Hydrophilic Bile Salts Protect Bile Duct Epithelium During Cold Preservation: A Scanning Electron Microscopy Study

Transcription

1 Hydrophilic Bile Salts Protect Bile Duct Epithelium During Cold Preservation: A Scanning Electron Microscopy Study Martin Hertl, * M. Catherine Hertl, * Dietrich Kluth, and Christoph E. Broelsch * Prolonged graft preservation is associated with postoperative bile duct strictures after liver transplantation. We previously showed that hydrophilic bile salts mitigate bile duct preservation injury in a pig model. Because this injury occurs at the epithelial level, scanning electron microscopy was performed to further characterize this effect in vitro. Swine livers were harvested after the intravenous infusion of 1 of 3 solutions: saline (n 7), tauroursodeoxycholate ([TUDC] hydrophilic; n 4), or taurodeoxycholate ([TDC] hydrophobic; n 4). Livers were perfused with University of Wisconsin solution. The bile ducts were flushed retrograde, and the liver was stored at 0 C to1 C for 20 hours. Bile duct samples were obtained at the time of harvest and 8, 12, 16, and 20 hours thereafter. In saline-infused controls at time 0, the epithelium was intact and composed of uniform cuboidal cells covered with fine regular microvilli. There were no spaces between individual cells. After 8 to 12 hours of preservation, cells were more irregular in shape, with loss of cell-cell contact. The cell surfaces showed fewer microvilli. Surface erosions suggested loss of cell-wall integrity. TUDC was protective, evidenced by normal-appearing cells with uniform microvilli after 16 hours. In contrast, TDC accelerated the injury process, causing cell-surface erosions, blebs, and loss of microvilli as early as time 0. Scanning electron microscopy is an excellent tool to study injury to bile duct epithelium. This study supports the hypothesis that hydrophilic bile salts protect bile ducts during preservation. To determine whether treatment with hydrophilic bile salts can prevent postoperative stricture, in vivo transplantation studies are needed. Copyright 2000 by the American Association for the Study of Liver Diseases P rolonged graft preservation is associated with postoperative bile duct strictures after liver transplantation. 1-7 Our hypothesis is that these strictures may be caused by damage to the bile duct epithelium during preservation. We have previously shown that such hydrophilic bile salts as tauroursodeoxycholate (TUDC) and dehydrocholate mitigate preservation injury to the bile ducts for 20 hours in a pig model, whereas such hydrophobic bile salts as taurodeoxycholate (TDC) accelerate the destructive process. 8 Because this injury occurs at the epithelial level, scanning electron microscopy was performed in vitro to evaluate epithelial changes during the preservation time. The time course of injury was also investigated because in the previous report, we collected samples for histological examination after 20 hours of preservation only. 8 Materials and Methods Fifteen male Landrace pigs weighing between 25 and 34 kg were acclimated for at least 1 week before surgery in our animal facility, where they received standard pig chow and water as they wanted. Animals were food-fasted overnight but were allowed water up to the procedure. The experimental protocol was approved by the Animal Ethics Committee of the University of Hamburg (Germany). Experimental Design In the control group, 0.9% saline was infused intravenously at a rate of 1.25 ml/min over 20 minutes into control animals (n 7). In a second group, TUDC (Calbiochem, San Diego, CA) was administered in the same volume at a rate of 2 mol/kg of body weight/min (n 4). In a third group, TDC (Calbiochem) was administered at the same rate and in the same volume as TUDC (n 4). The dose administered, as well as the duration of infusion, was derived from previous studies. 8 Surgical Procedure Surgery was performed as described previously. 8 Briefly, the abdomen was opened under general anesthesia. The cystic duct was ligated close to the gallbladder, and a cannula was introduced into the distal common bile duct and secured. The 20-minute intravenous infusion of TUDC, TDC, or 0.9% saline was then begun. Three minutes before crossclamping, while the infusion continued, 400 IU/kg of body weight of heparin was administered, and 3 minutes later, the abdominal aorta was cannulated with an 18 Charriere perfusion catheter, the intrathoracic aorta was cross-clamped, and the liver flushed with 1Lofice-cold University of Wisconsin (UW) solution (0 C to1 C). After harvest, the liver was flushed through the portal vein with 200 ml of ice-cold UW solution, and the vessels were prepared for transplantation as in the clinical setting. A cholecystectomy was performed, and the liver was stored in double sterile Lahey bags surrounded by UW solution and ice slush. From the *Departments of General Surgery and Pediatric Surgery, University Hospital Hamburg, Germany. Address reprint requests to Martin Hertl, MD, Department of General and Transplantation Surgery, University Hospital Essen, Hufelandstr 55, Essen, Germany. Copyright 2000 by the American Association for the Study of Liver Diseases /00/ $3.00/0 Liver Transplantation, Vol 6, No 2 (March), 2000: pp

2 208 Hertletal Table 1. Classification of Epithelial Preservation Injury to Pig Bile Ducts Assessed by Scanning Electron Microscopy Grade 1: No injury visible; abundant, well-preserved microvilli; tight cell-cell adhesions Grade 2: Fewer microvilli, occasional bleb formation, loosening of cell-cell junctions Grade 3a: Disruption of cell-cell contact, cell membrane erosions, absence of microvilli Grade 3b: Epithelial sloughing from the basal membrane Sample Procurement First-branch bile duct samples (segmental bile ducts from the left or right lobe of the liver) were obtained at the time of liver harvest and 8, 12, 16, and 20 hours thereafter. The biopsies were performed by cutting a 5-mm segment of the bile duct with scissors from areas located behind big portal venous branches. It was not necessary to trim the cut edges because the center of the specimen was examined by scanning electron microscopy. Care was taken to avoid obtaining samples from areas that had previously been biopsied to avoid contact with UW solution between biopsies. The minimal distance to a previously biopsied area was 5 cm. In practice, biopsy samples were obtained at different time points from different lobes. During the surgical procedure of biopsy, livers Table 2. Damage of the Biliary Epithelium of the Pig Bile Ducts Among the Experimental Groups Group Saline Animal a 3a-3b 3b Animal a 3a 3a-3b 3b Animal a 3a 3a-3b Animal a Animal a 3a-3b Animal a Animal a 3a 3a 3b TDC (n 4) 2 3a 3b 3b 3b TUDC (n 4) NOTE. Grading is according to Table 1. Note that in the TDC-infused group, severe damage was seen as early as time 0. Treatment with TUDC led to protection of the epithelium for up to 20 hours. Saline-infused control animals are listed individually because the damage in this group was variable. Because the damage was uniform in the TUDC and TDC groups, grading is representative for all animals of the respective groups. Figure 1. Low-power scanning electron microscopy of a bile duct of the saline-infused group. The sample was obtained immediately after flush-out of the liver with University of Wisconsin solution (time 0). Well-preserved epithelium, glandular orifices are not visible. (Original magnification 500.)

3 Bile Salts and Preservation 209 Figure 2. High-power scanning electron microscopy of a bile duct of Figure 1. (Original magnification 10,000.) Note microvilli and close approximation of cells. Figure 3. Scanning electron microscopy of an 8-hour preserved bile duct of a member of the saline group. Most cells are severely damaged, but some cells have preserved some level of integrity. (Original magnification 10,000.)

4 210 Hertletal Figure 4. Scanning electron microscopy of a 20-hour preserved bile duct of the tauroursodeoxycholate-infused group. Cell-cell spaces are bigger, blebbing is more disseminated, but microvilli are still largely intact. (Original magnification 10,000.) were kept submerged under the UW solution, which was surrounded by ice slush to ensure that the storage temperature remained constant at 0 C to1 C. For the duration of preservation, the liver was kept on ice in UW solution at 0 C to 1 C. Histological Examination Immediately after procurement, bile duct specimens were incised longitudinally under the microscope and divided into 2 pieces. To clean the epithelium from the thick mucus layer that sometimes covered it, the epithelial surface was flushed with saline solution from a syringe. The ducts were immediately immersed into 2.5% glutaraldehyde and processed for scanning electron microscopy. Scanning electron microscopy. Bile ducts were fixed in cold 2.5% glutaraldehyde solution for 24 hours, followed by postfixation with Bouin s solution. After alcohol dehydration and critical temperature drying, specimens were sputtered with gold and examined using a Leitz DSM 940 (Jena, Germany) scanning electron microscope. Specimens were examined by a blinded observer (D.K.) with extensive experience in scanning electron microscopy. Table 1 lists the different grades of injury that were identified. Only areas in the center of a specimen were evaluated to avoid misinterpretation because of mechanical artifacts. Results Table 2 lists the findings of saline and bile-salt infused livers. Preservation injury to the bile duct of saline controls was characterized by the following sequence of events; at harvest (time 0), the epithelium was intact and composed of uniform cuboidal cells covered with fine, regular microvilli (Figs. 1 and 2). There were no visible spaces between cells. After 8 hours of preservation, epithelial cells were more irregular in shape with less cell-cell contact. The cell surface was smoother, with fewer microvilli, and sometimes even showed fine holes or erosions, suggesting a loss of cell-wall integrity (Fig. 3). By 16 hours, all cells were uniformly damaged. The cell membranes were destroyed, and the cytoskeleton was visible. Cell-cell contact was absent, and microvilli were lost. In many areas, the epithelium was completely absent (not shown). Compared with the saline controls, TUDC was protective, evidenced by normal-appearing cells with uniform microvilli after 8 hours. Although blebbing appeared at 20 hours (Fig. 4), there were no erosions, and the epithelium was still attached to the basal membrane. In contrast, TDC treatment accelerated

5 Bile Salts and Preservation 211 Figure 5. Bile duct of a taurodeoxycholate-infused animal after 16 hours of preservation. The epithelium is gone except for remnants in the intramural glands. (Original magnification 10,000.) the injury process, causing cell-surface erosions, blebs, and loss of microvilli as early as time 0, leading to complete destruction at 8 hours. At 16 hours, the epithelium was gone, except for small islets around the intramural glands (Fig. 5). It appeared that in those areas, attachment of the epithelium to the underlying basal membrane was firmer. Discussion This study characterizes for the first time the morphological injury to the biliary epithelium during cold preservation in vitro and its prevention by using hydrophilic bile salts. The idea to administer hydrophilic bile salts to protect the liver during preservation was derived from studies that showed their protective effect in vivo in a number of chronic liver disease states. The mechanism by which hydrophilic bile salts act on the biliary epithelium is not precisely known. However, it is very likely that ursodeoxycholate (UDC) and probably also its conjugates are inserted into the cell membranes, thus providing stabilization. According to a hypothesis of Guelduetuna et al, 9,10 the apolar steroid part of UDC is bound in the center of the lipid double layer, whereas the conjugates are located near or in the surface of the membrane. With UDC inserted into the membrane, the ability of more hydrophobic bile salts, such as chenodeoxycholate, to solubilize cell membranes is diminished. This hypothesis is controversial, 11 but it would explain the findings in isolated human hepatocytes 12 and in red blood cells 13 that TUDC ameliorates the toxic effects of TDC and glycochenodeoxycholate (GCDC). Therefore, because red blood cells do not have a nucleus, it is unlikely that its main action is derived by changing the metabolism of the cells. Like cholesterol, the incorporation of UDC in the cell membrane maintains stability of the lipid double layer and prevents damage caused by chemical stress. 10 The tendency of UDC to form micelles makes it superior in regard to its membrane protection compared with even more hydrophilic triketo bile salts that are not able to form micelles because of their lack of polar and apolar regions. 14 Histological examination supported the results of our previous study. 8 In that study, we were able to show protection of the biliary epithelium for up to 20 hours by the infusion of hydrophilic bile salts into the donor animal. However, scanning electron microscopy was not performed; thus, the ultrastructural changes were not visualized. We did not find any study in the

6 212 Hertletal literature dealing with preservation injury of bile ducts on a scanning electron microscopic level. Benedetti et al 15 performed a study of bile duct fragments using transmission electron microscopy. Bile ducts were exposed to different concentrations of hydrophobic and hydrophilic bile salts that were conjugated and unconjugated. The most severe damage was produced by perfusing the ducts with unconjugated bile salts, whereas conjugated bile salts appeared to be nontoxic. These findings are in contrast to our study, in which TDC caused destruction of the biliary epithelium as early as time 0. Interestingly, the hepatocytes showed prominent damage with intracellular vacuole formation and morphological changes of the microvilli of the apical membrane. Sakato et al 16 studied the common bile duct of golden hamsters fed a lithogenic diet. An abundance of microvilli were seen on the epithelia of the control animals, whereas in treated hamsters, the number of mucus-producing goblet cells had increased significantly. They also found a correlation between the number of goblet cells and the amount of mucus secreted into the lumen of the bile ducts. We did not find goblet cells in the specimens at the scanning electron microscopy level, although in the former study, 8 goblet cells were present; thus, nonvisualization in the present study doesn t mean that they do not exist. TUDC, although increasing biliary secretion, may not induce increased mucus secretion. Conversely, TDC is very likely to induce goblet cell appearance because it is a hydrophobic and toxic drug. However, in our study, we failed to see this stimulation, probably because of the short time between exposure and cold preservation. In the study of Sakata et al, 16 the increase in number of goblet cells and mucus secretion was not seen before the second week of feeding a lithogenic diet. Infusion of TUDC did not change the number or appearance of microvilli, but preservation led to clubbing and reduction in number and density. TDC, as known from other studies, is highly toxic for the liver, and this effect is potentiated during cold preservation. In summary, scanning electron microscopy is an excellent tool to study injury to bile duct epithelium and, in this study, gives further evidence that hydrophilic bile salts protect the bile duct during preservation. In contrast, pretreatment of the liver with a hydrophobic bile salt such as TDC accelerates the injury, leading to visible damage of the epithelium as early as time 0. In vivo transplantation studies are needed to determine whether treatment with hydrophilic bile salts can prevent postoperative stricture formation. References 1. 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