Introduction

1. Introduction

The better preservation of immune function during laparoscopy compared with open surgery seems to be maintained under septic conditions but the underlying mechanisms that explain such results are still not completely understood. Several experimental evidences suggest that CO₂ pneumoperitoneum reduces the severity of sepsis and prolongs survival, compared with laparotomy in animal models of peritonitis [1–3]. However, little is known about the CO₂ pneumoperitoneum impact on early acute phase response in sepsis. Recently, we demonstrated that CO₂ pneumoperitoneum reduces inflammatory response by reducing early liver and lung interleukin (IL) 6 and tumor necrosis factor-alpha (TNF-α) expression in cecal ligation and puncture rat model [4]. Other studies have reported that CO₂ insufflation could also influence intra-abdominal bacterial contamination in murine models of abdominal sepsis [5, 6]. In this regard, β-arrestins play a critical role as modulators of inflammatory response. β-arrestins are peculiar adaptor proteins involved in G-protein-coupled receptor (GPCR) desensitization and are also implicated in regulation of Toll-like receptor signalling and proinflammatory gene expression. Both β-arrestins 1 and 2 differentially seem to regulate TLR4 signalling pathways. In particular, it has been suggested that β-arrestin 2 is a negative regulator of inflammatory response in CLP-induced mortality [7–9].

HMGB-1 is another potent proinflammatory cytokine involved in the delayed endotoxin lethality and systemic inflammatory response [10]. The majority of HMGB-1 secreted by activated monocyte/macrophages is from a preinformed cellular pool within the first 16 h [11]; HMGB-1 mRNA levels are increased in various tissues such as muscle, liver, and lung following endotoxemia [12]. Subsequently, increased cellular synthesis of HMGB-1 further reinforces their release into extracellular milieu [11]. In addition, cells undergoing unprogrammed cell death can passively release HMGB-1, which worsens and prolongs inflammation [13]. It has been reported that HMGB-1 can induce cellular activation and generate inflammatory responses by interaction of both TLR2 and TLR4 [14]. Finally, HMGB-1 seems to function as an “alarmin” signal to recruit, alert, and activate innate immune cells, sustaining a potentially injurious inflammatory response [15]. In light of these considerations and supported by our previous data, we performed this study, in order to better understand for the first time whether the role of CO₂ pneumoperitoneum might be related to a reduced bacterial count and consequently to a decreased activation of the inflammatory cascade via TLR activation in an experimental model of CLP. β-arrestin 2 and HMGB-1 protein expressions are investigated in liver, lung, and kidney as target sites of septic injury.

2. Materials and Methods

Materials and Methods

3. Results

Results

3.1. CO

CO

₂

 Pneumoperitoneum Reduces Bacterial Load in Peritoneal Fluid

The bacterial load in peritoneal cavity was determined by CFU count. Cecal ligation and puncture following LT produced a marked bacterial load in the peritoneal fluid compared with naïve animals (Figure 1). CO

CO

₂

 pneumoperitoneum (LS) significantly reduced bacterial loads in peritoneal cavity compared with animals subjected to laparotomy (LT) (

Figure 1

; 4 × 10

⁶

 versus 7 × 10

⁶

 CFU/mL, resp., 

P

 < 0.05) suggesting that an antimicrobial effect may concur to the protective effect of CO

₂

.

Figure 1

Bacterial count analysis in peritoneal fluid cultures (CFU/mL) from animals subjected to CLP followed by LT or LS procedures and naïve animals. Data represent the mean ± SD of seven animals. 

Figure 1. Bacterial count analysis in peritoneal fluid cultures (CFU/mL) from animals subjected to CLP followed by LT or LS procedures and naïve animals. Data represent the mean ± SD of seven animals. 

^**

 

P

 < 0.001 compared with naïve; 

^#

 

P < 0.05 compared with LT.

 < 0.05 compared with LT.

3.2. CO

CO

₂

 Pneumoperitoneum Regulates Liver, Lung, and Kidney Expression of 

β

-Arrestin 2

To determine whether β-arrestin 2 plays a key role in modulating TLR signalling pathway in CLP-induced inflammation, we first evaluated CO₂ effects on β-arrestin 2 expression in liver, lung, and kidney. Animals that underwent laparotomy (LT) showed significantly reduced β-arrestin 2 protein expression in these organs compared with naïve group (P < 0.05). CO₂ pneumoperitoneum preserved β-arrestin 2 expression in liver, lung, and kidney (LS), displaying higher levels of the protein compared with laparotomy (Figures 2(a), 2(b), and 2(c),  P < 0.05).

 

Figure 2

Representative western blot analysis of 

Figure 2. Representative western blot analysis of 

β

-arrestin 2 protein in liver (a), lung (b), and kidney (c) from animals subjected to CLP followed by LT or LS procedures and naïve animals. Data represent the mean ± SD of seven animals. 

^*

 

P

 < 0.05 compared with naïve; 

^#

 

P < 0.05 compared with LT.

 < 0.05 compared with LT.

3.3. CO

CO

₂

 Pneumoperitoneum Reduces Liver, Lung, and Kidney Expression of HMGB-1

HMGB-1 is known as late mediator in sepsis, but it can also be released early after a traumatic insult. Therefore, we studied the CO₂ pneumoperitoneum effects on CLP-induced HMGB-1 expression in liver, lung, and kidney of rats. Animals subjected to laparotomy showed a significant increase of HMGB-1 protein expression in liver, lung, and kidney (P < 0.05) compared with naïve animals (Figures 3(a), 3(b), and 3(c)). CO₂ pneumoperitoneum markedly reduced the expression of this cytokine in liver, lung, and kidney tissues (LS) with respect to the LT (P < 0.05), suggesting a CO₂ protective role during polymicrobial sepsis.

 

Figure 3

Representative western blot analysis of HMGB-1 protein in liver (a), lung (b), and kidney (c) from animals subjected to CLP followed by LT or LS procedures and naïve animals. Data represent the mean ± SD of seven animals. 

Figure 3. Representative western blot analysis of HMGB-1 protein in liver (a), lung (b), and kidney (c) from animals subjected to CLP followed by LT or LS procedures and naïve animals. Data represent the mean ± SD of seven animals. 

^*

 

P

 < 0.05 compared with naïve; 

^#

 

P < 0.05 compared with LT.

 < 0.05 compared with LT.

3.4. CO

CO

₂

 Pneumoperitoneum Reduces Liver and Lung Neutrophil Accumulation

MPO staining for the evaluation of neutrophil infiltrate showed a strong inflammatory reaction in liver and lung of LT animals (Figures 4(b) and 5(b)) compared with naïve animals (Figures 4(a) and 5(a)). By contrary, rats subjected to CO₂ pneumoperitoneum (LS) revealed a reduced accumulation of inflammatory cells in liver and lung (Figures 4(c) and 5(c)), suggesting a protective role of this procedure regarding CLP.

 

Figure 4

Immunohistochemical detection of myeloperoxidase in liver from naïve, LT, or LS animals. Samples collected from LT rats (b) show an increased inflammatory infiltrate and little, but still representative, edematous interstitial areas compared to naïve tissue samples (a). In contrast, samples from animals subjected to CO

Figure 4. Immunohistochemical detection of myeloperoxidase in liver from naïve, LT, or LS animals. Samples collected from LT rats (b) show an increased inflammatory infiltrate and little, but still representative, edematous interstitial areas compared to naïve tissue samples (a). In contrast, samples from animals subjected to CO

₂ pneumoperitoneum revealed a reduced accumulation of inflammatory cells (c). Original magnification ×40.

 pneumoperitoneum revealed a reduced accumulation of inflammatory cells (c). Original magnification ×40.

 

Figure 5

Immunohistochemical detection of myeloperoxidase in lung from naïve, LT, or LS animals. Samples collected from LT rats (b) show an increased inflammatory infiltrate and some grade of fibrosis of interalveolar septa compared to naïve tissue samples (a). In contrast, samples from animals subjected to CO

Figure 5. Immunohistochemical detection of myeloperoxidase in lung from naïve, LT, or LS animals. Samples collected from LT rats (b) show an increased inflammatory infiltrate and some grade of fibrosis of interalveolar septa compared to naïve tissue samples (a). In contrast, samples from animals subjected to CO

₂ pneumoperitoneum revealed a reduced accumulation of inflammatory cells, as well as a substantial reduction of pulmonary fibrosis (c). Original magnification ×40.

 pneumoperitoneum revealed a reduced accumulation of inflammatory cells, as well as a substantial reduction of pulmonary fibrosis (c). Original magnification ×40.

4. Discussion

Discussion

In today's surgical practice, laparoscopic treatment represents a regular part of therapeutic approach in septic surgical patients with localized and diffuse peritonitis [16]. Parallely, increasing studies delineate the physiopathological effects of pneumoperitoneum in septic conditions, where CO₂ seems to have a relevant biologic role. The immunological changes induced by pneumoperitoneum appear to contribute favourable outcomes to laparoscopy with increased survival in experimental model of sepsis [1–3]. However, this encouraging data is reflected on the final step of a common and complicated pathway. The upstream mechanisms by which CO₂ can affect the inflammatory process preserving immune function in septic state are not identified and not fully investigated. In sepsis, the massive deterioration of the immune response results in fact from an inappropriately amplified systemic inflammatory response to infection [17]. Exaggerated proinflammatory mediators production from macrophages, monocytes, or other host cells governs this crucial phase and can lead to the development of tissue damage until multiple organ failure and even death [18]. TLRs, expressed on the host cells, play a critical and primary role in the inflammatory responses [19]. Binding to TLR4 primes a series of signalling proteins that activate transcription factors and proinflammatory gene expression [20]. Recent studies have shown that β-arrestin 2, a member of the arrestin family, plays an important function in signal transduction pathway of TLR4 [7, 21, 22]. Similarly, HMGB-1, that has proinflammatory cytokine-like properties, can interact also with both TLR4 and TLR2 and prime inflammatory response that is similar to that initiated by LPS [14].

In a previous study, we have demonstrated an early and significant reduction of liver and lung TNF-α and IL-6 expression in CLP rat model undergoing CO₂ pneumoperitoneum. We also showed the correlation between early cytokine expression and histological alterations. In fact, in liver and lung specimens, these results were accompanied by a reduction in the inflammatory infiltrate, edema, venular ectasia, and cellular degeneration in animals subjected to CO₂ pneumoperitoneum compared with laparotomy. These data supported the possible protective role of CO₂ pneumoperitoneum in reducing the inflammatory cascade associated with sepsis via reduced expression of liver and lung proinflammatory cytokines [4]. Therefore, the main focus of the present study was to further investigate the possible upstream mechanisms of our preliminary findings consequent on CO₂ pneumoperitoneum in a murine model of polymicrobial sepsis that better reproduces human peritonitis. The current study is the first to explore the influence of CO₂ pneumoperitoneum on early β-arrestin 2 and HMGB-1 expressions in target organs of sepsis in correlation with peritoneal bacterial load and myeloperoxidase in a well-standardized rat model of abdominal sepsis. The data obtained by us demonstrate that CO₂ pneumoperitoneum exerts a dual beneficial effect both locally and on the organ level in CLP-induced inflammation.

Balagué et al. reported that the number of bacterial CFU obtained in peritoneal fluid was significantly lower in the laparoscopic groups than in the open group in a defined bacterial inoculum model [5]. In a similar experiment, Pitombo and coworkers demonstrate, 18 h after bacterial inoculum into the peritoneum, a significant increase of CFU/mL for the laparotomy group compared with CO₂ and control groups [6]. In our study, the microbiological evaluation of peritoneal fluid collected after CLP and pneumoperitoneum revealed that CO₂ significantly reduced the number of colony forming units in this group of animals compared to the LS group. These intriguing data induce thinking primarily that CO₂ can have a specific and local action. Its prominent role in bacterial control in the storage and preservation of food products is known [23–25]. The potential antimicrobial effect of the gas if translated in the current study could explain the correlation between microbial inactivation (reduced bacterial count) and intra-abdominal exposure to CO₂.

Looking at β-arrestin 2 results, we found a significant high expression in liver, lung, and kidney of animals subjected to laparoscopy following CLP compared with laparotomy animals and, in previous experiment, CLP-induced TNF-alpha and IL-6 expression turned out to be significantly higher in LT group with respect to the LS [4]. These data suggest that β-arrestin 2 is, in agreement with the literature, an essential negative regulator of inflammatory response in polymicrobial sepsis [7–9, 26, 27] via TLR signalling. In particular, our investigation reveals that CO₂ insufflation preserves β-arrestin 2 expression in the organs studied. This result if correlated with blunted CLP-induced increase in TNF-alpha and IL-6 in liver and lung tissues [4] supports the hypothesis that β-arrestin 2 improved expression is protective in a model of abdominal sepsis.

Regarding HMGB-1, we demonstrated, according to literature, an increase in liver, lung, and kidney expression of this proinflammatory protein in rats subjected to laparotomy following CLP. In contrast, after CO₂ insufflation, we had a significant reduction of this cytokine.

Our data also showed an increased neutrophil infiltration in liver and lung of LT animals. On the contrary, CO₂ pneumoperitoneum significantly reduced inflammatory cells, confirming its beneficial role in this animal model of peritonitis.

Altogether, our data might be taken into account in order to explain additional mechanism(s) of CO₂ beneficial effects in experimental sepsis. It is in fact likely that the reduced bacterial load induced by antimicrobial action of CO₂ leads to a reduced release of bacterial components as lipopolysaccharides. This could downregulate the Toll-like receptors activation that mediate antimicrobial responses. Consequently, the potential β-arrestin 2 and HMGB-1 interactions with TLRs could allow both a preservation of β-arrestin 2 and a reduction of HMGB-1 expression. We speculate that all these composite changes can correlate with a better anti-inflammatory effect exerted by β-arrestin 2 and in addition also justify the subsequent reduced proinflammatory cytokine expressions and tissue damage contributing to corroborating the previously reported results.

In conclusion, our study contributes to reinforcing the hypothesis that laparoscopic surgery better controls abdominal infection and favourably modifies the immune response reducing the inflammatory septic cascade. In particular, by our results, it can be evicted that CO₂ exerts a precocious and protective effect by reduced bacterial load with (and) likely Toll-like receptors activation which in turn lead to a preserved β-arrestin 2 expression, reduced HMGB-1 expression, and myeloperoxidase in this animal model of peritonitis.

Acknowledgment

References