Reprints Available Directly from the Publisher Photocopying Permitted by License Only Portal Hypertension Promotes Bacterial Translocation in Rats Mono-and Non Mono-associated with Escherichia Coli C25

The basis for the high incidence of infectious complications in portal hypertension (PHT) remains unclear. The hypothesis that PHT induces bacterial translocation (BT) was tested in a rat model with or without mono-association with streptomycin resistant Escherichia coli C25 and with or without hypovolemic shock. PHT was achieved by partial portal vein ligation and three weeks later hypovolemic shock (HS) was induced. Blood, liver, spleen and mesenteric lymph nodes cultures were performed twenty-four hours later. PHT promoted BT to mesenteric lymph nodes in indigenous flora (4/6 [67%]) and mono-associated animals (7/9 [78%]) compared to sham laparotomy and sham shock (SL + SS) animals (0/6 [0%] and 2/9 [22%] respectively) (p = 0.03). The combination of PHT and HS resulted in increased mortality in mono-associated (7/15 [47%]) and non mono-associated animals (8/15 [53%]). No significant translocation was noted in liver and spleen and bacteremia was found only in the PHT + HS mono-associated animals (4/8 [50%]). PHT induces BT to mesenteric lymph nodes and this may account for the high incidence of septic complications associated witti PHT. In this model, the addition of HS to PHT leads to an increased mortality but without uniform translocation of the gut flora beyond mesenteric lymph nodes.


INTRODUCTION
Sepsis and infections remain the most serious complications of patients with liver cirrhosis and portal hypertension (PHT)1. The incidence of bacteremia in patients with bleeding esophageal varices (portal hypertension and shock) has been reported to be as high flora (Escherichia coli, Proteus mirabilis, Enterobacter, Bacteroides).
Although immunosuppression related to cirrhosis (changes in the reticuloendothelial system of the liver, complement, polymorphonuclear leucocyte and monocyte function impairment) 3'' has been implicated as a contributing factor, the exact mechanism leading to infection in cirrhosis remains unknown.
Recent data suggest that antimicrobial prophylaxis in cirrhotics with gastrointestinal hemorrhage can reduce the incidence of bacterial infections6. Other studies have shown that selective intestinal decontamination prevents the incidence of spontaneous bacterial peritonitis in cirrhotic patients7.
In the following experiment, the ;hypothesis that portal hypertension (PHT) per se could induce bacterial translocation (BT)--the passage of live bacteria across the intestinal barriermwas tested. In addition, as part of the same experiment, some animals were submitted to hypovolemic shock, a common complication of PHT. The consequences of the combination of shock and portal hypertension on bacterial translocation were analyzed.

MATERIALS AND METHODS
Eighty-four male Wistar rats weighing 200-250gm were investigated in a model combining portal hypertension (PHT) and/or hypovolemic shock (HS) with or without mono-association with streptomycin resistant Escherichia coli C25. The experimental design is illustrated in Figure 1. The study was approved by the Animal Ethics Committee for Canton Berne. The animals were maintained in individual cages and allowed food and water ad libitum. All operations were done under pentobarbital anesthesia (0.05 g/kg intraperitoneally) and sterility was maintained during the operative procedures.
PHT was induced by partial portal vein ligation (PPL) as described by Chojkier and Groszmanna. The technique consisted of ligation of the portal vein over a 21 gauge blunt needle placed above the confluence of the splenic and superior mesenteric veins with 4.0 non-absorbable suture. The-needle was withdrawn immediately after ligation and patency of the portal vein was confirmed. Sham laparotomy (SL) consisted only of portal vein mobilization. Portal hypertension was studied in a pilot Study including six PPL and 7 SL animals followed Over three weeks. Body weight did not differ significantly between the two groups.
PHT was confirmed by a rise in portal pressure in PPL rats (18.9 3.7 mmHg) compared to SL rats (9.1 3.2 mmHg) and verified bythe presence of venous collaterals when the animals were sacrificed 21 days later.
In order to achieve bacterial mono-association, the indigenous intestinal flora was first cleared by adding bacitracin (0.4 mg/ml) and streptomycin (0.4.mg/ml) to the water from the fourteenth day on. Thereafter, all material in contact with the animals was sterilized (cages, food, water). On day eighteen, the animals were colonized by giving 100ml of water containing an overnight culture of Escherichia coli C25. Twenty days after PPL or SL the animals were anesthetized and the fight femoral artery was cannulated. The rats were submitted to 70 minutes of hypovolemic shock (HS) or sham shock (SS) as described by Baker and Deitch9. HS was induced by withdrawing blood into a syringe containing 100 IU of heparin in 0.3 ml of saline until the systolic pressure reached 30mmHg. Continuous hypotension was monitored with a pressure transducer (Statham P23XL, Spectramed, USA) connected to a dynagraph recorder. the systolic blood pressure rose 10 mmHg or more. SS consisted of arterial cannulation and pressure measurement, for 70 minutes. At the end of the HS, all shed blood was reinfused and one ml of blood was drawn for endotoxin measurements. Blood samples for endotoxins were centrifuged at 5000 g for 15 minutes, and the plasma was frozen immediately at -70C for turbidimetric limulus amebocyte lysate analysis (LAL) (Pyroquant Diagnostik GmbH 6082 Walldorf, Germany) as previously described10. Twenty-four hours later the animals were killed. Blood samples from the portal vein and inferior vena cava were taken for endotoxin analysis. Five ml of caval blood were sampled for aerobic cultures (Organon Technika, Durham, NC). The paracolic mesenteric lymph nodes (MLN), spleen and left lobe of liver were weighed and 1 gm samples placed in sterile hom-.ogenizing tubes containing 5 ml of normal saline and homogenized with a sterile teflon plunger. Dilutions of the homogenized tissues were spread on agar plates and the cultures read 24 hours later. Selective agars for coliform bacteria (violet bile glucose agar, Fakola AG, Basel, Switzerland) and gram negatives (violet bile agar, Fakola AG) and blood agar were used for cultures in non mono-associated animals and McConkey's agar was used for E. coli mono-associated animals. In addition, one gram of cecal content was homogenized with 10ml of normal saline in PHT + SS and SL + SS animals and quantitative cultures (colony forming units [CFU-I) performed in order to provide a control culture of the endoluminal flora.
Morphologic alterations of the intestinal mucosa of the ileum and cecum were analyzed by light microscopy. The tissues were fixed in 4% formaldehyde, embedded in paraffin and stained with hematoxylineosin.
The following variables were studied:

RESULTS
Mortality was significantly increased when HS was combined with PHT in mono-associated and indigenous flora animals (Table 1). Portal hypertension led to significant BT to mesenteric lymph nodes in both groups ( Table 2). Positive blood cultures were found in PHT + HS (Gram + rods, Gramrods, Corynebacterium, species and yeasts) in the mono-associated group only (Table 3). Liver and spleen cultures remained sterile in non mono-associated animals while one animal in the PHT + SS mono-associated group had a positive spleen culture (E. coli).
Cecal cultures showed no difference in the number of Gram negative bacteria between SL and PHT groups    (Table 4). Speculations as to the mechanism ,by which PHT leads to bacterial translocation in this model remain open, but some causes can be excluded. In this experiment BT was tested in animals with indigenous flora and after mono-association with Escherichia coli C25 in order to control for possible variations in the gut flora between groups. There was significant BT to MLN in PHT animals whether or not these were associated with Escherichia coli C25.
In this investigation, no significant microscopic mucosal alterations account for the persistance of bacteria in MLN after three weeks. This finding results either from the persistence of BT to the MLN through a normal appearing mucosa or from defective clearance of the bacteria translocated immediately after PPL. Venous hypertension and possibly an alteration in the mesenteric lymphatic system might have contributed to this finding.
In a recent study on BT and PHT, almost 100% positive vena cava cultures were obtained in portal hypertensive rats 2 weeks following PPL22. In the present model of chronic PHT, no increased incidence of positive blood cultures was observed in PHT alone at 3 weeks. The present study suggests an effective systemic clearance of bacteria by the reticuloendothelial system including the liver and appears to confirm the clinical observation of rare positive systemic blood cultures in uncomplicated PHT2. In the clinical conditions of cirrhosis and ascites, impaired immune syso tem 13 and hepatic functions 6 are important additional factors contributing to the high incidence of bacteremia and sepsis. Previous studies have shown a high incidence of shock related bacteremia9'21, but this study showed bacteremia only in mono-associated animals with PHT and HS. In the present study, the hypovolemic shock time was 70 minutes and shorter durations of shock were not studied. This led to a high mortality with only the most resistant animals being available for cultures twenty-four hours later. In addition, blood cultures performed after 24 hours may have been an underestimate of the true incidence of shock related bacteremia.
Significant endotoxemia was noted after HS only.
The addition of PHT to HS seemed to be protective as the rise in endotoxin levels in this group was not significant. The absence of significant endotoxemia in the PHT group suggests that endotoxins did not contribute, at least on a chronic basis, to BT to MLN.
The finding that PHT induces BT to MLN provides further explanation for results of recent studies which showed obstructive jaundice 24