Dig Dis Sci DOI 10.1007/s10620-014-3381-2

ORIGINAL ARTICLE

Bacterial Translocation and Endotoxemia After Pringle Maneuver in Cirrhotic Rats Yonghui Su • Haiyan Pan • Zhaowang Guo Wenying Zhou • Baimeng Zhang



Received: 18 February 2013 / Accepted: 4 April 2014 Ó Springer Science+Business Media New York 2014

Abstract Background Pringle maneuver (Pm) is advocated for the reduction of blood loss during liver surgery, while postoperative infections continue to be a frequent complication after hepatic resection and liver transplantation. Aim To investigate the effect of the Pringle maneuver on systemic bacterial translocation and endotoxemia in cirrhotic rats and cirrhotic rats with selective intestinal decontamination. Methods A total of 100 male Sprague–Dawley cirrhotic rats were randomly divided into five equal groups: sham operation, Pm of 10 min, Pm of 20 min, Pm of 30 min, and pretreatment. Tissue samples from mesenteric lymph nodes, liver, lungs, portal, and vena cava vein blood were obtained for culture after 30 min and 24 h of the operation. Endotoxin levels were measured in portal vein and vena cava blood. Results Portal vein and vena cava blood endotoxin concentrations increased significantly after 30 min, especially 24 h of operation in the Pm of 20 min and Pm of 30 min groups. A significant increase in contaminated mesenteric lymph nodes, liver, portal, and vena cava blood was noted 24 h later. The incidence of gut bacterial translocation increased with the duration extension of Pm. Escherichia coli was the most common bacteria isolated from the tissues. There was a significant decrease of portal vein and vena cava blood endotoxin concentrations and the incidence of bacterial translocation by selective intestinal decontamination.

Y. Su (&)  H. Pan  Z. Guo  W. Zhou  B. Zhang Department of General Surgery, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, Guangdong, China e-mail: [email protected]

Conclusions There is endotoxemia immediately after Pringle maneuver and gut bacteria translocation 24 h later. The incidence of gut bacterial translocation increases with the duration extension of Pm. Selective intestinal decontamination can decrease bacteria translocation and endotoxemia. Keywords Cirrhotic  Bacterial translocation  Endotoxemia  Pringle maneuver  Selective intestinal decontamination

Introduction Postoperative infections continue to be a frequent complication after hepatic resection and liver transplantation. Infectious complications have been reported in 47–80 % of liver transplant recipients by a mean of 1.0–2.5 episodes of infections per patient. These infections significantly impacted postoperative mortality, and 54 % of patients with major bacterial infections died [1]. During the past decade, post-hepatectomy infections have reportedly occurred in 4–20 % of cases, comprising half of the postoperative complications. Postoperative mortality associated with these infections is as high as 40 %. In particular, Enterobacter sp. and Enterococcus sp. have been shown to be common pathogens responsible for postoperative bacteremia after hepatic resection and were thought to be secondary to gut mucosal and/or biliary translocation [2, 3]. Intraoperative occlusion of the hepatoduodenal ligament together with the common bile duct [i.e., Pringle maneuver (Pm)] is advocated for the reduction of blood loss during liver surgery. There is also a period of portal vein clamping during liver transplantation operation. Most patients who undergo liver transplantation or hepatic resection have a background of cirrhosis.

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Dig Dis Sci

This study aimed to evaluate the effect of the Pringle maneuver (Pm) on systemic bacterial translocation (BT), endotoxemia in cirrhotic rats and cirrhotic rats with selective intestinal decontamination, and to find some interventions on decreasing the infection rate after liver surgery.

Materials and Methods

abdominal incisions were closed in two layers with 3–0 silk. Tissue samples were obtained (a) in 50 % of each group of animals 30 min after performing the laparotomy and (b) in the remaining 50 % of each group of animals 24 h later. Tissue samples were obtained for aerobic and anaerobic culture from mesenteric lymph nodes (MLNs), liver, lungs, portal vein, and vena cava blood. Blood was obtained from the portal vein and the vena cava for examining systemic endotoxin concentrations. Finally, tissue samples of the liver were examined histologically.

Animal Model Design Bacterial Culture and Identification The experimental protocols were approved by the Animal Review Board of the Fifth Affiliated Hospital of Sun Yatsen University, Zhuhai, China. One hundred male Sprague–Dawley (SD) rats, weighing 200–250 g, were used for this study. The animals were housed in stainless-steel cages, three rats per cage, under controlled temperature (21 °C) and humidity conditions on a 12-h light/dark cycle. They were maintained on standard laboratory diet with tap water and libitum throughout the experiment, except for an overnight fast before surgery. Cirrhosis was induced in 120 rats by subcutaneous injection of 60 % CCl4-olive oil solution every 4 days at an initial dose of 0.5 ml/100 g. Subsequent dosage was adjusted with body weight changes at a dose of 0.3 ml/100 g for a total of 15 times. Fifteen rats died during the induction of cirrhosis with an average mortality of 12.5 %. A final total of 100 cirrhotic rats were used for further study. The animals were divided randomly into five groups according to the treatment they received: sham operation group (n = 20); Pm of 10 min group (n = 20): occluding the hepatoduodenal ligament by using a small atraumatic clamp for 10 min; Pm of 20 min group (n = 20): occluding the hepatoduodenal ligament for 20 min; Pm of 30 min group (n = 20): occluding the hepatoduodenal ligament for 30 min; and pretreatment group (n = 20): norfloxacin was given by gavage before laparotomy for 3 days at a dose of 10 mg kg-1 day-1, and then, the hepatoduodenal ligament was occluded by using a small atraumatic clamp for 30 min. Surgical Procedures All surgical procedures were performed under strictly sterile conditions, using light ether anesthesia. The animals were placed on a warm pad throughout the experiment. All the rats underwent laparotomy on day 0. A 1 cm midline incision in the upper abdomen was performed, and the hepatoduodenal ligament was isolated. In the sham operation group, the hepatoduodenal ligament was mobilized but not clamped. In the remaining groups, the hepatoduodenal ligament was occluded by using a small atraumatic clamp with the time according to the design. The

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Two grams of liver, lung, and small bowel mesentery (including MLNs) was sharply excised in all animals. The tissue was then homogenized and placed in a sterile tube containing 2 ml of sterile saline (1 g/ml of the original sample). A total of 100 ll of each were inoculated into 5 % blood and McConkey’s agar plates, and they were incubated for 24–48 h at 35 °C, under aerobic conditions with 5–6 % CO2. A second tissue sample was inoculated into 2 ml of thioglycolate broth, was subcultured into anaerobic blood agar plates by the same procedure, and was incubated under anaerobic conditions. One milliliter of blood from the portal vein and vena cava was drawn for culture. The blood was inoculated into 4.5 ml of common broth for 16 h bacterial enrichment, and then, 100 ll of the enrichment solution was inoculated into 5 % blood and McConkey’s agar plates, and they were incubated for 24–48 h at 35 °C, under aerobic conditions with 5–6 % CO2. If there was no bacterial growth, cultures were regarded as negative results, but if there was bacterial growth, further identified was required. First, Gram-stained smears were performed to determine whether they were coccus or bacillus and G? or G-. Second, biochemical and serological identifications were performed by using standard and routine methods. Endotoxin Measurements In all groups of animals, the portal vein and the vena cava were punctured and samples of 1 mL blood were obtained for estimation of endotoxins, respectively. The serum was separated by centrifugation at 8,000g for 10 min. The serum endotoxin concentration was determined by a limulus amebocyte lysates (LAL) test with rat ET ELISA (Rapidbio Co., USA). Liver Fibrosis Evaluation Midsections of the left lobes of the livers were processed for light microscopy. This processing consisted of fixing

Dig Dis Sci Table 1 Histological scoring for liver fibrosis

Table 2 Positive cultures of portal, vena cava vein, and other tissues 24 h after laparotomy

Ishak grade

Score

No fibrosis

0

Fibrous expansion of some portal areas, with or without short fibrous septa

1

Fibrous expansion of most portal areas, with or without short fibrous septa Fibrous expansion of most portal areas with occasional portal to portal bridging

2

Sham operation (%)

3

Fibrous expansion of portal areas with marked bridging (portal to portal as well as portal to central)

4

Marked bridging (portal–portal and/or portal–central) with occasional nodules (incomplete cirrhosis) Cirrhosis, probable, or definite

5

Groups

n

Positive cultures MLNs

Liver

Lung

Portal vein

Vena cava

10

1 (10)

0 (0)

0 (0)

0 (0)

0 (0)

Pm of 10 min (%)

10

3 (30)

0 (0)

0 (0)

1 (10)

1 (10)

Pm of 20 min (%)

10

7 (70)a

4 (40)

0 (0)

6 (60)a

4 (40)

Pm of 30 min (%)

10

8 (80)a

5 (50)a

2 (20)

5 (50)a

3 (30)

Pretreatment (%)

10

1 (10)b

0 (0)b

0 (0)

1 (10)

0 (0)

a

6

Compared with the sham operation group, P \ 0.05 Compared with the Pm of 30 min group, P \ 0.05 (Fishers’ exact test)

b

Results

(P \ 0.05 compared with the sham operation group), respectively. In the Pm of 30 min group, positive cultures for bacteria of gut origin were found in 80 % of MLNs (P \ 0.05 compared with the sham operation group), 50 % of liver tissue (P \ 0.05 compared with the sham operation group), and 50 % of portal vein (P \ 0.05 compared to the sham operation group), respectively (Table 2). However, in the pretreatment group, specimens collected 24 h after laparotomy revealed that there were less positive cultures for bacteria of gut origin than those of the Pm of 30 min group (Table 2). In samples collected 30 min after laparotomy, there was one positive culture of MLNs in the sham operation group and the pretreatment group, and two positive cultures of MLNs in the Pm of 10 min group, Pm of 20 min group, and Pm of 30 min group. The portal vein, vena cava, and the remaining tissues showed no positive cultures for bacteria of gut origin.

Liver Fibrosis Evaluation in Cirrhotic Rats

Bacterial Species Isolated in the Cultures

Severity of liver fibrosis was assessed by a histological scoring scale adapted from Ishak et al. [4]. Tissues were scored on a scale of 0–6 (Table 1). In almost all groups of rats, the liver fibrosis progression reached stage 5–6. There was no significant difference in liver fibrosis histological scores among the groups.

Table 3 shows the bacterial species isolated in the various tissue specimens analyzed. Escherichia coli, E. cloacae, Proteus sp, Enterococcus, and Klebsiella sp were identified in the portal vein, vena cava, MLNs, liver, and lung specimens 24 h after laparotomy in each subsequent group (Table 3). In contrast to these findings, in all of the abovementioned specimens obtained from each group 30 min after laparotomy, only E. coli could be isolated.

the specimens in a 5 % neutral formol solution, embedding the specimens in paraffin, cutting 5-mm-thick sections and staining the sections with hematoxylin and eosin. The tissue slices were scanned and scored blindly by two experts. The degree of fibrosis, classified on a scale of 0–6 according to Ishak et al. [4], was estimated in each slide. Statistical Analysis Categorical variables were compared by Fishers’ exact test, and continuous variables were compared by Student’s t test. Histological scores of liver fibrosis were compared by the Kruskal–Wallis test. The results were considered statistically significant when P B 0.05.

Portal Vein, Vena Cava, and Other Tissues Cultures Table 2 shows the results of positive cultures of aerobic bacteria in specimens obtained from Portal vein, vena cava, MLNs, liver, and lung in all groups. Anaerobes were not isolated from the tissue samples. Specimens collected 24 h after laparotomy and Pringle maneuver in the Pm of 20 min group revealed positive cultures for bacteria of gut origin in 70 % of MLNs (P \ 0.05 compared with the sham operation group) and in 60 % of portal vein blood

Endotoxin Concentrations Figures 1 and 2 show the endotoxin concentrations, which were measured in blood collected from the portal vein and vena cava 30 min and 24 h after laparotomy in all groups studied. Animals treated with the Pringle maneuver of 20 or 30 min (Pm of 20 min group and Pm of 30 min group) presented significantly higher endotoxin concentrations in

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Dig Dis Sci Table 3 Bacterial species isolated in portal, vena cava vein, and other tissues cultures 24 h after laparotomy Groups

n

MLNs

Liver

Lung

Portal vein

Vena cava

Sham operation

10

E. coli(1)

0

0

0

0

Pm of 10 min

10

E. coli(3)

0

0

E. coli(1)

E. coli(1)

E. coli(3) Pm of 20 min

10

E. coli(6)

E. coli(4)

0

Proteus. sp(1) Pm of 30 min

10

Pretreatment

10

E. coli(6)

E. coli(3)

Proteus. sp(1)

Proteus. sp(2)

vena cava vein

0.6 0.5

portal vein

Pm of 10min Pm of 20min Pm of 30min pretreatment

Fig. 1 Portal vein and vena cava blood endotoxin concentrations 30 min after laparotomy. Triangle compared with the sham operation group, P \ 0.01. Asterisk compared with the Pm of 30 min group, P \ 0.01 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

vena cava vein portal vein

sham operation

Pm of 10min Pm of 20min Pm of 30min pretreatment

Fig. 2 Portal vein and vena cava blood endotoxin concentrations 24 h after laparotomy. Triangle compared with the sham operation group, P \ 0.01. Asterisk compared with the Pm of 30 min group, P \ 0.01

both the portal vein and vena cava blood compared with those in the sham operation group 30 min and 24 h after laparotomy (P \ 0.01). However, in the pretreatment group, both portal vein and vena cava blood endotoxin concentrations were significantly lower compared with those in the pretreatment group (P \ 0.01).

Discussion We examined the effects of Pringle maneuver on bacterial translocation and endotoxemia in cirrhotic rats. The results

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Proteus. sp(1)

Proteus. sp(1)

Enterococcus(1)

0.2 0.1 sham operation

E. coli(2)

E. coli(1)

0.3

0

Proteus. sp(1)

E. coli(3) Enterococcus(1)

0.4

E. coli(3)

E. cloacae(1)

Klebsiella. sp(1)

0.8 0.7

E. coli(2)

Proteus. sp(2)

of the present study demonstrate that a normothermic Pringle maneuver of 20 min duration results in immediate and delayed significant endotoxemia in portal vein and vena cava blood. Additionally, delayed bacterial translocation to MLNs, liver, and portal vein was detected. The incidence of gut bacteria translocation and endotoxin concentration increased with the duration extension of Pm. E. coli was the most common bacteria isolated from the tissues. There was a significant decrease in endotoxin concentrations in portal vein and vena cava blood and the incidence of bacteria translocation by selective intestinal decontamination. Intestinal bacterial translocation, first proposed by the Berg in 1979, is defined as the migration of viable microorganisms from the intestinal lumen to mesenteric lymph nodes and other extra-intestinal organs and sites [5]. Bacterial translocation increases in conditions associated with a high risk of infections by Gram-negative bacteria and multiple organ failure, such as hemorrhagic shock, intestinal obstruction, major burn injury, and serious trauma [6]. Almost 100 years ago, Pringle described a new technique to reduce blood loss during liver surgery [7]. Since then, the Pringle maneuver has become a routine procedure and a preferred method to avoid massive hemorrhage during partial liver resection for a large spectrum of nonmalignant and malignant diseases. Postoperative infections continue to be a frequent complication after hepatic resection and liver transplantation. Our study aimed to evaluate the effect of Pringle maneuver on systemic bacterial translocation and endotoxemia in cirrhotic rats. In our experiment, animals submitted to the Pringle maneuver of 20 min presented significantly higher concentrations of endotoxin in portal vein and vena cava compared with sham operation group 30 min and 24 h after laparotomy. Bacterial translocation to MLNs, liver, and portal vein could also be detected 24 h after laparotomy. The reasons for increased bacterial translocation and endotoxemia after Pringle maneuver are unclear. Intestinal ischemia/reperfusion (I/R) injury after Pringle maneuver causes acute hydrostatic gut edema,

Dig Dis Sci

delayed intestinal transit, increased mucosal permeability to macromolecules and disruption of the intestinal mucosal barrier [8–10]. Studies have reported that the degree of intestinal damage due to venous occlusion is greater than the damage resulting from arteriovenous and arterial occlusion [11, 12]. In addition, liver cirrhosis itself is an immunocompromised state. Clearance of translocated organisms may be impaired, given the depressed reticuloendothelial system phagocytic activity and impaired chemotaxis, phagocytosis, and intracellular killing by polymorphonuclear leukocytes and monocytes [13, 14]. There is also intestinal bacterial overgrowth, especially the increase of intestinal aerobic Gram-negative bacteria in cirrhosis [15]. All these conditions might at least partially explain the higher endotoxin concentrations and bacterial translocation after the hepatoduodenal ligament occlusion in the present study. However, no significantly increasing of endotoxin concentration and bacterial translocation was found after hepatic vascular occlusion of 10 min. The reason for these findings could be because the intestinal mucosal barrier function was not damaged much after Pringle maneuver of 10 min. Intestinal anaerobic bacteria outnumber aerobic bacteria by 100:1–1,000:1, despite the fact that anaerobes translocate rarely. Gram-negative bacteria (specifically E. coli, Klebsiella pneumoniae, and other Enterobacteriaceae) have been found to be the most adept at translocating to extraintestinal organs [16–18]. E. coli has been shown to translocate more efficiently, probably as a result of a greater ability to adhere to the intestinal mucosa [19]. Our study also showed that E. coli was the most common bacterial translocating to extra-intestinal organs 24 h after Pringle maneuver, and mesenteric lymph nodes were the most common tissues with positive culturings. However, with the increase of portal triad clamping time, other extraintestinal organs and tissues such as the liver, portal vein, and vena cava also had positive bacterial cultures. In addition to E. coli, Proteus, Enterococcus, Klebsiella pneumoniae, Enterobacter, and E. cloacae also translocated to extra-intestinal organs. These results suggest that in the treatment of infectious complications after liver surgery, antibiotics which are sensitive to E. coli and other Gram-negative bacteria should be adopted firstly. Although the species of bacteria in the intestine are more than 500, only a small number of bacteria can translocate to extra-intestinal organs [20]. Using oral antibiotics, the intestinal bacteria species which lead to translocation are reduced or eliminated, and the occurrence of bacterial translocation can be inhibited. The most current clinical study of antibiotic on the prevention of bacterial translocation is norfloxacin, because norfloxacin is not well absorbed in the intestine, and it has antibacterial activity to these bacterias that lead to translocation [21,

22]. In our study, oral administration of norfloxacin was used for selective intestinal decontamination in cirrhotic rats. Either 24 h or 30 min after laparotomy, endotoxin concentrations in the portal vein and vena cava in the pretreatment group were significantly lower than those in the Pm of 30 min group. In addition, 24 h after laparotomy, there were less positive culturings of extra-intestinal tissues in the pretreatment group than those in the Pm of 30 min group. This indicates that selective intestinal decontamination can reduce the occurrence of bacterial translocation and endotoxemia, and provide a reference value for prevention of infectious complications after liver surgery. In summary, our study reveals that there is endotoxemia immediately after Pringle maneuver and gut bacteria translocation 24 h later. The incidence of gut bacteria translocation turns higher with the duration extension of Pringle maneuver. Selective intestinal decontamination is effective in preventing bacteria translocation and endotoxemia. Further studies with the model presented here should provide useful information for understanding mechanisms of bacterial translocation after Pringle maneuver. Conflict of interest of interest.

The authors declare that they have no conflict

References 1. Singh N. Infectious diseases in the liver transplant recipient. Semin Gastrointes Dis. 1998;9:136–146. 2. Garwood RA, Sawyer RG, Thompson L, Adams RB. Infectious complications after hepatic resection. Am Surg. 2004;70:787–792. 3. Shigeta H, Nagino M, Kamiya J, et al. Bacteremia after hepatectomy: an analysis of a single center, 10-year experience with 407 patients. Langenbecks Arch Surg. 2002;387:l17–l124. 4. Ishak K, Baptista A, Bianchi L, et al. Histological grading and staging of chronic hepatitis. Hepatol. 1995;22:696–699. 5. Berg RD, Garlington AW. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in agnotobiotic mouse model. Infect Immun. 1979;23:403–411. 6. Edmiston CE Jr, Condon RE. Bacterial translocation. Surg Gyn Obstet. 1991;173:73–83. 7. Pringle JH. Notes on the arrest of hepatic hemorrhage due to trauma. Ann Surg. 1908;48:541–549. 8. Marubayashi S, Dohi K, Ochi K, Kawasaki T. Role of free radical in ischaemia rat liver cell injury prevention of damage by atocophervol administration. Sugery. 1986;99:184–192. 9. Moore-Olufemi SD, Xue H, Attuwaybi BO, Fischer U, Harari Y, Oliver DH, et al. Resuscitation-induced gut edema and intestinal dysfunction. Tramma 2005; 58:264–270. 10. Sun Z, Wang X, Deng X et al. Phagocytic and intestinal endothelial and epithelial barrier function during the early stage of small intestinal ischemia and reperfusion injury. Shock 2000; 13:209–216. 11. Guzma´n-de la Garza FJ, Ca´mara-Lemarroy CR, Alarco´n-Galva´n G, Cordero-Pe´rez P, Mun˜oz-Espinosa LE, Ferna´ndez-Garza NE. Different patterns of intestinal response to injury after arterial, venous or arteriovenous occlusion in rats. World J Gastroenterol. 2009; 15:3901–3907.

123

Dig Dis Sci 12. Yano K, Hata Y, Matsuka K, Ito O, Matsuda H. Time limits for intestinal ischemia and congestion: an experimental study in rats. Ann Plast Surg. 1994;32:310–314. 13. Rimola A, Soto R, Bory F, Arroyo V, Piera C, Rodes J. Reticuloendothelial system phagocyticactivity in cirrhosis and its relation to bacterial infections and prognosis. Hepatology. 1984;4:53–58. 14. Guarner C, Runyon BA. Macrophage function in cirrhosis and the risk of bacterial infection. Hepatology. 1995;22:367–369. 15. Sa´nchez E, Casafont F, Guerra A, de Benito I, Pons-Romero F. Role of intestinal bacterial overgrowth and intestinal motility in bacterial translocation in experimental cirrhosis. Rev Esp Enferm Dig. 2005;97:805–814. 16. Reiner W, Guadalupe GT. Bacterial translocation in cirrhosis. Hepatohogy 2005; 41:422–433. 17. Steffen EK, Berg RD, Deitch EA. Comparison of translocation rates of various indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node. J Infect Dis. 1988;157:1032–1038.

123

18. Wells CL. Colonization and translocation of intestinal bacterial flora. Transpl Proc. 1996;28:2653–2656. 19. Ljungdahl M, Lundholm M, Katouli M, et al. Bacterial translocation in experimental shock is dependent on the strains in the intestinal flora. Scand J Gstroenterol. 2000;35:389–397. 20. Bernard B, Grange´ JD, Khac EN, Amiot X, Opolon P, Poynard T. Antibiotic prophylaxis for the prevention of bacterial infections in cirrhotic patients with gastrointestinal bleeding: a meta-analysis. Hepatohogy. 1999;29:1655–1661. 21. Almeida J, Galhenage S, Yu J, Kurtovic J, Riordan SM. Gut flora and bacterial translocation in chronic liver disease. World J Gastroenterol. 2006;12:1493–1502. 22. Carbonell N, Pauwels A, Serfaty L, Fourdan O, Levy VG, Poupon R. Improved survival after variceal bleeding in patients with cirrhosis over the past two decades. Hepatology. 2004;40: 652–659.

Bacterial translocation and endotoxemia after pringle maneuver in cirrhotic rats.

Pringle maneuver (Pm) is advocated for the reduction of blood loss during liver surgery, while postoperative infections continue to be a frequent comp...
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