Rene Tolba

Experimental liver fibrosis research: update on animal models, legal issues and translational aspects.

Liver fibrosis is defined as excessive extracellular matrix deposition and is based on complex interactions between matrix-producing hepatic stellate cells and an abundance of liver-resident and infiltrating cells. Investigation of these processes requires in vitro and in vivo experimental work in animals. However, the use of animals in translational research will be increasingly challenged, at least in countries of the European Union, because of the adoption of new animal welfare rules in 2013. These rules will create an urgent need for optimized standard operating procedures regarding animal experimentation and improved international communication in the liver fibrosis community. This review gives an update on current animal models, techniques and underlying pathomechanisms with the aim of fostering a critical discussion of the limitations and potential of up-to-date animal experimentation. We discuss potential complications in experimental liver fibrosis and provide examples of how the findings of studies in which these models are used can be translated to human disease and therapy. In this review, we want to motivate the international community to design more standardized animal models which might help to address the legally requested replacement, refinement and reduction of animals in fibrosis research.

Immune Cell–Poor Melanomas Benefit from PD-1 Blockade after Targeted Type I IFN Activation

UNLABELLED Infiltration of human melanomas with cytotoxic immune cells correlates with spontaneous type I IFN activation and a favorable prognosis. Therapeutic blockade of immune-inhibitory receptors in patients with preexisting lymphocytic infiltrates prolongs survival, but new complementary strategies are needed to activate cellular antitumor immunity in immune cell-poor melanomas. Here, we show that primary melanomas in Hgf-Cdk4(R24C) mice, which imitate human immune cell-poor melanomas with a poor outcome, escape IFN-induced immune surveillance and editing. Peritumoral injections of immunostimulatory RNA initiated a cytotoxic inflammatory response in the tumor microenvironment and significantly impaired tumor growth. This critically required the coordinated induction of type I IFN responses by dendritic, myeloid, natural killer, and T cells. Importantly, antibody-mediated blockade of the IFN-induced immune-inhibitory interaction between PD-L1 and PD-1 receptors further prolonged the survival. These results highlight important interconnections between type I IFNs and immune-inhibitory receptors in melanoma pathogenesis, which serve as targets for combination immunotherapies. SIGNIFICANCE Using a genetically engineered mouse melanoma model, we demonstrate that targeted activation of the type I IFN system with immunostimulatory RNA in combination with blockade of immune-inhibitory receptors is a rational strategy to expose immune cell-poor tumors to cellular immune surveillance.

Increasing concentrations of prothrombin complex concentrate induce disseminated intravascular coagulation in a pig model of coagulopathy with blunt liver injury

Despite increasing use of prothrombin complex concentrate (PCC) to treat hemorrhage-associated coagulopathy, few studies have investigated PCC in trauma, and there is a particular lack of safety data. This study was performed to evaluate PCC therapy in a porcine model of coagulopathy with blunt liver injury. Coagulopathy was induced in 27 anesthetized pigs by replacing approximately 70% blood volume with hydroxyethyl starch 130/0.4 and Ringers lactate solution; erythrocytes were collected and retransfused. Ten minutes after trauma, animals randomly received PCC (35 or 50 IU/kg) or saline. Coagulation parameters including thromboelastometry, thrombin generation, and blood loss were monitored for 2 hours. Internal organs were examined macroscopically and histologically to determine the presence of emboli and assess liver injury. Total blood loss was significantly lower and survival was higher in both PCC groups versus the control group (P < .05). These outcomes appeared to be dose-independent. Thromboembolism was found in all animals treated with 50 IU/kg PCC; 44% also showed signs of disseminated intravascular coagulation. Liver injury was similar in all animals. In conclusion, 35 IU/kg PCC safely improved coagulation and attenuated blood loss. However, the higher dose of PCC (50 IU/kg) appeared to increase the risk of thromboembolism and disseminated intravascular coagulation.

Cold preservation of fatty liver grafts : prevention of functional and ultrastructural impairments by venous oxygen persufflation

BACKGROUND/AIMS The incidence of steatosis in livers retrieved for organ transplantation is up to 30%. Due to the shortage of donor organs, many of these livers are accepted for clinical transplantation, although a high rate of graft dysfunction is associated with ischemic preservation of steatotic livers. The present study was intended to reduce the ischemia/reperfusion injury of steatotic grafts by the use of venous systemic oxygen persufflation during cold storage. METHODS A histologically-documented mild to moderate steatosis was induced in livers of Wistar rats by fasting for 2 days and subsequent feeding of a fat-free diet enriched in carbohydrates. Fatty livers were retrieved and flushed via the portal vein with 60 ml of HTK. In group A, livers were then stored ischemically at 4 degrees C for 24 h. Livers of group B were additionally connected to a gaseous oxygen supply and persufflated with O2 via the venous vascular system during the cold storage period. Viability of the livers was then assessed upon isolated perfusion in vitro with oxygenated Krebs-Henseleit buffer. RESULTS Venous systemic oxygen sufflation resulted in a relevant and significant reduction of parenchymal (ALT: 132+/-90 vs 434+/-172 U/l; p<0.01) and mitochondrial (GLDH: 116+/-57 vs 633+/-241 U/l; p<0.001) enzyme release during reperfusion. Moreover, Kupffer cell activation, as evaluated from acid phosphatase activity in the perfusate, was reduced to about 1/3 (4.0+/-1.3 vs 11.9+/-5.3 U/l; p<0.01). Electron microscopic analysis revealed that the liver mitochondria and sinusoidal endothelial lining were better preserved after oxygen persufflation, which was in line with the data on enzyme release and the increased portal perfusion pressure in the untreated group, while normal values were found after venous systemic oxygen sufflation. CONCLUSION Venous oxygen persufflation may thus represent a useful tool for the safe and improved preservation of ischemia-sensitive steatotic livers.

Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation

There is a clear clinical requirement for the design and development of living, functional, small-calibre arterial grafts. Here, we investigate the potential use of a small diameter, tissue-engineered artery in a pre-clinical study in the carotid artery position of sheep. Small-calibre ( approximately 5 mm) vascular composite grafts were molded using a fibrin scaffold supported by a poly(L/D)lactide 96/4 (P(L/D)LA 96/4) mesh, and seeded with autologous arterial-derived cells prior to 28 days of dynamic conditioning. Conditioned grafts were subsequently implanted for up to 6 months as interposed carotid artery grafts in the same animals from which the cells were harvested. Explanted grafts (n = 6) were patent in each of the study groups (1 month, 3 months, 6 months), with a significant stenosis in one explant (3 months). There was a complete absence of thrombus formation on the luminal surface of grafts, with no evidence for aneurysm formation or calcification after 6 months in vivo. Histological analyses revealed remodeling of the fibrin scaffold with mature autologous proteins, and excellent cell distribution within the graft wall. Positive vWf and eNOS staining, in addition to scanning electron microscopy, revealed a confluent monolayer of endothelial cells lining the luminal surface of the grafts. The present study demonstrates the successful production and mid-term application of an autologous, fibrin-based small-calibre vascular graft in the arterial circulation, and highlights the potential for the creation of autologous implantable arterial grafts in a number of settings.

The isolated perfused rat liver: standardization of a time-honoured model

For many years, the isolated perfused rat liver (IPRL) model has been used to investigate the physiology and pathophysiology of the rat liver. This in vitro model provides the opportunity to assess cellular injury and liver function in an isolated setting. This review offers an update of recent developments regarding the IPRL set-up as well as the viability parameters that are used, with regards to liver preservation and ischaemia and reperfusion mechanisms. A review of the literature was performed into studies regarding liver preservation or liver ischaemia and reperfusion. An overview of the literature is given with particular emphasis on perfusate type and volume, reperfusion pressure, flow, temperature, duration of perfusion, oxygenation and on applicable viability parameters (liver damage and function). The choice of IPRL set-up depends on the question examined and on the parameters of interest. A standard technique is cannulation of the portal vein, bile duct and caval vein with pressure-controlled perfusion at 20 cm H2O (15 mmHg) to reach a perfusion flow of approximately 3 mL/min/g liver weight. The preferred perfusion solution is Krebs–Henseleit buffer, without albumin. The usual volume is 150–300 cm3, oxygenated to a pO2 of more than 500 mmHg. The temperature of the perfusate is maintained at 37°C. Standardized markers should be used to allow comparison with other experiments.

The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves.

The use of bioengineered nerve guides as alternatives for autologous nerve transplantation (ANT) is a promising strategy for the repair of peripheral nerve defects. In the present investigation, we present a collagen-based micro-structured nerve guide (Perimaix) for the repair of 2 cm rat sciatic nerve defects. Perimaix is an open-porous biodegradable nerve guide containing continuous, longitudinally orientated channels for orientated nerve growth. The effects of these nerve guides on axon regeneration by six weeks after implantation have been compared with those of ANT. Investigation of the regenerated sciatic nerve indicated that Perimaix strongly supported directed axon regeneration. When seeded with cultivated rat Schwann cells (SC), the Perimaix nerve guide was found to be almost as supportive of axon regeneration as ANT. The use of SC from transgenic green-fluorescent-protein (GFP) rats allowed us to detect the viability of donor SC at 1 week and 6 weeks after transplantation. The GFP-positive SC were aligned in a columnar fashion within the longitudinally orientated micro-channels. This cellular arrangement was not only observed prior to implantation, but also at one week and 6 weeks after implantation. It may be concluded that Perimaix nerve guides hold great promise for the repair of peripheral nerve defects.

Systemic antigen cross-presented by liver sinusoidal endothelial cells induces liver-specific CD8 T-cell retention and tolerization†

Peripheral CD8 T‐cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T‐cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen‐presenting cells in the liver or spleen, leads to cross‐presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen‐specific naïve CD8 T‐cell retention in the liver but not in other organs. Using bone‐marrow chimeras and a novel transgenic mouse model (Tie2‐H‐2Kb mice) with endothelial cell‐specific MHC I expression, we provide evidence that cross‐presentation by organ‐resident and radiation‐resistant LSECs in vivo was both essential and sufficient to cause antigen‐specific retention of naïve CD8 T‐cells under noninflammatory conditions. This was followed by sustained CD8 T‐cell proliferation and expansion in vivo, but ultimately led to the development of T‐cell tolerance. Conclusion: Our results show that cross‐presentation of circulating antigens by LSECs caused antigen‐specific retention of naïve CD8 T‐cells and identify antigen‐specific T‐cell adhesion as the first step in the induction of T‐cell tolerance. (HEPATOLOGY 2009.)

Additive manufacturing for microvascular reconstruction of the mandible in 20 patients

OBJECTIVES The aim of this study was to evaluate the use of model mandibles made preoperatively by additive manufacturing, which were used to prebend reconstruction plates prior to mandibular resection and reconstruction with microvascular bony flaps. MATERIALS AND METHODS Computer Tomography (CT) or Cone Beam Tomography (CBT) scans acquired preoperatively were used to obtain DICOM data sets to produce a model of the mandible using rapid prototyping. This model was used as a template to prebend and then sterilize a 2.3 or 2.7 reconstruction plate, which was used to reconstruct the mandible with a microvascular bony flap. This technique was used in 20 consecutive patients who required mandibular resection and reconstruction because of a tumour or osteoradionecrosis. RESULTS The prebent plate was used in all patients intraoperatively without the need for any further bending. The average time to bend a plate on a nonsterile model was 0.42 h (range 0.25-0.68 h). This is felt to represent the minimum amount of time saved during the operation. Additive manufacture of the mandible prior to resection and reconstruction with a microvascular flap is a useful technique which reduces the operating time.

Effects of different fibrinogen concentrations on blood loss and coagulation parameters in a pig model of coagulopathy with blunt liver injury

IntroductionThe early application of fibrinogen could potentially reverse haemodilution-induced coagulopathy, although the impact of varying concentrations of fibrinogen to reverse dilutional coagulopathy has not been studied in vivo. We postulated that fibrinogen concentration is correlated with blood loss in a pig model of coagulopathy with blunt liver injury.MethodsCoagulopathy was induced in 18 anaesthetized pigs (32 ± 1.6 kg body weight) by replacing 80% of blood volume with hydroxyethylstarch 130/0.4 and Ringers lactated solution, and re-transfusion of erythrocytes. Animals were randomly assigned to receive either 70 mg kg-1 (F-70) or 200 mg kg-1 (F-200) fibrinogen or placebo before inducing blunt liver injury using a force of 225 ± 26 Newton. Haemodynamics, coagulation parameters and blood loss were monitored for 2 hours. After death, histological examination of internal organs was performed to assess the presence of emboli and the equality of liver injury.ResultsPlasma dilution caused severe coagulopathy. Measured by thromboelastography fibrinogen restored coagulation dose-dependently. Total blood loss was significantly lower and survival better in both fibrinogen groups as compared to controls (P < 0.05). Between the F-70 (1317 ± 113 ml) and the F-200 group (1155 ± 232 ml) no significant difference in total blood loss could be observed, despite improved coagulation parameters in the F-200 group (P < 0.05). Microscopy revealed even injury pattern and no (micro) thrombi for either group.ConclusionsRestoring fibrinogen with 70 or 200 mg kg-1 after severe dilutional coagulopathy safely improved coagulation and attenuated blood loss after experimental blunt liver trauma. The higher dosage of fibrinogen was not associated with a further reduction in blood loss.