours after CLP the mice (6 in each group) were anesthetized (IP injection of ketamine [60 mg/g BW] plus xylazine [10 mg/g BW]) and blood was collected by cardiac puncture. The blood samples were allowed to clot for 1 h at room temperature before centrifuging for 20 min at 2000 g. The serum was removed, aliquoted and frozen at 80 8C. Analysis was performed on freshly thawed aliquots. Where necessary, the samples were diluted using the manufacture’s reagents and instructions.
[()TD$FIG]
Fig. 1 – Schematic diagram of the study protocol.
224
burns 37 (2011) 222–226
[()TD$FIG]
Fig. 2 – Survival curves for mice treated at 24 h (panel A) and 12 h (panel B) intervals with Simvastatin or placebo. As these results illustrate, there was a clear survival advantage of Simvastatin treatment and statistical analysis by the Log-Rank (Mantel-Cox) method demonstrated a significant effect ( p < 0.01). There was greater survival in the Simvastatin treated animals at 24 and 72 h compared with animals that received placebo ( p < 0.01). In contrast, survival was not significantly improved in animals treated with a daily dose of Simvastatin vs. placebo.
2.6.
Data analysis
The animals were inspected several times each day and number of survivors in each group was recorded. The LogRank (Mantel-Cox) method was used for statistical analysis. Pvalues of <0.05 were considered to be statistically significant. The IL-6 data was analyzed by ANOVA.
3.
Results
We then proceeded to examine the effect of burn and CLP. A schematic diagram of the study protocol is shown in Fig. 1. Fig. 2 illustrates, the effect of Simvastatin and placebo treatment on the survival of mice with full-thickness (30% TBSA) burn and sepsis produced by CLP at 7 days after burn; treatment with simvastatin and placebo was initiated 2 h after burn. In the animals treated at 12-h intervals, statistical analysis by the Log-Rank (Mantel-Cox) method demonstrated
significantly greater survival in the Simvastatin treated animals at 24 and 72 h compared with animals that received placebo ( p < 0.01). At 48 h there was tendency for greater survival in the Simvastatin treated animals, however, the difference was not statistically significant ( p = 0.15). In contrast, survival was not significantly improved in animals treated with a daily dose of Simvastatin vs. placebo (24 h, p > 0.2; 48 h, p > 0.5; 72 h, p > 0.2). As can be seen in Table 1, a 10-fold reduction in the dose of statin administered q12h resulted in the loss of the protective effect of statin pretreatment ( p < 0.01). In contrast, a 10-fold increase in statin dose seemed to provide even better protection than the standard statin dose ( p < 0.001). Administration of a single dose of statin at 2 or 4 h after CLP did not provide a protective effect compared to the 7 days of statin pretreatment ( p < 0.005). Reduction of the statin pretreatment schedule to 4 days of statin after burn continued to demonstrate a protective effect against CLP induced sepsis carried out on day 7; similar to 7 days of statin treatment.
Table 1 – Effect of statin dose and time of administration on burn induced increase in CLP induced mortality. Group (n = 12) Standard Burn+ CLP + standard statin dosea Statinb 1/10 dose Statinc 10 dose Statind single dose 2 h after CLP Statind single dose 4 h after CLP Statin standard dose begun 4 days after burne Statin standard dose begun 6 days after burne a b c d e
Survival after CLP 24 h
Survival after CLP 48 h
Survival after CLP 72 h
12/12 9/12 12/12 6/12 6/12 12/12 7/12
12/12 6/12 12/12 5/12 5/12 12/12 5/12
7/12 3/12 12/12 3/12 3/12 7/12 2/12
Statin (0.2 mg/g), q12h 7 days prior to CLP. Statin (0.02 mg/g), q12h 7 days prior to CLP. Statin (2.0 mg/g), q12h 7 days prior to CLP. Statin (0.2 mg/g) given as single dose to mice burned 7 days prior to CLP. Statin (0.2 mg/g), q12h 4 days prior to CLP.
Survival after CLP 96 h 6/12 0/12 12/12 1/12 1/12 6/12 0/12
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However, statin pretreatment started at 6 days after burn did not show any protective effect against CLP induced sepsis carried out on day 7. In contrast the effect of statin, injection of placebo had not effect on survival (data not shown). Seven days following burn, there was a significant increase in IL-6 in the serum of burned mice compared to sham controls (93 7 pg/mL vs. 13 2 pg/mL, mean SEM, p < 0.01, n = 6 each group). Statin treatment significantly reduced the burn induced increase in IL-6 (21 4 pg/mL vs. 93 7 pg/mL, p < 0.01, n = 6 each group). At 18 hrs after CLP induced sepsis, IL-6 serum levels were elevated in both the burned and burned plus statin treated groups (6324 1462 vs. 8841 3032 pg/mL, n = 6 each group) and the difference was not statistically significant.
4.
Discussion
Sepsis can be broadly defined as the presence of pus-forming bacteria or their toxins in the blood or tissues. Sepsis remains the leading cause of death in patients treated in intensive care units with mortality ranging from 30 to 70% [1,2]. The present study was designed to address two questions: (1) Does statin treatment, which has been shown to ameliorate the increased mortality produced by CLP protect in the burn plus CLP murine model? (2) Does statin treatment effect the elevation of serum IL-6 levels produced by burn or burn combined with CLP? Ward et al. has recently reviewed the use of CLP in rodents [14]. These authors stated that cecal ligation and puncture in rodents has become the most widely used model for experimental sepsis and is currently considered as the ‘‘gold standard’’ in sepsis research [14]. These authors also stated that despite its clinical relevance and widespread use in sepsis research, one of the major concerns of the CLP model is consistency [14]. We used the 7-day burn model since it is associated with increased infection related mortality of burned mice and also produces elevations in serum Il-6 (18, this laboratory, and unpublished observations). It should be noted that we used statin initially dissolved in ethanol and then diluted in saline. We did not measure blood ethanol levels in any of the mice since we did not want subject the burned mice with CLP to any further stress. However, based on calculation of the amount of ethanol that might be in the blood of the mice, assuming 7% total blood volume, there should be less than 1 mg% ethanol in the blood of the treated mice, a level that should not alter the inflammatory response. This is supported by the work of Arbabi et al. [15] which demonstrated that ethanol did not inhibit LPS stimulation of TNF production at concentrations less than 100 mg%. We first examined whether we could confirm the increased mortality associated with sepsis in our murine model, and if so, would statin treatment ameliorate this increased mortality. We chose the dosing route/regimen of statin to replicate previous work by Merx et al. [13] which demonstrated that statins are protective in the CLP model. Our studies confirmed that statin treatment improved survival in un-burned CLP mice. This was done to establish the validity of our sepsis model. We then demonstrated that the mortality of the mice burned 7 days previously and then subjected to CLP sepsis was greater than that of the mice with CLP sepsis only. Similar
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results of increased susceptiablity to CLP sepsis in mice burned 7 days previously has been reported by Schwacha et al. [5] and by Shelley et al. [16] in mice burned 10 days previously. We next examined the effect of the statin treatment on survival of burned mice with sepsis. These studies demonstrated that statin treatment has a dose dependent protective effect against CLP induced sepsis. Hence, a lower dose of statin was less protective while a higher dose was more protective. The statin effect appeared to be temporally related since initiation of statin pretreatment 4 days after burn prior to CLP treatment on day 7 still appeared to be protective. However, pretreatment with statin at 6 days after burn prior to CLP treatment on day 7 after burn did not have a protective effect. The present data suggest the q12 and q24 simvastatin group have basically the same survival curve with about 30– 40% survival after 80 h. This is not significantly different than the q24 placebo. However it appeared that q12 placebo administration had significantly worse outcome. The reasons for this observation is unclear, but may be related to the pharmacokinetics of statins in burned mice. Since statins are cleared by extensive first pass effects at the intestinal and/or hepatic level [17] and burn associated changes at the gut/liver level have been demonstrated in previous work in this laboratory [18], there may be differences in the clearance and/or metabolism of statin which need to be investigated. Diagnosis of sepsis in burn patients can be difficult to distinguish from the usual hyper dynamic, hyperthermia, hypermetabolic post-burn state. In addition, blood cultures are commonly negative in burn patients who go on to develop sepsis, and elevations of temperature may not be proportional to the degree of infection. Since it has been suggested that blood levels of IL-6 relate to the survival of burned patients with sepsis [19], we measured IL-6 levels in the burned mice with statin treatment before and after CLP. These studies confirmed that IL-6 is elevated in mice at 7 days after burn. Statin treatment significantly reduced this elevation in the burned mice, consistent with the hypothesis that statins exert anti-inflammatory effect(s) in the burned mice. There is considerable evidence for a statin-mediated regulation of the inflammatory pathways in atherogenesis [20]. CLP treatment of burned mice produced significant elevations in Il-6 levels. However, the Il-6 levels in statin treated burned mice with CLP and burned mice with CLP without statin treatment were not significantly different. It should be noted that these samples were collected prior to any mortality in either group, suggesting that statin pretreatment associated increased survival of burned mice may not be related to sepsis induced changes in IL-6 levels alone. In support this position that the absolute serum level of IL-6 may not be a predictor of survival in the CLP model, there is literature demonstrating that survival of IL-6 knockout mice (that cannot produce Il-6) subjected to CLP, is the same as wild type mice that mount robust elevations of IL-6 [21]. It is also important to note that Dobesh et al. [11] demonstrated that statin treatment in patients prior to the development of severe sepsis appears to produce a significant reduction in mortality compared with patients that were not treated with statins. In addition, it has been reported by Fogerty et al. that treatment with statins prior to injury
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significantly decreased mortality and showed a trend toward reduced septic shock in a retrospective study of 223 elderly burn patients [22]. The model described in this study may play an important role in determining the mechanism(s) by which statins prevent burn induced sepsis.
5.
Conclusions
The results of these studies suggest that statin treatment reduces mortality in mice with burn and CLP and that this effect may not be mediated via modulation of IL-6 levels. Future studies of the levels of other cytokines after burn and CLP are warranted.
Conflict of interest statement None of the authors have financial and personal relationships with other people or organizations that could inappropriately influence (bias) this work.
Acknowledgments This work was supported in part by grants from the National Institutes of Health (NIGMS P50 GM21000) and Shriners Hospitals for Children.
references
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