The Effect of Peritoneal Prednisolone Lavage in Bacterial Peritonitis: An Experimental Study

Objective. This study investigated and compared the effects of antibiotic and steroid lavage on survival and cytokine levels in an experimental abdominal sepsis model. Background. In abdominal sepsis, abdominal lavage with saline or antibiotic solutions is a well-documented intervention known to have positive impact on survival; however, the effects of steroid lavage in abdominal sepsis have not yet been investigated. Materials and Methods. Ninety-six Wistar rats were divided into 4 groups (n = 24). Abdominal sepsis was induced by cecal ligation and puncture. Six hours after laparotomy, the authors performed a relaparatomy followed by cecal resection and an abdominal lavage. Abdominal lavage was performed using saline in group 1, equal volumes of cefazolin sodium in group 2, low-dose methylprednisolone (1 mg/kg) in group 3, and high-dose methylprednisolone (2 mg/kg) in group 4. After division of 2 subgroups from each of the 4 groups, the first of the rats (n = 12) were euthanized 6 hours later for evaluation of cytokines (ie, interleukin [IL] 1β, 2, 4, 10, and tumor necrosis factor alpha [TNF-α]), and the others were followed for 30 days for analysis of mortality rates. Results. The mortality rate of the rats in group 2 was significantly higher than group 4, which had no mortality (P = 0.032). Although insignificant, the lowest mean value of IL-1β, IL-2, and TNF-α were in group 1, and the highest was in group 2. The lowest IL-4 level was in group 3, and the highest level was in group 2 (P = 0.41). Interleukin-10 levels were significantly lower in group 4 and higher in group 2 (P = 0.014). Conclusion. The authors state that peritoneal lavage with prednisolone improved survival rates with increasing doses in abdominal sepsis. 

Sepsis is a lethal condition affecting multiple organ systems which causes hemodynamic changes and multiple organ failure. Besides the improvements in antimicrobial therapy and in the understanding of pathogenesis of the disease, sepsis and septic shock are still the most common cause of hospital mortality.¹ One of the important steps in treating sepsis is the control of the septic source. Research^2,3 has defined the effects of the mediators and cytokines on the pathophysiology of sepsis. Consequently, it is understood that sepsis is a clinical response mechanism of the host against the infectious agents and mediated by a series of cytokines. Some studies^4,5 claim increased plasma levels of certain inflammatory mediators correlate with the severity of sepsis; in turn, the increased plasma level correlates with the mortality rates.

There are numerous studies attempting to treat sepsis and related conditions, and the role of corticosteroids in the treatment of sepsis is still controversial. Although some of the studies^2,4 have documented the beneficial role of steroids in sepsis treatment, others^6,7 have declared harmful effects. In abdominal sepsis, abdominal lavage with saline or antibiotic solutions are well-documented as having a positive impact on survival.⁷ But, according to the authors’ knowledge, the effects of steroid lavage in abdominal sepsis have yet to be investigated. In this study, the authors investigated and compared the effects of antibiotic and steroid lavage on survival and cytokine levels in an experimental abdominal sepsis model.

After the approval of the Institutional Ethical Committee of Gaziosmanpasa University (Tokat, Turkey), the study was conducted based on the experimental protocols. Ninety-six adult male Wistar rats, provided by Gaziosmanpasa University’s experimental research laboratory, were randomly divided into 4 groups (n = 24). All operative processes and follow-up were held at University Animal Studies Research Center at Gaziosmanpasa University. Rats were housed in separate wire cages, 2-3 animals per cage, with free access to food and water under standard laboratory conditions (ie, room temperature 23°C and 12-hour light/dark cycles). Rats were fasted for 12 hours before surgery, but had free access to water. Anesthesia was conducted by intraperitoneal injection of ketamine hydrochloride (75 mg/kg) and xylazine (10 mg/kg). The anterior abdominal wall was shaved with an electric razor and scrubbed with 10% povidone-iodine. In semisterile conditions, a 3-cm median laparotomy was performed. Abdominal sepsis was induced by cecal ligation and puncture model as described by Wichterman et al.⁸ All animals were resuscitated with 5 mL/kg saline. Six hours after laparotomy, the authors performed a relaparatomy followed by cecal resection and an abdominal lavage. Abdominal lavage was performed using isotonic saline (20 mL) in group 1, equal volumes of cefazolin sodium (6 mg/kg, Cefamezin, Eczacıbasi, İstanbul, Turkey) in group 2, low-dose methylprednisolone (1 mg/kg, Prednol, Mustafa Nevzat, İstanbul, Turkey) in group 3, and high-dose methylprednisolone (2 mg/kg, Prednol, Mustafa Nevzat, Istanbul, Turkey) in group 4. Thereafter, the fascia and skin were closed by running monofilament sutures of 4-0. All animals were resuscitated with 5 mL saline after relaparatomy. Each group of 24 were randomly divided into 2 subgroups (n = 12). Six hours after relaparotomy, blood samples were taken from 12 rats in each group for analysis of cytokine levels, and rats were euthanized with high-dose thiopental sodium (200 mg/kg). The remaining 12 rats in each group had a follow-up period of 30 days for evaluating survival rates; afterwards, the surviving rats were euthanized.

Cytokine level measurement. Blood samples were centrifuged and stored at -20°C until the analysis was performed. Interleukin (IL) 1β, 2, 4, 10, and tumor necrosis factor alpha (TNF-α) levels were measured using a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Rat IL-1β, IL-2, IL-4, IL-10, and TNF-α ELISA kits (Biosource International Inc., Camarillo, CA) were used.

Statistical analysis. Statistical evaluation of numeric variables was performed by one-way analysis of variance (ANOVA), followed by a post hoc Tukey test. Non-numeric variables were evaluated with the Chi-squared and Mann-Whitney U tests. A value of P < 0.05 was considered to be statistically significant. 

Three rats from group 1, 4 rats from group 2, and 1 rat from group 3 died within the first week. In group 4, the authors did not observe any mortality (Figure 1). During the autopsies, the authors found that all deceased rats had dilated bowel loops and increased volumes of peritoneal fluid. Gross macroscopic findings for all mortal cases are shown in Table 1. The second group had significantly higher mortality rates compared to group 4 (P = 0.032). Mortality rates between other groups were statistically insignificant (P > 0.05). 

Interleukin 1β, 2, 4, 10, and TNF-α levels in all groups are shown in Table 2. The lowest mean value of IL-1β, IL-2, and TNF-α levels were detected in group 1 (48.7 ± 11.1 pg/L, 202.9 ± 46.8 pg/L, and 247.3 ± 164.4 pg/L, respectively). In groups 2, 4, and 3, the highest mean values were observed (75.6 ± 36.5 pg/L, 264.9 ± 114.9 pg/L, and 284.8 ± 275.9 pg/L, respectively), but the difference was not statistically significant between these 3 groups (P = 0.15, 0.33, and 0.98, respectively). Although the lowest IL-4 levels were detected in group 3 (26.9 ± 7.6 pg/L), the highest levels were seen in group 2 (35.6 ± 18.2 pg/L), but with a statistically insignificant difference (P = 0.41). The lowest IL-10 levels were detected in group 4 (170.7±142.1 pg/L), and the highest levels in group 2 (538.2 ± 466.1 pg/L), with statistically significant difference between them (P = 0.014). Of all the cytokines measured, significant treatment effects were found only for IL-10 levels, with lower IL-10 levels 6 hours after treatment predicting survival of the rats receiving high-dose prednisolone. 

Sepsis causes high rates of mortality (45% to 82%), without a single definitive treatment.⁹ Effective treatment relies on early recognition and immediate treatment of the disease. Considering the large volume of literature and controversial topics about sepsis, current knowledge indicates the disease is highly complex^4,5,8,9 Clinical and experimental studies^8,9,10 attempt to clarify some of the important data concerning the pathophysiological aspects of sepsis. Sepsis is defined as the systemic immune response to severe infection, and it seems that both pro- and anti-inflammatory cytokines are produced more in this entity. Systemic inflammatory response syndrome (SIRS) is a condition caused by widespread inflammation as a result of the immune system’s response to an infection or disease such as acute pancreatitis, trauma, burns, infections, and it is widely accepted as the proinflammatory phase of sepsis.¹⁰ Thereafter, it is followed by the second phase known as compensatory anti-inflammatory response syndrome (CARS).¹⁰ The initial inflammatory response to endotoxin exposure in sepsis is a dynamic process that changes over time, but secondary changes are not associated with rapid changes in plasma levels of the cytokines.¹¹ Because of this, the authors obtained and compared cytokine levels 6 hours after sepsis-inducing surgery. 

Onset of clinical sepsis is largely dependent on the balance between proinflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-8; and anti-inflammatory cytokines like IL-10, soluble TNF receptor I and II (TNFRI and II), and IL-1 receptor antagonists and secretory IL-1 receptor antagonist (IL-1RA, sIL-1Ra, respectively).¹⁰ Inflammatory and anti-inflammatory cytokines have to reach certain levels for ongoing inflammation, and the severity of the inflammation is defined by the balance between pro- and anti-inflammatory cytokines. The changes between these ratios can cause uncontrolled severe or insufficient inflammatory response. High levels of anti-inflammatory cytokines like IL-10 have been associated with the prognosis of sepsis.¹² High circulating levels of TNF-α also correlate with high mortality rates and poor clinical outcome.¹³ Some studies^5,10 performed in patients with sepsis support prognostic potential of the cytokines (eg, IL-1, TNF-α) or the role as indicators of the severity of the inflammatory response in sepsis. 

In abdominal sepsis, the cytokine response is related to the amount of bacteria adhering to the peritoneal surface. Therefore, reducing the number of microorganisms within the peritoneal cavity with the addition of antibacterial-targeted therapy should be considered.¹⁴ In the study groups, the mean levels of the proinflammatory cytokines were similar. Group 2, the antibiotic-treated group, experienced higher levels of IL-10, confirming the correlation between higher levels of IL-10 and mortality. This finding parallels previous research⁵ that shows there are higher mortality rates in subjects with elevated regulatory cytokine levels. On the other hand, IL-10 levels were significantly lower in groups treated with prednisolone, and the IL-10 levels decreased with the increasing doses of prednisolone. Steroids protect the excessive reaction of the organism, which in turn regulates the host defense in all levels — mainly effects by inhibiting cytokine production. Interleukin-10 has been found to inhibit lipopolysaccharide (LPS)-induced tissue factor expression by monocytes in vitro. In addition to its well-described inhibitory effects on cytokine release, IL-10 modulates the fibrinolytic system and inhibits the coagulant responses during endotoxemia.¹⁵ The authors believe that decreasing IL-10 levels with the increasing prednisolone doses prevented insufficient inflammatory reaction and caused decreased mortality. 

Several treatment strategies include corticosteroids, ant-inflammatory agents, endotoxin antibodies, and specific cytokines have been investigated for modulating the inflammatory response associated with sepsis.¹¹ Adrenal gland hormones—mainly cortisol—are natural modulators of sepsis. Plasma-cortisol levels rise during critical infectious diseases to regulate the inflammatory cascade. However, with the progression of sepsis, the adrenal gland becomes affected and adrenal insufficiency develops.¹⁶ From this point of view, corticosteroid treatment in sepsis has been the main focus of numerous studies^6,16 since the 1970s. Utilization of steroids in sepsis is still controversial. Some studies¹⁶ claim there are no beneficial effects of steroids in sepsis, even in high doses. On the other hand, some studies¹⁷ have documented the beneficial effects of the steroids, if applied at the earlier periods of sepsis. Even if the effect of the steroids on sepsis is not universally accepted, some rational mechanisms have been defined. One of the most important effects of steroids is based on the regulatory roles on cardiovascular tonus. Steroids regulate the vasomotor tonus and reinforce the response of the cardiac contractility and vascular tonus by inducing catecholamines, which prevent β-receptor desensitization and increase the number of receptors.¹⁸ Actually, abdominal sepsis increases the net flow across the peritoneum to 300 mL/hour to 500 mL/hour in humans. This net flow is a physiological response of the organism to clean and flush the abdomen.¹⁹

Peritoneal lavage for treatment of abdominal sepsis has been performed in several studies^7,20 Several solutions such as saline, distilled water, antibiotics, or disinfection solutions were used.^7,20 However, the ideal peritoneal lavage solution has not been defined to date. The ideal solution should be effective with no toxicity and side effects.⁷ Indeed, results of peritoneal lavage in abdominal sepsis are controversial. In abdominal surgery, peritoneal lavage using sterile saline is almost routinely used to reduce peritoneal adhesion formation, most likely by altering the peritoneal fibrinolytic activity. 

This study had some limitations. However, in clinical practice, the first line antibiotic in the presence of abdominal sepsis is not cefazolin only. Rather, broad-spectrum antibiotic combinations with coverage for anaerobic organisms are recommended for empiric treatment, such as cefazolin, cefuroxime, ceftriaxone, cefotaxime, ciprofloxacin, or levofloxacin, each in combination with metronidazole. In cases of complicated abdominal sepsis, the causative agents are multibacterial, comprised mainly of gram-negative and anaerobic bacteria. Therefore, metronidazole should have been added as it is suggested; however, in this case, since the timing of the onset of sepsis is very early (ie, within 6 hours of wound induction) the authors believe that antibiotic coverage of only gram-negative bacteria might be enough; and cefazoline has a response to gram-negative bacteria. Also, the authors did not use intravenous antibiotic treatment for the rats. Rather, they compared the peritoneal lavage solutions with different ingredients, including saline, cefazolin, and steroids. The aim was to understand the effect of peritoneal lavage solutions on bacterial peritonitis. The administration of prednisolone in this abdominal sepsis model doesn’t reflect a real-world situation; however, the administration of prednisolone alone helped to understand the effect of corticosteroids without masking the effects with antibiotics.

In this study, the authors compared saline, cefazolin sodium, and 2 different doses of methylprednisolone on mortality rates and cytokine levels in an intra-abdominal sepsis model, and hypothesized that peritoneal lavage by antibiotic- or steroid-containing solutions might further assist in peritoneal cleansing. Various studies^21-23 have indicated the beneficial effects of systemic steroids on inflammation, cytokine response, and outcome in major abdominal surgery. The authors have shown that in a septic abdomen lavage with intraperitoneal prednisolone improved survival rates. Since only 2 doses of steroids were tested, it is difficult to draw a conclusion that increased doses have a better response. However, at present it shows that increased doses of steroids might result in better response rates in abdominal sepsis. These findings warrant further studies that use titrated doses of steroids. To the authors’ knowledge, this is the first study comparing the effects of these solutions on mortality rates and cytokine levels associated with intra-abdominal sepsis. The authors state that peritoneal cleansing by high-dose prednisolone significantly decreases mortality rates. This work shows the merit of future research exploring the authors’ hypothesis that early IL-10 levels may alert clinical professionals to the need for more rigorous action to prevent mortality.

Affiliations: Faculty of Medicine, Department of Family Medicine, Gaziosmanpasa University, Tokat, Turkey; Faculty of Medicine, Department of General Surgery, Gaziosmanpasa University; and Faculty of Medicine, Department of Biochemistry, Gaziosmanpasa University

Correspondence:
Zeki Ozsoy, MD 
Assistant Professor of Surgery
Faculty of Medicine
Department of General Surgery
Gaziosmanpasa University 
60100 Tokat, Turkey
zekiserkanozsoy@hotmail.com

Disclosure: The authors disclose no financial or other conflicts of interest.