Role of Vascular Endothelial Growth Factor in Protection of Intrahepatic Cholangiocytes Mediated by Hypoxic Preconditioning After Liver Transplantation in Rats

Objective

To investigate the effect on intrahepatic cholangiocytes mediated by hypoxic preconditioning (HP) after liver transplantation and the role of vascular endothelial growth factor (VEGF).

Materials and Methods

This experiment was based on a model of rat orthotopic liver autotransplantation. Sprague-Dawley rats were randomly divided into 3 groups: normal control, autotransplantation (AT), and HP. The HP group was subjected to 8% oxygen atmosphere for 90 minutes before surgery. At 6, 12, 24, and 48 hours after autotransplantation, the rats were killed for testing .Serum total bilirubin, direct bilirubin, and alkaline phosphatase concentrations were determined. The microstructure of cholangiocytes and the ultramicrostructure of cholangioles were determined. Immunohistochemistry was used to detect the expression of VEGF and the proliferation rate of cholangiocytes.

Results

Total bilirubin, direct bilirubin, and alkaline phosphatase concentrations in the AT group increased considerably more than in the HP group during the entire interval (P < .05). Light microscopy demonstrated that the microstructure of cholangiocytes in the AT group was damaged more seriously than in the HP group. At transmission electron microscopy, the ultramicrostructure of cholangioles was changed more obviously than in the HP group. The expression of VEGF on cholangiocytes and the proliferation rate of cholangiocytes were higher in the HP group than in the AT group over the entire experiment (P < .05).

Conclusion

Hypoxic preconditioning has a protective effect on cholangiocytes after liver autotransplantation. The mechanism may be related to HP-induced overexpression of VEGF on cholangiocytes.     

Protective role of nitric oxide induced by ischemic preconditioning on cold ischemic-reperfusion injury of rat liver graft

Aims

To investigate the protective role of nitric oxide (NO) induced by ischemic preconditioning (IP) on cold ischemic-reperfusion (IR) injury of rat liver grafts.

Methods

One hundred twenty-eight male Sprague Dawley rats used for orthotopic liver transplantation were randomly divided into four groups (n = 32): administering heparin before ischemic reperfusion (control group); IP with 10-minute ischemia and 10-minute reperfusion before IR (IP group); adenosine before IR (Ade group); and L-NAME (NG-nitro-L-arginine methyl ester) + IP before IR (NAME group). Half of each group were used to investigate 1-week recipient survival rate, and another to obtain blood and hepatic tissue samples after 2-hour reperfusion.

Results

One-week survival bile production, serum NO, and antioxidase activity were higher but serum alanine aminotransferase, tumor necrosis factor-α, and superoxide levels in hepatic tissue were lower in the IP group and Ade group versus the control group or NAME group. Liver sinusoidal endothelial cells in the IP and Ade groups showed less injury than the other groups.

Conclusion

NO induced by IP can improve 1-week survival and rat liver function as well as protect liver sinusoidal endothelial cells.     

Protective effect of deferoxamine on experimental spinal cord injury in rat

Objective

To observe the protective effect of deferoxamine on experimental spinal cord injury (SCI) in rats.

Methods

Sprague-Dawley rats were randomly divided into the following four groups. Control group: rats were performed laminectomy only; SCI group: rats were performed laminectomy with SCI; DFO group: rats were injected intraperitoneally a bolus of 100 mg/kg deferoxamine after SCI; vehicle group: rats were injected intraperitoneally 0.9% saline after SCI. The SCI of animal model was made by using a modified Allen's method on T₁₀. Six rats of each group were sacrificed at 4 h after injured, and the levels of free iron and malondialdehyde (MDA) of involved spinal cord segments were measured by bleomycin assay and the thiobarbituric acid (TBA) separately. The recovery of function was assessed by Modified Tarlov's scale and inclined plane method at 7, 14, 21 d after SCI. The histologic changes of the damaged spinal cord were also examined at 7 d after SCI.

Results

Following SCI, the levels of free iron and MDA were increased significantly and the Modified Tarlov's score and inclined plane angles decreased in SCI group and vehicle group. In DFO group, the levels of free iron and MDA were not increased, but the Modified Tarlov's score and inclined plane angles decreased, the histological findings were improved as well.

Conclusion

Deferoxamine can reduce the levels of free iron and lipid peroxidation, and improve the hind limb functional status of rats with spinal cord injury.     

Total parathyroidectomy without autotransplantation in dialysis patients and renal transplant recipients, long-term follow-up evaluation

Background

Persistent secondary hyperparathyroidism not responding to medication is treated successfully with surgical excision of parathyroid glands (total parathyroidectomy [PTX]). PTX without autotransplantation of parathyroid glands excludes the risk for recurrence of hyperparathyroidism.

Methods

During the years 2002 to 2005, 36 total parathyroidectomies were performed in 33 patients: 21 dialysis patients because of end-stage renal disease and 12 renal transplant recipients.

Results

PTX without autotransplantation was performed successfully in 33 patients, whereas 3 patients were reoperated for remaining parathyroid glands. Immediate improvement of clinical symptoms and a decrease of serum calcium and parathormone levels were observed after surgical procedures. Oral replacement treatment with vitamin D (1a-calcidiol) and calcium was commenced and long-term follow-up evaluation (23.5 ± 7.6 mo) showed that calcium homeostasis was controlled adequately.

Conclusions

PTX without autotransplantation is a safe and effective surgical procedure for the treatment of resistant secondary hyperparathyroidism with immediate response of clinical symptoms. Replacement treatment with vitamin D and calcium provides satisfactory coverage of individual needs.     

Effects of orthotopic liver transplantation in inbred rats on bone biomechanical properties

Aim

To determine the effects of orthotopic liver transplantation in inbred rats on the mechanical properties of bones at different anatomic sites.

Methods

The 24 rats that survived liver transplantation were paired with sham-operated rats of similar body weight. Six months after surgery, the lumbar vertebra, the proximal femur, and the middle femoral shaft were measured for their biomechanical properties and bone mineral density.

Results

The ultimate force, the ultimate stress, the Young modulus and the bone mineral density of both the proximal femur and the middle femur shaft of the rats were significantly reduced in the liver transplant group. However, no significant change was observed in the various parameters that indicate the biomechanical properties and the bone mineral density of the lumbar vertebra.

Conclusions

Orthotopic liver transplantation impairs the biomechanical properties of the proximal femur and the middle femoral shaft. Orthotopic liver transplantation itself is one of the risk factors for posttransplant fracture.     

A new liver autotransplantation technique using subnormothermic machine perfusion for organ preservation in a porcine model

Background

Hepatic resection is the gold standard of therapy for primary and secondary liver tumors, but few patients are eligible for this procedure because of the extent of their neoplasms. Improvements in surgical experience of liver transplantation (OLT), hepatic resection and preservation with sub-normothermic machine perfusion (MP) have prompted the development of a new model of large animal autotransplantation.

Methods

Landrace pigs were used in this experiment. After intubation, hepatectomy was performed according to the classic technique. The intrahepatic caval vein was replaced with a homologous tract of porcine thoracic aorta. The liver was perfused with hypothermic Celsior solution followed by MP at 20°C with oxygenated Krebs solution. An hepatectomy was performed during the period of preservation, which lasted 120 minutes, then the liver was reimplanted into the same animal in a 90° counterclockwise rotated position. The anastomoses were performed in the classic sequence. Samples of intravascular fluid, blood and liver biopsies were obtained at the end of the period of preservation in MP and again at 1 and 3 hours after liver reperfusion to evaluate graft function and microscopic damage.

Results

All animals survived the procedure. The peak of aspartate aminotransferase was recorded 60 minutes after reperfusion and the peak of alanine aminotransferase and lactate dehydrogenase after 180 minutes. Histopathologic examination under the light microscope identified no necrosis or congestion. Intraoperative echo-color Doppler documented good patency of the anastomosis and normal venous drainage.

Conclusion

This system made it possible to perform hepatic resections and vascular reconstructions ex situ while preserving the organ with mechanical perfusion (ex vivo, ex situ surgery). Improving surgical techniques regarding autotransplantation and our understanding of ischemia-reperfusion damage may enable the development of interesting scenarios for aggressive surgical treatment or radiochemotherapy options to treat primary and secondary liver tumors unsuitable for conventional in situ surgery.     

The Effect of Ischemic Preconditioning of the Pancreas on Severity of Ischemia/Reperfusion-Induced Pancreatitis After a Long Period of Ischemia in the Rat

Background

The role of ischemia/reperfusion injury in the pathogenesis of acute pancreatitis is still ill-defined. It is accepted, however, that ischemia/reperfusion induces the development of postimplantation pancreatitis that is responsible for considerable morbidity. Preconditioning by brief exposure to ischemia protects the organ against damage evoked by subsequent severe ischemia. This study was undertaken to examine whether two brief ischemic periods protect the pancreas against severe ischemia/reperfusion-induced pancreatitis.

Materials and methods

This study was performed on 30 rats in three groups. The first group (control) underwent a laparatomy without clamping of any artery. The second group underwent 30-minute clamping of the inferior splenic artery followed by 1-hour reperfusion of the pancreas, and the third group underwent clamping of inferior splenic artery (2 × 5 minutes with 5-minute interval) as ischemic preconditioning and then 30-minute clamping of inferior splenic artery followed by 1-hour reperfusion.

Results

Exposure to 30-minute pancreatic ischemia followed by 1-hour reperfusion led to the development of severe alterations greater than the other group that underwent ischemic preconditioning and then ischemia/reperfusion. Ischemia preconditioning applied prior to induction of pancreatitis reduced plasma lipase and interleukin-1β concentrations as well as less histological signs of pancreatic damage.

Conclusion

We concluded that pancreatic ischemic preconditioning reduced the severity of ischemia/reperfusion-induced pancreatitis. This effect seemed to be related at least in part to the release of the proinflammatory mediator interleukin-1β.     

Management of the primary tumor in patients with metastatic pancreatic neuroendocrine tumor: a contemporary single-institution review

Background

Pancreatic nonfunctioning neuroendocrine tumors (PNFNETs) are an uncommon malignancy and often present with metastatic disease. There is a lack of information on the management of the primary tumor in patients who present with unresectable synchronous hepatic metastases.

Methods

A retrospective review (2001-2008) of PNFNETs was conducted. Patients were divided into 3 groups: PNFNET without evidence of hepatic metastasis (group A), PNFNET with metastatic disease involving less than 50% of the liver (group B), and PNFNET with metastatic disease involving more than 50% of the liver (group C). Clinical data and outcomes were analyzed.

Results

Thirty-five patients with PNFNET were identified (group A = 15, group B = 11, group C = 9). Resection of the pancreatic tumor was performed in 26 patients. With a mean follow-up period of 30 months, death from disease progression occurred in 1 patient in group A, none in group B, and in 7 in group C.

Conclusions

In selected patients, resection of the primary pancreatic tumor even in the setting of unresectable but limited hepatic metastases may be indicated.     

Delayed Ischemic Preconditioning Protects Against Liver Ischemia-Reperfusion Injury In Vivo

Objectives

Ischemic preconditioning (IP) affords resistance to liver ischemia-reperfusion (IR) injury, providing an early phase of protection. Development of delayed IP against IR injury was assessed using partial IR in rat liver.

Methods

The IP manuver (10 minutes of ischemia and up to 72 hours of reperfusion) was induced before 1 hour of ischemia and 20 hours of reperfusion. At the end of the reperfusion period, blood and liver samples were analyzed for serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), haptoglobin and tumor necrosis factor-α (TNF-α) levels, hepatic histology, protein carbonyl and glutathione (GSH) contents as well as nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) DNA binding.

Results

The IP manuver significantly increased protein carbonyl/GSH ratios (275%), serum ALT (42%), and AST (58%); these changes normalized after 12 hours. Serum AST, ALT, and LDH levels were significantly increased by IR (4-, 5.6-, and 7.0-fold, respectively), with significant changes in liver histology, protein carbonyl/GSH ratio (481% enhancement), and serum TNF-α (6.1-fold increase). Delayed IP in IR animals reduced serum AST (66%), ALT (57%), and LDH (90%) and liver GSH depletion (89%), with normalization of protein carbonyl content, serum TNF-α levels, and liver histology. Enhanced AP-1/NF-κB DNA binding ratios and diminished haptoglobin expression induced by IR were normalized by IP.

Conclusion

These data support that delayed IP suppresses IR-induced liver injury, oxidative stress, and TNF-α response, which coincide with recovery of IR-altered signaling functions represented by normal AP-1/NF-κB DNA binding ratios and acute phase responses.     

Growth hormone selectively improves intestinal cholesterol absorption after jejunoileal autotransplantation in pigs

Background/Purpose

Small bowel transplantation impairs enteric function and causes malabsorption of cholesterol and bile acids. Growth hormone stimulates intestinal absorptive function. The authors hypothesized that long-term growth hormone therapy could improve absorption of bile acids and cholesterol after autotransplantation of the jejunoileum.

Methods

Sixteen pigs with similar food, cholesterol, and fat intake underwent either sham laparotomy or a model of jejunoileal autotransplantation, including extrinsic autonomic denervation, lymphatic interruption, and in situ cold ischemia. Five randomly chosen autotransplanted animals received daily growth hormone treatment for 8 weeks. Serum lipids, absorption, and excretion of cholesterol, bile acids, and fat were determined after 8 weeks. Mucosal morphometrics, proliferation, and enzyme activities were determined. Plasma cholesterol precursors and plant sterols, respective markers of cholesterol synthesis and absorption, were measured after 2 and 8 weeks.

Results

After jejunoileal autotransplantation, growth hormone treatment significantly increased body weight gain, cholesterol absorption efficiency from 45.1% to 62.1%, plasma campesterol to cholesterol proportions, and biliary secretion of cholesterol. With or without growth hormone treatment, autotransplantation significantly increased fecal bile acid excretion, plasma cholesterol precursors, fecal bacterially modified neutral sterols, mucosal thickness of the ileum (but not jejunum), and intestinal transit time when compared with sham-operated animals. Crypt cell proliferation, mucosal enzyme activities, and microvilli showed no differences between the groups.

Conclusions

These findings suggest that growth hormone treatment selectively improves cholesterol, but not bile acid absorption, after autotransplantation of the jejunoileum.     

Surgical treatment of biliary tract complications after liver transplantation

Objective

Biliary strictures are the most common biliary tract complication after liver transplantation. There are scarce data on the results of hepaticojejunostomy (HJ) in the management of biliary complications after orthotopic liver transplantation (OLT). Thus, the role of surgery in this setting remains to be established. The aim of this study was to evaluate the results of surgical treatment of patients with biliary complications at our institution.

Patients and Methods

We reviewed 1000 consecutive liver transplantations performed at our institution from 1984 to 2007. We used a prospectively recorded database to identify patients who underwent HJ to treat any biliary tract complication.

Results

Overall, 62 patients (6.2%) underwent HJ, 40 for an anastomotic and 7 for a non-anastomotic stricture as well as 15 for biliary leaks. Postoperative morbidity was 16%, and postoperative mortality 1.6%. There were 7 cases of anastomotic stenosis (11.3%). Four patients (5%) required retransplantation.

Conclusions

HJ is a safe procedure to manage biliary complications after OLT. It may be the first treatment choice especially for cases with anastomotic strictures.     

Favorable Outcome of Orthotopic Liver Transplantation in Very High-Risk Situations

Background

Favorable outcomes of marginal liver grafts depends on optimal perioperative control and good recipient parameters. The aim of this study was to assess the results of marginal liver grafts associated with prolonged ischemic times and high-risk recipients.

Methods

We retrospectively reviewed data from patients who underwent orthotopic liver transplantation between 2001 and 2005. The patients were divided into two groups: group 1 received marginal liver grafts with ischemia times ≥ 12 hours and recipient United Network for Organ Sharing (UNOS) status 1, 2A, and 2B. Patients who had marginal liver grafts with ischemic times less than 12 hours and/or better UNOS status were classified as group 2. We compared initial graft function as well as patient and graft survivals at 1 year between the two groups.

Results

Among 31 patients who were reviewed, four were excluded because of incomplete data and 24/27 received marginal liver grafts. Seven patients were classified into group 1, and 17 into group 2. The initial poor function rate was 85.7% (6/7 patients) and 76.47% (13/17 patients) in groups 1 and 2, respectively. The 1-year survival rate in group 1 was 85.7% (6/7 patients) and 94.12% (16/17 patients) in group 2.

Conclusion

Marginal liver grafts can be used with favorable outcomes even in high-risk situations, such as prolonged ischemia times and high-risk recipients.     

Early Expression of Hepatocyte Growth Factor, Interleukin-6, and Transforming Growth Factor-β1 and -β2 in Symptomatic Infection in Patients Who Have Undergone Liver Transplantation

Introduction

Early septic complications may be a deciding factor for successful recovery among patients who have undergone orthotopic liver transplantation. Therefore, monitoring liver function parameters plays an important role in postoperative treatment to achieve an early diagnosis of postsurgical complications. We ought to measure standard liver function parameters and the expression levels for selected cytokines among patients exhibiting symptoms of infection after orthotopic liver transplantation.

Materials and methods

The study was performed on 30 patients who were divided into two groups: SI-0 consisted of patients free of infection, and SI-1, those who had symptoms of infection. We determined standard liver function parameters and expression of hepatocyte growth factor (HGF), interleukin (IL)-6, transforming growth factor (TGF)-β1, and TGF-β2.

Results

There were no significant differences in standard liver function parameters between the two groups of patients. There were no significant differences in the levels of expression for the cytokines in question between the two groups of patients.

Conclusions

Although standard liver function parameters provide diagnostically valuable information on the patient's condition, they cannot be used to determine the extent of systemic infection among patients showing signs of infection after liver transplantation. Determining gene expression levels in circulating lymphocytes is a sensitive method to monitor patients' condition after liver transplantation. The expression levels of HGF, IL-6, TGF-β1, and TGF-β2 in circulating lymphocytes were not sufficiently specific to diagnose transitory postsurgical complications such as symptomatic infection.     

Telbivudin as prophylaxis for hepatitis B virus recurrence after liver transplantation: a case series in single-center experience

Background

Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) represents a severe condition that requires prophylaxis with specific immunoglobulin and lamivudine. Few studies have addressed the efficiency of other effective antiviral drugs posttransplantation or their impact on early renal function after transplantation. Herein, we have reported experience among seven transplanted patients prescribed Telbivudin (600 mg/d) while on the waiting list followed by treatment for 3 months after OLT.

Methods

Our series consisted of men with HBV-related end-stage liver disease. Once the patient started antiviral treatment, the viral load decreased rapidly while on the waiting list. All patients were evaluated for liver and renal functions immunosuppressive drug trough levels, CPK before (T0), as well as at 1 month (T1), and 3 months after liver transplant (T3).

Results

All patients received a CNI-based regimen. Their mean creatinine clearance (MDRD) was 72.5 mL/min at T0, 69.2 mL/min at T1, and 71.0 mL/min at T3. Neither CPK or serum transaminase levels increased throughout the study. Once HBV-DNA was cleared while on the waiting list, it remained negative throughout the follow-up period.

Conclusion

Telbivudin prophylaxis for HBV was safe and effective without any significant deleterious effect on liver or renal function tests after liver transplantation.     

New alternatives to the treatment of acute liver failure

Introduction

Acute-on-chronic liver failure (ACLF) is defined as an acute deterioration of a chronic liver disease. The most effective treatment in these patients is orthotopic liver transplantation (OLT), which is highly limited by the donor shortage. The aim of this study was to increase the usefulness of hepatocyte transplantation (HT) as a bridge or alternative to OLT.

Methods

During the last 2 years, we have performed HT in 3 patients with ACLF. The diagnosis was graft cirrhosis due to hepatitis C virus in 2 of them, who were already included on waiting lists for retransplantation, and the third, unknown alcoholic cirrhosis.

Results

After the first HT infusion, we observed an improvement in the clinical condition in all patients, hyperammonemia, and a partial correction of the degree of encephalopathy; 1 patient was retransplanted 6 days after the first HT.

Discussion

The main indications for HT are inborn errors of metabolism in children. Other indications especially in adults, are acute liver failure, ACLF in patients with end-stage-liver disease who are a waiting OLT, and acute liver failure after an hepatectomy. HT may be a new treatment to improve the clinical condition in patients awaiting OLT.     

High-mobility group box 1 protein activates hepatic stellate cells in vitro

Objectives

Our previous study noticed remarkably elevated titers of anti-high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis.

Methods

The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure α-smooth muscle actin (α-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs).

Results

HMGB1 at concentrations >1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. α-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment.

Conclusion

Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.     

Thromboelastography-Guided Transfusion Decreases Intraoperative Blood Transfusion During Orthotopic Liver Transplantation: Randomized Clinical Trial

Objective

To test in a prospective randomized study the hypothesis that use of thromboelastography (TEG) decreases blood transfusion during major surgery.

Material and Methods

Twenty-eight patients undergoing orthotopic liver transplantation were recruited over 2 years. Patients were randomized into 2 groups: those monitored during surgery using point-of-care TEG analysis, and those monitored using standard laboratory measures of blood coagulation. Specific trigger points for transfusion were established in each group.

Results

In patients monitored via TEG, significantly less fresh-frozen plasma was used (mean [SD], 12.8 [7.0] units vs 21.5 [12.7] units). There was a trend toward less blood loss in the TEG-monitored patients; however, the difference was not significant. There were no differences in total fluid administration and 3-year survival.

Conclusion

Thromboelastography-guided transfusion decreases transfusion of fresh- frozen plasma in patients undergoing orthotopic liver transplantation, but does not affect 3-year survival.