Hans-Ullrich Spiegel

Animal models of sepsis.

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.

Involvement of endothelin/ nitric oxide balance in hepatic ischemia/ reperfusion injury

Introduction: Endothelin (ET) and nitric oxide (NO) act as opponents in the regulation of the hepatic microcirculation. During ischemia/reperfusion (I/R) ET levels are increased, whereas no rise in NO levels is observed. This imbalance may be responsible for microcirculatory disturbances. The aim of this study was to restore the delicate ET/NO balance to maintain the integrity of the hepatic microcirculation and to reduce I/R injury. Methods: Ischemia was induced by crossclamping of the hepatoduodenal ligament for 30 min with portal decompression using a splenocaval shunt (56 Wistar rats, 200–250 g). Sham operation, ischemia and treatment groups with the endothelin receptor antagonist (ERA) bosentan (1 mg/kg body weight i.v.) and the NO donor l-arginine (400 mg/kg body weight i.v.) were performed. For assessment of the microcirculation, sinusoidal perfusion rate, diameters of sinusoids and postsinusoidal venules, leukocyte endothelium interactions and velocity of free-flowing leukocytes were investigated by means of in vivo microscopy 30–90 min after reperfusion. Local hepatic tissue pO2 was measured prior to ischemia, 30 min and 60 min after reperfusion and aspartate aminotransferase (AST)/alanine aminotransferase (ALT) levels were investigated 2 h and 6 h after reperfusion. Results: After ischemia, sinusoidal and venular diameters were reduced to 76% and 85%, respectively, of sham operation group values (P<0.05), but were maintained at baseline in ERA (98/102%) and NO (102/105%) groups (P<0.05). Increased postischemic leukocyte sticking in sinusoids (144%) and venules (435%) was reduced by therapy to 110/253% (ERA) and 111/ 324% (NO), respectively (P<0.05). Perfusion rate was increased to 93% and 94% compared with 82% in the ischemia group (P<0.05). Concomitant with the improved microcirculation in therapy groups, local hepatic tissue pO2 was improved 30 min after reperfusion in the ERA (11.0 mmHg) and the l-arginine group (11.5 mmHg) relative to the ischemic group (6.9 mmHg) (P<0.05). In addition, postischemic AST/ALT increase was reduced by therapy. Conclusion: Our results indicate that maintenance of ET/NO balance by blocking ET receptors, as well as providing a NO donor, protects the liver microcirculation and reduces hepatic I/R injury.

Exogenous L-Arginine Protects Liver Microcirculation from Ischemia Reperfusion Injury

Hepatic ischemia followed by reperfusion evokes an imbalance between the vasoregulatory compounds endothelin and nitric oxide (NO) followed by constriction of the vascular bed, leading to microcirculatory disturbances and reduced blood flow, thereby causing hypoxia and liver damage. The aim of this study was to protect the liver microcirculation by maintaining this delicate balance. Material and Methods: In an in vivo ischemia/reperfusion model with portal decompression by a splenocaval shunt, hepatic ischemia was induced for 30 min by Pringle’s maneuver. The effect of the NO donor L-arginine (400 mg/kg b.w. i.v.) was assessed by in vivo microscopy. Microhemodynamic studies, including the sinusoidal perfusion rate, diameters of hepatic sinusoids and postsinusoidal venules, leukocyte endothelium interactions and leukocyte velocity were performed. Microcirculatory data were compared with local tissue pO2 and serum transaminase levels. Results: After ischemia the diameters of sinusoids and postsinusoidal venules were significantly reduced to 76 ± 7 and 86 ± 10% respectively in the nontreatment group, but dilated to 102 ± 3 and 105 ± 7% in the group treated with L-arginine (p < 0.001). The percentage of permanently adherent leukocytes in sinusoids and venules was increased by ischemia, but L-arginine reversed this increase (p < 0.001). Perfusion rate was improved to 90 ± 2 compared with 83 ± 5% in the untreated group (p < 0.01). Systemic arterial blood pressure was not affected by administration of the NO donor. The postischemic increase in serum transaminase levels was diminished in the treatment group (ASAT: p < 0.05). Local postischemic hepatic tissue pO2 was significantly decreased to 45% of basal values after 30 min and to 55% after 60 min of reperfusion (p < 0.05). Administration of L-arginine results in a significant increase in local tissue pO2 to 86 and 106% of basal values respectively (p < 0.05). Conclusion: These data indicate that L-arginine improves hepatic microcirculation after warm ischemia by increasing sinusoidal perfusion rate and by diminishing leukocyte endothelium interactions. Maintained integrity of microcirculation is associated with sufficient oxygen supply and improved hepatocellular function.

Growth factors and gastrointestinal anastomotic healing

BACKGROUND Failure of anastomotic healing in the gastrointestinal tract is a major source of surgery-related morbidity, repeated surgical procedures, and impaired quality of life. Growth factors have been shown to be involved in healing processes in various tissues including the gastrointestinal tract. This opens the perspective to use growth factors therapeutically to support impaired anastomotic healing. The aim of the present study was to review the particular role of several growth factors in different phases of anastomotic healing, experimental approaches of growth factor application, and to discuss possibilities and limitations of growth factor-directed interventions in gastrointestinal surgery. MATERIALS AND METHODS A PubMed search was performed to examine the potential role of fibroblast growth factor, epidermal growth factor, heparin binding EGF-like growth factor, transforming growth factor β, insulin-like growth factor I, vascular endothelial growth factor, and platelet-derived growth factor during anastomotic healing. RESULTS Growth factors show beneficial effects on a broad range of cell types and regulate various processes during all phases of tissue healing. Despite extensive research in the field of growth factors, additional evidence is needed before translating into a clinical setting. CONCLUSIONS Future research should focus on adequate sustained but limited drug delivery. Undesired side effects, such as formation of strictures, development of peritoneal adhesions, and potential induction of malignancies, have to be reflected. Although growth factor application is currently far from clinical routine in gastrointestinal surgery, it might find application in selected patients at risk for impaired anastomotic healing, such as patients with long-time steroid therapy, immunosuppressives, inflammatory disorders, sepsis, hemodynamic shock, malnutrition, or neoadjuvant radiochemotherapy.

Influence of Heme Oxygenase-1 on Microcirculation After Kidney Transplantation

BACKGROUND Cytoprotective proteins, such as heme oxygenase-1 (HO-1), play a decisive role in ischemia-reperfusion injury during kidney transplantation. The aim of this study was to investigate the impact of heme oxygenase-1 on microcirculation and on ischemia-reperfusion injury in an isogenic kidney transplantation rat model. MATERIALS AND METHODS Seventy male Lewis rats were distributed into three groups. In Group 1(control), the kidneys were only mobilized. In Groups 2 and 3, bilateral nephrectomy was performed, and a kidney from another Lewis rat was orthotopically transplanted on the left side. The donor animals in Group 3 received preconditioning with the HO-1 inductor hemin. 24 h after reperfusion graft function and morphology were examined. Microcirculation was investigated by in vivo microscopy of the renal surface 1 h after reperfusion. RESULTS HO-1 preconditioning led to significantly lower serum creatinine and serum urea, as well as less histological damage and inducible nitric oxide synthase expression. Microcirculation was improved by a significant enlargement of the vascular diameter and an increase of the capillary flow. CONCLUSIONS Treatment with hemin improves microcirculation by induction of HO-1 and reduces ischemia-reperfusion injury after kidney transplantation. HO-1 induction was shown to be a promising approach in the preconditioning of donor kidneys.

Organ Preservation with EC, HTK, and UW Solutions in Orthotopic Liver Transplantation in Syngeneic Rats. Part I: Functional Parameters

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.

Orthotopic Rat Liver Transplantation and Bile Duct Reconstruction by a Splint Technique

The aim of the present study is to investigate the impact of bile duct reconstruction by a splint technique, a method which has not been sufficiently researched in animals after liver transplantation. Three experimental groups were set up: I = control, sham operation; II = bile duct reconstruction; III = orthotopic rat liver transplantation (ORLT). After bile duct reconstruction, serum levels of ASAT and ALAT in group II revealed a peak on the first postoperative day. The transplanted animals (group III) showed a second peak in liver enzyme levels on the fifth postoperative day; it was significantly higher than in group II. Serum bilirubin was more elevated in the transplant group, with a peak on day 7. Morphological investigations at the end of surgery revealed only intralobular necrosis and reactive changes in the liver capsule (group II); after transplantation (group III), there was also interstitial and intracellular edema, fatty degeneration and disintegration of the sinusoidal lining. One month later, necrosis, bile duct proliferation, cholestasis, cholangitis and vascular alterations were found in groups II and III. Furthermore, an increased rate of hepatocellular and bile duct proliferation was observed. These findings are partly due to the bile duct reconstruction. We recommend that a bile duct reconstruction control group should be included in ORLT experiments.

sTNF-RII: is it useful for the early diagnosis of rejection and for prognosis after renal transplantation?

Abstract Changes in soluble tumour necrosis factor receptor II (sTNF‐RII) correlate with transplant rejection, and it increases in the course of sepsis. These changes might help to identify rejection early, and thus lead to more effective treatment. Serum and urine sTNF‐RII levels were measured in 70 patients during the first 3 weeks after kidney transplantation and correlated with clinical and laboratory findings. Retrospectively, three groups were identified: I. stable transplant function (n= 23), II. at least one rejection episode (n=38) and III. other complications (infection or reperfusion injury) (n= 9). The pre‐operative maximum for serum sTNF‐RII was 22.4±10.7 ng/ml. In group I it decreased to 9.5±6.7 ng/ml on day 6 after transplantation (P<0.01), while in group II sTNF‐RII serum levels were significantly higher on day 6 (24.9±15.0 ng/ml, P<0.01). High levels of sTNF‐RII in serum (>40 ng/ml for at least 2 days) predicted a higher risk of an unfavourable outcome. High serum levels of sTNF‐RII are not specific but seem to be a prognostic indicator of a complicated course; sTNF‐RII in urine has no diagnostic value.

ATIII Attenuates Endotoxemia Induced Healing Impairment in the Colon

BACKGROUND Intra-abdominal infections are considered a contributing factor to the impairment of anastomotic healing in patients undergoing surgical procedures of digestive system. Antithrombin (ATIII) is known to improve the microcirculation in sepsis. We hypothesized that it may also positively influence the healing of the colon anastomoses under endotoxemia. MATERIALS AND METHODS Ninety Balb/c mice (n = 10 per group on day 2, 4, and 7) were randomly assigned to three groups: Control (N), Sepsis (S) (administration of lipopolysaccharides (LPS) dosed at 2 mg/kg bodyweight, 18 h before colon surgery), and Sepsis with ATIII therapy (SIII) (administration of LPS and ATIII). All the animals underwent colonic anastomoses. Immediately after their completion, microcirculatory parameters were measured, and both macroscopic and histological parameters were assessed on day 2, 4, and 7 postoperation. Additionally, immunohistology studies were performed for CD31, ssDNA, and iNOS, along with an examination for bacterial translocation to the mesenteric lymph nodes. RESULTS Compared with group S, the functional capillary network was denser in the control group N (P < 0.001) and group SIII (P < 0.01). Mean bursting pressures were significantly lower in group S compared with group N, on day 2, 4, and 7, and with group SIII on day 2 and 7. At the anastomosis, the inflammatory infiltrate in group S was denser compared with groups N (P < 0.001) and SIII (P < 0.01). Furthermore, the apoptotic rate was higher, and the vascular density was lower on day 7 in group S compared with groups SIII and N (P < 0.05). Bacterial translocation decreased over time (P < 0.05) with no significant differences between the groups. CONCLUSION ATIII improved the anastomotic microcirculatory parameters and anastomotic healing in mice with endotoxemia, though the improvement failed to achieve the levels of the control mice.

Interleukin-6, interleukin-8, and interleukin-10 in kidney transplantation: Improved risk strategy?

GENETIC FACTORS and environmental influences determine immune defenses. Early immunologic reactions after transplantation may provide a clue to the further evolution of the transplanted organ. The major factors that determine the long-term results of kidney transplantation are rejection and infection. Early and adequate therapy favorably influences the subsequent course. Early identification of patients at risk for septic complications or rejection, therefore, is important. Interleukins are mediators of intercellular communication, triggering the immunologic responses of alloantigen recognition and defense, or tolerance. Although certain cytokines display stimulatory effects in vitro, others appear to attenuate T-cell responsiveness. In vivo the impact of these complementary and apparently competitive effects is still incompletely understood. Interleukins-6 (IL-6), a potent inducer of acute-phase proteins, has been shown to repress tumor necrosis factor (TNF) synthesis by monocytes. IL-6 is increased in peripheral blood during infection or transplant rejection. Interleukin-8 (IL-8), an acute-phase cytokine mediating infection and tumor defenses via neutrophil and T-cell chemoattraction; is also observed to be increased during transplant rejection, especially in tissues with irreversible rejection. Similarly, it is increased among patients with a fatal outcome after posttransplantation sepsis. Interleukin-10 (IL-10), a regulatory cytokine of inflammatory responses that is produced by peripheral blood mononuclear cells to induce T cell anergy and inhibit activated monocytes and macrophages, acts as a general inhibitor of proliferative and cytokine responses of both Th1 and Th2 type lymphocytes. It has been proposed as a mediator of transplant tolerance, but has been observed to be increased prior to rejection. Because acute rejection is the most common cause of transplant dysfunction, its early diagnosis and differentiation from other causes has been the goal of many investigations. It also may be assumed that evidence of an early immunologic reaction toward the stimulus “transplantation” might provide a clue concerning the later course. The aim of this study was to assess the prognostic value of IL-6, IL-8, and IL-10 levels in blood and urine as early markers of the evolution of recipient responses and transplant function after transplantation. PATIENTS AND METHODS