Effect of ursodeoxycholate in University of Wisconsin solution on long term preservation of rat livers.

OBJECTIVE: To find out whether the addition of ursodeoxycholate (UDC) to University of Wisconsin (UW) solution reduces injury during preservation of the liver. DESIGN: Controlled laboratory study. SETTING: University hospital, Germany. MATERIALS: Preserved livers from 12 rats. METHODS: Livers were flushed with 10 ml of ice-cold (0-1 degrees C) UW or iso-osmolar UW containing 5 mmol/l UDC. After preservation for 30 hours at 0-1 degrees C and 1 hour at 22 degrees C the livers were reperfused for 4 hours. Aspartate amino-transaminase, alanine aminotransferase, glutamate lactate dehydrogenase, and white blood cell (WBC) and platelet counts were measured during reperfusion. Bile flow and total bile salt secretion rate were also measured. RESULTS: Addition of UDC to UW reduced the release of liver enzymes, slightly increased platelet adherence, and reduced bile flow and bile salt secretion rate. CONCLUSION: The membrane-stabilising effect of UDC may explain the reduction in enzyme release. However, liver function, as expressed by bile salt secretion rate and bile flow was adversely affected.

Hydrophilic bile salts protect bile duct epithelium during cold preservation: a scanning electron microscopy study.

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 degrees C to 1 degrees 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.

In vivo protection of the pig liver against ischemia/reperfusion injury by tauroursodeoxycholate.

INTRODUCTION: Tauroursodeoxycholate (TUDC) is used routinely in the treatment of cholestatic liver disease. The present study was designed to determine whether it would mitigate ischemia/reperfusion injury in an in vivo pig liver-transplantation model. METHODS: Transplantation was performed in 12 animals after a preservation time of 8 h. In the control group (n=6), 0.9% saline was infused into the donor. In the experimental group (n=6), TUDC was given intravenously at a rate of 2 micromol/kg body weight per minute. In the recipient, infusion was started at the time of reperfusion; saline was infused for 400 min in the control group, TUDC for the same duration at a rate of 0.2 micromol/kg body weight per minute in the experimental group. Blood was drawn for determination of liver enzymes. Bile samples were collected and bile flow (BF) and bile salt secretion rate (BSSR) were determined. RESULTS: One-week survival was 92% and not different among groups. Liver enzymes were lower in the TUDC group than the saline group. Prior to TUDC infusion in the donor animals, there were no differences in BF and BSSR. After infusion of TUDC, BF and BSSR were highly significantly different than the control group. DISCUSSION: Infusion of TUDC in pig livers protects against ischemia/reperfusion injury in vivo. This might be due to the membrane-stabilizing effect of TUDC. Preconditioning of liver grafts with TUDC could potentially lead to improved liver function post-transplantation.

Tauroursodeoxycholate ameliorates reperfusion injury after pig liver transplantation.

Reperfusion injury is a serious problem after clinical liver transplantation, often leading to dys- or even non-function of grafts. The present study was designed to determine whether the hydrophilic bile salt tauroursodeoxycholate (TUDC), known to be hepatoprotective in cholestatic liver disease, mitigates reperfusion injury in an in vivo pig liver transplantation model. Liver transplantation was performed in 12 pigs after a preservation time of 8 h. TUDC was administered to donor and recipient animals, and saline to controls. Blood was drawn at different time points for determination of liver enzymes. Bile samples were collected, and bile flow (BF), and bile salt secretion rate (BSSR) determined. Samples of liver tissue and bile ducts were taken for assessment by light and electron microscopy. Liver enzymes were significantly lower in the TUDC group. BF and BSSR were significantly higher. Microscopy revealed better preservation of bile duct architecture of the TUDC-infused animals. We can conclude that infusions of TUDC in pig livers ameliorate reperfusion injury in vivo. The molecular basis for this finding may be the membrane stabilizing effect of TUDC. Further studies are warranted to clarify its effect.

Feminizing Sertoli cell tumors associated with Peutz-Jeghers syndrome: an increasingly recognized cause of prepubertal gynecomastia.

Testicular sex cord tumors with annular tubules are an increasingly recognized cause of prepubertal gynecomastia typically accompanied by accelerated linear growth and advanced bone maturation. Serum estrogen levels may be elevated. Testicular ultrasound and biopsy are diagnostic, and mastectomy is indicated. Although these tumors can occur independently, causing gynecomastia in 10 percent of cases, they usually occur in patients with Peutz-Jeghers syndrome. In any Peutz-Jeghers syndrome patient developing gynecomastia, a testicular tumor should be sought. Conversely, because a significant proportion of all reported prepubertal gynecomastia patients have Peutz-Jeghers syndrome with testicular tumors, this syndrome must be considered for all young boys in whom the cause of gynecomastia is not otherwise apparent. When Peutz-Jeghers syndrome is suspected, gastroscopy, colonoscopy, and testicular biopsies can be performed under one anesthetic at the time of mastectomy.

Intrafascicular injection of ammonium sulfate and bupivacaine in peripheral nerves of neonatal and juvenile rats.

BACKGROUND AND OBJECTIVES: Regional nerve blocks are often used for the treatment of postoperative pain in children. Ammonium sulfate is a non-narcotic anesthetic agent, which has been reported to provide pain relief lasting days to weeks, with few reported side effects in adult studies. Prior to considering clinical use in children, the neurotoxicity of ammonium sulfate in 4-day and 3-week old rats was assessed and compared with that of bupivacaine. METHODS: Each rat received a posterior tibial nerve intrafascicular injection (0.01 mL in 4-day-old and 0.02 mL in 3-week-old rats) using either 10% ammonium sulfate (n = 24 per age group), 0.5% bupivacaine (n = 18 per age group), 0.9% saline (n = 18 per age group), or 5% phenol (n = 18 per age group). A functional assessment by serial walking track analysis and a morphologic assessment by neurohistology were made. RESULTS: No abnormalities in serial walking track analysis and no structural nerve damage were detected after ammonium sulfate, bupivacaine, or saline injection. Bupivacaine caused mild focal changes in both age groups, which recovered by 8 weeks. CONCLUSIONS: Intrafascicular injection of ammonium sulfate was as safe as bupivacaine in this animal model. Further animal studies must be made before human trials are initiated.

The effect of ammonium sulfate injection on peripheral nerve.

Local anesthetic drugs with prolonged nerve-block effect would have clinical application for postoperative or neuromatous pain relief. This study evaluated the possibility of peripheral nerve neurotoxicity by injection of 10 percent ammonium sulfate. Both intrafascicular and extrafascicular injection of 10 percent ammonium sulfate were tested in the rat sciatic nerve model. One percent lidocaine HCl, 5 percent phenol, and normal saline were similarly injected for comparison. Using histologic studies and motor function evaluation with walking-track analysis, 10 percent ammonium sulfate was found to be neurotoxic when it is injected intrafascicularly; however, extrafascicular injection of this drug did not cause significant nerve injury. The neurotoxicity of the 10 percent ammonium sulfate solution was intermediate between the neurotoxicity of 0.1 percent lidocaine hydrochloride and the marked neurotoxicity of 5 percent phenol solution.

Microbial growth inside saline-filled breast implants.

In vitro and in vivo experiments were conducted to determine whether intraluminal saline in breast implants can support the growth of common wound-infecting microorganisms over a prolonged period of time. The bacteria tested were Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Corynebacterium jeikeium, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Three fungal species also were tested: Aspergillus fumigatus, Paecilomyces variotii, and Candida albicans. In the in vitro study, four organisms survived in flasks of sterile saline for the 2 weeks in which serial cultures were performed: K. pneumoniae, C. albicans, A. fumigatus, and P. variotii. In the in vivo study, 61 white rabbits (122 implants) received both an experimental implant inoculated with one of the test organisms and a control implant containing only sterile saline. They were sacrificed at 1-, 3-, or 6-month scheduled endpoints. None of the control implants containing sterile saline had positive cultures. In contrast, the intraluminal saline was culture positive for 7 of the 10 inoculated organisms after varying lengths of time: S. epidermidis, E. coli, E. cloacae, K. pneumoniae, P. aeruginosa, A. fumigatus, and P. variotii. Samples of capsular tissue also were cultured. Of the 122 capsular tissue specimens, 21 (17 percent) had positive cultures and surrounded both inoculated and sterile implants. In most instances, capsules that were culture positive contained an organism different from the one that had been inoculated in the group. In only 3 cases was the same organism cultured from both the periprosthetic tissue and the intraluminal saline, and these may represent instances of the inoculated organism migrating through the implants filler valves. The data show that several types of bacteria (particularly gram-negative species) and fungi can grow and reproduce in a restricted saline environment for extended periods of time.

Peripheral nerve allograft preservation improves regeneration and decreases systemic cyclosporin A requirements.

Peripheral nerve allografting is limited by the need for long-term systemic immunosuppression. The purpose of this study was to determine if nerve allograft preservation reduced the requirements for systemic Cyclosporin A (CsA) immunosuppression. One hundred twenty Lewis rats were randomized to one of seven experimental groups. Group 1 received a 2-cm Lewis posterior tibial nerve autograft. Groups 2-7 received 2-cm ACI posterior tibial nerve allografts. The allograft group was then further subdivided into three groups of two receiving daily subcutaneous injections of 0, 2.5, or 5.0 mg/kg of CsA for 12 weeks. Within each CsA dose, one group received a fresh while the other received a preserved allograft. Preserved grafts were stored in University of Wisconsin solution for 7 days at 5 degrees C prior to implantation. Animals from each group were sacrificed at 6, 12, and 20 weeks postoperatively. Evaluations included histomorphometry, electrophysiology, and serial walking track analysis. Histology revealed varying degrees of nerve regeneration in all groups at 6, 12, and 20 weeks. For a given CsA dose, the group receiving the preserved graft revealed evidence of better nerve regeneration by all histomorphometric parameters including fiber width and density, percentage neural tissue, and total fiber number. There was no statistical difference in walking track analysis between groups at 4 weeks. By 20 weeks, functional recovery statistically poorer than autograft was seen only in the fresh allograft groups receiving 0 or 2.5 mg/kg of CsA. Identical electrophysiologic findings were seen at 20 weeks. These results suggest that nerve graft preservation may decrease systemic immunosuppression requirements while improving functional recovery. As well, storage of nerve grafts is feasible and would facilitate elective surgery and less costly reconstructive repair.

Wire mesh as a post-operative physiotherapy assistive device following peripheral nerve graft repair in the rat.

Traditional methods of assessing nerve recovery following injury, including histomorphometry and electrophysiology, do not necessarily correlate with return of motor and sensory function. Accordingly, many investigators have used walking track analysis as an assessment of global functional recovery following sciatic, peroneal or tibial nerve injury. However, walking track reliability may be compromised by the development of flexion contractures secondary to neurologic loss. To prevent this, regular manual physiotherapy is recommended which is a time-consuming and often frustrating process for both the animal and investigator. We report the use of a wire mesh as a simple post-operative assistive device to provide constant physiotherapy. Twelve Lewis rats were randomized to one of two experimental groups. Each rat received a 2 cm posterior tibial nerve autograft. Postoperatively, animals in group 1 received manual physiotherapy, consisting of repeated flexion and extension exercises of the ankle, knee and hip every two weeks. Group 2 rats were permitted to climb freely on a 30 x 18 cm piece of wire mesh placed at a 45 degree angle within their cage. Group 2 rats received no manual physiotherapy throughout the course of the study. Serial walking tracks were performed every four weeks until sacrifice at sixteen weeks. There was no development of flexion contractures in the injured hind limbs of either group. There was no morbidity such as blisters associated with the use of the wire mesh. There was no statistical difference in walking track recovery between groups at any time period. However, a trend towards better functional recovery was seen in the group receiving constant physiotherapy via the wire mesh. The use of a wire mesh as a post-operative assistive device is an inexpensive, simple and reliable method to provide continuous physiotherapy to animals following denervation.

The role of conduits in nerve repair: a review.

The restoration of effective and meaningful axonal function following peripheral nerve injury continues to be a considerable clinical challenge. The use of conduits to bridge the gap between severed ends is a contemporary experimental maneuver that isolates the microenvironment of regenerating axons. Entubulation has allowed analysis and manipulation of putative influences upon nerve regeneration. A review is provided of the research efforts that have explored the neurobiological and mechanical factors that guide nerve regeneration within conduits. Levels of specificity, from tissue specific growth to end-organ specific growth, are outlined within the framework of the theories of Neurotropism, Contact Guidance and Neurotrophism. Included are investigations utilizing different conduit materials and the few clinical applications of these conduits. A number of chamber manipulations, extra-cellular matrix substrates and growth factors and their molecular receptors have been implicated in enhanced regeneration specificity. This information has been extended to the conduit model. The interposition of healthy nerve segments into conduits is proposed as a means of extending the length of successful nerve regeneration.

The dose-related effect of monoclonal antibodies against adhesion molecules ICAM-1 and LFA-1 on peripheral nerve allograft rejection in a rat model.

Donor-specific immunosuppression using anti-intercellular adhesion molecule-1 (ICAM-1) and anti-lymphocyte function-associated antigen-1 (LFA-1) has been shown to inhibit nerve allograft rejection without side effects. This dose-response study evaluated several dosing regimens using a 2-week course of three monoclonal antibodies (mAbs) against ICAM-1 and LFA-1 in combination on peripheral nerve allograft rejection in a rat model. Assessments of regeneration included walking track, electrophysiological, and histomorphologic analyses. Donor (ACI)-specific tolerance induction was assessed. Toxicity and mAb serum levels were monitored. At 18 weeks post engraftment, intermediate and high-dose groups were histologically indistinguishable from isograft controls, and superior to the untreated allograft group which demonstrated a significantly lower percent nerve tissue than all other groups. There were no differences in print length factor after 12 weeks or conduction velocity at sacrifice between any groups. Tolerance induction was not demonstrated. During mAb administration, animals in higher dose groups experienced temporary systemic side effects. This study demonstrated that a short course of mAb therapy directed against ICAM-1/LFA-1 inhibits rejection in rat peripheral nerve allografts by an unknown mechanism. The use of immune modulation in nerve transplantation may eliminate the need for systemic immunosuppression.

Paecilomyces variotii contamination in the lumen of a saline-filled breast implant.

This report describes a case of gross contamination with the filamentous fungus P. variotii cultured from an intraluminal saline breast implant removed from a patient 14 months after implantation because of severe capsular contracture. We suspect the fungal contamination occurred when a container of saline was left open in the operating room prior to filling and placement of the implant. This case may be the first documented report of microbial growth and reproduction in the internal environment of a saline implant. We assume that organisms such as P. variotii can survive--and accumulate biomass--on the minute amounts of substrates that diffuse across an implant envelope.

Monoclonal antibodies against ICAM-1 and LFA-1 prolong nerve allograft survival.

In a rat nerve allograft model, specific immunosuppression was approached with monoclonal antibodies (MAbs) against cell-surface molecules. After engraftment, recipients were treated with antiintercellular adhesion molecule-1 (ICAM-1) and antilymphocyte function-associated antigen-1 (LFA-1) MAbs for 14 days. Functional recovery was evaluated biweekly. Electrophysiological and histological assessments were performed at 6 and 16 weeks. Immunologic responsiveness in the recipients was assessed with a cytotoxic T lymphocyte (CTL) assay at 16 weeks and skin grafts at 18 weeks. The untreated allograft group demonstrated complete disruption of fascicular architecture with poor nerve regeneration. The MAb-treated allografts maintained well-organized nerve architecture with a dense population of well-myelinated fibers. These animals showed functional and electrophysiological recovery. Suppression of CTL activity was nerve donor specific and the survival time of nerve donor skin grafts was prolonged, suggesting induction of alloantigen-specific tolerance. MAbs therapy directed against ICAM-1/LFA-1 presents a new approach for the management of the peripheral nerve allograft response.

Immunosuppressive effect of monoclonal antibodies to ICAM-1 and LFA-1 on peripheral nerve allograft in mice.

This study evaluated the immunosuppressive effect of monoclonal antibodies against cell surface molecules in a murine peripheral nerve allograft model. After nerve allografting, 18 recipients were treated with both anti-intercellular adhesion molecule-1 (ICAM-1) and anti-lymphocyte function-associated molecule-1 (LFA-1) monoclonal antibodies in low or high dose. Nerve allografts were harvested at 8 weeks for histologic and morphometric evaluation. Recipients were subsequently challenged with skin grafts at 9 weeks and a cytotoxic assay at 12 weeks. The majority of the antibody-treated allografts (13 of 18) showed excellent regeneration comparable to the autografts with preservation of the normal nerve architecture and scant cellular infiltrate. All untreated allografts demonstrated severe structural disorganization with cellular infiltrate consistent with acute rejection. In the high dose group, the mean skin graft survival time from nerve donor mice, but not third-party mice, was significantly prolonged. (17.5 vs. 11.3 days). Similarly, the cytotoxic activity against nerve donor alloantigen was significantly suppressed. These preliminary findings suggest that antibody therapy alone can facilitate nerve regeneration in a murine nerve allograft model.