Burkhard Stoffels

A Single Intraperitoneal Dose of Carbon Monoxide-Saturated Ringer's Lactate Solution Ameliorates Postoperative Ileus in Mice

Treatment with inhaled carbon monoxide (CO) has been shown to ameliorate bowel dysmotility caused by surgical manipulation of the gut in experimental animals. We hypothesized that administration of CO dissolved in lactated Ringers solution (CO-LR) might provide similar protection to that observed with the inhaled gas while obviating some of its inherent problems. Postoperative gut dysmotility (ileus) was induced in mice by surgical manipulation of the small intestine. Some mice were treated with a single intraperitoneal dose of CO-LR immediately after the surgical procedure, whereas other mice received only the LR vehicle. Twenty-four hours later, intestinal transit of a nonabsorbable marker (70-kDa fluorescein isothiocyanate-labeled dextran) was delayed in mice subjected to intestinal manipulation but not the sham procedure. Gut manipulation also was associated with increased expression within the muscularis propria of transcripts for interleukin-1β, cyclooxygenase-2, inducible nitric-oxide synthase, intracellular adhesion molecule-1, and Toll-like receptor-4, as well as infiltration of the muscularis propria with polymorphonuclear leukocytes and activation of mitogen-activated protein kinases and nuclear factor-κB. All of these effects were attenuated by treatment with CO-LR. The salutary effect of CO-LR on gut motility, as well as many of the anti-inflammatory effects of CO-LR, was diminished by treatment with a soluble guanylyl cyclase (sGC) inhibitor, suggesting that the effects of CO are mediated via activation of sGC. These data support the view that a single intraperitoneal dose of CO-LR ameliorates postoperative ileus in mice by inhibiting the inflammatory response in the gut wall induced by surgical manipulation, possibly in a sGC-dependent fashion.

Proinflammatory Role of Leukocyte-Derived Egr-1 in the Development of Murine Postoperative Ileus

BACKGROUND & AIMS Early growth response gene-1 (Egr-1) is an important inflammatory transcription factor. We hypothesize that leukocyte-derived Egr-1 plays a key inflammatory role in causing postoperative ileus. METHODS Wild-type, Egr-1 knockout, and chimera mice (constructed by irradiation followed by injection with Egr-1(+/+) or Egr-1(-/-) bone marrow) were subjected to surgical manipulation of the gastrointestinal tract to induce ileus. Reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry quantified and localized Egr-1. Lumenal transit of nonabsorbable fluorescein isothiocyanate-labeled dextran and in vitro organ bath techniques measured functional gastrointestinal motility. Inflammatory mediator expressions were measured by Griess reaction, enzyme-linked immunosorbent assay, and multiplex Luminex assay. RESULTS Intestinal manipulation rapidly and significantly induced Egr-1 messenger RNA and protein within the inflamed muscularis externa. Egr-1 was colocalized early to smooth muscle and enteric neurons and later in extravasated monocytes after surgery when postoperative ileus was functionally prominent. The functional severity of postoperative ileus was significantly ameliorated in mice deficient in Egr-1(-/-) and chimera wild-type mice transplanted with Egr-1(-/-) bone marrow, whereas knockout mice with Egr-1(+/+) bone marrow again displayed significant ileus. Motility was mechanistically associated in Egr-1(-/-) gene deficiency with a down-regulation in the release of nitric oxide, prostanoids, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, interleukin-6, interleukin-1, and granulocyte colony-stimulating factor, as well as a decrease in the recruitment of leukocytes into the manipulated muscle wall of the intestine compared with wild-type mice. CONCLUSIONS Leukocyte-derived Egr-1 plays an early critical inflammatory role in the initiation of the postoperative inflammatory response, which leads to a prolonged decreased in gastrointestinal motility after intestinal surgery.

Role of interleukin 10 in murine postoperative ileus

Background and aims: Intestinal manipulation triggers an inflammatory cascade within the muscularis causing postoperative ileus (POI). The aim of this study was to investigate the recovery and therapeutic potential of interleukin 10 (IL10) for POI. Methods: POI was induced by bowel surgical manipulation (SM) in wild-type, IL10–/– and recombinant murine IL10 (rmIL10)-treated mice. Immunohistochemistry localised IL10 in the muscularis externa, histochemistry quantified neutrophil recruitment, and quantitative PCR quantified alterations in mRNA. Luminex multiplex analysis, Griess reaction and ELISA measured proteins, nitric oxide (NO) and prostanoid release from the muscularis externa, respectively, in 24 h organ culture. Gastrointestinal transit and jejunal circular muscle organ bath techniques assessed gastrointestinal function. Results: In IL10 knockouts compared with the wild type, the expression of numerous proinflammatory mRNAs (IL6, IL1β, chemokine C-C motif ligand 2 (CCL2) and haem oxygenase-1) and proteins (IL6, IL1α, IL12, IL17, interferon γ, tumour necrosis factor α, CCL2, interferon-inducible protein-10 and granulocyte–macrophage colony-stimulating factor (GM-CSF)) were accentuated, and release of muscle inhibitors NO and prostanoids was increased; motility never recovered from manipulation and mortality rate was 87.5%. In wild types, complete functional recovery occurred in 7 days with no mortality. SM delay in transit and suppression in jejunal circular muscle contractions were significantly improved by rmIL10 treatment. Upregulation in IL1β, IL6 and CCL2 mRNAs and inflammatory mediators (IL1α, IL6, CCL2, macrophage inflammatory protein-1α, GM-CSF, NO and prostaglandin) after SM were significantly less with rmIL10 treatment, which resulted in a decrease in neutrophil recruitment compared with SM controls. Conclusion: IL10 plays an obligatory role in postoperative intestinal recovery, and exogenous IL10 prevents its development. Pre-emptive exogenous recombinant human IL10 could be a treatment for the prevention of clinical POI.

Anti‐inflammatory role of glycine in reducing rodent postoperative inflammatory ileus

Background  Inflammatory events within the intestinal muscularis, including macrophage activation and leukocyte recruitment, have been demonstrated to participate in causing postoperative ileus. Recently, glycine has gained attention due to its beneficial immunomodulatory effects in transplantation, shock and sepsis.

IL-10 administration attenuates pulmonary neutrophil infiltration and alters pulmonary iNOS activation following hemorrhagic shock

Abstract.Objective and design:Several studies report immuno-modulatory effects of endogenous IL-10 after trauma. This study investigates the effect of IL-10 administration on systemic and pulmonary inflammation in hemorrhagic shock.Material and Methods:Male C57/BL6 mice (4–6 animals per group) were subjected to volume controlled hemorrhagic shock for 3 hrs followed by resuscitation. Animals were either subcutaneously injected with 0.9 % saline (Shock group) or with recombinant mouse IL-10 (Shock+IL-10 group) 1 h before and 1 h after the induction of hemorrhagic shock. Serum TNF-α, IL-6, and keratinocyte (KC) concentrations were measured with the LuminexTM multiplexing platform. Acute pulmonary inflammation was assessed by pulmonary myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activity.Results:IL-10 administration significantly decreased serum TNF-α (10.30 ± 1.68 vs 37.42 ± 10.64; p < 0.05), IL-6 (44.22 ± 6.65 vs 85.24 ± 7.94; p < 0.05), and KC (276.74 ± 52.67 vs 465.61 ± 58.98; p < 0.05) levels following hemorrhagic shock. Further, pulmonary MPO activity was significantly lower (2698.85 ± 431.10 vs 4580.67 ± 294.38; p < 0.05) and pulmonary iNOS activity upregulated.Conclusion:These findings suggest that administration of IL-10 modulates the degree of hemorrhage-induced systemic and pulmonary inflammation and support the notion of a central role for iNOS in acute lung injury.

IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock

In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3h followed by resuscitation. Animals were either sacrificed 3 or 24h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the Luminex multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock.

Alvimopan and COX-2 inhibition reverse opioid and inflammatory components of postoperative ileus

Abstract  Our objective was to investigate the therapeutic potential of peripheral opioid antagonism with alvimopan and anti‐inflammatory cyclooxygenase 2 (COX‐2) inhibition in an animal model of postoperative ileus with pain management. Intestinal manipulation was conducted in mice and rats with or without postoperative morphine injection. Rodents were orally fed non‐digestible fluorescein (FITC)‐labelled dextran and transit measured after a period of 90 min. The immunomodulatory effects of morphine and alvimopan were determined on nitric oxide released from the organ cultured muscularis externa. Surgical manipulation of the intestine resulted in a delay in gastrointestinal transit after 24 h that worsened with exogenous morphine. Alvimopan did not significantly alter transit of control or manipulated animals, but significantly antagonized the transit delaying effects of morphine. However, when the inflammatory component was robust enough to obscure a further opioid induced delay in gastrointestinal transit, alvimopan ceased to be effective in improving postoperative intestinal function. Cyclooxygenase 2 inhibition significantly diminished the inflammatory component of postoperative ileus. Surgical manipulation resulted in an increased release of nitric oxide from the inflamed isolated muscularis externa in 24‐h organ culture which was not altered by morphine or alvimopan. Two distinct mechanisms exist which participate in postoperative bowel dysfunction: a local inflammatory response which is antagonized by COX‐2 inhibition, and a morphine‐induced alteration in neural function which can be blocked with alvimopan.

Differential Molecular and Cellular Immune Mechanisms of Postoperative and Lps-Induced Ileus in Mice and Rats

Ileus is caused by the initiation of a complex cascade of molecular and cellular inflammatory responses within the intestinal muscularis, which might be species specific. Our objective was to investigate a possible immunological divergence in the mechanisms of postoperative- and endotoxin-induced ileus in C57BL/6 mice and Sprague-Dawley rats. Gastrointestinal transit (GIT) was measured at 24 h after the injurious stimulus. MPO-staining and F4/80 immunohistochemistry were used to quantify polymorphonuclear and monocyte infiltration of jejunal muscularis whole-mounts, and intestinal muscularis MCP-1, ICAM-1 and iNOS gene expression was assessed by RT-PCR. Intestinal muscularis subjected to in vivo surgical manipulation (SM) or LPS treatment was cultured for 24 h, and the liberation of nitric oxide and chemokines/cytokines into the culture medium was analyzed by Griess reaction and Luminex multiplex assay. Intestinal SM and lipopolysaccharide (LPS) (15 mg/kg) caused a significant delay in gastrointestinal transit, which was more severe in mice compared to rats in both injury models. Both SM- and LPS-triggered neutrophil and monocytic extravasation into the rat jejunal muscularis exceeded the cellular infiltration seen in mice. These results correlated with significantly greater increases in rat muscularis MCP-1 (syn. CCL2), ICAM-1 and iNOS message with more subsequent NO production after SM or LPS compared to mouse. The cultured muscularis obtained from SM mice released significantly more inflammatory proteins such as TNF-α, IL-1-α, IL-4 and GM-CSF compared to the manipulated rat muscularis. In contrast, LPS initiated the secretion of significantly more IL-1β by the inflamed rat muscularis compared to the mouse, but GM-CSF (syn. CSF2) liberation from mouse muscularis was markedly higher compared to LPS-treated rat muscularis. The data indicate that mechanistically the development of ileus in rat is mediated predominately through a leukocytic pathway consisting of chemotaxis, cellular extravasation and NO liberation. Whereas, the more intense mouse ileus evolves via a potent but injury-specific local cytokine response.

Functional assessment of intestinal motility and gut wall inflammation in rodents: analyses in a standardized model of intestinal manipulation.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.(1,2) These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.(3) The inflammation is characterized by liberation of proinflammatory mediators such as IL-6(4) or IL-1β or inhibitory neurotransmitters like nitric oxide (NO).(5) Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.(2) Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.(6) In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Komplikationen durch immunsuppressive Therapie nach Lebertransplantation – eine retrospektive 12-Jahres-Analyse des Transplantationszentrums Bonn

Hintergrund: Seit der ersten orthotopen Lebertransplantation (oLTx) 1963 hat sich die oLTX als Standardverfahren der Therapie progredienter Leberkrankheiten etabliert. Die Überlebensraten wurden kontinuierlich verbessert. Nebenwirkungen immunsuppressiver Therapie treten seither zunehmend in den Vordergrund. Besonders arterielle Hypertonie (AH), Post-Transplant Diabetes Mellitus (PTDM) und Einschränkungen der Nierenfunktion (Niereninsuffizienz (NI)) sind relevante Probleme, die die Lebensqualität und Prognose der Patienten einschränken und hohe Kosten verursachen. Patienten und Methoden: In einer retrospektiven Analyse wurden die Daten von 248 oLTx der Chirurgischen Universitätsklinik Bonn in den Jahren 01/1992 bis 01/2005 ausgewertet. Ergebnisse: In Abhängigkeit von der jeweiligen Immunsuppression zeigte sich eine erhöhte postoperative Inzidenz für AH (prä- 25% vs. postoperativ 50%; Cyclosporin A (CyA) 47% vs. Tacrolimus (FK) 46%) und NI (prä- 29% vs. postoperativ 67%; CyA 65% vs. FK 57%). Bei PTDM lag eine hohe postoperative Inzidenz von 50%, aber kein signifikanter Unterschied zwischen den verschiedenen immunsuppressiven Therapien vor (CyA 45% vs. FK 51%). Schlussfolgerungen: Postoperative Nierenfunktionseinschränkungen und weitere Komplikationen nach oLTx sind häufig bedingt durch die Calcineurin-Inhibitor (CNI)-basierte immunsuppressive Therapie. Durch Modifikation des immunsuppressiven Therapieregimes (Einsatz von Mycophenolsäure + CyA oder FK und Corticosteroiden) konnten viele der CNI-bedingten Nebenwirkungen vermieden oder minimiert werden. mTOR (mammalian target of rapamycin)-Inhibitoren oder monoklonale Antikörper gegen IL-2-Rezeptoren stellen alternative Therapieoptionen ohne negative Auswirkungen auf die Nierenfunktion dar.