Ralf Bahde

Impact of rapamycin on liver regeneration

The remarkable capacity of the liver to regenerate after injury and the prospects of organ self-renewal have attracted much interest in the understanding and modulation of the underlying molecular events. We investigated the effect of mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on liver by correlating intravital microscopy, immunohistochemistry, and reverse transcriptase polymerase chain reaction in a rat model of 2/3 hepatectomy. RAPA significantly retarded proliferation of hepatocytes, endothelial cells, and hepatic stellate cells (HSCs) mostly between days 2 and 4 after hepatectomy and down-regulated major cytokines and growth factors (tumor necrosis factor alpha, hepatocyte growth factor, platelet-derived growth factor, platelet-derived growth factor receptor, insulin-like growth factor-1, transforming growth factor beta 1) important for liver regeneration. These effects were almost absent at later time points. RAPA also had a transient, but broad effect on angiogenesis, and impaired sinusoidal density as well as mRNA levels of vascular endothelial growth factor, vascular endothelial growth factor receptor 1, vascular endothelial growth factor receptor 2, and angiopoietin-1. Activation of HSC was also transiently suppressed as observed by smooth muscle protein 1 alpha protein expression and intercellular adhesion molecule-1 mRNA levels. The rate of apoptosis in liver was significantly increased by RAPA between day 3 and day 7. The effect of RAPA on liver repair, angiogenesis, and HSC activation is confined to the phase of active cell proliferation. This transient effect might allow further exploration of mTOR inhibitors in clinical situations that involve liver regeneration, and seems to have implications beyond immunosuppression.

Type of Steatosis Influences Microcirculation and Fibrogenesis in Different Rat Strains

ABSTRACT This study investigates the impact of rat strain on the development of nonalcoholic fatty liver disease (NAFLD) focusing on morphological features and microcirculation. Male rats of Lewis, Wistar, and Sprague Dawley (n = 6 per strain and group) were randomized into a high-fat group which was fed with a special high-fat nutrition for a 3-week period and a control group which received standard nutrition. Intravital microscopy was used for the evaluation of microcirculation and correlated to morphological changes using a fatty liver scoring system. All three strains receiving a high-fat diet developed a grade 3 steatosis (>66% liver cell steatosis). Whereas Lewis showed a solely microvesicular steatosis, Wistar developed a mixed form and Sprague Dawley showed a pure macrovesicular steatosis and the highest degree of fibrosis and hepatocyte damage. Microcirculatory results revealed that sinusoidal density was already affected by a microvesicular steatosis and decreased with increasing macrovesicular proportion (Lewis: 18%, Wistar: 31%, Sprague Dawley: 23%). The degree of steatosis correlates with reduced blood flow velocity in central veins as well as in sinusoids (Lewis: 28%, Wistar: 39%, Sprague Dawley 44%). The densities of hepatocytes and hepatic stellate cells were only impaired once macrovesicular cell steatosis (Wistar and Sprague Dawley) was present. The development of NAFLD in the rat revealed strain-specific morphological features correlating with microcirculatory changes that should be considered in further studies using these models.

The microRNA-200 family--a potential diagnostic marker in hepatocellular carcinoma?

Hepatocellular carcinoma (HCC) represents the main cause of death among patients with cirrhotic liver disease, but little is known about mechanisms of cirrhosis associated carcinogenesis. We investigated the diagnostic impact of microRNA‐200 (miR‐200) family members as important epigenetic regulators of epithelial–mesenchymal transition (EMT) to differentiate between patients with HCC and liver cirrhosis.

Influence of Heme Oxygenase-1 on Microcirculation After Kidney Transplantation

BACKGROUNDnCytoprotective proteins, such as heme oxygenase-1 (HO-1), play a decisive role in ischemia-reperfusion injury during kidney transplantation. The aim of this study was to investigate the impact of heme oxygenase-1 on microcirculation and on ischemia-reperfusion injury in an isogenic kidney transplantation rat model.nnnMATERIALS AND METHODSnSeventy male Lewis rats were distributed into three groups. In Group 1(control), the kidneys were only mobilized. In Groups 2 and 3, bilateral nephrectomy was performed, and a kidney from another Lewis rat was orthotopically transplanted on the left side. The donor animals in Group 3 received preconditioning with the HO-1 inductor hemin. 24 h after reperfusion graft function and morphology were examined. Microcirculation was investigated by in vivo microscopy of the renal surface 1 h after reperfusion.nnnRESULTSnHO-1 preconditioning led to significantly lower serum creatinine and serum urea, as well as less histological damage and inducible nitric oxide synthase expression. Microcirculation was improved by a significant enlargement of the vascular diameter and an increase of the capillary flow.nnnCONCLUSIONSnTreatment with hemin improves microcirculation by induction of HO-1 and reduces ischemia-reperfusion injury after kidney transplantation. HO-1 induction was shown to be a promising approach in the preconditioning of donor kidneys.

Prognostic factors for kidney allograft survival in the Eurotransplant Senior Program.

BACKGROUNDnThe shortage of organ donors has led to the introduction of the Eurotransplant Senior Program (ESP) to optimize the allocation of kidneys from elderly donors by age-matching. In the face of a rapidly aging population, identification of prognostic factors for kidney allograft survival within the ESP population will be of enormous significance.nnnMATERIAL AND METHODSnDonor and recipient data from 89 patients transplanted under the ESP protocol between 1999 and 2007 were retrospectively analyzed. Data were correlated with initial graft function, graft survival, acute rejection episodes, serum creatinine levels, glomerular filtration rates, and patient survival using univariate and multivariate analysis. Maximum follow-up was 5 years.nnnRESULTSnCold ischemia time (CIT) >16 hours, body mass index (BMI) ≥25 kg/m(2), and kidney re-transplantation were significant risk factors for delayed graft function (DGF). Odds ratio for primary non-function was significantly increased with prolonged CIT, BMI ≥25 kg/m(2), and duration of renal replacement therapy >69 months. CIT >15 h, DGF, and kidney re-transplantation were associated with poor graft survival (P<0.05).nnnCONCLUSIONSnRisk reduction (e.g., aiming at CIT <15 h) and close surveillance of patients at risk appear to be crucial for allograft survival in the ESP.

Attenuated Cold Storage Injury of Rat Livers Using a Modified HTK Solution

BACKGROUNDnPreservation injury is a main factor leading to graft failure in liver transplantation. The aim of the study was to minimize preservation injury by modifications of the histidine-tryptophan-ketoglutarate (HTK) solution (incorporation of N-acetyl-histidine, aspartate, glycine, alanine, and arginine).nnnMATERIALS AND METHODSnThe study was carried out in rats and subdivided into four parts. (1) Systemic toxicity of the modified HTK solution in comparison to the standard HTK solution was tested. (2) Hemodynamic and microcirculatory parameters were analyzed after i.v. injection of the respective solution. (3) Preservation injury after cold storage for different periods of time was assessed microscopically. (4) Reperfusion injury was analyzed in the isolated perfused liver by enzyme release and bile production.nnnRESULTSnBlood values, hemodynamic and microcirculatory parameters after i.v. and i.p. application did not significantly differ from control. The modified HTK solution led to an attenuated preservation injury after cold preservation for 24 h compared with standard HTK solution. There was a significantly decreased lactate dehydrogenase release after ischemia for 72 h and reperfusion using the modified solution. After 24 h cold storage and reperfusion the apoptosis index was reduced and bile production significantly increased.nnnCONCLUSIONnOptimizing the HTK solution may be a promising therapeutic strategy for attenuation of cold storage injury.

Gender and strain-specific differences in the development of steatosis in rats

Non-alcoholic fatty liver disease (NAFLD) is a common problem with a wide variety of phenotypes. While its pathogenesis is still not fully understood, several risk factors for disease progression have been identified. Therefore, defining adequate animal models may serve to unreveal the pathogenesis in NAFLD. We studied Lewis and Sprague-Dawley rats of both genders (n = 6) fed standard (Std) or high-fat (HF) diet for three weeks. Disease stage was assessed by haematoxylin–eosin, Azan Heidenheim and Oil-Red staining, apoptosis by single-stranded DNA (ssDNA) detection and liver regeneration by Ki-67 staining. Serum markers of liver injury and lipid metabolism including adipocytokines were analysed. Livers of both strains and genders fed with HF diet demonstrated evidence of steatosis. Lewis rats developed microvesicular steatosis whereas Sprague-Dawley rats presented macrovesicular steatosis accompanied by pronounced fibrosis. Female gender of both strains was associated with lower steatosis grade and higher proliferation rate (P < 0.05). Gender-specific differences were most prominent in Lewis rats on a HF diet, where females showed lower alkaline phosphatase, cholesterol, triglyceride and leptin levels and a more favourable low-density lipoprotein/high-density lipoprotein ratio than males (P < 0.05). Reverse transcriptase-polymerase chain reaction analysis was performed to demonstrate changes in expression of various genes important for liver regeneration, fibrosis and steatosis. HF diet induced downregulation of proangiogenic genes such as vascular endothelial growth factor receptor 1 and 2 (P < 0.05) in males was not present in females. In conclusion, strain and gender served major roles in disease progression. These differences should be considered when designing studies and may offer new ways to advance therapeutic strategies.

Microcirculatory dysfunction in endotoxemic bowel anastomosis: the pathogenetic contribution of microcirculatory dysfunction to endotoxemia-induced healing impairment.

BACKGROUNDnAn established intra-abdominal infection as in perforated diverticulitis is considered to contribute to anastomotic healing impairment. Since microvascular dysfunction in sepsis is known to be due to organ failure, the influence of inflammation on the anastomotic microcirculation needs further investigation.nnnMATERIAL AND METHODSnSixty BALB/c mice (n = 10 per group and day 2, 4, and 7) were randomized to two groups: Control and Sepsis (lipopolysaccharide administration 2 mg/kg bodyweight 18 h before colon surgery). All animals underwent colonic anastomosis. Immediately after its completion intravital fluorescence microscopy of the anastomosis was performed, and both macroscopic and histological parameters were assessed on days 2, 4, and 7 postoperatively. Additionally, immunohistology was performed for CD31 (platelet endothelial cell adhesion molecule-1), single-strand DNA, and inducible nitric oxide synthase.nnnRESULTSnAs compared to Control the functional capillary network of the perianastomotic region was decreased in Sepsis (P < 0.001) as well as the hemoglobin O(2) saturation in the antimesenteric region of the anastomosis (P < 0.05). Bursting pressure was significantly decreased in Sepsis compared to Control at days 2, 4, and 7. On day 7 there were significant differences between the two groups in the anastomotic region: neutrophil infiltration in Sepsis was higher (P < 0.001); vascular density and differentiation in Sepsis was lower (P < 0.01, P < 0.05, respectively); and apoptosis was higher in Sepsis (P < 0.05).nnnCONCLUSIONnThe inflammatory state increases microvascular dysfunction at the anastomosis resulting in healing impairment.

Repeated Transplantation of Hepatocytes Prevents Fulminant Hepatitis in a Rat Model of Wilson's Disease

The outcome of consecutive hepatocyte transplants was explored in a rat model of Wilsons disease before the onset of fulminant hepatitis without preconditioning regimens. Rats received a high‐copper diet in order to induce a rapid induction of liver failure. Sham‐operated rats (15/15) developed jaundice and fulminant hepatitis, and they died within 4 weeks of first transplantation. Despite the continuation of a high dietary copper challenge, long‐term survival was observed for a notable proportion of the transplanted animals (7/18). All survivors displayed normalized levels of hepatitis‐associated serum markers and ceruloplasmin oxidase activity by posttransplant days 50 and 98, respectively. The liver copper concentrations, the liver histology, and the expression of marker genes were significantly restored within 4 months of transplantation in comparison with the control group. The high expression of a copper transporter gene (ATPase Cu++ transporting beta polypeptide) in the livers of the survivors indicated a high rate of repopulation by donor hepatocytes. Our data suggest that repeated cell transplantation can overcome the limitations of a single therapy session in rats with severe hepatic disease by functionally restoring the host liver without preconditioning. Liver Transpl 18:248–259, 2012.

Role of angiotensin-1 receptor blockade in cirrhotic liver resection

Background: The regeneration capacity of cirrhotic livers might be affected by angiotensin‐1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated.