Gallstones are common in patients throughout the western world and are found in about 10% of the adult population 1. Gallstone related disease is one of the most frequent medical problems demanding surgical intervention. In Denmark, 130 per 100.000 inhabitants are cholecystectomized each year 2, most often because of biliary attacks of pain. The annual incidence of acute cholecystitis, the second largest group undergoing cholecystectomy, is approximately 20 per 100.000 inhabitants 2. In a subgroup of patients with acute cholecystitis, surgical intervention is hazardous due to poor performance status. In these patients, the standard treatment options have traditionally been conservative treatment or percutaneous transperitoneal cholecystostomy (PTCS) 34567. A significant drawback with both these treatment modalities is a high rate of recurrences, reported to range from 15% to 47% 89. In recent years, an alternative treatment option, endoscopic gallbladder drainage (EGBD), has emerged 1011121314. One theoretical advantage of EGBD compared with PTCS, is that treatment can be sustained for longer periods, because there is not, as in PTCS, an external component of the drain. This possibility for prolonged treatment raises the hope that EGBD may be a more definitive treatment of cholecystitis than PTCS.

Calculous cholecystitis is thought to develop when the cystic duct becomes obstructed by an impacting gallstone. Secondary bacterial contamination of the stagnant bile seems an obvious mechanism for exacerbation in the development of acute cholecystitis. Cultures of gallbladder bile however, are only positive in 15% to 30% of cases 15, suggesting that the inflammatory process most often could be of another nature, some suggest inflammation to be caused by a chemical agent 16. The predominant microorganisms isolated from gallbladder bile in patients with acute cholecystitis are Escherichia coli (60%) and Klebsiella pneumoniae (22%) 15.

Occlusion of the arterial blood supply to the gallbladder may be a fundamental element in the pathogenesis of acute acalculous cholecystitis 17.

Several groups have tried to induce acute cholecystitis in animal models by combining surgical induced cholestasis with either bacterial infection, chemical irritants and/or gallbladder ischaemia1618.

We wanted to induce a severe condition of acute cholecystitis in order to demonstrate the effect of the intervention. Therefore we combined the insults, ligating the cystic duct and artery and inoculation of bacteria.

The aim of the present study was to investigate whether internal drainage of the gallbladder could protect against the development of acute cholecystitis in a pig model.

The research protocol was approved by the local research committee (registration number: 2008/561-1489) in accordance with the Danish regulations on animal experiments. Twenty female pigs (Danish Landrace/Yorkshire) with a body weight of approximately 65 kg (Research Centre Foulum under the Danish Institute of Agricultural Sciences) were used for the experiment. Animals were randomized in blocks of four. The pigs were divided into two groups and all of the pigs had acute cholecystitis induced as described below. Drained pigs had an internal double pigtail catheter from the gallbladder to the duodenum. In the undrained pigs the catheter was placed as in drained pigs, but was then immediately removed.

Acute cholecystitis was induced using a combination of previously described models 1619202122 with slight modifications, thus comprising:

1) Ligation of the cystic artery.

2) Ligation of the cystic duct.

3) Injection of bacteria into the gallbladder lumen.

Of the 20 pigs included in the study, 16 survived the full four days after the initial operation leaving eight pigs in each group. Four pigs died, three because of cholascos, one due to gastric retention. There were no significant differences in any blood tests, temperature or weight between the groups.

The present study in a pig model shows that internal drainage from the gallbladder to the duodenum protects against development of acute cholecystitis judged by a histopathological scoring system. Internal drainage could not prevent the bile from still being infected with bacteria inoculated into the gallbladder lumen four days earlier. There was, however, a distinct difference in the macroscopic appearance in the sense that the bile in the internal drainage group was yellow and transparent whereas it was dark and non-transparent in the undrained pigs.

Multiple factors have been hypothesized to be involved in the development of acute cholecystitis and, therefore, have been used in different animal models. Obstruction of the cystic duct by an impacting gallstone has been the primary causative factor to be proposed, whilst other factors to be implicated include impairment of the gallbladder blood supply, secondary bacterial infection, and abnormal concentrations of bile constituents leading to a chemical inflammation 1619202122. In the actual study, acute cholecystitis was induced combining previous described models with modifications comprising ligation of the cystic artery, ligation of the cystic duct and injection of a 2 ml suspension of bacteria (1 ml of Escherichia coli 10⁵/ml and 1 ml of Klebsiella pneumonia 10⁵/ml) 1619202122. The severeness of the combined insults might explain the high mortality.

Sections from the gallbladders were scored using a previously described histopathological scoring system and showed pronounced signs of inflammation in the group without internal drainage, indicating that the model used for inducing acute cholecystitis in the actual study was adequate 21.

Using a histopathological scoring system based on the presence, extent and severity of hemorrhage, edema, formation of pregranulation tissue, lymphatic dilatation, and PMC infiltration in the gallbladder wall, we found a highly significant protective effect of internal drainage from the gallbladder to the duodenum in the development of acute cholecystitis. These findings support the theory that a gallstone impacting the cystic duct plays a crucial role as a pathogenetic mechanism in the development of acute cholecystitis and it may be an important factor in the maintenance of this disease state. There were only mild degrees of inflammation identifiable microscopically in the internal drainage group, in spite of the fact, that these animals had the cystic artery ligated and had bacteria inoculated into the gallbladder. This suggests that the latter factors are of relatively less importance as single pathogenetic insults compared to outflow impediment in the development of acute cholecystitis. The findings in the actual study also allows us to speculate that internal drainage might be a way to treat already evolved acute cholecystitis.

Surprisingly, bile cultures sampled day 0 showed bacterial growth in nine of 16 cases. This could not be due to contamination from the inoculate, since extraction of bile was done prior to injection of bacteria, but we offer two other explanations:

1. Retrograde contamination from the duodenum while placing the stent, since this was done prior to extraction of bile or

2. In a high proportion of normal pigs, bile is colonized with bacteria from the intestinal tract.

On day four, all bile cultures were contaminated with bacteria in both of the groups, suggesting that infected bile as a sole factor is insufficient to produce acute cholecystitis. These findings also support the above mentioned theory that gallstone impaction in the cystic duct is the key pathogenetical mechanism in the development acute cholecystitis, i.e. acute cholecystitis will not develop if the bile drainage through the cystic duct is preserved in spite of bacterial infection.

Fourteen of 16 pigs showed bacterial growth in blood cultures on day four. Some of the strains cultured (i.e. Staphylococci and Steptococci) could be due to contamination from the skin. However, Escherichia coli and Klebsiella pneumonia demonstrated in blood cultures most probably originated from the gallbladder as these were the predominant bacteria cultured from bile. It is not possible to determine whether the positive blood cultures had any effect on the well-being of the animals in the present study as we had no control animals with negative blood cultures.

In conclusion, the present study demonstrated that internal drainage from the gallbladder to the duodenum prevents the development of acute cholecystitis. Since development of acute cholecystitis can be prevented by internal drainage, future studies must demonstrate whether internal drainage can be applicable as a therapeutic intervention in patients suffering from accumulated biliary attacks of pain or in patients already suffering from acute cholecystitis.

The authors declare that they have no competing interests.

DWK participated in the design of the study, carried out the surgery, drafted the manuscript and performed the statistical analysis. FVM participated in the design of the study and helped to draft the manuscript. JKM was responsible for analysis of cultured bacteria. SJHD did the histological examination and helped to draft the manuscript. PFJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.