Biomedical subjects

Thomas M van Gulik

Publications and source records attributed to Thomas M van Gulik.

At least 19 recordsLinked to original sources

PubMed2007

Steatosis as a risk factor in liver surgery.

OBJECTIVE: To review present knowledge of the influence of hepatic steatosis in liver surgery as derived from experimental and clinical studies. SUMMARY BACKGROUND DATA: Hepatic steatosis is the most common chronic liver disease in the Western world, and it is associated with obesity, diabetes, and metabolic syndrome. Fatty accumulation affects hepatocyte homeostasis and potentially impairs recovery of steatotic livers after resection. This is reflected clinically in increased mortality and morbidity after liver resection in patients with any grade of steatosis. Because of the epidemic increase of obesity, hepatic steatosis will play an even more significant role in liver surgery. METHODS: A literature review was performed using MEDLINE and key words related to experimental and clinical studies concerning steatosis. RESULTS: Experimental studies show the increased vulnerability of steatotic livers to various insults, attributed to underlying metabolic and pathologic derangements induced by fatty accumulation. In clinical studies, the severity of steatosis has an important impact on patient outcome and mortality. Even the mildest form of steatosis increases the risk of postoperative complications. CONCLUSIONS: Hepatic steatosis is a major factor determining patient outcome after surgery. Further research is needed to clarify the clinical relevance of all forms and severity grades of steatosis for patient outcome. Standardized grading and diagnostic methods need to be used in future clinical trials to be able to compare outcomes of different studies.

Fatty Liver↗

PubMed2007

Severe steatosis increases hepatocellular injury and impairs liver regeneration in a rat model of partial hepatectomy.

OBJECTIVE: The aim of this study was to assess the influence of severe steatosis with inflammation on hepatocellular recovery after 70% hepatectomy in a rat model of diet-induced steatosis. BACKGROUND: Patients with steatosis have an increased risk of inflammatory complications after liver resection. This might be attributable to Kupffer cell-mediated inflammation in steatotic livers causing progressive injury. METHODS: Male Wistar rats were fed a standard methionine- and choline-deficient diet for 1 or 5 weeks. A 70% partial hepatectomy (PH) was performed, after which rats were killed at 24, 48, or 72 hours. The extent of steatosis and inflammation was determined by assessment of hepatic triglycerides, cytokine content, and histopathology. Outcome parameters were: liver regeneration (MIB-5 proliferation rate, mitotic index, and regenerating liver mass), hepatocellular injury (plasma aminotransferases, lipid peroxidation, histopathology, and apoptosis), Kupffer cell-mediated proinflammatory response (TNF-alpha, IL-1beta, IL-6, IL-10 in plasma and liver) and antioxidant content (total glutathione). RESULTS: Methionine- and choline-deficient diet induced uncomplicated steatosis after 1 week (<30% hepatocytes affected without inflammation) and severe steatosis after 5 weeks (>60% hepatocytes affected, including prominent inflammation) as confirmed by histopathology. After PH, liver regeneration was impaired at all time points in the severe steatosis group as compared with the mild and control groups (P < 0.05). Hepatocellular injury was significantly increased in the severe steatosis group at all time points (P < 0.05). Kupffer cell-mediated inflammatory responses were aggravated in the severe steatosis group along with decreased antioxidant content (P < 0.05). Necrosis was the main type of cell death in severe steatotic livers compared with mainly apoptotic cell death in mild steatotic and normal livers. CONCLUSION: Steatosis with prominent inflammation impaired liver regeneration probably because of increased hepatocellular lipid peroxidation and damage in concert with Kupffer cell-mediated proinflammatory responses. These results suggest an increased risk of performing extensive liver resection in the presence of severe steatosis.

Animals↗

PubMed2006

Current role of portosystemic shunt surgery in the management of hepatic venous outflow obstruction.

BACKGROUND: Hepatic venous outflow obstruction (HVOO) is a rare disorder that occurs predominantly due to a hypercoagulable state. The syndrome may result from hepatic vein obstruction, inferior vena cava obstruction or a combination of both and manifests with post-sinusoidal portal hypertension. The presentation may be fulminant with poor prognosis or as either acute, subacute or chronic forms with relatively better prognosis. Portosystemic shunt surgery (PSS) has thus far been the mainstay of treatment for HVOO. However, over the last decade, transjugular intrahepatic portosystemic shunt (TIPS) and liver transplantation have emerged as viable options. This review aims to evaluate the available treatment options and the current relevance of PSS for the management of HVOO. METHODS: A literature review on investigations and treatment was performed using Medline and additional library searches. RESULTS: Portosystemic shunts form the mainstay of treatment for patients with subacute presentation (preserved liver function) with medically intractable ascites or recurrent variceal hemorrhage. Excellent results with 5-year survival of more than 90% have been reported from specialized centers. The main limitation for PSS is the reported perioperative mortality of 10-20% and a declining technical expertise for such surgery. Liver transplantation with disease-specific 5-year survival between 50 and 95% is presently the treatment of choice for patients with fulminant presentation, end-stage liver disease (ESLD), unshuntable anatomy or decompensation after PSS. TIPS may be preferable for sick patients with acute presentation with isolated hepatic vein thrombosis or as a temporizing measure for those with ESLD awaiting transplantation. The drawback of TIPS is late shunt dysfunction that occurs in more than 50% of patients at 1 year. CONCLUSIONS: Due to rarity of the disorder there is a lack of trials comparing the different treatment modalities. Hence, the current treatment recommendations are based on retrospective studies. In a select group of HVOO patients (subacute presentation with preserved liver function), PSS remains the treatment of choice with excellent long-term results.

Budd-Chiari Syndrome↗

PubMed2006

Histopathologic evaluation of an animal model for Barrett's esophagus and adenocarcinoma of the distal esophagus.

INTRODUCTION: Barrett's esophagus and adenocarcinoma of the esophagus are related to long-standing duodeno-gastroesophageal reflux. The development of an animal model in which Barrett's esophagus and/or carcinoma is induced by duodeno-(gastro-)esophageal reflux could provide better understanding of the pathogenesis of the metaplasia-dysplasia-carcinoma sequence and would create the possibility of investigating new treatment strategies for this aggressive disease. MATERIALS AND METHODS: Two rat models were analyzed. In the first experiment, 44 male Sprague Dawley rats underwent end-to-side esophagojejunostomy with gastric resection, to ensure duodenoesophageal reflux without gastric acid. In the second experiment a side-to-side esophago-gastrojejunostomy was performed in 30 rats, ensuring duodeno-gastroesophageal reflux. In both experiments animals were not exposed to any exogenous carcinogens during the experiment. Sequential morphological changes (i.e., esophagitis, intestinal metaplasia, dysplasia, and carcinoma) were studied after 4, 6, and 12 months. To analyze histopathologic characteristics, evaluation of the hematoxylin and eosin specimens was combined with immunohistochemical stainings for high-iron diamine-alcian blue, alcian blue/periodic acid-Schiff, the proliferation marker PCNA, and mutations in the tumor suppressor gene p53. RESULTS: In the first experiment, only 11 animals survived the postoperative period. These animals had to be sacrificed at a median of 11 weeks due to persistent weight loss and failure to thrive. Severe ulcerative esophagitis was seen in all animals, with a 2-mm segment of metaplastic epithelium found at the anastomosis. In four animals a large, well-differentiated, mucinous tumor without malignant characteristics was observed. In the second experiment, eight animals died postoperatively. Twelve animals were sacrificed according to protocol at 4 or 6 months. In these animals, extensive esophagitis with squamous cell hyperplasia was found. In addition, a short (2 mm) segment of metaplastic epithelium was observed, without dysplasia. The remaining animals survived 1 year. After 1 year, 9 of the 10 animals had developed a glandular metaplastic segment (median length, 10 mm), which was histologically and immunohistologically characteristic for the specialized columnar epithelium of Barrett's esophagus without signs of dysplasia. Finally, in seven animals a mucinous tumor with cytologic characteristics of a well-differentiated mucinous adenocarcinoma was found without infiltrative growth. These tumors were always found at the site of the anastomosis, originated in the submucosa, and did not reach either the luminal surface or the muscular layer. The mucinous lesions were not positive for p53, and PCNA was only slightly increased. Although they showed cytological characteristics of malignancy, histopathologic evaluation was more suggestive of a reactive mucous producing lesion fitting the diagnosis "esophagitis cystica profunda." CONCLUSION: This study demonstrates the development of a long Barrett's segment in an animal duodeno-gastroesophageal reflux model. Although mucinous tumors resembling adenocarcinomas develop around the anastomosis, these are probably not reflux induced and are more likely to be reactive lesions. However, the true nature of these tumors remains to be elucidated.

Adenocarcinoma↗

PubMed2006

Combining partial liver resection and local ablation of liver tumours: a preliminary Dutch experience.

BACKGROUND: The combination of partial liver resection and radiofrequency ablation (RFA) is a novel concept in the treatment of unresectable liver malignancies. The aim of this study is to evaluate the results of this combined strategy in the Netherlands. METHODS: Thirty-five patients treated with a combination of partial liver resection and RFA were identified from a prospectively registered pooled multicentre database. All patients were operated between June 1999 and November 2003 in 8 medical centres in The Netherlands. Main outcome parameters were morbidity, mortality, local success rate, and survival. RESULTS: Thirty-seven operations were performed in 35 patients. The group consisted of 20 male and 15 female patients with a median age of 59 years (range 41-76). Seventy-six lesions were resected and RFA was performed to ablate 82 unresectable liver tumours. Twelve patients developed a total of 24 complications, resulting in an overall perioperative morbidity rate of 32%. In two patients major complications resulted in postoperative death (postoperative mortality rate 5.4%). Local success rate after RFA was 88% and the estimated 1-, 2- and 3-year overall survival rates were 84%, 70% and 43%, respectively. CONCLUSION: This strategy should only be performed following strict patient selection and within the context of prospective clinical trials.

Journal Article↗

PubMed2006

Improved outcome of resection of hilar cholangiocarcinoma (Klatskin tumor).

BACKGROUND: Treatment of hilar cholangiocarcinoma (Klatskin tumors) has changed in many aspects. A more extensive surgical approach, as proposed by Japanese surgeons, has been applied in our center over the last 5 years; it combines hilar resection with partial hepatectomy for most tumors. The aim of this study was to assess the outcome of a 15-year evolution in the surgical treatment of Klatskin tumors. METHODS: A total of 99 consecutive patients underwent resection for hilar cholangiocarcinoma in three 5-year time periods: periods 1 (1988-1993; n=45), 2 (1993-1998; n=25), and 3 (1998-2003; n=29). Outcome was evaluated by assessment of completeness of resection, postoperative morbidity and mortality, and survival. RESULTS: The proportion of margin negative resections increased significantly from 13% in period 1 to 59% in period 3 (P<.05). Two-year survival increased significantly from 33%+/-7% and 39%+/-10% in periods 1 and 2 to 60%+/-11% in period 3 (P<.05). Postoperative morbidity and mortality were considerable but did not increase with this changed surgical strategy (68% and 10%, respectively, in period 3). Lymph node metastasis was, next to period of resection, also associated with survival in univariate analysis. CONCLUSIONS: Mainly in the last 5-year period (1998-2003), when the Japanese surgical approach was followed, more hilar resections were combined with partial liver resections that included segments 1 and 4, thus leading to more R0 resections. This, together with a decrease in lymph node metastases, resulted in improved survival without significantly affecting postoperative morbidity or mortality.

Adult↗

PubMed2006

Factors influencing the local failure rate of radiofrequency ablation of colorectal liver metastases.

BACKGROUND: The prognosis of patients with colorectal cancer is poor, especially when there is distant metastatic disease. Local ablation of tumor by radiofrequency ablation (RFA) has emerged as a safe and effective new treatment modality, but its long-term efficacy may be hindered by renewed local tumor growth at the site of RFA. The objectives of this study were to identify risk factors for local RFA failure and to define exclusion criteria for RFA treatment of colorectal liver metastases. METHODS: A total of 199 lesions in 87 patients were ablated with RFA. Factors influencing local failure rates were identified and compared with data from the literature. RESULTS: The local failure rate was 47.2%, and the average time to local disease progression was 6.5 months. Factors that significantly correlated with increased failure rates were metachronous occurrence of liver metastases, large mean lesion size, and central tumor location. CONCLUSIONS: Because accurate electrode placement is pivotal in achieving adequate tumor necrosis, RFA should not be performed percutaneously when electrode placement is impaired. We suggest that lesions >5 cm and lesions located near great vessels or adjacent organs should be treated with open RFA, thus allowing vascular inflow occlusion and complete mobilization of the liver. Lesions that are difficult to reach by electrodes should be approached by an open procedure.

Adult↗

PubMed2006

Hepatobiliary function assessed by 99mTc-mebrofenin cholescintigraphy in the evaluation of severity of steatosis in a rat model.

PURPOSE: This study evaluated the utility of non-invasive assessment of hepatobiliary function by 99mTc-mebrofenin cholescintigraphy in a rat model of diet-induced steatosis. METHODS: Male Wistar rats (250-300 g) were fed a standard methionine- and choline-deficient (MCD) diet for up to 5 weeks, thereby inducing hepatic fat accumulation, progressive inflammation and fibrogenesis corresponding with clinical steatosis. 99mTc-mebrofenin pinhole scintigraphy was used to evaluate the hepatocyte mebrofenin uptake rate, the time of maximum hepatic uptake (T(peak)) and the time required for peak activity to decrease by 50% (T(1/2peak)). Scintigraphic parameters were correlated with biochemical and serological parameters and with liver histopathology. RESULTS: MCD diet induced mild steatosis after 1 week and severe steatosis with prominent inflammation after 5 weeks. T(peak), T(1/2peak) prolonged and the uptake rate decreased significantly, while the severity of steatosis increased (p<0.05). There was a strong, significant correlation between the severity of steatosis (histopathology, hepatic triglyceride content) and the 99mTc-mebrofenin uptake rate (r2=0.83, p<0.0001 and r2=0.82, p<0.0001, respectively). In addition, the uptake rate correlated significantly with the increased inflammation (plasma and hepatic TNF-alpha, r2=0.72, p<0.0001 and r2=0.52, p=0.001, respectively). The correlation of the uptake rate with hepatocellular damage was weak (AST and ALT, r2=0.29 and 0.32, respectively), but correlation with synthetic function was strong (prothrombin time, r2=0.70, p<0.001). CONCLUSION: Hepatobiliary function assessed by 99mTc-mebrofenin scintigraphy correlates with the extent and progression of steatosis. These results suggest a potential role for mebrofenin scintigraphy as a non-invasive functional follow-up method for disease progression in steatotic patients.

Aniline Compounds↗

PubMed2006

Systematic review of preservation methods and clinical outcome of infrainguinal vascular allografts.

OBJECTIVE: We systematically reviewed clinical studies on the use of venous and arterial allografts for infrainguinal revascularization. We attempted to find evidence for the best infrainguinal vascular allograft by a systematic review of the available literature. METHODS: An electronic search of the MEDLINE, EMBASE, and Cochrane databases was used to determine key articles from studies on the different types of vascular allografts used in infrainguinal reconstruction from 1966 to 2004. Articles were independently reviewed by using previously defined inclusion and exclusion criteria. Study results were gathered with cumulative primary patency as the primary end point. Secondary end points were major complications, graft disintegration, and major limb loss. Quantitative analysis was performed on the prospective randomized trials, and linear regression analysis was performed on cumulative primary patency. Fontaine's classification system was applied. RESULTS: No systematic review of randomized controlled trials was found. Five randomized controlled trials, 3 prospective cohort or case series, and 15 retrospective case series with 3,837 vascular allografts were found. Methods of allograft preservation were cryopreservation (5 studies), cold storage (3 studies), and glutaraldehyde preservation of human umbilical veins (15 studies). One-year cumulative primary patency rates were 13% to 79% for cryopreservation, 63% to 80% for cold storage, and 40% to 91% for glutaraldehyde. The weighted mean 1-year cumulative primary graft patency rate was 41% for cryopreservation, 71% for cold storage, and 70% for glutaraldehyde allografts. Four randomized trials on femoropopliteal bypasses demonstrated higher patency rates of glutaraldehyde-preserved human umbilical veins than polytetrafluoroethylene grafts. Statistical heterogeneity between studies (I(2) = 91.4%) was too high to perform a formal meta-analysis. The rate of major limb loss was 20% to 58% for cryopreservation, 10% to 69% for cold storage, and 0% to 65% for glutaraldehyde, and the percentage of graft disintegration was 2% to 6% for cryopreservation, 4% to 15% for cold storage, and 0% to 11% for glutaraldehyde. CONCLUSIONS: A firm conclusion could not be made because there were no studies available in which direct comparison was performed between different preservation methods of vascular allografts. In addition, heterogeneity of the individual studies hampered direct comparison of different types of vascular allografts. However, the overall graft performance of glutaraldehyde-preserved human umbilical vein allografts may be superior to that of other vascular allografts.

Arterial Occlusive Diseases↗

PubMed2006

Hypoxia/reoxygenation impairs glucose absorption and cAMP-mediated secretion more profoundly than glutamine absorption and Ca2+/PKC-mediated secretion in rat ileum in vitro.

BACKGROUND: Intestinal ischemia and reperfusion may lead to profuse secretion of water and electrolytes. The underlying mechanisms have been related to increased hydrostatic pressure, to denudation of intestinal villi, and, recently, to adenosine-mediated enhancement of chloride secretion. METHODS: We studied the effects of hypoxia and reoxygenation on baseline electrophysiologic parameters; on glucose- and glutamine-induced absorption; on secretion induced by carbachol, histamine, and forskolin; and on epithelial barrier function to disodium-fluorescein and horseradish peroxidase in rat ileum mounted in Ussing chambers. RESULTS: We observed that 30 minutes of hypoxia followed by 60 minutes of reoxygenation differentially affected glucose and glutamine absorption to 11% and 42%, respectively, of control values. Cyclic adenosine monophosphate-mediated secretion induced by forskolin was reduced to 9% of controls. In contrast, Ca(2+)/protein kinase C-mediated secretion induced by carbachol or histamine was reduced only to 35% to 48% of controls. Furthermore, the epithelium fully was capable of maintaining its barrier function to small and large permeability probes, even after 90 minutes of hypoxia. CONCLUSIONS: We conclude that hypoxia and reoxygenation differentially impair nutrient absorption, corroborating recent absorption data in in vivo models of ischemia, and that it differentially affects secretory capacity in crypts, dependent on the intracellular messenger pathway. The relative persistence of Ca(2+)/protein kinase C-mediated secretion to hypoxia and reoxygenation indicates that secretagogues that activate this pathway play a significant role in the intraluminal fluid sequestration and diarrhea observed after intestinal ischemia and reperfusion.

Absorption↗

PubMed2006

Peritoneal lavage with activated protein C alters compartmentalized coagulation and fibrinolysis and improves survival in polymicrobial peritonitis.

OBJECTIVE: During peritonitis, intra-abdominal fibrin entraps bacteria and hampers their elimination. Systemic administration of anticoagulant activated protein C improves survival in patients with severe sepsis, but its precise mode of action is unclear. This study in polymicrobial peritonitis assessed the effects of local activated protein C administration in peritoneal lavage fluid on coagulation, fibrinolysis, and survival. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: C57BL/6 mice. INTERVENTIONS: Twenty-four hours after induction of peritonitis by cecal ligation and puncture, mice underwent peritoneal lavage with activated protein C (1.0 microg/mL) or saline. Peritoneal lavage fluid, blood, and lungs were sampled after 24, 48, or 72 hrs (n = 8/group/time point). For survival analysis, maximum observation was 96 hrs (n = 22/group). Clotting time, tissue factor expression, thrombin-antithrombin complexes, fibrin degradation products (D-dimers), plasminogen activator, and plasminogen activator inhibitor were used to assess coagulation and fibrinolysis responses. MEASUREMENTS AND MAIN RESULTS: Activated protein C lavage reduced abdominal bacterial load, abdominal and pulmonary clotting times, D-dimers (p < .05 vs. saline), pulmonary tissue factor expression, and fibrin depositions, without clear effects on systemic thrombin generation. Activated protein C lavage decreased plasma and abdominal tissue plasminogen activator levels with increased inhibitor plasminogen activator inhibitor-1 levels (p < .05) but had reverse effects on pulmonary fibrinolysis. Survival improved from 55% (saline) to 80% after intra-abdominal activated protein C administration (p = .03). CONCLUSIONS: Peritoneal lavage with activated protein C may rebalance coagulation and fibrinolysis within compartments and improve survival in polymicrobial peritonitis.

Animals↗

PubMed2006

Portal vein ligation is as effective as sequential portal vein and hepatic artery ligation in inducing contralateral liver hypertrophy in a rat model.

PURPOSE: Dual embolization of the hepatic artery and portal vein (PV) has been proposed to enhance contralateral liver regeneration before resection. The aim of this study was to evaluate the effect of PV ligation compared with simultaneous or sequential dual ligation on regeneration, proinflammatory response, and liver damage. MATERIALS AND METHODS: Single hepatic artery ligation (HAL), PV ligation (70%), or dual ligation of the hepatic artery and PV (70%) simultaneously or sequentially within a 48-hour interval was performed in a rat model. Liver regeneration, proinflammatory mediators, hepatocellular synthetic function and injury, histopathology, and apoptosis were assessed at a maximum of 14 days after surgery. RESULTS: Sequential dual ligation resulted in a faster increase in hepatocyte proliferation at 24 hours without additional increase in liver mass compared with PV ligation after 14 days. Both dual ligations significantly increased proinflammatory response in plasma and in the regenerating liver compared with PV ligation alone. Fourteen days after PV ligation, the hepatic parenchyma was completely restored, whereas fibronecrosis was seen in the sequentially dual-ligated groups and complete necrosis was seen in simultaneously ligated groups. Increased apoptosis in the regenerating liver and prolonged hepatic dysfunction were observed after both dual ligations. CONCLUSIONS: PV ligation is as effective as dual ligation in inducing liver regeneration. No additional benefit of arterial ligation was observed.

Animals↗

PubMed2006

Management of spontaneous haemorrhage and rupture of hepatocellular adenomas. A single centre experience.

BACKGROUND: Hepatocellular adenomas (HCA) may present with spontaneous haemorrhage and rupture. The aim of this study was to assess management in 22 patients treated for haemorrhage and/or rupture of HCA. PATIENTS AND METHODS: Between May 1990 and July 2005, 22 female patients were diagnosed with acute haemorrhage and/or rupture of lesions highly suspicious of HCA. Preoperative imaging diagnostics and pathologic specimens were reviewed. RESULTS: Twelve haemodynamically stable and four unstable patients could be treated conservatively. One patient underwent acute partial liver resection, whereas four patients underwent laparotomy with initial packing of the liver. In one patient, selective embolisation of the left hepatic artery was performed. Fifteen patients eventually underwent resection after a mean time of 8 months after initial treatment. Six patients did not undergo resection and showed no complications or rebleeding after a mean follow-up of 24.6 months. Only in seven patients, histopathological examination showed HCA, and in one patient, HCA with focal nodular hyperplasia. CONCLUSION: HCA with haemorrhage and/or rupture does not necessarily require immediate liver resection. Conservative treatment is justified in stable patients. In case of an instable patient with or without hemoperitoneum, laparotomy with packing or selective embolisation can stop the bleeding.

Adenoma, Liver Cell↗

PubMed2006

Liver protection by hypothermic perfusion at different temperatures during total vascular exclusion.

INTRODUCTION: In situ hypothermic perfusion (HP) can be applied to attenuate ischemia and reperfusion (I/R) injury during liver resection under total vascular exclusion (TVE). This study examines the protective effect of cooling by HP at 20 and 28 degrees C as compared with no HP during TVE in a porcine liver I/R model. METHODS: Twenty-one pigs underwent 60 min TVE of the liver followed by 24 h reperfusion. HP was performed via the portal vein using ringerlactate solution of 4 degrees C. Pigs were assigned to three groups: TVE without HP (no-HP, n=9), TVE with HP at 28 degrees C (HP-28, n=6) and TVE with HP at 20 degrees C (HP-20, n=6). RESULTS: Perfusion volumes during TVE were 5.1+/-0.5 and 17.3+/-1.7 l in HP-28 and HP-20, respectively (P<0.05). Aspartate aminotransferase (AST) after 24 h reperfusion was 1172+/-440 U/l in no-HP as compared with 223+/-69 and 180+/-22 U/l in HP-28 and HP-20, respectively (P<0.05). No differences in liver function or histopathology were found between the HP-28 and HP-20 groups. CONCLUSIONS: HP at 20 degrees C is equally effective in preserving liver function and preventing hepatocellular injury under TVE as compared with HP at 28 degrees C. HP at 28 degrees C is advised, because of the lesser perfusion volume necessary for cooling of the liver.

Animals↗

PubMed2006

Wash-out of the non-heart-beating donor liver: a matter of flush solution and temperature?

BACKGROUND AND AIMS: Ischemically damaged donor livers are prone to graft non-function. This can in part be explained by a suboptimal wash-out during procurement. An enriched machine perfusion (MP) preservation solution for livers, named Polysol, was developed. The aim of this study was to investigate the type of flush solution, temperature and anticoagulant content on the wash-out of the non-heart-beating donor (NHBD) rat liver. METHODS: Rat livers were flushed after 30 min warm ischemia. After excision, livers were reperfused at 37 degrees C, with analysis of damage and function, concerning (1) solutions (University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK) and Polysol); (2) temperature (4 degrees C, 18 degrees C and 37 degrees C); (3) addition of heparin and (4) wash-out followed by 24 h MP. RESULTS: (1) Reperfusion results were inferior in the UW group; (2) less damage and improved function were seen after wash-out using Polysol at 37 degrees C; (3) No effects were seen of the addition of heparin to Polysol; (4) MP after wash-out using HTK resulted in more liver damage and decreased liver function as compared with wash-out using Polysol. CONCLUSIONS: Polysol is applicable as a flush solution for the NHBD liver, resulting in equal to better wash-out as compared with UW and HTK. The best temperature for this NHBD wash-out is 37 degrees C.

Adenosine↗

PubMed2006

Assessment of in vitro applicability of reversibly immortalized NKNT-3 cells and clonal derivatives.

In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4-20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.

Cell Culture Techniques↗

PubMed2005

Mild hypothermic preservation for transport purposes of the AMC bioartificial liver charged with porcine hepatocytes.

BACKGROUND: Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. METHODS: Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37 degrees C. Bioreactors in the transport group were kept at 37 degrees C during day 1, at 15 degrees C during day 2, and again at 37 degrees C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15 degrees C and 26 degrees C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. RESULTS: A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. CONCLUSIONS: Mild hypothermic preservation at temperatures as low as 15 degrees C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.

Albumins↗

PubMed2005

Evidence for Galalpha(1-3)Gal expression on primary porcine hepatocytes: implications for bioartificial liver systems.

BACKGROUND/AIMS: To bridge acute liver failure (ALF) patients to orthotopic liver transplantation, several bioartificial liver (BAL) systems have been developed. The bio-component of most BAL systems consists mainly of porcine hepatocytes. Plasma or blood of ALF patients is perfused through the BAL thereby contacting porcine hepatocytes. Xenogeneic BAL systems may suffer from hyperacute rejection similar to whole-organ xenotransplants. Hyperacute rejection is mediated by antibodies directed against Galalpha(1-3)Gal, a carbohydrate structure present on most mammalian cells. Galalpha(1-3)Gal is produced by the enzyme alpha1,3-galactosyltansferase (alphaGal-T). Conflicting data have been published concerning Galalpha(1-3)Gal expression on hepatocytes in intact porcine liver. We investigated whether isolated porcine hepatocytes express Galalpha(1-3)Gal. METHODS: Immunofluorescence, flow cytometry, RT-PCR and enzyme activity assays were performed on freshly isolated and cultured porcine hepatocytes and liver biopsies. Anti-Galalpha(1-3)Gal antibodies were measured in plasma from patients treated with BAL by ELISA. RESULTS: Isolated porcine hepatocytes express (alphaGal-T) at low levels and Galalpha(1-3)Gal is present in low quantities on these cells, in contrast to hepatocytes in situ. Furthermore, IgG and IgM anti-Galalpha(1-3)Gal are depleted from the plasma of ALF patients during BAL treatment. CONCLUSIONS: Isolation and culture of porcine hepatocytes induce Galalpha(1-3)Gal expression, which may elicit immunological responses potentially compromising BAL functionality.

Animals↗