Christian Grosse-Siestrup

In vitro models to study hepatotoxicity.

Drug discovery and development consists of a series of processes starting with the demonstration of pharmacologica l effects in experimental cell and animal models and ending with drug safety and effi cacy studies in patients. A main limitation is often the unacceptabl e level of toxicity with the liver as the primary target organ. Therefore, approaches to study hepatic toxicity in the early phase of drug discovery represent an important step towards rational drug development. A variety of in vitro liver model s have been developed in the past years. Next to their use in drug development, they can also be applied to study environmental toxins and their hepatotoxicity. The 3 main approache s are ex vivo isolated and perfused organ models, precision-cut liver slices and cell culture models. Although the advantage of whole organ perfusions is based on the assessment of physiologic parameters such as bile production and morphologic parameters such as tissue histology, cell culture models can be effi ciently used to assess cellular metabolism, cytotoxicity and genotoxicity. The advantage of precision-cut liver slices is based on the juxtaposition of cellular assays and tissue morphology. None of these model s can be compared as they all focus on different fi elds of hepatoxicolog y. For the future, the ideal setup for testing the hepatic toxicity of a new compound could of primary studies in cell or slice cultures to assess cellular effects and secondary studies using ex vivo perfused organs to examine gross organ function parameters and histology.

A model of isolated autologously hemoperfused porcine slaughterhouse kidneys.

Background: The rapidly evolving field of transplantation research with a focus on ischemic and reperfusion injuries has gained importance since the methodology of organ preservation significantly limits graft survival. Numerous models of isolated perfused kidneys have been established in the past years but limitations such as organ size, perfusate and ethical standards have restricted a widespread research in this area. Methods: A model of hemoperfused isolated porcine slaughterhouse kidneys was established which encompasses the advantages of autologous blood as optimal perfusate and a reduction of animal experiments. Results: The size and geometry of the porcine kidney is more comparable to human conditions and various renal functions, blood parameters and morphology can easily be accessed in the present model. Stable organ function can be maintained over 2 h with an amount of 500–1,000 ml of autologous blood which is metabolically controlled via a dialysis system. Conclusion: In summary, the present model describes a new and economic approach for targeting renal function in transplantation models by combining autologous blood as optimal perfusate with a well-defined organ geometry and function and slaughterhouse animals as a source.

Hydroxyethyl starch 130 kd/0.4 and albumin improve CVVH biocompatibility whereas gelatin and hydroxyethyl starch 200 kd/0.5 lead to adverse side effects of CVVH in anesthetized pigs.

Both fluid management and renal replacement therapies play a fundamental role in the treatment of critically ill patients. In a recent in vitro study, we have shown specific interactions of different colloids and the hemocompatibility of hemofilters. The present study was performed to compare the five most common fluids for volume resuscitation, i.e., normal saline (SAL), hydroxyethyl starch 130 kd/0.4 (HES130), hydroxyethyl starch 200 kd/0.5 (HES200), albumin (ALB), and gelatin (GEL) with respect to their interaction with continuous venovenous hemofiltration (CVVH) in anesthetized domestic pigs. Methods Animals (n = 63) were allocated randomly to the fluid type and the respective subgroups, which were divided into control and CVVH groups (n = 6 ndash; 7 per group). Bolus infusion of group specific fluid was followed by a bolus of heparin and the initiation of hemofiltration in CVVH groups. Thereafter, fluids were infused at constant rates, and heparin application was adjusted to keep the activated clotting time at 200 to 250 s. Hemodynamics, airway pressures, pulmonary gas exchange, diuresis, creatinine clearance, and blood cell counts were investigated during the entire procedure (10 ndash; 12 hours). Results Basics of in vivo effects of SAL, HES130, and ALB were not altered during CVVH. HES130 and ALB enabled stable hemocompatibility, diuresis, and hemodynamics in the respective groups. In contrast, organ functions were significantly different between control and CVVH groups when animals were treated with GEL or HES200. In particular, during CVVH, HES200 led to reduced platelet counts, deteriorated hemodynamics, and increasing airway pressures during CVVH. GEL led to increasing airway pressures, a decrease in pulmonary gas exchange, deteriorated hemodynamics, altered renal histomorphology, reduced platelet counts, and reduced hemoglobin. Conclusions Direct in vivo effects of colloids in anaesthetized and ventilated pigs are not predictable for their effects during CVVH. Interaction between CVVH and every volume substitute occur in a highly specific manner. This observation could be helpful to explain contradictory study results and should be considered for future study designs.

Isolated Hemoperfused Slaughterhouse Livers as a Valid Model to Study Hepatotoxicity

Different models of isolated and perfused livers and precision cut liver slices have been developed for studies on liver toxicology the past years. As most of these models were limited by nonphysiologi c settings, a new model of normothermic hemoperfused isolated porcine slaughterhous e livers to examine hepatotoxicity was established encompassing the advantages of slaughterhouse organs to reduce animal experiments and autologous blood as an optimal perfusate. As model compound, the analgesic substance diclofenac was used and the effects of this drug on organ function parameters were compared to an untreated control group. Using an amount of 2,000 ml, the organs were perfused over 180 minutes, metabolically controlled via a dialysis and oxygenation system and various hematological and hepatic parameters were examined. In contrast to the untreated control organs, significant differences were found in the diclofenac group for parameters such as lactate, creatinine, ALT, bicarbonate, or bile flow. In summary, the presently established model of isolated hemoperfused slaughterhouse livers displays a useful new approach to assess hepatotoxicity of different substances on the organ level. As a major economic advantage in comparison to setups using laboratory animals, the new model can be run with blood and organs obtained from slaughterhouse animals.

An Improved Model of Isolated Hemoperfused Porcine Livers Using Pneumatically Driven Pulsating Blood Pumps

Existing liver perfusion models are largely limited by high degrees of ischemic and reperfusion injury and the lack of standardization. To establish a highly standardized perfusion model and minimize reperfusion injury, a porcine liver perfusion model was developed using an artificial heart pump (Buecherl Artificial Heart). This model is characterized by pneumatically driven and pressure controlled blood pumps with pulsating flow characteristics. The perfusion parameters and the integrity of the perfused organ were assessed using hemodynamic and hepatic function tests. In eight porcine liver perfusion experiments the system allowed maintaining stable and physiologic organ function over 3 hours by bile production (5.5 ±3.1 ml/30 minutes, resp. 22.9 ±8.4 ml cumulative at 180 minutes), oxygen consumption (2.2 ±0.2 ml/min/100 g overall mean) and significantly better liver enzyme levels (AST 19.5 ± 10.1 U/l/100 g, ALT 2.1 ± 0.8 U/l/min, LDH 57.8 ± 24.2 U/l/100 g) compared to previous studies. It was also possible to reduce the circulating blood volume to 1,000 ml and to create a compact perfusion system that is adoptable to other organ systems such as the kidneys. The compact size and the absence of magnetic components also allow a use for advanced imaging techniques. In conclusion this optimized perfusion system provides a sound basis for future studies in the area of hepatotoxicity and pharmacology.

Protective Effects of B2 Preservation Solution in Comparison to a Standard Solution (Histidine-Tryptophan-Ketoglutarate/Bretschneider) in a Model of Isolated Autologous Hemoperfused Porcine Kidney

Reperfusion injuries after organ transplantation affect graft function and influence long-term graft survival. As hypothermic storage, which minimizes the extent of unspecific tissue injury after ischemia and reperfusion, is significantly influenced by the composition of preservation solutions, strategies to optimize the different components may lead to longer graft survival. In the present study the effects of the preservation solution B2 on early renal function and histopathological changes were compared to histidine-tryptophan-ketoglutarate solution (HTK, Bretschneider) in a model of isolated blood-perfused porcine kidneys. B2-preserved kidneys displayed a lower renal resistance and significantly better creatinine clearance as compared to HTK. Mean differences were also found for filtration fraction and sodium fraction reabsorption. The functional data were also related to histopathological changes. Together, these data indicate that the recently developed preservation solution B2 offers new principles of preservation and is a useful preservation solution for experimental isolated perfused kidney models. B2 may also be an interesting model for optimizing preservation within other organ perfusion models.

How Can We Achieve Infection-Resistant Percutaneous Energy Transfer?

Clinical records show ever increasing functional times of rotary blood pumps implanted in patients. With longer functional time, the problem of driveline infection is becoming more urgent. No material or scaffold has been found, which allows a permanent and stable ingrowth of skin cells that would prevent (pathogenic) germs entering the body. Usually, the epithelial cells die at the exit site and new cells form a sulcus around the driveline, which grows deeper and finally becomes infected. The purpose of this project is to present a solution to this problem by elaborating a new mechanism, the active skin-penetrating device. The device is composed of a tube with a 5-mm diameter, a protective sleeve that surrounds the catheter exit site, and an active traction device. The protective sleeve is made of thin polyurethane covered with polyethylenterephtalat (PET, i.e. Dacron) fibers to permit the attachment of keratinocytes, similar to the standard driveline. The active traction device exerts a constant pull on the protective sleeve. The ingrown keratinocytes slowly give way and the protective sleeve gradually moves out of the body at a rate of a few millimeters per week. Meanwhile, the keratinocytes transform into horny cells and are then shed as in natural skin. Therefore, the formation of a sulcus is avoided, and the protective sleeve remains infection-free. In a first proof of the concept, four of the new devices and 10 control devices were implanted in goats. The devices remained infection-free for a period of 420 days, whereas four of the 10 control devices became infected. On the basis of these experiments, the active skin-penetrating device has been further developed and is being tested again in goats in a refined version. The results so far indicate that with the active-skin penetrating device an infection-resistant percutaneous energy transfer can be achieved for a prolonged period of time.