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Hepatic ischemia/reperfusion injury has so far been investigated in various experimental models. A clinical transfer of experimental results is, however, problematic because of anatomical and physiological differences and also the inevitable simplification of experimental work. The choice of model must therefore be adapted to the clinical question to be answered. The simplest procedure for inducing normothermic ischemia is to clamp the hepatoduodenal ligament. Models that do not avert portal congestion are regarded as unsuitable. Our current understanding of the pathogenesis of ischemia/reperfusion injury depends mainly on studies whose authors have investigated either global liver ischemia with a portocaval shunt, spleen transposition and in the isolated perfused system, or partial ischemia. This review is a critical examination of various approaches to the study of normothermic hepatic ischemia in experimental animals.

The aim of this study was to develop and establish a new system of laparoscopic fluorometry for the purpose of investigating the intestinal microcirculation. In 25 pigs (German Landrace, 16-25 kg body weight), ischemia was established in two segments (A, irreversible; B, reversible ischemia; C, internal control) of the small intestine by a laparoscopic technique. Microcirculation in the segments was assessed by laparoscopy at a second-look operation 24 h later by means of the fluorescence system Endoscan. The fluorescence of the three bowel segments was measured by arbitrary dye fluorescence units (DFU) 15 min after starting reperfusion, before and after injection of sodium fluorescein (NaFlu, 0.25 mg/kg body weight). The dividing line between viable and nonviable bowel tissue was established from the inflow and outflow rates of NaFlu with the aid of ROC (receiver operating characteristic) curves. The specificity and sensitivity of the new method were evaluated by correlating the results with the viability of each intestinal segment as predicted by three laparoscopically experienced surgeons and by histological examination. By means of the calculated separation sharpness (fluorescence index at 2 min >0.5, outflow factor of NaFlu at 10 min >20%), the overall predictions of intestinal viability in all 25 animals achieved a sensitivity of 93.5% and a specificity of 94.1% by laparoscopic fluorometry, versus a sensitivity of 70.8% and a specificity of 87.5% for the prediction of bowel viability by ordinary laparoscopic technique. Used as an adjunct to conventional laparoscopy, laparoscopic fluorometry brought significant gains in sensitivity and specificity in the distinction between reversible and irreversible intestinal ischemia.

Knowledge of sepsis is growing rapidly and new pathogenetic concepts and therapeutic strategies evolve. The animal models of sepsis catalyze this development. Any model of this complex disease is inevitably a compromise between clinical realism and experimental simplification. Against the background of current pathogenetic concepts this review tries to analyze the validity and clinical relevance of each model. Endotoxemia and bacteremia represent models without an infectious focus. They reproduce many characteristics of sepsis and are highly controlled and standardized. However, they reflect a primarily systemic challenge and create neither an infectious focus nor the protracted immune reaction that characterizes sepsis. In this respect, any model with an infectious focus is decisively closer to clinical reality. In these models the peritoneal cavity is contaminated either by bacteria or inoculated feces or perforation of the bowel wall. Both the bolus injection and the implantation of carriers loaded with bacteria or feces are used. In fecal spesis and perforation models the complete spectrum of enteric pathogens is present in the septic focus and infective selection is undisturbed. Here the pathophysiologic and immunologic features of clinical sepsis are successfully reproduced. However, presumably due to inadequate control of the bacterial challenge, only poor interlaboratory standardization is possible. As to optimize models for the clinical reality the choice of an appropriate class of models is crucial. Moreover the incorporation of clinical therapy such as volume resuscitation, antibiotic therapy and surgical treatment of the septic focus is indispensable. Finally, the importance of simulation of comorbidities cannot be overemphasized.

The aim of this study was to explore the complex role of endothelins (ETs) in hepatic ischemia-reperfusion injury and to minimize this type of injury by nonselective ET receptor blockade. In an in vivo rat model, hepatic ischemia was induced for 30 min. The rats were divided into three groups: (1) sham operated, (2) untreated ischemic, and (3) group treated with the nonselective ET receptor antagonist bosentan (1 mg/kg body weight iv). Blockage of the ET system during ischemia-reperfusion was assessed by: (1) in vivo microscopic analysis, (2) measurement of local tissue PO2, (3) laser Doppler flowmetry, (4) transaminases, and (5) tumor necrosis factor (TNF)- serum levels. During liver ischemia, anoxia (mean liver pO2 decreased from 14.7 to 1.5 mm Hg) and TNF- (levels rose from 0 pg/mL to 145.3 pg/mL at the end ofischemia) were associated with the release of ETs. Immunoreactive ET-1 (ir-ET-1) plasma levels (basal levels: 12.1+/-1.8 pg/mL) went up by 2.6-fold (32.1+/-6.8 pg/mL) after 15 min and by 11.7-fold (142.1+/-32.6 pg/mL) after 120 min of reperfusion. Increased plasma levels of ir-ET-1 were associated with sinusoidal constriction to 77.6+/-7.1% of basal diameters. This constriction led to significant decreases in perfusion rate (77+/-3%), local tissue PO2 (6.9+/-2.7 mm Hg), and erythrocyte flux (61.7+/-13.8% of basal values). Hepatocellular damage, evaluated via the serum level of aspartate aminotransferase (AST, increase to 393.5+/-68.3 U/L, preoperative 23.9+/-2.0 U/L) was detectable 6 h after reperfusion (p < .05). Administration of bosentan before 30 min of ischemia significantly reduced ischemia-reperfusion injury and was associated with an increase of ir-ET-1 levels to 110.8+/-12.0 pg/mL and 94.1+/-25.0 pg/mL after 15 and 120 min of reperfusion. Sinusoidal diameters were maintained at nearly 100% in the treatment group instead of 77%, while perfusion rate (88+/-2%) and tissue PO2 (12.1+/-1.0 mm Hg) rose significantly in contrast with the nontreatment group (p < .05). Hepatocellular damage was reduced (AST levels after 6 h of reperfusion 244.0+/-34.4 U/L, p <.05), and leukocyte sticking and rolling were diminished (p < .01). In the treatment group, bosentan values of 5.6+/-0.7 and 2.9+/-0.4 ng/mL after 15 and 120 min of reperfusion were measured. In conclusion the release of endothelins is combined with microcirculatory disturbances and local hypoxia, thereby causing liver damage. By protecting the liver microcirculation, ET receptor blockade of both receptors at a low dose increased blood and oxygen supply to the liver and reduced hepatocellular injury. These results constitute the bases for further studies and transfer into clinical practice.

When biomechanical properties of tendons are studied, the technique of clamping the tendons in the testing machine presents a methodological challenge, especially when murine tendons are examined. These short tendons tend to rupture at the transition line to the fixation, leading to false interpretations. Therefore a new clamping technique for investigation of healthy murine Achilles tendons (n = 50) was developed, in which the intramuscular tendon fibers were fixed between two paper strips and the calcaneus was wedged into a conical slot in a wooden block and then mounted in the testing machine (n = 20). This technique was compared with the conventional clamping technique that fixes both ends of the tendon by clamps (n = 15) and an earlier described method that used glue or plastic cement for the fixation of the intramuscular tendon fibers and calcaneus in the testing machine (n = 15). When tested by the new clamping technique, 17 tendons ruptured intratendinously at a mean tensile force of 8.4 +/- 1.1 N. Three Achilles tendons (17%) tore at the site of paper fixation and had to be excluded from investigation. Data from 73% of the measurements fixed by gluing had to be excluded because slippage of the proximal tendon fibers and contamination of the tendon with glue occurred. All the conventionally clamped tendons ruptured at the site of fixation at a mean tensile force of 6.1 +/- 2.3 N (p < .05). This was 30% lower than with the new clamping technique. Thus, the newly developed clamping technique enables investigators to obtain more valid biomechanical studies of the murine Achilles tendon.

The introduction of orthotopic liver transplantation in the management of acute liver failure has dramatically increased the survival rates of patients at the cost of removing the patient's native liver and life-long dependence on immunosuppression. However, it is well known that in many patients with acute liver failure the diseased liver has the potential to recover. Death in these patients is often due to increased intra-cranial pressure or infection. Liver bridging techniques are assigned to temporarily provide liver function and enable the native liver to recover in patients with acute liver failure. They represent an attractive alternative to conventional liver transplantation in the management of acute liver failure, since after recovery of the native liver the patient is freed from immuno-suppression with all associated side-effects and risks. Auxiliary liver transplantation, artificial liver support devices and hepatocyte transplantation represent different ways of bridging liver function in acute liver failure. The aim of this review is to present the ideas and principles of these three different liver bridging techniques. We will discuss the relative importance and the future potential of theses bridging techniques in the treatment of acute liver failure by comparing the experimental and clinical results.

One of the changes produced by ischemia and reperfusion is endothelin (ET)-mediated constriction of the hepatic vascular bed. This leads to microcirculatory disturbances and reduced blood flow, thereby causing local hypoxia and liver damage. Our aim was to induce stepwise changes of microvascular vessel diameters so as to define the best protective vessel width that could be produced by drug therapy and thereby to minimize ischemia/reperfusion injury. The mixed ET receptor antagonist bosentan was used in different dosages in a rat liver ischemia/reperfusion model with splenocaval shunt. In vivo microscopy was performed 30-90 min after reperfusion and local hepatic tissue pO2 was determined, together with aspartate aminotransferase/alanine aminotransferase (AST/ALT). After ischemia, sinusoidal diameters were significantly reduced to 76 +/- 7% of those in the control group. After the administration of bosentan in dosages of 0.1, 1, and 10 mg/kg body weight iv and 10 mg/kg body weight intraportally we found diameters of 83 +/- 4, 98 +/- 2, 109 +/- 6, and 137 +/- 19%, respectively. Perfusion rate and leukocyte-endothelium interactions showed dependence on sinusoidal diameters, with the best results in the group where preischemic sinusoidal vessel width had been maintained. Local tissue pO2 and transaminase levels also showed that oxygen supply was sufficient and that hepatocellular injury was most minimized in this group. Graduated blocking of ET receptors allows stepwise regulation of sinusoidal and postsinusoidal venular vessel width and offers a treatment strategy for pathophysiological situations that are associated with ET-induced vasoconstriction. The results suggest that maintenance of preischemic microvascular diameter is the best therapeutic approach.

The bronchosecretolytic agent ambroxol added to histidine-tryptophan-ketoglutarate (HTK) solution has recently been shown to protect cold stored rat hepatocytes. The aim of the present study was to confirm these observations in a rat liver transplantation model. Before orthotopic liver transplantation, donor livers from 30 syngeneic Wistar rats were assigned to three groups (n = 10): (A) in situ flush (ISF) and 1/2-h cold storage (CS) with HTK solution, (B) ISF and 3-h CS with HTK, and (C) ISF and 3-h CS with HTK + 10(-3) mol/L ambroxol. The efficacy of the drug was evaluated by postoperative survival (> 14 days) and liver enzyme release (ALT), bile flow, histomorphological injury, and malondialdehyde (MDA) level in the grafts 15 min after reperfusion. After 1/2-h CS with HTK solution (A), 90% of the transplanted rats survived. In comparison with donor conditions, bile flow in the reperfused grafts decreased to 87 +/- 5.3%, whereas postoperative ALT levels slightly increased. After 3-h HTK preservation (B), the survival rate decreased to 60%, while ALT values markedly increased and bile flow after reperfusion declined to 82 +/- 6.6%. Ambroxol added to HTK solution (C) enhanced bile flow to 106 +/- 3.4%(p < .05), and reduced ALT and MDA levels and histomorphological injury of the transplanted livers, so that its beneficial effect in organ preservation has been confirmed in the transplant model. However, survival rate was not improved by the agent, probably because of the low cold ischemia tolerance of the Wistar rat livers used.

In vivo microscopy is an excellent technique for investigating the microcirculation and until recently the only one that allowed direct visualization. Rat liver has been widely studied, because microcirculatory disorders play a pivotal role in the pathogenesis of organ failure during hepatic ischemia, transplantation, hemorrhagic shock, endotoxemia, and sepsis. The state of the microcirculation is an important prognostic factor for the reestablishment of organ function after these injuries. This article introduces the most common procedures for in vivo microscopy of the rat liver, summarizes the available fluorescent dyes, and gives an overview of criteria for the expression and evaluation of microscopic findings. Particular emphasis is given to a description of the different parameters assessed by direct observation of hepatic microcirculation, such as perfusion rate, leukocyte-endothelium interactions, leukocyte velocities, and phagocytic activity. Examples of normal range values are given. This overview is intended to help those wanting to introduce this method into their research and who are embarking on intravital microscopy for the first time, and to enable them to decide which techniques are appropriate for answering special questions.

The quality of organ preservation is of major importance in minimizing the incidence of primary graft nonfunction and organ rejection. For this study a new semiquantitative score was developed that grades morphologic tissue alterations in the liver according to their frequency and severity. It was applied to assess commonly used perfusion solutions for their efficacy in preventing early and late tissue damage after rat liver transplantation. For transplantation the livers were stored in Euro-Collins (EC, group I; n = 11), histidine-tryptophan-alpha-ketoglutarate (HTK, group II; n = 11), or University of Wisconsin solution (UW, group III; n = 11). Rat liver transplantation was performed with graft arterialization by the method of Engemann. Biopsies were taken for morphological examination and semiquantitative scoring during the donor operation, after 4 h of cold storage, 1 h after reperfusion, and 4 weeks postoperatively. An immunohistological bromodeoxyuridine (BrdU) assay was also performed on the day of dissection to assess the rate of hepatic proliferation. Semiquantitative morphological analysis gave widely differing results in all experimental groups after 4 h of ischemia. There was less intracellular and interstitial edema, fatty degeneration, intralobular necrosis, and hepatocellular proliferation in the HTK group than in the other groups. Neither after cold ischemia nor 1 h after reperfusion did Kupffer-cell activation occur; this is known to play a major role in the development ofischemia and reperfusion injury. Furthermore, late changes such as bile-duct proliferation and vascular and sinusoidal alterations appeared less frequently in this group. The hepato-protective powers of HTK solution might therefore be due to decreased Kupffer-cell activation.

Liver transplantation in rats is frequently used as a transplantation model. Although liver transplantation in larger laboratory animals such as dogs and pigs is technically easier, the rat has become the most important subject for experimental liver transplantation because of the availability of genetically defined animals. Numerous surgical techniques have been developed that permit the investigator to carry out studies with high clinical relevance. In this article the principal models of orthotopic rat liver transplantation and their technical modifications of vessel anastomoses, rearterialization, and bile duct reconstruction techniques are reviewed. More than 20 transplantation models are described in detail and demonstrated with clear illustrations. Finally, the advantages and uses of all the surgical procedures (e.g., suture and cuff anastomoses, bile duct anastomoses, and rearterialization techniques), specific problems, and survival criteria are discussed and the experiences of investigators who applied these techniques are analyzed. In conclusion, an overview and critical evaluation of all surgical techniques of orthotopic rat liver transplantation are given, together with instructions for learning these techniques.

Blood samples taken directly from the hepatic veins are often required for evaluation of liver function or ischemia/reperfusion injury and for direct determination of metabolic products of the liver. In the rat, blood samples are usually obtained by direct puncture of the hepatic veins. Successful catheterization of these small vessels is only possible under direct vision because great dexterity is needed to persuade the catheter to enter the hepatic vein. In this report a procedure for hepatic vein catheterization under direct vision is described in the rat. After performing a median laparotomy, a flexible catheter (Buchbinder, 3.0 F/1 mm, as used in cardiology) with a bent end is inserted into the infrahepatic vena cava. Under direct vision through the thin walls of the inferior vena cava the bent end of the catheter is slowly pushed up until the tip slides into a hepatic vein, which opens into the inferior vena cava at a right angle. Selective catheterization of the desired hepatic vein is possible by rotating the catheter and changing the position of its tip. This is a reliable new technique of hepatic vein catheterization in rats, by which blood samples can be collected directly from the hepatic veins.

Ischemic injury to the liver is known to influence the outcome of liver transplantation. In this study the efficacy of Euro-Collins (EC), histidine-tryptophan-ketoglutarate (HTK), and University of Wisconsin (UW) preservation solution was analyzed in the model of orthotopic liver transplantation in syngeneic rats. The study design was as follows: Group I, Euro-Collins solution (n = 11); Group II, Histidine-Tryptophan-Ketoglutarate solution (n = 11); Group III, University of Wisconsin solution (n = 11). The rat liver transplantation was performed with arterialization of the graft as described by Engemann. The postoperative follow-up was 28 days. The perfusion flow rate of the preservation solution measured during organ perfusion revealed lowest levels in the UW group and comparable levels in Groups I and II. Postoperative graft function was monitored by measuring liver enzymes (aspartate amino-transferase, ASAT, alanine aminotransferase, ALAT), bilirubin and bile production. The survival rate was 10/11 in each group. Liver enzymes and bilirubin increased postoperatively and went back to normal within 2 or 3 weeks. In contrast to bilirubin, the liver enzymes showed a biphasic increase with maxima on the 1st and 5th days (range: ALAT, 220-264 U/L; ASAT, 145-177 U/L). Bile production was observed in all groups, but was significantly higher after UW-preservation (P < .005). Analysis of inflammatory cells revealed high concentrations of intrasinusoidal leukocytes and lymphocytes in the graft with a maximum on the 5th day.

The extent of callus formation about a bone fracture depends on the rigidity of fracture fixation. The mechanism that converts the mechanical stimulus into the biologic response is unknown. On the basis of existing literature, an attempt has been made to define a model that explains this mechanobiologic transduction. Once integrity of the bone has been disrupted, a sequence of biochemical and cellular events commences that induces inflammatory reactions. Messengers (e.g., metabolites of the clotting or complement system, eicosanoids, or growth factors) are released or activated. They control the migration, proliferation, and protein synthesis of cells that are essential for angiogenesis and connective tissue formation. The key component in this inflammatory sequence seems to be the macrophage. Growth factors (e.g., released by macrophages) stimulate endothelial cells to form capillaries and mesenchymal cells to synthesize their matrix. In mechanically neutral areas, the fracture cavity is revascularized and osteoblasts proliferate and form bone. In mechanically instable fracture areas, spreading capillaries are disrupted by shear forces. In these areas, therefore, the milieu becomes hypoxic again. This milieu seems to support the differentiation of chondrocytes that stabilize the fracture by cartilage formation. If the strength of repair tissue is surpassed, the disrupture of the repair tissue triggers the mechanisms of inflammation again and additional cells immigrate and proliferate. Their protein synthesis increases repair callus. The increase of callus formation, however, stops when the tissue is capable of resisting motion. Links to the callus formation in osteitis are shown.

Endothelin evokes strong and longlasting constriction of postischemic sinusoids, leading to microcirculatory disturbances and local hypoxia, thereby causing liver damage. The aim of the study was to avoid the constrictive response of sinusoids by blocking endothelin receptors. In an in vivo ischemia-reperfusion model (21 female Wistar rats, 250-300 g) with portal decompression by a splenocaval shunt, hepatic ischemia was induced for 30 min by cross clamping of the hepatoduodenal ligament. The endothelin receptor antagonist bosentan (10 mg/kg bw IV) was administered before ischemia. The effect of the receptor antagonist was assessed by serum levels of aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) that were determined prior to ischemia, 2 and 6 h postoperatively. The local tissue pO2 was measured prior to inducing ischemia, 30 and 60 min after reperfusion. Application of 10 mg/kg bw endothelin receptor antagonist (ERA) intravenously did not influence the systemic blood pressure. The postischemic increase in serum ASAT and ALAT levels was diminished after receptor antagonist treatment (ASAT: p < .05). Local postischemic hepatic tissue pO2 was significantly decreased to 45% of basal values after 30 min and to 54.8% after 60 min of reperfusion (p < .05). Application of ERA results in a significant increase in local tissue pO2 to 110.9% of basal values after 30 min and to 90.7% after 60 min of reperfusion (p < .05). These data indicate that the endothelin receptor antagonist treatment results in a prevention of postischemic sinusoidal constriction avoiding hypoxia and leading to improved hepatocellular recovery.

For reduction of radical formation during reperfusion, a lower oxygen supply by limiting the reperfusion flow rate should be beneficial for the organ. Thus, indomethacin was given prior to reperfusion for induction of temporary postischemic vasoconstriction. In an in vivo model (female Wistar rats, 200-250 g, n = 16) with portal decompression by a splenocaval shunt, hepatic ischemia was induced for 30 min by cross-clamping of the hepatoduodenal ligament followed by portal intravenous injection of indomethacin (2 mg/kg body wt) at the end of ischemia. Liver injury was assessed by serum levels of Aspartat-aminotransferase (ASAT) and Alaninaminotransferase (ALAT) that were determined prior to ischemia, on days 2, 4, 6 and 21 postoperatively. The local tissue pO2 was measured preischemically, 1 h after reperfusion and on day 21. Application of indomethacin significantly reduced the local tissue-pO2 by about 50% after 1 h of reperfusion (p < .05). The increase in serum ASAT levels on day 2 was significantly diminished after indomethacin application (p < .05). ALAT values on day 2 showed a significant increase in the control group but did not differ from baseline in the indomethacin group. These data support the hypothesis that temporarily limited reperfusion results in an amelioration of reperfusion injury, although further studies with more selectively vasoactive agents must still be performed since indomethacin also has a major effect on the eicosanoid metabolism.

The implantation of antibiotic-containing cement beads became standard adjuvant local antibiotic therapy of chronic osteomyelitis. The new developed bioabsorbable drug carrier system of polyglycolic acid with the antibiotic Ciprofloxacin was tested in vitro and in vivo. The goal of this study was to determinate the penetration depth of Ciprofloxacin into bone cortex and marrow. Two monofil, PGA cylinders, 3.2 x 5 mm in size, containing 3 mg of Ciprofloxacin each, were implanted into the proximal part of both femora of 18 rabbits (New Zealand white). After sacrifice, the concentration of Ciprofloxacin in micrograms/g cortex and marrow was measured with high-performance liquid chromatography in relation to the distance from the test material at day 2, as well as at 1, 2, 3, 4, and 6 weeks postimplantation. For a distance up to 5 mm, marrow levels of the drug exceeded cortical levels at day 2 (5000 to 240 micrograms/g). At a distance of 5-10 mm, cortex levels were similar to marrow levels after 2 weeks and were higher than marrow levels at week 3. This observation could be made at a distance between 10 and 15 mm only after 2 days. Later, marrow concentrations again exceeded that in cortex. At a distance of more than 15 mm, antibiotic levels were low and approximated. After 6 weeks, at 5 mm distance, a bactericidal drug concentration of about 2 micrograms/g in bone marrow could be measured. Drug penetration into cortical bone in bactericidal concentrations of about 2 micrograms/g was achieved at up to 30 mm in the first few days.(ABSTRACT TRUNCATED AT 250 WORDS)

A model of orthotopic liver transplantation in pigs was developed to investigate reperfusion injury of the liver. To have direct access to the liver for organ monitoring a relaparotomy was performed 6 h after revascularization. The details of modification in anesthesia and surgical technique are given. Salient features of the technique include (1) the use of an extremely short cuff of the donor suprahepatic caval vein, (2) the use of a central venous catheter fixed in a hepatic vein, and (3) the use of an aortic graft for standardized arterial reconstruction. Twenty-nine transplantations were performed. The total anesthetic time was 13.28 +/- 1.45 h and the operative time was 10.06 +/- 0.77 h. The animals withstood this long procedure remarkably well.

In an animal study (7 mongrel dogs) the effects of neuroleptanalgesia (NLA) and combinations of NLA with nitrous oxide (N2O) and isoflurane on the macro- and microcirculation of the liver were investigated. Measurements were made in three steps. After NLA alone the dogs were supplementarily ventilated with nitrous oxide/oxygen at a ratio of 2:1. During the last step, 1 MAC isoflurane was added to the inspired gas. From the portal vein, arterial and mixed-venous systems' hemodynamic parameters, blood gases, and acid-base balance were recorded. As a parameter of oxygenation the tissue PO2 of the liver was measured with a multiwire surface electrode. During NLA stable hemodynamic conditions and a balanced acid-base status were observed. The nitrous oxide combination resulted in an increase of the mean pulmonary artery pressure of 16%. The addition of isoflurane had a negative inotropic effect: The heart index decreased to 74% of the starting value and the total peripheral resistance (TPR) increased by 27%. The summarized PO2 histograms under NLA and NLA/N2O showed arithmetic mean values of 34.1 and 35.2 mm Hg, respectively. The addition of isoflurane resulted in a left shift and a decrease of the mean value to 28.6 mm Hg. This histogram corresponds exactly to the oxygen pressure distribution in the dog liver during piritramide basic anesthesia. It seems that NLA and the combination of NLA/N2O increase the liver perfusion with a higher portal-venous and tissue PO2. This effect can be explained only by a massive change of visceral circulation. It is canceled by the addition of isoflurane.