Hepatocyte Transplantation to the Liver via the Splenic Artery in a Juvenile Large Animal Model.

Hepatocyte transplantation (HcTx) is a promising approach for the treatment of metabolic diseases in newborns and children. The most common application route is the portal vein, which is difficult to access in the newborn. Transfemoral access to the splenic artery for HcTx has been evaluated in adults, with trials suggesting hepatocyte translocation from the spleen to the liver with a reduced risk for thromboembolic complications. Using juvenile Göttingen minipigs, we aimed to evaluate feasibility of hepatocyte transplantation by transfemoral splenic artery catheterization, while providing insight on engraftment, translocation, viability, and thromboembolic complications. Four Göttingen Minipigs weighing 5.6 kg to 12.6 kg were infused with human hepatocytes (two infusions per cycle, 1.00E08 cells per kg body weight). Immunosuppression consisted of tacrolimus and prednisolone. The animals were sacrificed directly after cell infusion (n=2), 2 days (n=1), or 14 days after infusion (n=1). The splenic and portal venous blood flow was controlled via color-coded Doppler sonography. Computed tomography was performed on days 6 and 18 after the first infusion. Tissue samples were stained in search of human hepatocytes. Catheter placement was feasible in all cases without procedure-associated complications. Repetitive cell transplantations were possible without serious adverse effects associated with hepatocyte transplantation. Immunohistochemical staining has proven cell relocation to the portal venous system and liver parenchyma. However, cells were neither present in the liver nor the spleen 18 days after HcTx. Immunological analyses showed a response of the adaptive immune system to the human cells. We show that interventional cell application via the femoral artery is feasible in a juvenile large animal model of HcTx. Moreover, cells are able to pass through the spleen to relocate in the liver after splenic artery infusion. Further studies are necessary to compare this approach with umbilical or transhepatic hepatocyte administration.

Computed tomography-based survey of the vascular anatomy of the juvenile Göttingen minipig.

Over the past 50 years, image-guided procedures have been established for a wide range of applications. The development and clinical translation of new treatment regimens necessitate the availability of suitable animal models. The juvenile Göttingen minipig presents a favourable profile as a model for human infants. However, no information can be found regarding the vascular system of juvenile minipigs in the literature. Such information is imperative for planning the accessibility of target structures by catheterization. We present here a complete mapping of the arterial system of the juvenile minipig based on contrast-enhanced computed tomography. Four female animals weighing 6.13 ± 0.72 kg were used for the analyses. Imaging was performed under anaesthesia, and the measurement of the vascular structures was performed independently by four investigators. Our dataset forms a basis for future interventional studies in juvenile minipigs, and enables planning and refinement of future experiments according to the 3R (replacement, reduction and refinement) principles of animal research.

Total body Na(+)-depletion without hyponatraemia can trigger overtraining-like symptoms with sleeping disorders and increasing blood pressure: explorative case and literature study.

Exhausting physical exercise and insufficient nutritional intake impairing immunological and neuro-endocrine pathways are the most discussed issues in research on overtraining syndrome (OTS). Interestingly, depletion of the total body sodium (Na(+))-content which occurs in case of various diseases with completely different aetiologies is associated with a symptom pattern strikingly comparable to overreaching (OR) and/or OTS. The transient dilution based hyponatraemia gained attention due to its impact on reduced performance and the death of various endurance athletes. But the stepwise depletion of the total body (tissue) Na(+)-content is a completely different pathophysiology and is still relatively unknown. That is because depleted tissue Na(+)-content is hard to detect. The complex, dominant mechanisms for the maintenance of plasma homeostasis are concealing the Na(+)-depletion in the tissues quite successfully in a stage when symptoms already may be prominent. Furthermore, we are all programmed to think about sedentary people who are rather at risk to have a salt (Na(+)) intake which is far too high. But either, competitive top athletes and engaged recreational athletes have high losses of electrolytes with sweat and might be prone to a stepwise Na(+)-depletion. All the more because they also try to have a balanced, health sodium reduced diet. One person of our research group who is used to a rather low sodium-nutrition repeatedly experienced OR-(short term-OTS)-symptoms when training loads of recreational sport activities were increasing. Getting aware about identical symptoms between OR and total body Na(+)-depletion in another professional context the decision for a self experiment was settled. Under a given training protocol changing symptoms under low sodium-nutrition were recorded. When OR-like symptoms became prominent the training loads were maintained but stringent Na(+)-substitution was performed instead of the usually recommended resting period. As experienced before, typical symptoms such as sleeping disorders, harassed feeling, high diuresis, thirst and increasing blood pressure developed within 2 weeks with the increased training loads and the usual low Na(+)-nutrition. This was before plasma sodium decreased below the physiological range. High Na(+)-substitution instead of a resting period enabled the recovery from OR symptoms within some days. Out of various articles we choose and report some interesting further medical phenomenon where our hypothesis of Na(+)-depletion as a trigger mechanism might give new ideas for identifying pathophysiological mechanisms. The hypothesis: Tissue Na(+)-depletion triggers OR- and OTS- development via the renin-angiotensin-aldosterone system which initiate at first a stimulation and then exhaustion of the sympathetic system.

The effect of prone versus supine positioning of Goettingen minipigs on lung density as viewed by computed tomography.

Minipigs are frequently used for scientific research as they are easy to handle and the dimensions of their vascular system do not change after 20 months of age. Although surgical interventions under anaesthesia are often performed in the supine position the effects of this positioning on lung functionality in minipigs have not been systematically described. This study aimed to analyse the influence of supine positioning on the macrostructure of the lung and the pulmonary density by the use of computed tomography imaging in pre-adult Göttingen minipigs. Twelve pre-adult female minipigs were used in the study and lung density was investigated in both the prone and the supine positions. The time between the scans in prone and supine positions was less than 5 minutes (296 ± 6 sec). In the prone position lung density did not differ between the dorsal and ventral part of the lung (-641 ± 72 Hounsfield units [HU]). However in the supine position there was a ventrodorsal gradient of decreasing density (ventral part of the lung: -497 ± 106 HU, dorsal part of the lung: -723 ± 51 HU). The changes in lung density were not accompanied by changes in lung volume (829 ± 191 ml). These results suggest an influence of the body position on the ventilation/perfusion (V(A)/Q) matching of the lung which could possibly result in lowered lung oxygenation as well as in an increased heart activity in the supine position. Additionally, due to the steep course of the vena cava caudalis from the caval foramen in the diaphragm across to the heart (in contrast to the more shallow course in the prone position) the activity of the heart necessary to pump the venous blood to the right atrium has to be higher in the supine position than in the prone position. In pigs the capacity of the heart to increase frequency is limited due to a diastolic/systolic (D/S) ratio <1. Supine positioning may possibly increase their risk of cardiovascular complications.

Pig specific vascular anatomy allows acute infrarenal aortic occlusion without hind limb ischemia and stepwise occlusion without clinical signs.

OBJECTIVE: In a porcine, aortic graft model we found 5 animals to develop and survive unnoticed, complete infrarenal aortic occlusion and 2 pigs with an acute occlusion but rather unspecific clinical symptoms. We investigated the pigs' vascular system to classify the anatomic capabilities to compensate for an acute abdominal aortic occlusion. DESIGN OF STUDY: Retrospective analysis of CT scans and clinical data to specify unexpected results in a case series of infrarenal occlusion in a porcine model. SETTING: Collaborative study of experimental and clinical departments. SUBJECTS: Fifteen healthy female minipigs. INTERVENTIONS: All pigs underwent an infrarenal aortic graft intervention. Anesthesia and perioperative management of the animals were preformed along the standard operating procedures of the local Department of Experimental Medicine. All animals received perioperative antibiotics, ASS, and carprofen for postoperative analgesia. Arterial pressure, heart rate, body temperature, and diuresis were monitored during surgery and therapeutic interventions. MAIN OUTCOME MEASURES: Contrast media based computed tomography (CT) with total body scans were performed at 1, 4, 10, 12 weeks after surgery. Comparable scans of cardiovascular healthy subjects (humans and pigs) and patients with a Leriche's syndrome were analyzed. RESULTS: Neither acute (within the first 12 h after surgery) nor stepwise total aortic occlusion show unmistakable clinical signs. In pigs, the epigastric artery (EGA) - which is in connection with suprarenal lumbal arteries, subclavian and external iliac artery - is highly developed associated to the high number of mammary glands of about 7 on one side. In humans, the ratio of aortic to EGA-diameter is 1 : 0.15. In minipigs we found a ratio of 1 : 0.43 which changed during aortic occlusion resulting in a ratio of 1 : 0.58. Pigs with a slowly developing occlusion demonstrated an enlargement of the ureteric artery of about 210% completing a sufficient collateral system. CONCLUSION: While in the human Leriche's syndrome months are needed to enlarge the EGAs for a partial collateral support of an infrarenal aortic occlusion the pig's EGA is a naturally sufficient collateral system capable to cover immediately for an acute infrarenal aortic occlusion. Further collateral enlargement even provides a permanent, sufficient hind limb perfusion in pigs. As the sufficient collateral system probably reduce pressure and shear rates in the infrarenal aortic segment after cross clamping, pigs might have a higher predisposition to produce early thrombosis related graft occlusions tan humans.

Liver volume measurement: reason of the difference between in vivo CT-volumetry and intraoperative ex vivo determination and how to cope it.

PURPOSE: Volumetric assessment of the liver regularly yields discrepant results between pre- and intraoperatively determined volumes. Nevertheless, the main factor responsible for this discrepancy remains still unclear. The aim of this study was to systematically determine the difference between in vivo CT-volumetry and ex vivo volumetry in a pig animal model. MATERIAL AND METHODS: Eleven pigs were studied. Liver density assessment, CT-volumetry and water displacement volumetry was performed after surgical removal of the complete liver. Known possible errors of volume determination like resection or segmentation borders were eliminated in this model. Regression analysis was performed and differences between CT-volumetry and water displacement determined. RESULTS: Median liver density was 1.07g/ml. Regression analysis showed a high correlation of r(2) = 0.985 between CT-volumetry and water displacement. CT-volumetry was found to be 13% higher than water displacement volumetry (p<0.0001). CONCLUSION: In this study the only relevant factor leading to the difference between in vivo CT-volumetry and ex vivo water displacement volumetry seems to be blood perfusion of the liver. The systematic difference of 13 percent has to be taken in account when dealing with those measures.

Ventral recumbency is crucial for fast and safe orotracheal intubation in laboratory swine.

The aim of this study was to find the fastest, easiest and safest method of achieving orotracheal intubation for general anaesthesia in laboratory pigs. Twenty-one Yorkshire x Landrace crossbreed male castrated pigs (32.9 +/- 4.8 kg) were investigated. Dorsal and ventral recumbency are the alternatives most frequently described for animal positioning during intubation procedures. Based on standardized induction of general anaesthesia using pentobarbital and remifentanil, the dorsoventral and ventrodorsal positions were compared with regard to the time needed, changes in oxygenation and circulatory response. Positioning was found to be crucial for fast orotracheal intubation. The time required for safe intubation is significantly shorter with the ventrodorsal position (17.3 s) in comparison with the dorsoventral position (58.4 s; P < 0.001). Hypoxia did not occur in either group. A significant drop in systolic blood pressure was observed in both groups. Diastolic and mean arterial pressures were not influenced by intubation. A significant increase in heart rate was observed in pigs intubated in ventral recumbency, but not after intubation in the dorsal position. Preoxygenation before intubation is vitally important for preventing hypoxia. With regard to clinical practice, the haemodynamic changes observed in this investigation do not appear to be relevant, as the mean arterial pressure was not altered and heart rates only increased moderately. It may be concluded that the ventrodorsal position can be recommended for orotracheal intubation in pigs as the first choice for providing a smooth and fast airway.

Buffer capacity of 4% succinylated gelatin does not provide any advantages over acidic 6% hydroxyethyl starch 130/0.4 for acid-base balance during experimental mixed acidaemia in a porcine model.

BACKGROUND AND OBJECTIVE: Four percent gelatine is an alkaline compound due to NH2 groups, whereas 6% hydroxyethyl starch 130/0.4 (HES130) has acidic features. We investigated whether these solutions lead to differences in acid-base balance in pigs during acidaemia and correction of pH. METHODS: Anaesthetized pigs were randomized to HES130 or gelatine infusion (n = 5 per group). Animals received acid infusion (0.4 M solution of lactic acid and HCl diluted in normal saline) and low tidal volume ventilation (6-7 mL kg(-1), PaCO2 of 80-85 mmHg, pH 7.19-7.24). Measurements were made before and after induction of acidaemia, before and after correction of pH with haemofiltration (continuous venovenous haemofiltration) and tris-hydroxymethylaminomethane infusion. We measured parameters describing acid-base balance according to Stewart's approach, ketone body formation, oxygen delivery, haemodynamics, diuresis and urinary pH. RESULTS: Acid-base balance did not differ significantly between the groups. In HES130-treated pigs, the haemodilution-based drop of haemoglobin (1.4 +/- 1.0 g dL(-1), median +/- SD) was paralleled by an increase in the cardiac output (0.5 +/- 0.4 L min(-1). Lacking increases in cardiac output, gelatine-treated pigs demonstrated a reduction in oxygen delivery (149.4 +/- 106.0 mL min(-1)). Tris-hydroxymethylaminomethane volumes required for pH titration to desired values were significantly higher in the gelatine group (0.7 +/- 0.1 mL kg(-1) h(-1) vs. HES130: 0.5 +/- 0.2 mL kg(-1) h(-1)). CONCLUSION: The buffer capacity of gelatine did not lead to favourable differences in acid-base balance in comparison to HES130.

Hepatitis B in chronic kidney disease: moving toward effective prevention.

Control of hepatitis B virus (HBV) infection has been a continuous challenge in the management of patients with advanced kidney disease. HBV infections are still difficult to treat, so the main objective is disease prevention by timely immunization with hepatitis B surface antigen vaccines. In the general population, a simple two- or three-standard-dose vaccination strategy has been proven to be highly successful. In contrast, an intensified and more tailored approach is evolving in patients with advanced renal failure.

Thermography as potential real-time technique to assess changes in flow distribution in hemofiltration.

Flow distributions are critical determinants in the function of hemofilters. Despite their importance, however, flow distributions cannot currently be measured in filters during experimental or clinical applications. Here, we demonstrate that the thermal conduction properties of extracorporeal circuits may provide a tool to overcome this limitation. More specifically, we show that thermography provides an indirect approach to visualize differences in regional perfusion rates through temperature profiles on the filter surface. Thermograms were recorded using a TVS700 system (Ca. Goratec) during recirculating in vitro hemofiltration of porcine blood. Different test protocols were executed to characterize the contribution of thermal conduction and convection to the measurable changes in the temperature at the surface of the filter housing. For comparison and validation, these experiments were supplemented by computer tomography (CT) of filters after dye injection. Thermography enabled real-time visualization of the flow distributions in a hemofilter. Moreover, 'point' trends taken from different regions of the filter provided quantitative information about changes of flow distributions in response to changing experimental conditions. Our preliminary data suggest that thermography is a promising new approach for assessing the principles and time-related changes in flow distributions in hemofiltration. As expected, resolution is lower than that in CT measurements and further studies will be necessary to determine the smallest temperature gradient that still identifies differences in regional perfusion rates. Given its potential to develop into an inexpensive tool for the 'bedside' level monitoring of flow distributions during clinical studies, further investigation of thermography is highly desirable.

Effects of propofol and fentanyl on duodenal motility activity in pigs.

To investigate the effects of propofol and fentanyl on the postprandial duodenal motility the intraluminal impedance technique was used. Six pigs were instrumented with a central venous catheter, a percutaneous enterogastrostomy (PEG), and an impedance catheter, which was introduced via the PEG into the duodenum through endoscopy. Over the following 3 d, duodenal motility was measured for 8-hour periods. Measurements were taken on each subject under 3 different sets of conditions: in the conscious unrestrained pig, during propofol sedation, and during sedation with propofol-fentanyl. Both, after morning feeding and during gastric nutrition via the PEG, duodenal feeding patterns and duodenal phase II of the migrating motor cycle were shortened during propofol and propofol-fentanyl sedation. In contrast, the duration of phase I was prolonged by propofol and propofol-fentanyl. In conclusion, either propofol or propofolfentanyl sedation shortens duodenal feeding patterns, as well as phase II of the migrating motor cycle.

Effects of a single dose of ketamine on duodenal motility activity in pigs.

In order to investigate the effects of a single dose of ketamine on duodenal motility, the present study focused on the electric impedance technique. Five pigs (32 to 40 kg, CVC group) were instrumented with a central venous catheter 1 d before measurements. The next day, general anesthesia was started and maintained via central venous catheter by propofol and fentanyl. In contrast, the pigs of the KETA group (n = 5) received ketamine intramuscularly prior to the induction of anesthesia by the injection of propofol-fentanyl via an ear vein. An intraluminal impedance catheter was manually introduced into the proximal duodenum. Measurements were recorded for 4 h. The KETA group showed a median duration of phase II that was shortened by 35%, while phase I was prolonged by 73% (P < 0.05). In conclusion, when gastrointestinal motility has to be investigated, the effects of a single dose of ketamine, even for premedication, should be taken into consideration.

Influence of differences in body weight and volume management on experimental results in porcine models.

BACKGROUND: In contrast to humans, young pigs naturally have a low COPpl (12-16 mmHg versus 22-26 mmHg in young humans). Thus, behavior occurring when volume management is performed similar to human medicine might be different. Potentially underestimated intra- to extravascular fluid and solute shifts could influence time course of variables investigated. That is why we studied whether differences in the basic protocol for infusion therapy and different levels of COP according to age or body weight, respectively - impair reproducibility and reliability of experimental results. METHODS: Group A [n=6, lower body weight (LBW < 22 kg)] was treated with unrestricted infusion rates (UIR) adjusted to maintain a constant blood pressure; group B [n=6, higher body weight (HBW > 28 kg)] was treated with a restricted continuous infusion rate for fluid balance (RIR); group C (n=6) combined HBW and UIR protocol. Blood pressure, plasma solutes, diuresis, and peritoneal fluid were analysed. Statistical analysis was performed using Kruskal-Wallis test and Wilcoxon test. RESULTS: UIR-treated pigs with LBW (group A) developed ascites and demonstrated time dependent decreases of plasma solute concentrations whereas in pigs of group C mainly diuresis was increased and subcutaneous edema occurred. None of the protocols enabled constant blood pressure. DISCUSSION: In young pigs (LBW), an adaptive volume substitution using crystalloids to standardize blood pressure may induce fluid extravasation; in turn data may not show statistical significance, stable hemodynamics may not be achieved and changes in plasma solute concentrations may lead to false interpretations. RIR-strategy provided reproducible, plausible results and thus should be recommended in combination with pigs >29 kg BW for the use of porcine models.

Maximal flow rates and sieving coefficients in different plasmafilters: effects of increased membrane surfaces and effective length under standardized in vitro conditions.

In Europe, capillary membrane filters are the favored method for plasmapheresis and detoxification systems including a plasma separation unit. Using capillary membrane filters clearance depends on the filtration rate and the sieving coefficient (Qs) for the plasma substrates. We investigated whether the increase of the effective membrane surface of plasmafilters enables higher clearance rates than a small plasmafilter. Heparinized porcine blood (5 I.U./l) of healthy pigs was used for an in vitro circuit. Filter types used were from GAMBRO: n = 5 PF1000N (effective membrane surface: 0.15 m(2)), n = 4 PF2000N (0.35) and from ASAHI: n = 4 PLASMAFLO OP-05W (L) (0.5). Spontaneous hemolysis was chosen to indicate the individual threshold limit for maximal flow rates. Sieving coefficient was determined for ten parameters at lowest and highest blood flow (Qb) and filtration rates (Qf). PF1000N enabled maximal flow rates as high as PF2000N and PLASMAFLO (Qb/Qf in ml/min: 200/40). Qs for most substrates were similar between the filter types and the flow rates. Compared to PLASMAFLO, the filter type with the largest membrane surface, the PF1000N provided significantly better Qs for triglycerides and albumin and a trend for a better elimination of cholesterol, GGT, and LDH. The sieving properties of the PF2000N ranged between PF1000N and PLASMAFLO. Under standardized in vitro conditions, the larger plasmafilters tested did not improve the overall performance of the plasmapheresis procedure. Thus, enlargement of the membrane surface such as provided by two commercially available and clinically well-established plasmafilters could not be proven to enable higher clearance rates.

The influence of HES on the filtration properties of capillary membrane plasmaseparation.

Plasmaseparation is a treatment under discussion for critically ill patients, especially in sepsis and multiorgan failure. These patients receive a variety of different fluid substitutes, including hydroxyethylstarch (HES). HES is known to influence rheological properties, but nothing is known about the possible interactions between HES and the plamaseparation procedure. We used an in vitro plasmaseparation circuit with heparinized porcine blood. Before priming the system, 2 liters of blood were supplemented by adding 100 ml of either NaCl 0.9% or HES (n=6 in each group). We monitored the transmembrane (TMP) and the filtration pressure (PF) and measured free plasma hemoglobin (free Hb) and platelet counts before and after the two hours plasmaseparation procedure. The final transmembrane pressure was significantly higher with HES substitution. In the HES group we found negative filtration pressures from the very beginning with a significant further decrease toward the end of the experiments. A significant increase in free Hb and decrease in platelet counts were noted only in the HES group. Volume substitution with HES leads to impaired filtration properties and deteriorated hemocompatibility in in vitro plasmaseparation. Further studies have yet to evaluate whether or not the effects described also occur under clinical conditions.

The influence of hypoalbuminemia on maximal flow rates and transmembrane pressure during plasmapheresis--an in vitro study.

BACKGROUND: Plasmapheresis has been used for the treatment of acute liver failure (ALF). In these patients, hypoalbuminemia is often observed. Since albumin improves the disaggregability of erythrocytes, hypoalbuminemia might deteriorate rheology and thus influence the overall performance of plasmapheresis. METHODS: Hypoalbuminemia was mimicked by using porcine blood because of its physiologically low albumin/globulin ratio (AGR). Filters (n = 16) were integrated in a closed extracorporeal in vitro system. In the control group (n = 8), native porcine blood (AGR 0.8) was used. In the study group (n = 8), we used porcine blood supplemented with human albumin to obtain the human AGR value of 1.2. Two different heparinization protocols were compared in each group (2.5 IU/ml: n = 4 with albumin and n = 4 without albumin versus 5 IU/ml: n = 4 with and n = 4 without albumin). RESULTS: In both heparinization protocols the higher AGR led to lower transmembrane pressure (TMP) levels compared to the lower AGR. The reduced TMPs enabled higher blood flow and filtration rates. CONCLUSION: Maintenance of a physiological AGR in ALF patients might improve the performance of plasmapheresis and--as opposed to raised heparinization--contribute to a safer application.

Adsorption of xenobiotics to plastic tubing incorporated into dynamic in vitro systems used in pharmacological research--limits and progress.

Commonly used materials incorporated into dynamic culture systems typically show the feature of adsorption of lipophilic xenobiotics. Yet, this phenomenon is strongly limiting the use of dynamic culture models and ex vivo organ perfusions in pharmacological and toxicological research. The aim of the study was to characterize different materials with respect to their capacity for drug adsorption and to find methods or materials to reduce the loss of substrate by adsorption in order to improve the use of dynamic in vitro systems. The adsorption of different xenobiotics (lidocaine, midazolam, lormetazepam, phenobarbital, testosterone, ethoxyresoroufine) to tubes used in dynamic in vitro systems (polyvinyl-chloride, silicone) were investigated and compared to a new material (silicone-caoutchouc-mixture). In addition, the role of protein deposition onto the tubing was studied and it was investigated whether it was possible to reach saturation of the inner tube surface by pre-loading it with the test compound. We found that silicone tubes provided the highest comfort with respect to handling and reusability, but they also demonstrated the highest capacity for substrate adsorption. Polyvinyl-chloride was the second best in handling but also demonstrated a high complexity in its adsorption behavior. The silicone-caoutchouc-mixture reached acceptable experimental results with respect to its handling and demonstrated a very low capacity for substrate adsorption.

Enhancing filtration rates by the use of blood flow around the capillaries of plasmafilters: an in vitro study.

As the low clearance rate of plasmaseparation limits its use in the treatment of patients suffering from liver failure, sepsis or MOF, we intend to develop strategies for a plasmaseparation unit which increases plasmafiltration rates. Our first question focused on whether commercially available plasmaseparation filters, and in particular their membranes, are suitable for the inversion of blood and plasma compartments. This experimental study was performed using in vitro systems. Commercially available plasmafilters PF2000N (Gambro) and Plasmaflo (Asahi) were compared in both their normal operating mode with blood flow through the capillary lumen, and in the inverse mode. Inverse mode means that blood flows through the outer space of the capillaries while plasma was obtained from the lumen. Heparinised porcine blood (5 I.U./ml) was used in a heated, recirculating in vitro circuit. Our main results were that the normal use of both filter types Plasmaflo and PF2000N enabled maximal blood flows (Qb) of 200 ml/min and filtration rates (Qf) of 25-40 ml/min. Operating the filters in the inverse mode enabled Qb up to 500 ml/min and Qf up to 100 ml/min. Hemolysis, platelet counts and coagulation parameters did not differ significantly regardless of whether the normal or inverse mode was used. The tested plasmafiltration membranes appear to be suitable for use in inverse mode. Although in our experiments, hemocompatibility tests did not indicate severe problems induced by the module geometry, the development of a module specially constructed for blood flow outside of the hollow fibers appears to be necessary in order to minimise shunts and low perfusion areas.

Development of a special catheterbag to enable artificial organ evaluation in conscious, unrestrained pigs: technical note.

Pigs are widely used as models for a variety of human diseases, because many of their physiological functions closely resemble those of humans. However, information on instrumentation techniques is still scarce. In particular, experiments in conscious pigs focused on extracorporeal circuits are connected to a variety of methodical problems with respect to the handling of the animals. Usually, pigs are placed in restraint-slings during the application of an extracorporeal system. However, this method of restraint may lead to excessive mental distress even in trained animals. The latter might influence the results and certainly affects principles of animal welfare. Our own experiences with instrumented, conscious, but unrestrained dogs encouraged us to modify methods used for the fixation of in-dwelling central venous catheters in dogs with special regard to the species specific behaviour and phenotype of pigs. A cord retractable leash (CRL) was used for maintaining a safe distance between the animal and the outer ends of the catheters. To prevent dehiscences of the required fixation sutures a new catheter bag (CB) was designed to counteract tension forces caused by the CRL's spring-mechanism. The combination of both the CRL and CB enabled us to conduct safe experiments with conscious, unrestrained pigs. We alleviated the mental distress these animals were exposed to in comparison to former methods based on restraint of the animals.

Comparative analysis of metabolism of medium- and plasma perfused primary pig hepatocytes cultured around a 3-D membrane network.

Culture media are frequently used in the evaluation of metabolical functions of hepatocytes in hybrid liver support systems (hLSS). However, media compositions differ substantially from those of plasma. Therefore, our study was designed to investigate whether current in vitro studies with medium are suitable to assess the metabolical competence of hLSS-cultures during clinical application as well as to explore whether the cell nutrition with medium provides a suitable modus operandi for stand by cultivation. Paired bioreactor cultures were perfused with either Williams' Medium E (MPB) or human plasma (PPB). About 6x108 primary pig hepatocytes (>97% viability) were cultured in three laboratory scale bioreactors designed according to Gerlach's bioreactor-concept. Different perfusion protocols were initiated after a standardised period allowing for cell attachment and reorganisation in aggregates. Whereas patterns of enzyme release were similar in both protocols the metabolical behaviour was different between MPB (anabolic state) and PPB (catabolic state). Furthermore, compared to MPB the lidocaine-MEGX-tests for PPB demonstrated lower MEGX-concentrations and a different reaction pattern. We conclude that the nutrition of hepatocytes with medium during the stand by period itself might influence the cell function and subsequently the efficacy of the hLSS-treatment during clinical application.