Ginkgo biloba extract (EGb-761) increases peripheral and cerebral blood flow and microcirculation and improves myocardial ischemia reperfusion injury. Some major biochemical/pharmacological activities of EGb-761 are free radical scavenger activity, thereby decreasing tissue levels of ROS and inhibition of membrane lipid peroxidation and anti-PAF activity, contributing to improvements in cerebral insufficiency ¹⁴. EGb-761 has protective effect on hepatic endothelial cells and hepatic microcirculation in rats with chronic liver injury induced by CCl₄ ^15,16. These properties of natural antioxidant Egb 761 are thought to provide many benefical effects free radical injuries.

The goal of treatment in patients with OJ is to relieve the obstruction and obtain the bile flow into gastrointestinal tract. Before and after surgical intervention, reduction of the structural and functional damage in the liver is also an important part of therapy. To the best of our knowledge, there has been no study about the effects of Egb-761 on OJ in the literature. Therefore, we investigated the effects of Egb-761 on the liver and intestine of rats in which OJ was induced.

All surgical procedures and sampling were performed under general anesthesia. Anesthesia was induced by intramuscular injection of ketamin HCL 12.5mg/100gr (Ketalar flk, Eczacibasi, Turkey) and Xylazine HCl 1mg/100gr (Rompon flk, Bayer). Laparatomy was performed with an average of 2 cm middle line incision under aseptic condition. In control group were applied no surgical procedure. In sham-operated group, only the common bile duct (CBD) was identified and mobilized but not ligated. In other treatment groups, common bile duct was dissected and ligated (CBDL) with 4/0 silk for twice and excised between the two knots in order to avoid recanalisation. Abdominal incision were closed with 3/0 silk sutures.

The rats in the control and sham groups received no medical treatment. Group 3 received 1 ml normal saline, Group 4 were given 50 mg/kg dose EGb-761 (Tebokan drops 50 ml Abdi Ibrahim, Turkey) via orogastric intubation twice daily for 2 days before and after the operation. Following this treatment, 1 ml of E.coli suspension containing 1 x 10¹⁰ bacteria in phosphate buffer saline was administered to each group through orogastric intubation on the postoperative day 2. Twelve hours after the administration of E.coli suspension, samples were obtained and the rats were sacrificed.

Following the laparatomy, blood samples were taken by cardiac puncture which were preserved at 20 ºC until assayed. Distal terminal ileum and the caudad (omental) lobes were excised and soaked in 10% formaldehyde for histopathological examination.

For PCR analysis, DNA was isolated from the blood samples of the rats using a commercial DNA extraction kit (Wizard Genomic DNA Purification System, Promega Corp., Madison, WI). The PCR protocols were used as described previously (17). Ten microliters of the amplification products were run on a 2% agarose gel and the product was visualized by ethidium bromide staining (Figure- 1).

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Figure 1: Agarose gel electrophoresis of polymerase chain reaction products (PCR) products. Lane 1; a 760 base pair PCR product amplified from DNA obtained from standard cultures, Lane 2; PCR product of DNA obtained from the control group rat, Lane 3; a 760 base pair PCR product amplified from DNA of the rat treated with EGb761, Lane 4; PCR control containing dH2O. Lane 5; a 760 base pair PCR product amplified from DNA of the rat treated with normal saline solution, Lane M; 100 bp DNA ladder.

During analyses, previously confirmed colonies obtained from standard cultures were used as positive control for E. coli. The sensitivity of PCR was determined with 10-fold dilution of the DNA extracted from the isolated colonies E. coli. In order to overcome both false positivity and negativity, we included negative and positive controls in each experiment.

Liver and terminal ileum tissue samples were fixed in formaldehyde solution and divided into 0.5 cm sections. After the standard laboratory proceedings, 5 µm slices were prepared for subsequent histopathological examination, by fixing the sections in paraffin and staining them with hemotoxiline-eosin. Liver tissue samples were examined by light microscopy and the results were scored in aspects hepatocyte bloating, multinucleation, degeneration, cell necrosis, Kupffer cell proliferation, cholestasis, portal edema, portal region extension, portal PNL infiltration, bile duct proliferation, bile duct dilatation and portal fibrosis: 0 (n/a), 1 (mild), 2 (moderate), 3 (intensive) and 4 (severe).

Terminal ileum samples were examined by light microscopy and the results were scored in aspects of mucosal damage, PNL infiltration in lamina propria, lenfoid follicular hyperplasia, goblet cell reduction, increase in the number of veins and capillary expansion: 0 (n/a), 1 (mild), 2 (moderate), 3 (intensive) and 4 (severe).

Serum urea, creatinin, total bilirubin, gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and alanin aminotransferase (ALT) were analyzed outomatically using a kit Olympus Diagnostica GmbH'dan (Wendenstrabe, Hamburg-Deutschland).

The data were given as avarage value ± standart error. For the statistical comparison of biochemical and histopathological results of trial groups, “Bonferroni adjusted Mann Whitney U test” was used and P<0.025 was considered to be significant. “Pronounced chi-square test” was used for the comparison of microbiological results and P<0.05 was considered to be significant.

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Table 1: Presence of Escherichia coli genetic material in blood samples determined by polymerase chain reaction assay.

Biochemical results: Urine and creatine values of all groups were very close and did not significantly differ. In CBDL groups, ALT, ALP, GGT, and billirubin levels were found to be significantly increased compared to control and sham-operated groups (P< 0.025). The increase in ALT and GGT levels of EGb-761 group was significantly less than G3 (p< 0.025) (Table-2).

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Table 2: Serum biochemistry values

Histopathological results: No change in cellular or portal level was observed in liver sections of rats in control and sham-operated groups. In jaundiced groups, pathological changes in liver were smaller in groups G4, according to G3. This difference was statistically significant (p< 0.025). Bile duct proliferation and dilatation and hepatocyte degeneration were more rarely seen in G4 (Figure 2).

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Figure 2: Histopathologic evaluation of study groups.

During microscopical evaluation of terminal ileum, no pathology was detected in control and sham-operated groups. Mild lenfoid follicular hyperplasia was observed only in one sample obtained from sham-operated group. In jaundiced groups, mild to severe mucosal damage was observed. There was a statistically significant reduction in mucosal damage in groups treated with EGb-761 compared to normal saline group (p<0.025) (Figure-2).

Traditional methods used for detection of BT in experimental animals include demonstration of live bacteria in blood and in organs/tissues distal from intestines through culturing; or measurement of radioactivity in organs following the administration of radioisotope marked bacteria/endotoxin through enteric route ²¹. Translocated bacteria cannot be always grown successfully in a suitable culture medium or it may take a long period of time. The radioactive method carries the risk of giving false negative and false positive results. PCR method to amplify nucleic acid of microorganisms has been introduced. PCR method is a quite specific technique and is not affected by conditions of sampling and shipping ²². We therefore believe that PCR method is an effective method for the detection and identification of bacterial translocation.

Retention and accumulation of toxic hydrophobic bile salts in hepatocytes may cause hepatocyte toxicity by inducing apoptosis ^1,2. Without proper treatment in rats with CBDL, hepatocellular injury is an invariant feature of cholestasis causing liver dysfunction, promoting fibrogenesis, and ultimately leading to liver failure. Injury of the liver has been suggested to be mediated by increased xanthine dehydrogenase and oxidase activity with production of oxygen free radicals and reduced inactivation of xanthine oxidase activity³.

Bile acids reduce the bacterial overgrowth both clinically and experimentally. Bile acids act as detergent in bacteria cell wall and inactivate endotoxins. In the absence of bile flow an increase in gram (-) aerobic bacteria population (by biliary diversion) has been shown. Endotoxins increased in the absence of bile flow damage intestinal mucosa by stimulating ileal xanthine dehydrogenase and oxidase activity ²³. Xu et al. ¹³ have been show that blood flow to the distal ileum and caecum is selectively reduced by 35-50% following endotoxin administration, whereas blood flow to the rest of the intestine, liver, spleen, and pancreas is normal. In addition, small bowel permeability is increased at sites of decreased blood flow (ileum) but not at sites of normal blood flow (jejunum). Other studies have reported significantly reduced blood flow in the mesenteric artery during experimental endotoxaemia ²⁴ or sepsis ^25, even when cardiac output is normal or elevated. Changes in gastrointestinal morphology associated with obstructive jaundice are most marked in the distal ileum ^8-12. Some authors have described sub epithelial edema, with lifting of the epithelium from the lamina propria ^8,9, dilated lymph vessels and edematous sub epithelial regions, with infiltration of inflammatory cells in the villi ^10-12.

The studies have shown that mucosal damage facilitates BT by giving harm to intestinal barrier function. Both non-specific defense mechanisms and immune system depression have a role in the development of BT in obstructive jaundice ^19-23. The response of T lymphocytes to pathogens decreases in rats after CBDL operation, while T suppressor cell activity increases and interleukin 2 production decreases ²⁶ and chemotaxis and fagocytosis becomes depressed ²⁷.The present study has demonstrated an increased incidence of BT with obstructive jaundice. The physiopathological mechanism of BT has not been studied.

EGb-761 prevents extreme free radical formation by inhibiting lipid peroxidation. Preventive effect of EGb-761 on free radical formation in brain ischemia, destruction of cell membrane structure and vascular endothelium damage is well known ¹⁴. Additionally flow cytometry and DNA fragmentation studies have shown that EGb-761 decreases apoptosis caused by hydroxyl radicals ²⁸. In a study that chronic liver injury was induced by CCl₄, EGb-761 has protective effect on hepatic endothelial cells and hepatic microcirculation in rats. The mechanisms may involve its inhibition on ET-1, PAF and lipid peroxidation ¹⁶. In another study on ischemia reperfusion injury of intestine concluded that EGb-761 pre-treatment before ischemia-reperfusion decreased malondialdehyde and myeloperoxidase levels and attenuated the mucosal damage ²⁹. We suggest that EGb-761 does not reduce the development rate of bacterial translocation in rats with CBDL. When focusing on the histopathological changes in terminal ileum observed in our study, there was a significant reduction in mucosal damage in EGb-761 groups compared to the control group.

According to the histopathological findings in the present study, it was shown that EGB-761 administration decreased the damage in liver and ileum of obstructive jaundice. Level of histopathological changes may be connected to the number of translocated E. coli together with presence of E. coli in the rats. However, we detected presence only of E. coli from the rats by PCR method. Therefore, additional studies are required to investigate the relationship between the number of translocated E. coli and level of histopathological changes.

Serum liver enzymes (ALT, ALP, GGT) were significantly higher in CBDL Groups than in the sham and control groups (P<0.025). GGT and ALP are better indicators of liver damage due to OJ. Significant decrease was observed in GGT and ALP values in EGb-761 group in comparison to normal saline group (P<0.025). We concluded that EGb-761 treatment decreased the liver damage due to OJ.

We conclude that in rats which OJ is induced, EGb-761 administration decreases the damage in liver and terminal ileum. However, reduction of bacterial translocation rate in rats was not statistically significant. It is expected that this study will lead to further studies on the effects of EGb-761 on morbidity and mortality following surgery in the presence of OJ.

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