Microsurgical training course for clinicians and scientists: a 10-year experience at the Münster University Hospital.

BACKGROUND: Microsurgical techniques are an important part of clinical and experimental research. Here we present our step-by-step microsurgery training course developed at the Münster University Hospital. The goal of this course was to create a short, modular curriculum with clearly described and easy to follow working steps in accordance with the Guidelines for Training in Surgical Research in Animals by the Academy of Surgical Research. METHODS: Over the course of 10 years, we conducted an annual 2.5 day (20 h) microsurgical training course with a total of 120 participants. RESULTS: Prior to the course, 90% of the participants reported to have never performed a microanastomosis before. During the 10 years a total of 84.2% of the participants performed microanastomoses without assistance, 15% required assistance and only 0.8% failed. CONCLUSIONS: Our step-by-step microsurgery training course gives a brief overview of the didactic basics and the organization of a microsurgical training course and could serve as a guide for teaching microsurgical skills. During the 2.5-day curriculum, it was possible to teach, and for participants to subsequently perform a microsurgical anastomosis. The independent reproducibility of the learned material after the course is not yet known, therefore further investigations are necessary. With this step-by-step curriculum, we were able to conduct a successful training program, shown by the fact that each participant is able to perform microvascular anastomoses on a reproducible basis.

Dietary copper triggers onset of fulminant hepatitis in the Long-Evans cinnamon rat model.

AIM: To investigate the impact of dietary copper given at different time points on the onset of fulminant hepatitis. METHODS: The Long-Evans cinnamon (LEC) rat model of Wilson's disease (WD) was used to study the impact of high dietary copper (hCu) on the induction of fulminant hepatitis at early or late time points of life. High Cu diet was started in rat pups or in adults (month 5) for three months. Animals that received reduced dietary copper (rCu) throughout their lifetime served as a control. Hepatitis-associated serum markers (alanine aminotransferase, aspartate transaminase, bilirubin) were analyzed in animal groups receiving hCu or rCu. Liver copper content and liver histology were revealed at sacrifice. A set of 5 marker genes previously found to be affected in injured liver and which are related to angiogenesis (Vegfa), fat metabolism (Srebf1), extracellular matrix (Timp1), oxidative stress (Hmox1), and the cell cycle (Cdkn1a) were analyzed by real-time polymerase chain reaction. RESULTS: Regardless of the time point when hCu was started, LEC rats (35/36) developed fulminant hepatitis and died. Animals receiving rCu (36/36) remained healthy, did not develop hepatitis, and survived long term without symptoms of overt disease, although liver copper accumulated in adult animals (477 ± 75 µg/g). With regard to start of hCu, onset of fulminant hepatitis was significantly (P < 0.001) earlier in adults (35 ± 9 d) that showed pre-accumulation of liver copper as compared to the pup group (77 ± 15 d). Hepatitis-associated serum markers, liver copper and liver histology, as well as gene expression, were affected in LEC rats receiving hCu. However, except for early and rapid onset of hepatitis, biochemical and molecular markers were similar at the early and late time points of disease. CONCLUSION: Rapid onset of fulminant hepatitis in asymptomatic LEC rats with elevated liver copper suggests that there is a critical threshold of liver copper which is important to trigger the course of WD.

Longitudinal analysis of serum miR-122 in a rat model of Wilson's disease.

PURPOSE: MicroRNA-122 (miR-122) has recently been shown to represent a novel biomarker of liver disease. However, the presence of serum miR-122 after liver injury was mostly studied at singular time points. The course of serum miR-122 was determined at consecutive time points during the onset of disease. METHODS: Fulminant hepatitis was induced by a high-copper diet in Long-Evans Cinnamon (LEC) rats that were used as models for Wilson's disease (WD). Levels of serum miR-122, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and liver histology were determined. RESULTS: Toxic copper given to isolated hepatocytes induced release of miR-122 into the tissue culture medium. Levels of serum miR-122 were highly elevated (21.9 ± 5) in LEC rats after high-copper diet in fulminant hepatitis, whereas healthy rats showed low (<0.6) baseline levels of miR-122. Levels of miR-122 in the serum of LEC rats after high-copper diet continuously increased for about 4 weeks prior to the onset of fulminant hepatitis. In most of the animals (77.8%), significantly increased levels of miR-122 were detected about 2 weeks (13.7 ± 2 days) earlier as compared to hepatitis-associated serum markers ALT, AST, and bilirubin. Analysis of miR-122 in survivors after cell-based therapy of WD demonstrated a rapid decrease of miR-122 levels following hepatocyte transplantation. miR-122 expression in the serum was normalized to baseline levels in most of the (4/5) survivors. CONCLUSION: Our results suggest that longitudinal analysis of miR-122 allows detection of severe liver disease at an early stage and might be excellently suited to monitor therapy, at least when severe liver disease can be restored as observed after cell-based therapy of WD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12072-012-9348-5) contains supplementary material, which is available to authorized users.

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 Wilson's 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.

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. MATERIALS AND METHODS: In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl(4) . Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre-operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki-67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. RESULTS: Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time-points and of non-parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)-α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). CONCLUSION: Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.

A new technique for ureteral defect lesion reconstruction using an autologous vein graft and a biodegradable endoluminal stent.

PURPOSE: Ureteral defect lesions are severe complications caused by iatrogenic lesions or trauma. For ureteral defect lesions elaborate surgical intervention is needed, such as autotransplantation or ureteral replacement with small bowel. Thus, we developed a new technique for ureteral defect reconstruction in a pig model using an autologous vein graft splinted by an endoluminal biodegradable poly-L-lactic acid stent (Institute of Textile Technology and Process Engineering, Denkendorf, Germany). MATERIALS AND METHODS: In 42 pigs we removed the external jugular vein and used it as an autologous vein graft. After median laparotomy a 3 cm segment was resected from the proximal ureter and replaced by the vein with or without an endoluminal biodegradable poly-L-lactic acid stent. As controls, we used 14 pigs. We observed survival, kidney function, and neoureteral and kidney morphological changes for 7 days and for 6 months. RESULTS: After 6 months the stent material was completely broken down and the vein graft was relined with urothelium. It resembled native ureter with cytokeratin-7 positive columnar epithelium and newly formed capillaries in the ureteral wall. All animals had normal kidney function without renal pelvis congestion. CONCLUSIONS: This new technique for ureteral defect reconstruction using an autologous vein graft and a biodegradable endoluminal stent is feasible. It is an interesting alternative in the clinic due to the preservation of physiological urine passage and the antireflux mechanism.