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Cecal Ligation and Incision: An Acute Onset Model of Severe Sepsis in Rats
Journal of Surgical Research 151, 132–137 (2009) doi:10.1016/j.jss.2008.02.032
Cecal Ligation and Incision: An Acute Onset Model of Severe Sepsis in Rats 1 Patrick Scheiermann, M.D.,*,2 Sandra Hoegl, M.D.,*,† Marc Revermann, M.D.,* Devan Ahluwalia,* Johannes Zander, M.D.,‡ Kim A. Boost, M.D.,* Thach Nguyen, M.D.,* Bernhard Zwissler, M.D.,§ Heiko Muhl, Ph.D.,† and Christian Hofstetter, M.D.* *Department of Anesthesiology, Intensive Care Medicine, and Pain Therapy, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany; †Pharmazentrum/ZAFES, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany; ‡Institute of Medical Microbiology and Infection Control, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany; §Department of Anesthesiology, Hospital of the Ludwigs-Maximilians-University, Munich, Germany Submitted for publication October 18, 2007
Background. Sepsis is a leading cause of death among critically ill patients. Up to now, severe sepsis with acute onset in animals has been induced mainly through injection of single bacteria species or endotoxin and not through a surgical procedure, which might adequately mirror the situation in septic patients. We therefore aimed to establish a surgical model of severe sepsis in rodents fulfilling international sepsis criteria. Materials and methods. Twenty-eight anesthetized/ ventilated Sprague Dawley rats underwent laparotomy and cecal mobilization. The cecum was either replaced into the abdomen (SHAM, n ⴝ 14) or the cecum and the mesenteric blood vessels were ligated, and the cecum was opened through a 1.5 cm blade incision (cecal ligation and incision, CLI, n ⴝ 14). Results. Within 390 min, mortality was 0% (SHAM) and 50% (CLI), respectively. Compared with SHAM, CLI resulted in a 43% reduction of mean arterial blood pressure and in severe metabolic acidosis as measured by arterial base excess and pH. CLI led to a 15-fold increase in mononuclear cell population and to a 5-fold accumulation of nitrite in peritoneal lavage. Abdominal swabs from the Douglas cavity in CLI-animals showed gram-positive and gram-negative bacterial growth on agar compared with sterile swabs from SHAM-animals. In CLI-animals, plasma IL-1 level was 1 Parts of this study were presented at the 2007 Meeting of the German Sepsis-Society (DSG) in Weimar, Germany. 2 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology, Intensive Care Medicine, and Pain Therapy, Hospital of the Johann Wolfgang GoetheUniversity, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany. E-mail: [email protected]
0022-4804/09 $34.00 © 2009 Elsevier Inc. All rights reserved.
increased to 435 pg/mL (SHAM: 10 pg/mL) and plasma IL-6 level to 19718 pg/mL (SHAM: 832 pg/mL). Conclusions. CLI causes bacterial peritonitis with subsequent systemic inflammation and organ dysfunction. Thus, CLI mimics clinical sepsis and provides a surgical short term model of severe sepsis in rodents. © 2009 Elsevier Inc. All rights reserved. Key Words: polymicrobial peritonitis; severe sepsis; systemic inflammation; organ dysfunction; cytokines; rat. INTRODUCTION
Sepsis as defined by the 2001 International Sepsis Definitions Conference is a clinical syndrome consisting of systemic inflammation (systemic inflammatory response syndrome, SIRS) caused by an infectious origin [1]. Severe sepsis, in addition, includes organ dysfunction and is considered one of the leading causes of death among intensive care patients in Europe and the United States [2, 3]. Moreover, septic shock combines a severe sepsis with persistent arterial hypotension despite adequate fluid resuscitation [4]. To provide ways of improving patients’ outcome, researchers have already identified important mechanisms of pathophysiology in sepsis [5]. Yet, one of the main goals remains the establishment of animal models mirroring the clinical situation in septic patients. Rodents have by far been the most commonly used animals in sepsis research [6]. While mouse models provide a greater variety of molecular tools, experimental rat models are preferred for studying hemodynamics or organ pathophysiology. Lustig et al. only recently adopted the colon ascendens stent peritonitis to the rat [7]. Wichterman
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et al. presented a sepsis model of cecal ligation and puncture (CLP), which led to persistent fecal leakage from the cecum into the abdomen, thus providing a polymicrobial source of infection [6]. The latter model has been widely accepted among animal researchers ever since. However, sepsis progression in the CLP model is slow [8 –10] and is largely influenced by the puncture size [11, 12]. Up to this point, to our knowledge, severe sepsis with acute onset and high mortality as present in intensive care unit (ICU) patients [13] is induced mainly through intraperitoneal (i.p.) or intravenous (i.v.) injection of single bacteria species or endotoxin/lipopolysaccharide (LPS). Yet, Wichterman et al. and Fink et al. pointed out that sepsis and endotoxemia are not the same entity [6, 14]. Moreover, we also believe that the i.p./i.v. injection of single bacteria species is somewhat artificial and does not adequately mirror the situation in septic patients in ICU suffering from bacterial peritonitis. We therefore aimed at establishing an experimental model of severe sepsis characterized by acute onset and high mortality, fulfilling sepsis criteria as defined by the 2001 International Sepsis Definitions Conference [1]. We deliberately chose the CLP model by Wichterman et al. [6] as experimental base because this established animal model provides persistent fecal leakage into the abdomen, which we consider as absolutely necessary for polymicrobial contamination and sepsis development in rodent models involving bacterial peritonitis. MATERIALS AND METHODS Animals and Anesthesia All animal experiments in this prospective randomized study were approved by the governmental board for the care of animal subjects (Regierungspräsidium Darmstadt, Germany) and were in accordance with the National Institute of Health guidelines (National Academy of Sciences, Washington DC, 1996). Twenty-eight male Sprague Dawley rats (Harlan-Winkelmann, Borchen, Germany, mean body weight 532 ⫾ 56 g) were kept on a 12 h light/dark cycle with free access to food and water. Rats were anesthetized by i.p. injection of pentobarbital (10 mg/kg body weight; Narcoren, Halbergmoos, Germany) and fentanyl (0.05 mg/kg; Janssen-Cilag, Neuss, Germany). Unconscious rats were tested for sufficient depth of anesthesia by tail clamping, weighed, and then placed supine on a heating pad. A tracheotomy was performed and a 13-G cannula (i.d. 2.0 mm, o.d. 2.5 mm; Abbott, Wiesbaden, Germany) was endotracheally inserted. Subsequently, rats were ventilated with a neonatal ventilator (Stephanie, Stephan, Gackenbach, Germany) using pressure-controlled ventilation: inspiratory oxygen fraction 0.21, T I/E 1:2, p max 20 cm H 2O, PEEP 4 cm H 2O, tidal volume 16 –20 mL/kg, respiratory rate 31/min [15]. Respiratory settings were adjusted to maintain normocapnia according to arterial blood gas analyses (ABL500; Radiometer, Willich, Germany), which were obtained through fluid-filled polyurethane catheters (i.d. 0.58 mm, o.d. 0.96 mm, SIMS Portex, Hythe, United Kingdom) in the right femoral artery. A similar catheter was inserted into the right femoral vein for continuous i.v. infusion of 0.9% NaCl (12 mL/kg/h; B. Braun, Melsungen, Germany, as described elsewhere [16]), pentobarbital (0.6 mg/kg/h), and fentanyl (0.03 mg/kg/h). Body temperature was monitored by a rectally inserted probe.
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Surgical Procedure and Experimental Protocol Rats were randomly assigned to 2 groups of 14 rats each. After establishment of sufficient anesthesia and arterial baseline blood gas analysis, a 2 cm long midline laparotomy was performed in all rats. The cecum was carefully exteriorized in all animals by means of cotton sticks that had been placed in 0.9% NaCl solution before. In the SHAM-group, the cecum was replaced into the abdomen after gentle manipulation. In the other group, the cecum and the mesenteric blood vessels were ligated below the ileocecal valve (2/0-Mersilene; Ethicon, Norderstedt, Germany) to exclude bowel obstruction. Subsequently, the ligated cecum was opened through a 1.5 cm blade incision (no. 10; Feather, Osaka, Japan) on the antimesenteric side (cecal ligation and incision, CLI). The cecum was then carefully replaced into the abdomen. All surgical procedures were performed by the same investigator to minimize variability. In both groups, 2 mL/kg of 0.9% NaCl solution was given i.p. as fluid resuscitation before the abdominal wall was closed in 2 layers (3/0-vicryl; Ethicon Norderstedt, Germany). Mean arterial blood pressure (MAP), arterial base excess (BE) and arterial pH (pH) were measured hourly for 390 min. At the end of observation time or in case of prior death, all animals were exsanguinated and heparinized whole blood samples (heparin-natrium; Ratiopharm, Ulm, Germany) were obtained.
Abdominal Swabs, Peritoneal Lavage, and Isolation of Peritoneal Cells After postmortem laparotomy, abdominal swabs (Transystem; Hain Lifescience, Nehren, Germany) were obtained from the Douglas cavity. Subsequently, the abdominal cavity was lavaged with 2 aliquots of 20 mL phosphate-buffered saline (Invitrogen, Karlsruhe, Germany). Both rinses in all animals were performed in a standardized manner by the same investigator within 20 s. The obtained peritoneal lavage fluid (PL) was kept on ice and centrifuged at 1500 rpm for 10 min. The PL supernatant was stored at ⫺80°C for further analysis. The PL cell pellet was resuspended and washed twice in 10 mL phosphate-buffered saline. Peritoneal cells were identified as being mononuclear cells by light microscopy and were quantified using a Neubauer counting chamber. Abdominal swabs were processed directly on blood agar and Mueller-Hinten-Agar (Heipha, Ettelheim, Germany). Bacteria were identified according to antibiogram and growth on different types of nutrient agar.
Nitrite Assay, Plasma Samples, and Enzyme-Linked Immunosorbent Assay Nitrite release in PL supernatant was analyzed by the Griess reaction as described in detail elsewhere [17]. Absorbance at 540 nm/595 nm and comparison with a nitrite standard provided nitrite concentration in PL supernatant. Heparinized whole blood samples were centrifuged at 1700 rpm for 10 min. Thus, plasma was separated from blood cells and stored at ⫺80°C for further analysis. Plasma levels of IL-1 and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA; R and D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions.
Statistics Statistical analysis was performed with SigmaStat 3.1 (Systat Software; San Jose, CA). Unless otherwise stated, data are expressed as median (semi-interquartile range) using Mann-Whitney rank sum test. Survival differences between both groups were analyzed using Kaplan-Meier log-rank test. Differences between the groups were considered significant at P ⬍ 0.05 unless otherwise stated.
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to the SHAM-group (Table 1). In addition, nitrite content in the PL-fluid from CLI-animals was significantly increased compared with SHAM-animals (P ⫽ 0.002; Table 1). Blood smears obtained from CLI-animals at the end of the observation time or at the time of prior death showed reduced leukocyte population in light microscopy compared with blood smears from SHAM-animals (data not shown). Cytokine Plasma Levels
CLI resulted in a significant increase in plasma IL-1 levels compared with SHAM control (435 (156) versus 10 (35) pg/mL, respectively; P ⬍ 0.001; Fig. 5A). Likewise, plasma IL-6 levels in CLI-animals were significantly increased compared to SHAM-animals (19718 (9181) versus 832 (799) pg/mL, respectively; P ⬍ 0.001; Fig. 5B). FIG. 1. Survival rate (%) of rats within 390 min after laparotomy (SHAM, n ⫽ 14, solid line) or additional CLI (n ⫽ 14, dash line). Kaplan-Meier log-rank test; *P ⫽ 0.003 versus SHAM.
RESULTS Outcome and Hemodynamics
The first animal in the CLI-group died 150 min after laparotomy. After 390 min of observation time, overall mortality was significantly higher in CLI-animals than in SHAM-animals (50% versus 0%, respectively, P ⫽ 0.003; Fig. 1). At baseline, both groups did not differ in MAP, BE, and pH. In CLI-animals, MAP decreased significantly throughout observation time compared with SHAM-animals (Fig. 2). First significant changes between both groups were observed 210 min after the respective surgical procedure. Likewise, within the observation time, arterial BE and arterial pH decreased significantly in CLI-animals compared to SHAM-animals (P ⬍ 0.001; Fig. 3). No significant differences in body temperature, heart rate, tidal volume, and blood gas parameters (PaO 2, PaCO 2, HCO 3 –) were observed between the 2 groups (data not shown).
DISCUSSION
With the present study, we aimed at establishing an experimental model of severe sepsis characterized by acute onset and high mortality in the rat. Using the accepted CLP model [6] as experimental base, we induced gram-positive and gram-negative bacterial peritonitis and subsequent rapid sepsis progression by a cecal ligation and blade incision instead of puncture. Within the observation time of 390 min, CLI resulted in a mortality of 50% and in a decrease of MAP, BE, and pH. Moreover, CLI caused leukocyte influx into the peritoneum leading to accumulation of nitrite in PL supernatant. In addition, highly increased plasma levels of IL-1 and IL-6 were observed in CLI-induced septic animals.
Abdominal Situs and Peritoneal Characteristics
Postmortem laparotomy showed normal peritoneal bowel status in SHAM-animals (Fig. 4A) and fecal bowel content covering abdominal organs and intestines (Fig. 4B) in CLI-animals. Abdominal swabs from the Douglas cavity in SHAManimals proved to be sterile and showed E. faecalis, P. mirabilis, and E. coli growth on agar in the CLI-group (Table 1). In the PL fluid, total mononuclear cell population was significantly increased in the CLI-group compared
FIG. 2. MAP (mmHg, intra-arterial registration) of rats over 390 min of observation time after laparotomy (SHAM, n ⫽ 14, filled circles, solid line) or additional CLI (n ⫽ 14, open circles, dash line). Data are expressed as median (error bars ⫽ semi-interquartile range). Mann-Whitney rank sum test; *P ⬍ 0.05 versus SHAM.
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FIG. 3. Arterial BE [mmol/L, (A)] and arterial pH [pH, (B)] from blood gas analysis of rats over 390 min of observation time after laparotomy (SHAM, n ⫽ 14, filled circles, solid line) or additional CLI (n ⫽ 14, open circles, dash line). Data are expressed as median (error bars ⫽ semi-interquartile range). Mann-Whitney rank sum test; **P ⬍ 0.001 versus SHAM.
Sepsis is a complex syndrome with no absolute criteria, which have to be fulfilled to be diagnosed. The 2001 International Sepsis Definitions Conference therefore proposed a variety of possible criteria for sepsis [1]. These can be summarized as the presence (or strong suspicion) of an infectious origin of the disease combined with systemic inflammation. A severe sepsis includes organ dysfunction, whereas a septic shock represents persistent arterial hypotension despite adequate fluid resuscitation. Our main objective for conducting the present study was to provide an experimental animal model reflecting acute onset in severe sepsis. In fact, we have demonstrated that our model fulfils the above mentioned sepsis criteria. First, CLI, as applied in this study, leads to high mortality after 390 min, thus providing a progressive deterioration of the physical status of the animals, which thereby mimics the rapidly worsening situation in patients with septic shock. It is known that mortality in CLP models depends on the puncture size [11]. Yet, to our knowledge, there has not been a prospective, randomized study characterizing the short-term effects of cecal ligation and blade incision, which we consider as
an adequate experimental model reflecting a severe sepsis with acute onset as seen in ICU patients with bowel perforation and subsequent bacterial peritonitis. Several rodent models have been in use to match this clinical situation [18]. In conscious rats, implantation of a fibrin clot containing high doses of E. coli did not affect heart rate and MAP, but displayed a mortality rate of 50% to 80% within 2 days [19]. In mice, CLP and i.p. injection of LPS expressed different proinflammatory cytokine profiles leading in both models to higher mortality than in our model within a comparable time frame [20]. CLI does not lead to immediate septic shock and death after the surgical procedure, but is instead characterized by progressive decrease of arterial MAP, BE, and pH within the observation period. Organ perfusion highly depends on stable MAP [21] and persistent arterial hypotension in sepsis is associated with worsening of outcome [22]. Moreover, 2 of the main conditions for basic organ functions are tightly regulated arterial BE and pH [23]. More specifically, arterial BE can predict the outcome of patients admitted to ICU [24] and is considered to be a sensitive parameter of acidosis in shock [25]. Reversing acidosis will reduce
FIG. 4. Abdominal situs of rats postmortem at 390 min after laparotomy [SHAM, (A)] or additional CLI (B). Notice the feces covering organs and intestine after CLI (filled arrows). The photographs represent typical findings of n ⫽ 14 experiments for each group. (Color version of figure is available online.)
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TABLE 1 Peritoneal Characteristics* ,**
SHAM CLI
Bacterial growth from abdominal swabs on agar
Population of peritoneal mononuclear cells (⫻10 6)
Nitrite (moL)
Sterile E. faecalis, E. coli, P. mirabilis
3.5 (0.8) 42.2 (32.8)*
1.31 (0.11) 7.19 (2.1)**
Notes. Peritoneal characteristics of rats post mortem at 390 min after laparatomy (SHAM, n⫽8) or additional CLI (n⫽8). Bacterial growth shows representative data from n⫽3 experiments for each group. Data are expressed as median (semi-interquartile range). E. faecalis ⫽ Enterococcus faecalis, E. coli ⫽ Escherichia coli, P. mirabilis ⫽ Proteus mirabillis. * P ⬍ 0.05 vs. SHAM. ** P ⬍ 0.002 vs. SHAM.
mortality in sepsis [26] since severe metabolic acidosis results in a release of proinflammatory mediators and significantly hampers immune functions [27]. Hence, these 3 parameters are accepted characteristics of organ dysfunction as observed in sepsis [4]. For many years, a cornerstone of sepsis criteria has been the infectious origin of the disease [28]. Despite the fact that only the cecum has been deliberately damaged, CLI leads to a generalized (four quadrant) peritonitis. By taking abdominal swabs from the Douglas cavity and by observing the abdominal status after postmortem laparotomy, we have proven that CLI leads to gram-positive and gram-negative bacterial growth within the peritoneum, which is ultimately responsible for the pathophysiologic changes as presented in the data. As severe sepsis caused by both gram-positive and gram-negative bacteria is common [29], the CLI model mirrors this mixed bacterial source of infection. Bacterial challenge leads to leukocyte chemotaxis [30], which is reflected in the high number of mononuclear cells in the PL fluid from CLI-animals. The predominant cellular element responding to bacteria are neutrophil granulocytes and macrophages [31], which in turn produce nitrite upon stimulation [32]. An additional major source of nitrite derives from the bacterial reduction of nitrate [33]. Therefore, the high amount of nitrite release in the PL of CLI-animals
is most likely due to the presence of neutrophil granulocytes and bacteria within the peritoneal cavity. Another cornerstone of sepsis criteria is the systemic inflammatory response in the host. Plasma cytokine levels can reflect this inflammatory response and, moreover, cytokine imbalance is known to participate in organ dysfunction [34]. In addition, recent literature shows that plasma levels of IL-1 and IL-6 correlate positively with organ dysfunction in patients with severe sepsis [35]. For this reason, we did not measure other proinflammatory cytokine plasma levels (i.e., TNF-␣). Highly elevated plasma levels of IL-1 and IL-6 as observed in CLI-animals strongly argue in favor of systemic inflammation. Limitations of This Study
In the present severe sepsis model of CLI, the standard sepsis treatment (i.e., aggressive fluid resuscitation, use of vasoconstrictors) has not been applied. Yet, it has been shown that fluid management or pharmacologic agents directed at circulatory support heavily influence cytokine production and thereby may alter the host’s immune response [36, 37]. Since we aimed at providing a new animal model for the study of severe sepsis characterized by acute onset, we decided to eliminate all modulating agents and instead chose to provide a basic experimental model.
FIG. 5. Box plots (median, 25% and 75% quartile with 95% confidence intervals) showing IL-1 (A) and IL-6 (B) plasma levels (pg/mL) at 390 min after laparotomy (SHAM, n ⫽ 14) or additional cecal ligation and incision (CLI, n ⫽ 14). Mann-Whitney rank sum test; **P ⬍ 0.001 versus SHAM.
SCHEIERMANN ET AL.: CECAL LIGATION AND INCISION
In summary, the present experimental model is characterized by acute onset and high mortality and represents a new possibility to study severe sepsis in rodents. Further research by our group based on the present model will be directed at identifying pharmacologic agents, which might improve the outcome of septic animals by possibly altering the host’s immune response to the inflammatory challenge.
17.
18. 19.
20.
ACKNOWLEDGMENTS The authors thank Ms. Andrea Dolfen and the team from the Institute of Medical Microbiology and Infection Control for expert technical assistance.
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22.
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Cecal Ligation and Incision: An Acute Onset Model of Severe Sepsis in Rats 1 Patrick Scheiermann, M.D.,*,2 Sandra Hoegl, M.D.,*,† Marc Revermann, M.D.,* Devan Ahluwalia,* Johannes Zander, M.D.,‡ Kim A. Boost, M.D.,* Thach Nguyen, M.D.,* Bernhard Zwissler, M.D.,§ Heiko Muhl, Ph.D.,† and Christian Hofstetter, M.D.* *Department of Anesthesiology, Intensive Care Medicine, and Pain Therapy, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany; †Pharmazentrum/ZAFES, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany; ‡Institute of Medical Microbiology and Infection Control, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany; §Department of Anesthesiology, Hospital of the Ludwigs-Maximilians-University, Munich, Germany Submitted for publication October 18, 2007
Background. Sepsis is a leading cause of death among critically ill patients. Up to now, severe sepsis with acute onset in animals has been induced mainly through injection of single bacteria species or endotoxin and not through a surgical procedure, which might adequately mirror the situation in septic patients. We therefore aimed to establish a surgical model of severe sepsis in rodents fulfilling international sepsis criteria. Materials and methods. Twenty-eight anesthetized/ ventilated Sprague Dawley rats underwent laparotomy and cecal mobilization. The cecum was either replaced into the abdomen (SHAM, n ⴝ 14) or the cecum and the mesenteric blood vessels were ligated, and the cecum was opened through a 1.5 cm blade incision (cecal ligation and incision, CLI, n ⴝ 14). Results. Within 390 min, mortality was 0% (SHAM) and 50% (CLI), respectively. Compared with SHAM, CLI resulted in a 43% reduction of mean arterial blood pressure and in severe metabolic acidosis as measured by arterial base excess and pH. CLI led to a 15-fold increase in mononuclear cell population and to a 5-fold accumulation of nitrite in peritoneal lavage. Abdominal swabs from the Douglas cavity in CLI-animals showed gram-positive and gram-negative bacterial growth on agar compared with sterile swabs from SHAM-animals. In CLI-animals, plasma IL-1 level was 1 Parts of this study were presented at the 2007 Meeting of the German Sepsis-Society (DSG) in Weimar, Germany. 2 To whom correspondence and reprint requests should be addressed at Department of Anesthesiology, Intensive Care Medicine, and Pain Therapy, Hospital of the Johann Wolfgang GoetheUniversity, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany. E-mail: [email protected]
0022-4804/09 $34.00 © 2009 Elsevier Inc. All rights reserved.
increased to 435 pg/mL (SHAM: 10 pg/mL) and plasma IL-6 level to 19718 pg/mL (SHAM: 832 pg/mL). Conclusions. CLI causes bacterial peritonitis with subsequent systemic inflammation and organ dysfunction. Thus, CLI mimics clinical sepsis and provides a surgical short term model of severe sepsis in rodents. © 2009 Elsevier Inc. All rights reserved. Key Words: polymicrobial peritonitis; severe sepsis; systemic inflammation; organ dysfunction; cytokines; rat. INTRODUCTION
Sepsis as defined by the 2001 International Sepsis Definitions Conference is a clinical syndrome consisting of systemic inflammation (systemic inflammatory response syndrome, SIRS) caused by an infectious origin [1]. Severe sepsis, in addition, includes organ dysfunction and is considered one of the leading causes of death among intensive care patients in Europe and the United States [2, 3]. Moreover, septic shock combines a severe sepsis with persistent arterial hypotension despite adequate fluid resuscitation [4]. To provide ways of improving patients’ outcome, researchers have already identified important mechanisms of pathophysiology in sepsis [5]. Yet, one of the main goals remains the establishment of animal models mirroring the clinical situation in septic patients. Rodents have by far been the most commonly used animals in sepsis research [6]. While mouse models provide a greater variety of molecular tools, experimental rat models are preferred for studying hemodynamics or organ pathophysiology. Lustig et al. only recently adopted the colon ascendens stent peritonitis to the rat [7]. Wichterman
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et al. presented a sepsis model of cecal ligation and puncture (CLP), which led to persistent fecal leakage from the cecum into the abdomen, thus providing a polymicrobial source of infection [6]. The latter model has been widely accepted among animal researchers ever since. However, sepsis progression in the CLP model is slow [8 –10] and is largely influenced by the puncture size [11, 12]. Up to this point, to our knowledge, severe sepsis with acute onset and high mortality as present in intensive care unit (ICU) patients [13] is induced mainly through intraperitoneal (i.p.) or intravenous (i.v.) injection of single bacteria species or endotoxin/lipopolysaccharide (LPS). Yet, Wichterman et al. and Fink et al. pointed out that sepsis and endotoxemia are not the same entity [6, 14]. Moreover, we also believe that the i.p./i.v. injection of single bacteria species is somewhat artificial and does not adequately mirror the situation in septic patients in ICU suffering from bacterial peritonitis. We therefore aimed at establishing an experimental model of severe sepsis characterized by acute onset and high mortality, fulfilling sepsis criteria as defined by the 2001 International Sepsis Definitions Conference [1]. We deliberately chose the CLP model by Wichterman et al. [6] as experimental base because this established animal model provides persistent fecal leakage into the abdomen, which we consider as absolutely necessary for polymicrobial contamination and sepsis development in rodent models involving bacterial peritonitis. MATERIALS AND METHODS Animals and Anesthesia All animal experiments in this prospective randomized study were approved by the governmental board for the care of animal subjects (Regierungspräsidium Darmstadt, Germany) and were in accordance with the National Institute of Health guidelines (National Academy of Sciences, Washington DC, 1996). Twenty-eight male Sprague Dawley rats (Harlan-Winkelmann, Borchen, Germany, mean body weight 532 ⫾ 56 g) were kept on a 12 h light/dark cycle with free access to food and water. Rats were anesthetized by i.p. injection of pentobarbital (10 mg/kg body weight; Narcoren, Halbergmoos, Germany) and fentanyl (0.05 mg/kg; Janssen-Cilag, Neuss, Germany). Unconscious rats were tested for sufficient depth of anesthesia by tail clamping, weighed, and then placed supine on a heating pad. A tracheotomy was performed and a 13-G cannula (i.d. 2.0 mm, o.d. 2.5 mm; Abbott, Wiesbaden, Germany) was endotracheally inserted. Subsequently, rats were ventilated with a neonatal ventilator (Stephanie, Stephan, Gackenbach, Germany) using pressure-controlled ventilation: inspiratory oxygen fraction 0.21, T I/E 1:2, p max 20 cm H 2O, PEEP 4 cm H 2O, tidal volume 16 –20 mL/kg, respiratory rate 31/min [15]. Respiratory settings were adjusted to maintain normocapnia according to arterial blood gas analyses (ABL500; Radiometer, Willich, Germany), which were obtained through fluid-filled polyurethane catheters (i.d. 0.58 mm, o.d. 0.96 mm, SIMS Portex, Hythe, United Kingdom) in the right femoral artery. A similar catheter was inserted into the right femoral vein for continuous i.v. infusion of 0.9% NaCl (12 mL/kg/h; B. Braun, Melsungen, Germany, as described elsewhere [16]), pentobarbital (0.6 mg/kg/h), and fentanyl (0.03 mg/kg/h). Body temperature was monitored by a rectally inserted probe.
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Surgical Procedure and Experimental Protocol Rats were randomly assigned to 2 groups of 14 rats each. After establishment of sufficient anesthesia and arterial baseline blood gas analysis, a 2 cm long midline laparotomy was performed in all rats. The cecum was carefully exteriorized in all animals by means of cotton sticks that had been placed in 0.9% NaCl solution before. In the SHAM-group, the cecum was replaced into the abdomen after gentle manipulation. In the other group, the cecum and the mesenteric blood vessels were ligated below the ileocecal valve (2/0-Mersilene; Ethicon, Norderstedt, Germany) to exclude bowel obstruction. Subsequently, the ligated cecum was opened through a 1.5 cm blade incision (no. 10; Feather, Osaka, Japan) on the antimesenteric side (cecal ligation and incision, CLI). The cecum was then carefully replaced into the abdomen. All surgical procedures were performed by the same investigator to minimize variability. In both groups, 2 mL/kg of 0.9% NaCl solution was given i.p. as fluid resuscitation before the abdominal wall was closed in 2 layers (3/0-vicryl; Ethicon Norderstedt, Germany). Mean arterial blood pressure (MAP), arterial base excess (BE) and arterial pH (pH) were measured hourly for 390 min. At the end of observation time or in case of prior death, all animals were exsanguinated and heparinized whole blood samples (heparin-natrium; Ratiopharm, Ulm, Germany) were obtained.
Abdominal Swabs, Peritoneal Lavage, and Isolation of Peritoneal Cells After postmortem laparotomy, abdominal swabs (Transystem; Hain Lifescience, Nehren, Germany) were obtained from the Douglas cavity. Subsequently, the abdominal cavity was lavaged with 2 aliquots of 20 mL phosphate-buffered saline (Invitrogen, Karlsruhe, Germany). Both rinses in all animals were performed in a standardized manner by the same investigator within 20 s. The obtained peritoneal lavage fluid (PL) was kept on ice and centrifuged at 1500 rpm for 10 min. The PL supernatant was stored at ⫺80°C for further analysis. The PL cell pellet was resuspended and washed twice in 10 mL phosphate-buffered saline. Peritoneal cells were identified as being mononuclear cells by light microscopy and were quantified using a Neubauer counting chamber. Abdominal swabs were processed directly on blood agar and Mueller-Hinten-Agar (Heipha, Ettelheim, Germany). Bacteria were identified according to antibiogram and growth on different types of nutrient agar.
Nitrite Assay, Plasma Samples, and Enzyme-Linked Immunosorbent Assay Nitrite release in PL supernatant was analyzed by the Griess reaction as described in detail elsewhere [17]. Absorbance at 540 nm/595 nm and comparison with a nitrite standard provided nitrite concentration in PL supernatant. Heparinized whole blood samples were centrifuged at 1700 rpm for 10 min. Thus, plasma was separated from blood cells and stored at ⫺80°C for further analysis. Plasma levels of IL-1 and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA; R and D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions.
Statistics Statistical analysis was performed with SigmaStat 3.1 (Systat Software; San Jose, CA). Unless otherwise stated, data are expressed as median (semi-interquartile range) using Mann-Whitney rank sum test. Survival differences between both groups were analyzed using Kaplan-Meier log-rank test. Differences between the groups were considered significant at P ⬍ 0.05 unless otherwise stated.
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to the SHAM-group (Table 1). In addition, nitrite content in the PL-fluid from CLI-animals was significantly increased compared with SHAM-animals (P ⫽ 0.002; Table 1). Blood smears obtained from CLI-animals at the end of the observation time or at the time of prior death showed reduced leukocyte population in light microscopy compared with blood smears from SHAM-animals (data not shown). Cytokine Plasma Levels
CLI resulted in a significant increase in plasma IL-1 levels compared with SHAM control (435 (156) versus 10 (35) pg/mL, respectively; P ⬍ 0.001; Fig. 5A). Likewise, plasma IL-6 levels in CLI-animals were significantly increased compared to SHAM-animals (19718 (9181) versus 832 (799) pg/mL, respectively; P ⬍ 0.001; Fig. 5B). FIG. 1. Survival rate (%) of rats within 390 min after laparotomy (SHAM, n ⫽ 14, solid line) or additional CLI (n ⫽ 14, dash line). Kaplan-Meier log-rank test; *P ⫽ 0.003 versus SHAM.
RESULTS Outcome and Hemodynamics
The first animal in the CLI-group died 150 min after laparotomy. After 390 min of observation time, overall mortality was significantly higher in CLI-animals than in SHAM-animals (50% versus 0%, respectively, P ⫽ 0.003; Fig. 1). At baseline, both groups did not differ in MAP, BE, and pH. In CLI-animals, MAP decreased significantly throughout observation time compared with SHAM-animals (Fig. 2). First significant changes between both groups were observed 210 min after the respective surgical procedure. Likewise, within the observation time, arterial BE and arterial pH decreased significantly in CLI-animals compared to SHAM-animals (P ⬍ 0.001; Fig. 3). No significant differences in body temperature, heart rate, tidal volume, and blood gas parameters (PaO 2, PaCO 2, HCO 3 –) were observed between the 2 groups (data not shown).
DISCUSSION
With the present study, we aimed at establishing an experimental model of severe sepsis characterized by acute onset and high mortality in the rat. Using the accepted CLP model [6] as experimental base, we induced gram-positive and gram-negative bacterial peritonitis and subsequent rapid sepsis progression by a cecal ligation and blade incision instead of puncture. Within the observation time of 390 min, CLI resulted in a mortality of 50% and in a decrease of MAP, BE, and pH. Moreover, CLI caused leukocyte influx into the peritoneum leading to accumulation of nitrite in PL supernatant. In addition, highly increased plasma levels of IL-1 and IL-6 were observed in CLI-induced septic animals.
Abdominal Situs and Peritoneal Characteristics
Postmortem laparotomy showed normal peritoneal bowel status in SHAM-animals (Fig. 4A) and fecal bowel content covering abdominal organs and intestines (Fig. 4B) in CLI-animals. Abdominal swabs from the Douglas cavity in SHAManimals proved to be sterile and showed E. faecalis, P. mirabilis, and E. coli growth on agar in the CLI-group (Table 1). In the PL fluid, total mononuclear cell population was significantly increased in the CLI-group compared
FIG. 2. MAP (mmHg, intra-arterial registration) of rats over 390 min of observation time after laparotomy (SHAM, n ⫽ 14, filled circles, solid line) or additional CLI (n ⫽ 14, open circles, dash line). Data are expressed as median (error bars ⫽ semi-interquartile range). Mann-Whitney rank sum test; *P ⬍ 0.05 versus SHAM.
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FIG. 3. Arterial BE [mmol/L, (A)] and arterial pH [pH, (B)] from blood gas analysis of rats over 390 min of observation time after laparotomy (SHAM, n ⫽ 14, filled circles, solid line) or additional CLI (n ⫽ 14, open circles, dash line). Data are expressed as median (error bars ⫽ semi-interquartile range). Mann-Whitney rank sum test; **P ⬍ 0.001 versus SHAM.
Sepsis is a complex syndrome with no absolute criteria, which have to be fulfilled to be diagnosed. The 2001 International Sepsis Definitions Conference therefore proposed a variety of possible criteria for sepsis [1]. These can be summarized as the presence (or strong suspicion) of an infectious origin of the disease combined with systemic inflammation. A severe sepsis includes organ dysfunction, whereas a septic shock represents persistent arterial hypotension despite adequate fluid resuscitation. Our main objective for conducting the present study was to provide an experimental animal model reflecting acute onset in severe sepsis. In fact, we have demonstrated that our model fulfils the above mentioned sepsis criteria. First, CLI, as applied in this study, leads to high mortality after 390 min, thus providing a progressive deterioration of the physical status of the animals, which thereby mimics the rapidly worsening situation in patients with septic shock. It is known that mortality in CLP models depends on the puncture size [11]. Yet, to our knowledge, there has not been a prospective, randomized study characterizing the short-term effects of cecal ligation and blade incision, which we consider as
an adequate experimental model reflecting a severe sepsis with acute onset as seen in ICU patients with bowel perforation and subsequent bacterial peritonitis. Several rodent models have been in use to match this clinical situation [18]. In conscious rats, implantation of a fibrin clot containing high doses of E. coli did not affect heart rate and MAP, but displayed a mortality rate of 50% to 80% within 2 days [19]. In mice, CLP and i.p. injection of LPS expressed different proinflammatory cytokine profiles leading in both models to higher mortality than in our model within a comparable time frame [20]. CLI does not lead to immediate septic shock and death after the surgical procedure, but is instead characterized by progressive decrease of arterial MAP, BE, and pH within the observation period. Organ perfusion highly depends on stable MAP [21] and persistent arterial hypotension in sepsis is associated with worsening of outcome [22]. Moreover, 2 of the main conditions for basic organ functions are tightly regulated arterial BE and pH [23]. More specifically, arterial BE can predict the outcome of patients admitted to ICU [24] and is considered to be a sensitive parameter of acidosis in shock [25]. Reversing acidosis will reduce
FIG. 4. Abdominal situs of rats postmortem at 390 min after laparotomy [SHAM, (A)] or additional CLI (B). Notice the feces covering organs and intestine after CLI (filled arrows). The photographs represent typical findings of n ⫽ 14 experiments for each group. (Color version of figure is available online.)
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TABLE 1 Peritoneal Characteristics* ,**
SHAM CLI
Bacterial growth from abdominal swabs on agar
Population of peritoneal mononuclear cells (⫻10 6)
Nitrite (moL)
Sterile E. faecalis, E. coli, P. mirabilis
3.5 (0.8) 42.2 (32.8)*
1.31 (0.11) 7.19 (2.1)**
Notes. Peritoneal characteristics of rats post mortem at 390 min after laparatomy (SHAM, n⫽8) or additional CLI (n⫽8). Bacterial growth shows representative data from n⫽3 experiments for each group. Data are expressed as median (semi-interquartile range). E. faecalis ⫽ Enterococcus faecalis, E. coli ⫽ Escherichia coli, P. mirabilis ⫽ Proteus mirabillis. * P ⬍ 0.05 vs. SHAM. ** P ⬍ 0.002 vs. SHAM.
mortality in sepsis [26] since severe metabolic acidosis results in a release of proinflammatory mediators and significantly hampers immune functions [27]. Hence, these 3 parameters are accepted characteristics of organ dysfunction as observed in sepsis [4]. For many years, a cornerstone of sepsis criteria has been the infectious origin of the disease [28]. Despite the fact that only the cecum has been deliberately damaged, CLI leads to a generalized (four quadrant) peritonitis. By taking abdominal swabs from the Douglas cavity and by observing the abdominal status after postmortem laparotomy, we have proven that CLI leads to gram-positive and gram-negative bacterial growth within the peritoneum, which is ultimately responsible for the pathophysiologic changes as presented in the data. As severe sepsis caused by both gram-positive and gram-negative bacteria is common [29], the CLI model mirrors this mixed bacterial source of infection. Bacterial challenge leads to leukocyte chemotaxis [30], which is reflected in the high number of mononuclear cells in the PL fluid from CLI-animals. The predominant cellular element responding to bacteria are neutrophil granulocytes and macrophages [31], which in turn produce nitrite upon stimulation [32]. An additional major source of nitrite derives from the bacterial reduction of nitrate [33]. Therefore, the high amount of nitrite release in the PL of CLI-animals
is most likely due to the presence of neutrophil granulocytes and bacteria within the peritoneal cavity. Another cornerstone of sepsis criteria is the systemic inflammatory response in the host. Plasma cytokine levels can reflect this inflammatory response and, moreover, cytokine imbalance is known to participate in organ dysfunction [34]. In addition, recent literature shows that plasma levels of IL-1 and IL-6 correlate positively with organ dysfunction in patients with severe sepsis [35]. For this reason, we did not measure other proinflammatory cytokine plasma levels (i.e., TNF-␣). Highly elevated plasma levels of IL-1 and IL-6 as observed in CLI-animals strongly argue in favor of systemic inflammation. Limitations of This Study
In the present severe sepsis model of CLI, the standard sepsis treatment (i.e., aggressive fluid resuscitation, use of vasoconstrictors) has not been applied. Yet, it has been shown that fluid management or pharmacologic agents directed at circulatory support heavily influence cytokine production and thereby may alter the host’s immune response [36, 37]. Since we aimed at providing a new animal model for the study of severe sepsis characterized by acute onset, we decided to eliminate all modulating agents and instead chose to provide a basic experimental model.
FIG. 5. Box plots (median, 25% and 75% quartile with 95% confidence intervals) showing IL-1 (A) and IL-6 (B) plasma levels (pg/mL) at 390 min after laparotomy (SHAM, n ⫽ 14) or additional cecal ligation and incision (CLI, n ⫽ 14). Mann-Whitney rank sum test; **P ⬍ 0.001 versus SHAM.
SCHEIERMANN ET AL.: CECAL LIGATION AND INCISION
In summary, the present experimental model is characterized by acute onset and high mortality and represents a new possibility to study severe sepsis in rodents. Further research by our group based on the present model will be directed at identifying pharmacologic agents, which might improve the outcome of septic animals by possibly altering the host’s immune response to the inflammatory challenge.
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18. 19.
20.
ACKNOWLEDGMENTS The authors thank Ms. Andrea Dolfen and the team from the Institute of Medical Microbiology and Infection Control for expert technical assistance.
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