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Introduction Postoperative ileus (POI) is a frequent complication after abdominal surgery (AS). Until today, neither a prophylaxis nor an evidence-based therapy exists. This originates from the absence of objective parameters evaluating the severity and duration of POI resulting in clinical trials of modest quality. The SmartPill®, a capsule which frequently measures pH value, temperature and intraluminal pressure after swallowing, offers an elegant option for analysing gastrointestinal (GI) transit times and smooth muscle activity in vivo. As the use in patients in the first months after AS is not covered by the marketing authorisation, we aim to investigate the safety and feasibility of the SmartPill® immediately after surgery. Additionally, we analyse the influence of prokinetics and laxatives as well as standardised physiotherapy on postoperative bowel contractility, as scientific evidence of its effects is still lacking. Methods and analysis The PIDuSA study is a prospective, single-centre, two-arm, open-label trial. The SmartPill® will be applied to 55 patients undergoing AS having a high risk for POI and 10 patients undergoing extra-abdominal surgery rarely developing POI. The primary objective is the safety of the SmartPill® in patients after surgery on the basis of adverse device effects/serious adverse device effects (ADE/SADE). The sample size suggests that events with a probability of 3% could be seen with a certainty of 80% for at least once in the sample. Secondary objective is the analysis of postoperative intestinal activity in the GI tract in both groups. Furthermore, clinical signs of bowel motility disorders will be correlated to the data measured by the SmartPill® to evaluate its significance as an objective parameter for assessing POI severity. Additionally, effects of prokinetics, laxatives and physiotherapy on postoperative peristaltic activity recorded by the SmartPill® will be analysed. Ethics and dissemination The protocol was approved by the federal authority (94.1.05-5660-8976) and the local ethics committee (092/14-MPG). Findings will be disseminated through publications and conference presentations. Trial registration number NCT02329912; Pre-results.

Clinical evidence suggests that recurrent acute cellular rejection (ACR) may trigger chronic rejection and impair outcome after intestinal transplantation. To test this hypothesis and clarify underlying molecular mechanisms, orthotopic/allogenic intestinal transplantation was performed in rats. ACR was allowed to occur in a MHC-disparate combination (BN-LEW) and two rescue strategies (FK506monotherapy vs. FK506+infliximab) were tested against continuous immunosuppression without ACR, with observation for 7/14 and 21 days after transplantation. Both, FK506 and FK506+infliximab rescue therapy reversed ACR and resulted in improved histology and less cellular infiltration. Proinflammatory cytokines and chemotactic mediators in the muscle layer were significantly reduced in FK506 treated groups. Increased levels of CD4, FOXP3 and IL-17 (mRNA) were observed with infliximab. Contractile function improved significantly after FK506 rescue therapy, with a slight benefit from additional infliximab, but did not reach nontransplanted controls. Fibrosis onset was detected in both rescue groups by Sirius-Red staining with concomitant increase of the fibrogenic mediator VEGF. Recovery from ACR could be attained by both rescue therapy regimens, progressing steadily after initiation of immunosuppression. Reversal of ACR, however, resulted in early stage graft fibrosis. Additional infliximab treatment may enhance physiological recovery of the muscle layer and enteric nervous system independent of inflammatory reactions.

Background/Aims: Ischemia reperfusion (I/R) injury after small bowel transplantation leads to inflammatory reactions and loss of structural integrity with subsequent graft contractile dysfunction in the early postoperative phase. The natural tetrahydropyrimidine ectoine (1-,4-,5-,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid; THP) protects the ileal mucosa and muscularis against effects of I/R injury in an experimental model of isolated graft reperfusion. The effects of THP treatment were evaluated in an established experimental intestinal transplant model. Methods: Isogenic, orthotopic small bowel transplantation was performed in Lewis rats (6 h cold ischemia time). Perioperative THP treatment (intraluminal/intravascular) groups were compared to vehicle-treated animals (after 3 and 24 h) and non-transplanted controls (n = 5/group). Park’s score defined the effects of I/R injury. The infiltration of neutrophils, monocytes and macrophages, mRNA expression of IL-6 and TNF-α, serum levels of IL-6 and NO and smooth muscle contractility were evaluated. Results: Improved graft outcome after intraluminal and intravascular THP treatment was defined by considerably ameliorated neutrophil infiltration and less histological signs of I/R injury (p ≤ 0.05). In the presence of THP, mRNA expression of IL-6 and TNF-α and IL-6 and NO serum levels were reduced and smooth muscle function was improved. Conclusion: THP treatment offers protection against the effects of I/R injury in intestinal transplantation in vivo, however, only as supplementary treatment option.

Ischemia-reperfusion injury leads to impaired smooth muscle function and inflammatory reactions after intestinal transplantation. In previous studies, infliximab has been shown to effectively protect allogenic intestinal grafts in the early phase after transplantation with resulting improved contractility. This study was designed to reveal protective effects of infliximab on ischemia-reperfusion injury in isogenic transplantation.Isogenic, orthotopic small bowel transplantation was performed in Lewis rats (3 h cold ischemia). Five groups were defined: non-transplanted animals with no treatment (group 1), isogenic transplanted animals with vehicle treatment (groups 2/3) or with infliximab treatment (5 mg/kg body weight intravenously, directly after reperfusion; groups 4/5). The treated animals were sacrificed after 3 (group 2/4) or 24 h (group 3/5). Histological and immunohistochemical analysis, TUNEL staining, real-time RT-PCR, and contractility measurements in a standard organ bath were used for determination of ischemia-reperfusion injury.All transplanted animals showed reduced smooth muscle function, while no significant advantage of infliximab treatment was observed. Reduced infiltration of neutrophils was noted in the early phase in animals treated with infliximab. The structural integrity of the bowel and infiltration of ED1-positive monocytes and macrophages did not improve with infliximab treatment. At 3 h after reperfusion, mRNA expression of interleukin (IL)-6, TNF-α, IL-10, and iNOS and MCP-1 displayed increased activation in the infliximab group.The protective effects of infliximab in the early phase after experimental small bowel transplantation seem to be unrelated to ischemia-reperfusion injury. The promising effects in allogenic transplantation indicate the need for further experiments with infliximab as complementary treatment under standard immunosuppressive therapy. Further experiments should focus on additional infliximab treatment in the setting of acute rejection.

Mechanical trauma of the gut is an unavoidable event in abdominal surgery. Former studies demonstrated that intestinal manipulation induces a strong inflammation within the tunica muscularis. We hypothesized that mechanical strain initiates or aggravates proinflammatory responses in intestinal smooth muscle cells (iSMC) or macrophages. First, an appropriate isolation and culture method for neonatal rat iSMC was established. Purified iSMC and primary peritoneal macrophages (pMacs) were subjected to static or cyclic strain, and gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-6, and IL-1β was analyzed by quantitative PCR. Supernatants from stretched iSMC were transferred to untreated pMacs or contrariwise, and medium transfer-triggered inflammatory gene expression was measured in unstretched cells. Finally, we investigated the synergistic effect of static strain on LPS-induced proinflammatory gene expression. Although cyclic strain failed, static strain significantly induced iNOS, COX-2, and IL-1β mRNA in iSMC. pMacs showed an increase in all inflammatory genes investigated as well as macrophage inflammatory protein (MIP)-1α and MIP-2 mRNA after static strain. Both cell entities liberated unknown mediators in response to stretch that mutually stimulated iNOS gene expression. Finally, mechanostimulation amplified LPS-induced iNOS and IL-1β gene expression in iSMC as well as COX-2 and IL-6 mRNA in pMacs. In conclusion, static strain initiates proinflammatory gene expression in iSMC and pMacs and triggers a bidirectional paracrine communication between both cultured cell entities via the liberation of unknown mediators. Furthermore, static strain synergistically operates with Toll-like receptor 4 ligation in a cell-specific manner. Hence, this study demonstrates that mechanical strain functions as an immunomodulatory stimulus in abdominal cells.

Localized abdominal surgery can lead to disruption of motility in the entire gastrointestinal tract (postoperative ileus). Intestinal macrophages produce mediators that paralyze myocytes, but it is unclear how the macrophages are activated, especially those in unmanipulated intestinal areas. Here we show that intestinal surgery activates intestinal CD103(+)CD11b(+) dendritic cells (DCs) to produce interleukin-12 (IL-12). This promotes interferon-γ (IFN-γ) secretion by CCR9(+) memory T helper type 1 (T(H)1) cells which activates the macrophages. IL-12 also caused some T(H)1 cells to migrate from surgically manipulated sites through the bloodstream to unmanipulated intestinal areas where they induced ileus. Preventing T cell migration with the drug FTY720 or inhibition of IL-12, T-bet (T(H)1-specific T box transcription factor) or IFN-γ prevented postoperative ileus. CCR9(+) T(H)1 memory cells were detected in the venous blood of subjects 1 h after abdominal surgery. These findings indicate that postoperative ileus is a T(H)1 immune-mediated disease and identify potential targets for disease monitoring and therapy.

An in vivo model was used to investigate the role of cathepsins in mouse intestine after mechanical manipulation. Inspection of different intestine segments by immunofluorescence microscopy provided evidence for a local release of cathepsin B from cells of individual gut sections shortly after traumatic injury. Densitometry of immunoblots ruled out alterations in cathepsin B expression levels. Because similar results were obtained with both mouse and rat intestine trauma models, we were interested in identifying potential targets of released cathepsin B in early post-traumatic phases. Immunoblotting revealed initial declines followed by an increase in protein levels of claudin-1 and E-cadherin, indicating that tight junctions and cell-cell adhesions were only transiently compromised by surgical trauma. Apical aminopeptidase N and dipeptidyl peptidase IV were only slightly affected, whereas basolateral low-density lipoprotein receptors were strongly up-regulated in response to trauma. As potential targets of cathepsin B released from injured cells, we identified collagen IV and laminin of the basement membrane that was damaged during initial post-traumatic stages. Because increased collagen IV expression was observed in the intestine of cathepsin B-deficient animals, we propose a direct role of cathepsin B in that it contributes to acute post-traumatic extracellular matrix damage and may thereby facilitate onset of post-operative ileus.

Background & Aims Postoperative ileus, an iatrogenic complication of abdominal surgery, is mediated by severe inflammation of the tunica muscularis. Macrophages that reside in the muscularis have important roles in initiating the inflammation. We investigated whether activation of the p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase is involved in the genesis of postoperative ileus, and whether p38-MAPK inhibition by the macrophage-specific inhibitor semapimod prevents intestinal dysmotility. Methods Postoperative ileus was induced by intestinal manipulation of the small bowel in mice. Protein kinase phosphorylation was assessed by immunoblotting of muscularis externa preparations. Proinflammatory gene expression was quantified by real-time polymerase chain reaction. Myeloperoxidase histochemistry for neutrophils was performed in jejunal segments. Nitric oxide production was measured by Griess reaction in smooth-muscle organ culture supernatants. Jejunal contractility was assessed within an organ bath setup. Intestinal motility was analyzed by gastrointestinal and colonic transit measurements. Results High levels of p38-MAPK and stress-activated protein kinase phosphorylation were observed immediately after intestinal manipulation. Semapimod treatment led to a significant decrease of p38-MAPK phosphorylation in macrophages; proinflammatory gene expression of macrophage inflammatory protein-1α, interleukin-6, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1; and neutrophil infiltration. Furthermore, semapimod completely abrogated nitric oxide production within the tunica muscularis. Subsequently, semapimod prevented the suppression of smooth muscle contractility and small intestinal and colonic motility after intestinal manipulation. Conclusion A single preoperative semapimod administration prevents intestinal macrophage activation and subsequent gastrointestinal dysmotility induced by abdominal surgery. Semapimod inhibits p38-MAPK and nitric oxide production in macrophages, making it a promising strategy for prophylaxis of postoperative ileus.

The cholinergic anti-inflammatory pathway reduces systemic tumor necrosis factor (TNF) via acetylcholine-producing memory T cells in the spleen. These choline acetyltransferase (ChAT)-expressing T cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-enhanced green fluorescent protein (eGFP) reporter mice. CD4Cremice were crossed to ChATfl/flmice to achieve specific deletion of ChAT in CD4+T cells. We observed that the majority of ChAT-expressing T cells in the human and mouse intestine have characteristics of Th17 cells and coexpress IL17A, IL22, and RORC. The generation of ChAT-expressing T cells was skewed by dendritic cells after activation of their adrenergic receptor β2. To evaluate ChAT T cell function, we generated CD4-specific ChAT-deficient mice. CD4ChAT−/−mice showed a reduced level of epithelial antimicrobial peptides lysozyme, defensin A, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT−/−mice. We conclude that ChAT-expressing T cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT-expressing T cells may function to mediate the host AMP secretion, microbial growth and expansion.

In recent years, multimodal treatment approaches have led to an increased median survival time of patients with colorectal liver metastases. In particular, this results from new perioperative chemotherapy regimens, which in turn are accompanied by an increased risk of perioperative bleeding and/or liver failure due to the hepatotoxic side effects. Nineteen to 58 % of patients treated with oxaliplatin develop sinusoidal obstruction syndrome (SOS). The influence of preexisting SOS on liver surgery remains controversial. Animals were operated 4 days after SOS induction with monocrotaline and received either vascular occlusion in the form of Pringle maneuver (PM) or hepatectomy (LR; 70 %) or a combination of both (LR + PM). Postoperative liver function was assessed by determination of liver enzyme levels, bile production, and tissue oxygen saturation. Preexisting SOS impaired morbidity after liver resection, reflected by elevated liver enzyme levels, reduced bile secretion, and low liver tissue oxygenation levels. Mortality was increased by up to 25 %. Additional ischemia in the form of PM showed no further impact in the LR ± PM group compared to LR alone. PM without LR results in high enzyme distribution in the SOS group. SOS significantly affects the outcome after liver resection in our experimental rat model only without PM and showed no protective effect in ischemia in the form of PM.

Gut manipulation and ischemia/reperfusion evoke an inflammatory response within the intestinal muscularis that contributes to dysmotility. We hypothesize that resident macrophages play a key role in initiating the inflammatory cascade. Isogenic small bowel transplantation was performed in Lewis rats. The impact of recovery of organs on muscularis inflammation was investigated by comparing cold whole-body perfusion after versus prior to recovery. The role of macrophages was investigated by transplantation of macrophage-depleted gut. Leukocytes were counted using muscularis whole mounts. Mediator expression was determined by real-time RT-PCR. Contractility was assessed in a standard organ bath. Both organ recovery and ischemia/reperfusion induced leukocyte recruitment and a significant upregulation in IL-6, MCP-1, ICAM-1 and iNOS mRNAs. Although organ recovery in cold ischemia prevented early gene expression, peak expression was not changed by modification of the recovery technique. Compared to controls, transplanted animals showed a 65% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted isografts exhibited significant less leukocyte infiltration and only a 19% decrease in contractile activity. In summary, intestinal manipulation during recovery of organs initiates a functionally relevant inflammatory response within the intestinal muscularis that is massively intensified by the ischemia reperfusion injury. Resident muscularis macrophages participate in initiating this inflammatory response.

Background Laparoscopic Surgery has become a worldwide standard procedure for a variety of indications. This has been attributed to a milder postoperative inflammatory response by the innate immune system potentially mediated through immune mediators released by the visceral adipose tissue (VAT). However, an in vivo experimental evidence is lacking and is the issue of our present study. Methods Male Wistar rats (N = 24) underwent standardized surgical procedures of conventional cecum resection (CCR), conventional sham operation, laparoscopic cecum resection (LCR), or laparoscopic sham operation. Cytokine expression of leptin, resistin, and IL-6 was analyzed in VAT before and after resection by quantitative RT-PCR. Results Postoperative leptin gene expression was reduced in the CCR and LCR groups, while expression was not significantly affected in both sham groups compared to the preoperative levels. In contrast, IL-6 expression was not affected in the LCR group, but was significantly elevated in the CCR cohort. The IL-6 expression was significantly higher in CCR compared to LCR. Resistin expression levels did not differ between all groups. Conclusions Our study underlines the role of immunological involvement of VAT in the postoperative phase. Low leptin levels seem to act as a stimulator for energy uptake in order to cope with postoperative stress. A lower IL-6 expression in the LCR compared to the CCR group may indicate a weaker inflammatory activity potentially adding to the clinical benefits observed in patients undergoing LS.

Aims Postoperative ileus (POI) is a frequent complication after abdominal surgery, resulting from an inflammation of the muscularis externa (ME). So far no valid biomarker for occurrence, duration or intensity of POI exists. Extravasation of monocytes and neutrophils from blood circulating into the postoperative ME is well known as a hallmark of POI. In a previous study we demonstrated that a low abundant subset of T H 1 cells, activated by IL-12, can be detected in the peripheral blood of a small subset of patients in response to abdominal surgery. The aim of the present study was to investigate if these specific T H 1 cells, IL-12 or circulating leukocyte levels could act as a valid marker for POI occurrence. Main methods At different time points, blood samples of patients undergoing abdominal or extraabdominal surgery were collected. Serum levels of IL-12 or T H 1 cells as well as neutrophils and monocytes were analyzed. Data were compared between both groups and correlated with clinical signs of POI. Key findings Time until first flatus and defecation as well as solid food tolerances are delayed after abdominal compared to extraabdominal surgery. Circulating IL-12 levels and numbers of T H 1 cells, neutrophils and monocytes did not differ between both groups. Significance While previous experiments indicated that specific T H 1 cells play a crucial role in POI dissemination, our present data from a larger human cohort demonstrate that they do not seem to be suitable to distinguish between abdominal and extraabdominal surgery. Furthermore neither T H 1 cells nor leukocytes or serum IL-12 levels are appropriate biomarkers for POI in a clinical setting.

Resolution of inflammation is an active counter-regulatory mechanism involving polyunsaturated fatty acid-derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15-lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1+/Ly6C+ infiltrating monocytes rather than Ly6G+ neutrophils. Mass spectrometry analyses demonstrated that a 12/15-lipoxygenase increase was accompanied by production of docosahexaenoic acid-derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17-hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood-derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G+/Ly6C+ neutrophil extravasation after protectin DX treatment, whereas Ly6G-/Ly6C+ monocyte numbers were not affected. 12/15-lipoxygenase-deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid-rich lipid emulsion reduced postoperative leukocyte infiltration in wild-type mice but failed in 12/15-lipoxygenase-deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15-lipoxygenase-deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15-lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid-derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15-lipoxygenase pathway, may be instrumental in prevention of postoperative ileus.

Growing evidence shows that the soluble N-terminal form (sAPPalpha) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPPalpha, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPPalpha has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts.

sAPPalpha, the soluble form of the beta-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPalpha. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPalpha. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPalpha. We found that recombinant sAPPalpha facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPalpha to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPalpha. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPalpha suggesting that sAPPalpha mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were significantly reduced by about 59% after addition of recombinant sAPPalpha. These results show that sAPPalpha exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPalpha.

sAPP, the secretory domain of the β-amyloid precursor protein (APP), exerts a growth promoting and motogenic activity on keratinocytes. Here we report on the expression of APP and its homologue, the amyloid precursor like protein 2 (APLP2), during cutaneous wound repair using a full-thickness excisional wound healing model in mice. In unwounded skin APP was predominantly expressed in the basal cell layer. During wound healing increased suprabasal expression of APP was observed in all cell layers of the hyperproliferative epithelium at the wound margin. APP mRNA was increased up to 2.3-fold, whereas the APLP2 mRNA was decreased. Immunocytochemically, all proliferation competent keratinocytes of the normal as well as the wound site epidermis showed increased expression of APP but not of APLP2. Using culture models of keratinocyte differentiation the release of sAPP was found to be significantly higher in proliferating cells, i.e., when cultured at subconfluency or at low [Ca2+], than in quiescent, partially differentiated keratinocytes cultured at confluency or at high [Ca2+]. Our results suggest that sAPP secretion is presumably also increased in proliferation competent keratinocytes of the wound margin and that sAPP due to its growth promoting and motogenic function might participate in the control of epidermal wound repair.

Postoperative ileus, a major cause of morbidity after abdominal surgery, is characterized by intestinal dysmotility and inflammation. The aim was to investigate the involvement of sphingolipids in postoperative intestinal inflammation using a standardized rat model of intestinal surgical manipulation. Sphingolipid analysis (ESI-MS) of intestinal muscularis after manipulation revealed a time-dependent increase of sphingosine 1-phosphate (S1P) and of ceramide 1-phosphate (C1P). We therefore established a culture system of primary rat intestinal smooth muscle cells and examined the potential role of these sphingolipids in intestinal inflammation. Incubation of cells with either of the two sphingolipid-phosphates resulted in an elevated production of PGE(2). Further analysis revealed that S1P enhances cyclooxygenase 2 (COX-2) expression whereas C1P increases release of arachidonic acid, indicating an enhanced phospholipase A(2) activity. S1P-induced COX-2 expression was pertussis toxin sensitive, suggesting the involvement of Gi/o protein-coupled S1P receptors. Further downstream mediators of S1P induced COX-2 expression appear to be extracellular regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Collectively, our results demonstrate that intestinal smooth muscle cells represent a major target for both C1P and S1P activity. Thus, the sustained elevated concentration of the two bioactive sphingolipids in this tissue could at least in part explain postoperative intestinal dysmotility.

Introduction Intestinal manipulation leads to local bowel wall inflammation that subsequently spreads over the entire gastrointestinal tract. Previously, this gastrointestinal field effect had been demonstrated by us in a rodent model. We herein postulated an immunologic mechanism mediated by activated leukocytes. The aim of this study was to investigate the activation, maturation and migration of dendritic cells (DC) of the intestinal smooth muscle following surgical trauma and i.p. lipopolysaccharide challenge. Methods Mice underwent standardized intestinal manipulation or iP LPS administration and tissues (intestinal muscularis, Peyer’s patches, mesenteric lymph nodes, and spleen) were obtained at various times after manipulation. DC were isolated by tissue digestion and separated by CD11c-iMAG. The harvested DC were analyzed by FACS. The activation pattern of DC was analyzed by polymerase chain reaction. Results We found a significant increase in DC within the intestinal muscularis, the Peyer’s patches and the mesenteric lymph nodes at 6 and 12 hours following intestinal manipulation and injection of LPS. There was an upregulation of the costimulatory molecules major histocompatibility complex II, CD40, CD80, CD86, and CD205 in the DC after intestinal manipulation. CCR-2, CCR-5, CCR-7, CCL-19, and interleukin-12a were upregulated in a time- and tissue-dependent manner. Conclusion Intestinal manipulation or LPS challenge induced a recruitment of DC into the muscularis externa and mesenteric lymph nodes combined with an upregulation of costimulatory immunocompetent molecules and migratory surface markers in DCs. These findings demonstrate a precondition for an immunologic response and a possible immunologically mediated gastrointestinal field effect.

Background & Aims Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. Methods We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2 −/− , TLR-4 −/− , TLR-2/4 −/− , MyD88 −/− , MyD88/TLR adaptor molecule 1 −/− , interleukin-1 receptor [IL-1R1] −/− , and interleukin (IL)-18 −/− mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. Results Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1 −/− mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. Conclusions IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with the IL-1 signaling pathway are likely to be effective in the treatment of POI.

BACKGROUND Resident macrophages within the tunica muscularis are known to play a crucial role in initiating severe inflammation in response to ischemia reperfusion injury after intestinal transplantation contributing to graft dysmotility, bacterial translocation, and possibly, acute rejection. The p38 mitogen-activated protein kinase is a key player in the signaling of proinflammatory cytokine synthesis in macrophages. Therefore, we investigated the effects of CPSI-2364, an apparent macrophage-specific inhibitor of the p38 mitogen-activated protein kinase pathway in an isogenic intestinal rat transplantation model. METHODS Recipient and donor animals were treated perioperatively with CPSI-2364 (1 mg/kg, intravenously) or vehicle solution. Nontransplanted animals served as control. Animals were killed 30 min, 3 hr, and 18 hr after reperfusion. RESULTS CPSI-2364 treatment resulted in significantly less leukocyte infiltration and significantly improved graft motor function (18 hr). Messenger RNA expression of proinflammatory cytokines (interleukin 6) and kinetic active mediators (NO) was reduced by CPSI-2364 in the early phase after transplantation. Histologic evaluation revealed the protective effects of CPSI-2364 treatment by a significantly less destruction of mucosal integrity at all time points. Perioperative treatment with CPSI-2364 improves graft motor function through impaired inflammatory responses to ischemia reperfusion injury by inhibition of proinflammatory cytokines and suppression of nitric oxide production in macrophages. CONCLUSIONS CPSI-2364 presents as a promising complementary pharmacological approach preventing postoperative dysmotility for clinical intestinal transplantation.

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.

Background Active matrix metallopeptidase 9 (MMP-9) disruption of the extracellular matrix (ECM) plays an important role in inflammatory disorders. In this study, we investigated the inflammatory role of MMP-9 and the ECM breakdown product hyaluronan as a trigger for the postoperative intestinal inflammatory response of postoperative ileus. Methods We performed a standardized intestinal surgical manipulation on rats to produce ileus assessed by the oral non-digestible fluorescein isothiocyanate–dextran transit assay. We studied isolated intestinal muscularis extracts for mRNA expressions of interleukin 6 (IL-6), MMP-9 and CD44. We quantified peritoneal MMP-9 activity using zymography, and quantified peritoneal fluid and serum for hyaluronan and tissue inhibitor of metalloproteinase 1 levels by enzyme-linked immunosorbent assay (ELISA). We cultured peritoneal macrophages and exposed them to peritoneal fluid or synthetic hyaluronan for ELISA analysis of IL-6 and macrophage inflammatory protein-1α. Results Transit was significantly delayed after surgical manipulation, and extracts of the isolated jejunal and colonic muscularis demonstrated a significant induction of IL-6, MMP-9, and CD44 mRNAs compared with controls. Zymography confirmed significant MMP-9 activity in peritoneal fluid compared with controls. Enzyme-linked immunosorbent assay measurements showed a significant up-regulation in hyaluronan and tissue inhibitor of metalloproteinase 1 in the peritoneal fluid and serum. In addition, ELISA and reverse transcriptase–polymerase chain reaction measurements of peritoneal macrophages stimulated with postsurgical peritoneal fluid and synthetic hyaluronan resulted in higher expressions of IL-6 and macrophage inflammatory protein-1α in the macrophage supernatant. Conclusions Our results confirm that MMP-9 disruption in the ECM with hyaluronan release and muscularis CD44 receptor induction has the potential to trigger muscularis proinflammatory cascades that cause postoperative ileus. Matrix metallopeptidase 9 inhibition may be a novel therapeutic approach to limit postoperative ileus.

Background: Heme oxygenase (HO-1) protects against inflammation. In this study, we investigated the protective function of hemin-induced HO-1 against lipopolysaccharide (LPS)-induced ileus. Methods: Rats received LPS intraperitoneally 24 h after intraperitoneal hemin pretreatment or placebo. We also injected zinc protoporphyrin (ZnPP, 3rd group), an inhibitor of HO-1, intraperitoneally 2 h before LPS administration. To assess intestinal muscle function, we examined muscularis strip contractility in an organ bath and measured gastrointestinal transit in vivo. We investigated inflammation within the muscularis using polymerase chain reaction (interleukin [IL]-6, inducible nitric oxide synthase (iNOS), HO-1 and IL-10) 6 and 24 h after LPS. Results: Hemin significantly improved in vitro intestinal muscularis contractility (P < 0.001). In addition, hemin prevented LPS-induced dysmotility in vivo (gastrointestinal transit, geometric center: 8.39 � 0.33 versus 5.68 � 0.44; P < 0.001). In Zinc protoporphyrin (ZnPP)-treated animals, both parameters were significantly decreased compared with the hemin group. Messenger RNA expression demonstrated a significant reduction in IL-6 (6 h, hemin: 127.6 � 36.7 versus LPS: 14,431 � 5407; 24 h: 1.58 � 0.39 versus 11.15 � 2.59;P < 0.01) and iNOS (6 h: 2516 � 985 versus 50,771 � 13,321; 24 h: 55.11 � 10.55 versus 257.1 � 43.18; P < 0.001) in hemin-treated animals. Anti-inflammatoryHO-1messengerRNAlevels(6h,hemin:116.3 � 18.55versusLPS:26.02 � 3.64; 24h:18.46 � 2.69versus2.80 � 0.32;P

Postoperative ileus (POI) is an iatrogenic complication of abdominal surgery, mediated by a severe inflammation of the muscularis externa (ME). Previously, we demonstrated that intravenous application of the tetravalent guanylhydrazone semapimod (CNI-1493) prevents POI, but the underlying mode of action could not definitively be confirmed. Herein, we investigated the effect of a novel orally active salt of semapimod (CPSI-2364) on POI in rodents and distinguished between its inhibitory peripheral and stimulatory central nervous effects on anti-inflammatory vagus nerve signaling. Distribution of radiolabeled orally administered CPSI-2364 was analyzed by whole body autoradiography and liquid scintillation counting. POI was induced by intestinal manipulation with or without preoperative vagotomy. CPSI-2364 was administered preoperatively via gavage in a dose- and time-dependent manner. ME specimens were assessed for p38-MAP kinase activity by immunoblotting, neutrophil extravasation, and nitric oxide production. Furthermore, in vivo gastrointestinal (GIT) and colonic transit were measured. Autoradiography demonstrated a near-exclusive detection of CPSI-2364 within the gastrointestinal wall and contents. Preoperative CPSI-2364 application significantly reduced postoperative neutrophil counts, nitric oxide release, GIT deceleration, and delay of colonic transit time, while intraoperatively administered CPSI-2364 failed to improve POI. CPSI-2364 also prevents postoperative neutrophil increase and GIT deceleration in vagotomized mice. Orally administered CPSI-2364 shows a near-exclusive dispersal in the gastrointestinal tract and effectively reduces POI independently of central vagus nerve stimulation. Its efficacy after single oral dosage affirms CPSI-2364 treatment as a promising strategy for prophylaxis of POI.

CLINICAL BACKGROUND: In all patients undergoing abdominal surgery, a transient phase of interruption of bowel motility, named postoperative ileus (POI) occurs. POI is often accepted as an unavoidable "physiological" response and a self-limiting complication after surgery although it has a significant impact on patient morbidity with prolonged hospitalization and increased costs. Annual economic burden has been estimated as much as US $1.47 billion in the USA (Iyer et al. in J Manag Care Pharm 15(6):485-494, 2009).The pathophysiology has been elucidated within the last decades, demonstrating that both, neurogenic and inflammatory mechanisms are involved in response to the surgical trauma. It is now generally accepted that POI pathogenesis processes in two phases: a first neurogenic phase is accountable for the immediate postoperative impairment of bowel motility. This is followed by a second immunological phase that can last for days and mainly affects strength and length of POI. More recent findings demonstrate a bidirectional interaction between the nervous and the immune system, and this interaction significantly contributed to our present understanding of POI pathophysiology. Although nerval mechanisms have a significant impact in the early phase of POI, the contribution of the immune system and subsequently its manipulation has risen as the most promising strategy in prevention or treatment of the clinically relevant prolonged form of POI.The present manuscript will give an update on the inflammatory responses, the involved cell types, and participating immune mediators in POI.

Abdominal surgery results in an inflammation of the intestinal muscularis externa (ME), subsequently leading to postoperative ileus (POI). Polyunsaturated fatty acids (PUFA) are known to modulate inflammation. The aim of this study was to analyze the effect of preoperative parenteral administration of marine (n-3) or soybean (n-6) PUFA lipid emulsions (PUFA-LE) on POI and tissue fatty acid profiles. Rodents underwent intestinal manipulation (IM) after 5 days of parenteral administration of 10-mL/kg body weight saline, (n-3), or (n-6) PUFA-LE. Sham animals received saline treatment without IM. In rats, postoperative inflammation was quantified by ME neutrophil levels and NO production in organ culture, and ME function was determined by an in vitro contractility measurement. Additionally, in vivo gastrointestinal transit (GIT) was analyzed in mice. Lipopolysaccharide-induced IL-6 expression of rat bone marrow-derived mononuclear cells and ME was analyzed. Fatty acids were measured by gas chromatography in rat blood, bone marrow cells, and ME. The (n-3) PUFA-LE reduced neutrophil levels and NO production after IM and improved in vitro jejunal contractility and GIT time. The (n-6) PUFA-LE significantly reduced postoperative inflammation and tended to improve intestinal motility (P

Significant improvements of graft and patient survival have been achieved over the past 20 years in the field of intestinal transplantation. Tacrolimus monotherapy with corticosteroids, or in combination with sirolimus is the most commonly used immunosuppressive regimen. Early (24h) after experimental allogenic small bowel transplantation in rats, sirolimus reduces the cellular and molecular inflammatory response with subsequent graft dysmotility more efficiently than tacrolimus, with contrary effects at 7 days after transplantation. This study evaluates three immunosuppressive strategies in the post-acute phase after intestinal transplantation - tacrolimus or sirolimus monotherapy and the combination therapy of tacrolimus with infliximab. After orthotopic intestinal transplantation between Brown Norway and Lewis rats, animals received 14 days of immunosuppressive treatment. Histology, infiltration of neutrophils, monocytes and macrophages, cytokine and mediator mRNA expression (real time RT-PCR) and smooth muscle function in a standard organ bath were assessed at 14 days after transplantation in all treatment groups and isogenic controls. Allogenic transplanted rats without immunosuppressive therapy and non-transplanted animals served as further control. Tacrolimus prevented acute rejection and graft dysmotility more effectively (p ≥ 0.05) than sirolimus. Reduced mRNA expression levels of CD4, IFN-γ, IL-6, IL-10, iNOS, NFκB, TNF-α and MCP-1 (p ≤ 0.05) were observed in tacrolimus treated animals compared to sirolimus. Additional infliximab application did not influence the cellular and molecular inflammatory response in the post-acute phase after transplantation. In conclusion, the severe cellular and molecular inflammatory response in allogenic transplanted grafts without immunosuppressive therapy is ameliorated by all three immunosuppressive regimens, but tacrolimus was found to be more efficient than sirolimus at 14 days after transplantation. Our findings do not rule out the usage of sirolimus as single immunosuppressive therapy, but indicate and confirm the potency and effectivity of tacrolimus as basis immunosuppressant in the field of intestinal transplantation.

Prevention of perioperative activation of intestinal muscularis macrophages is a promising intervention to avoid post-traumatic gastrointestinal tract dysfunction. However, impaired macrophage function could have deleterious consequences on anastomotic healing, especially in complications aggravating the healing process itself, such as infectious problems either as preexisting local inflammation or infection (e.g., complicated diverticulitis) or endotoxemia due to early postoperative infections (e.g., pneumonia). Aim of this study was to investigate colonic anastomotic healing in macrophage-depleted mice in the presence of endotoxemia. Colonic anastomoses were performed, and mice were randomized into six groups (wild type; wild type with endotoxemia; pharmacological depletion of macrophages; pharmacological depletion with endotoxemia; genetically conditioned within the gut muscularis macrophage-deficient osteopetrotic mice; osteopetrotic mice with endotoxemia). Anastomotic tissues were removed 2, 5, and 10 days after surgery and used for functional, histological, biochemical, and molecular investigations. After pharmacological pretreatment, an almost complete depletion of macrophages was found in the muscularis up to 24 h postoperatively. Bursting pressure was significantly lower than 10 days after anastomotic procedure in osteopetrotic mice during endotoxemia, in marked contrast to transient pharmacologically macrophage-depleted mice. Pharmacological depletion during endotoxemia did not affect hydroxyproline concentration. Finally, in osteopetrotic mice during endotoxemia, collagen-3 expression was significantly lower compared to controls. In our current model, we demonstrate that perioperative pharmacological macrophage depletion and inactivation transiently diminishes muscularis macrophages and does not affect intestinal anastomotic healing in the presence of endotoxemia. However, a long-lasting macrophage absence or dysfunction impairs anastomotic healing and could be a risk factor for postoperative anastomotic leakage.

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.

Experimental and clinical studies on the sealing of colorectal anastomoses in order to reduce the rate of leakage have previously been performed with divergent results. However, comparatively few studies have been performed on anastomotic healing using a fibrin glue-coated patch. The aim of this experimental basic scientific study in mice was to investigate the effect of fibrin glue-coated collagen patches on the healing process of colonic anastomoses in situations of adverse healing process (technical deficiency and peritonitis).Colonic anastomoses were carried out in 206 mice and randomized into six groups (I: complete anastomoses, II: sealed complete anastomoses, III: incomplete anastomoses, IV: sealed incomplete anastomoses, V: complete anastomoses in the presence of bacterial peritonitis, VI: sealed complete anastomoses in the presence of bacterial peritonitis). Tissues from the anastomoses were removed and used for functional, histochemical, molecular, and biochemical investigations.The evaluation of postoperative course data revealed the beneficial effect of additional sealing with a fixed combination of collagen matrix-bound coagulation factors I and IIa (Tachosil(®), Nycomed Austria, Linz) in high-risk experimental anastomotic healing. Sealing incomplete anastomoses resulted in significantly lower lethality and leakage rates, as well as significantly higher bursting pressure values and histopathologic scores. Collagen 1 and 3 expressions and hydroxyproline concentrations are greatly increased with additional sealing in all high-risk anastomoses.In our current model, we demonstrate that additionally sealing high-risk experimental colonic anastomoses provides a positive effect on the healing process. The effect on the molecular level in particular seems to be essential and requires further experimental studies to evaluate the mechanism.

Background. The mechanical trauma of the gut is an unavoidable consequence of abdominal surgery leading to postoperative ileus (POI). Former studies revealed that activation of resident macrophages in the muscularis externa (ME) is an initial step in the inflammatory cascade resulting in massive inflammation of the bowel wall with intestinal dysmotility [1, 2]. The aim of this study was to investigate the efficacy of the macrophage-specific c-Raf-pathway inhibitor CPSI 2364 in preventing POI in swine. Additionally, we investigated disturbances of intestinal wound repair, as macrophage-function is essential in this process [3]. Materials and Methods. Swine were treated orally with placebo or 1mg/kg CPSI 2364 before standardized intestinal manipulation. 24 h later swine were sacrificed and the whole digestive tract was removed for further investigation. Inflammation within the ME of the small bowel was quantified using RT-PCR (MCP-1) and a myeloperoxidase-assay. To examine smooth muscle function, jejunal muscularis strips were exposed to an increasing concentration of a muscarinic agonist in an in vitro organ bath and contractility was analyzed. Furthermore intestinal transit time was measured in vivo. In a second experiment swine received an anastomosis of the colon to examine intestinal wound repair. On postoperative day 6 mRNA levels of wound healing parameters (VEGF, Collagen 1 and 3) and perianastomotic hydroxyproline concentration were examined. To assess mechanical strength bursting pressure was measured. Results. After treatment with CPSI 2364 a significant inflammatory reduction within the ME on mRNA- and cell-level could be demonstrated. Furthermore, smooth muscle function was improved, resulting in an accelerated intestinal transit time and an elevated contractility. Clinical course and perianastomotic mRNA-levels, hydroxyproline concentration or bursting pressure showed no evidence of impaired intestinal wound healing. Conclusion. Preoperative application of CPSI 2364 reduces postoperative inflammation within the ME subsequently preventing POI. A detrimental influence of CPSI 2364 on intestinal wound repair could not be demonstrated.

Introduction: Abdominal surgery results in the inflammation of the tunica muscularis (ME), subsequently leading to postoperative ileus (POI) [1]. Previously, we demonstrated that inhibition of macrophage function and cytokine production prevents rodents from POI [2]. Omega-3 and omega-6 polyunsaturated fatty acids (PUFA) are known to exert anti- or proinflammatory effects, respectively. The aim of this study was to analyze the effect of omega-3 and omega-6 PUFA on POI in rodents. Methods: Male rats and mice underwent intestinal manipulation (IM) after 5 days preoperative enteral or parenteral application of vehicle, omega-3 (Omegaven®) or omega-6 (Lipovenos®-MCT) PUFA-enriched emulsions (1 ml/100 g bodyweight, once daily). Erythrocyte membranes were analyzed by gas chromatography for fatty acid composition. Inflammatory activity was determined by detection of neutrophils 24 h after IM. Production of nitric oxide (NO) from ME specimen was determined by Griess reaction. Contractility of jejunal ME strips was measured in vitro in an organ bath setup. Gastrointestinal and colonic transits were determined in vivo. Results: Omegaven treatment significantly increased omega-3 to omega-6 PUFA rates in erythrocytes (1 : 9), compared to untreated controls (1 : 15). Furthermore, Omegaven reduced neutrophil levels (–35 %) and NO production (–38 %) after IM and improved contractility and gastrointestinal and colonic transit time. Interestingly, enteral Omegaven treatment showed increased NO production and tended toward increased neutrophils levels. In all experiments, Lipovenos-MCT treatment solely showed a trend to reduced inflammation and improved motility. Conclusion: Perioperative parenteral omega-3 PUFA enriched nutrition reduces intestinal inflammation and prevents postoperative ileus.

Background Postoperative ileus is mediated through a severe inflammation of the tunica muscularis. Inhibition of initially involved muscularis macrophages could be a promising clinical approach to prevent postoperative ileus. The aim of this study was to investigate whether pharmacologic or genetic depletion of these inflammatory cells influences anastomotic healing. Methods Standardized ileal anastomoses were performed and the mice were randomized into 4 groups: (1) wild type; (2) pharmacologically depleted and inactivated, by means of chlodronate liposomes and gadolinium chloride; (3) heterozygous osteopetrosis littermates; (4) genetically depleted osteopetrosis mutant mice. Tissues from the anastomoses were removed 2, 5, and 14 days after surgery and used for molecular (collagen 1 and 3, matrix metalloproteinases 2, 9, and 13 expressions), histochemical (anastomotic healing score, cross polarization microscopy) and functional (anastomotic bursting pressure) investigations. Results RT-PCR measurements demonstrated that the investigated genetic events were similar between controls and macrophage-depleted groups. Comparison of histologic healing scores and bursting pressure values showed no significant differences between the groups. Finally, cross polarization microscopy on picrosirius-red stained sections revealed no obvious disturbance in production and deposition of collagen. Conclusion In our current model we demonstrate that transient perioperative pharmacologic and genetic muscularis macrophage inhibition does not affect intestinal anastomotic healing. These results call for further investigations to establish a pharmacologic prophylaxis for the prevention of postoperative ileus.

Background The mechanical trauma of the gut is an unavoidable consequence of abdominal surgery leading to massive inflammation of the entire bowel wall and subsequently in postoperative ileus [1]. Further studies revealed the activation of the resident macrophages in the tunica muscularis as an initial step in the inflammatory cascade [2]. The aim of this study was to investigate the effectivity of the macrophage-specific c-Raf-Kinase Inhibitor CPSI 2364 in preventing postoperative ileus in rodents and swine.

Introduction: The mechanical trauma is an iatrogenic consequence of abdominal surgery that results in a massive inflammation of the entire bowel wall, subsequently leading to postoperative ileus 1, 2. In addition to the trauma, bacterial translocation and LPS challenge are discussed as initiator of this inflammation. Although several cell types are involved, resident muscularis macrophages and smooth muscle cells play a central role. The aim of this study was to investigate the cellular and molecular reactions of both cell types after LPS-challenge or mechanical trauma. Methods: Rat intestinal (iSMC) and vascular (vSMC) smooth muscle cells and peritoneal macrophages (pMacs) were repetitively stretched for 6 hours in the presence or absence of 100 ng/ml LPS. Gene expression of iNOS, COX-2 and interleukin-6 was analyzed by quantitative PCR. In another experimental setting, intercellular inflammatory stimulation was analyzed after transfer of conditioned culture media. Therefore, media from stretched cells were transferred to unstretched cells — from SMC to macrophages or vice versaand MIP-1a, MIP-2, COX-2, IL-6 and iNOS expression was analyzed after 6 hours. Results: Mechanical stretch does not alter iNOS, COX-2 or IL-6 gene expression in iSMC compared to unstretched controls. However, LPS-challenge induced a significant gene expression of IL-6 (3-fold) and iNOS (292-fold) in iSMC. LPS and stretch together led to and further significant increase of IL-6 (5-fold) and iNOS (1250-fold). However, IL-6 expression was not altered in vSMC cultures by stretch or LPS stimulation, whereas COX-2 expression was increased by stretch (7.8-fold) and enhanced by stretch with LPS (15.4-fold). Stretch together with LPS stimulation did not increase the LPS induced iNOS expression (141-fold) in vSMC. In pMacs, gene expression was not altered by mechanical stretch but all genes were strongly increased after LPS induction. LPS induced proinflammatory gene expression was significantly enhanced in combination with mechanical stretch. The transfer of conditioned media from stretched iSMC induced a significant increase in MIP-2, iNOS and COX-2 expression in pMacs. On the other way, no effect on gene expression was observed in iSMC after transfer of media from stretched pMacs. Conclusion: A mechanical trauma does not induce a proinflammatory gene expression in macrophages and iSMC but it enhances LPS induced strong inflammatory reactions in both cell types. However, in response to a mechanical trauma, iSMC liberate unknown mediators that initiate a proinflammatory reaction in pMacs. Interestingly, vascular and intestinal smooth muscle cells differ in their molecular responses to stretch. Finally, LPS stimulation and a mechanical trauma together induce a massive inflammatory reaction in intestinal smooth cells and macrophages. The investigation of the molecular basics could help to identify targets responsible for the overwhelming inflammation in postoperative inflammations.

Introduction: The mechanical trauma is an iatrogenic consequence of abdominal surgery that results in a massive inflammation of the entire bowel wall, subsequently leading to POI [1]. Former studies of our group demonstrate that a mechanical trauma in vitro results in a opposite proinflammatory activation of primary cultures of resident muscularis macrophages and smooth muscle cells (SMC) [2]. A major disadvantage of these primary cell cultures is the absence or limitation of proliferative capacity in macrophage or SMC cultures, respectively and the differentiation of SMC to myofibroblasts. Therefore, the aim of this study is to generate conditionally immortalized cell lines of both, macrophages and SMC. Methods: Two different expression vectors, encoding the large-T antigen of the SV40 virus or the v-myc oncogene of the ARK Virus were delivered by electroporation into primary macrophages and SMC. The integration of a 72bp enhancer sequence of the SV40 virus should enhance plasmids targeting to the nucleus. The conditional gene expression of the oncogenes is regulated by a tetracycline dependent promoter. Transfected cells were enriched by geneticin selection and analyzed after several passages in culture for the expression of cell specific differentiation markers. Results: The transfection of primary SMC is much more efficient compared to macrophages. Large-T transgenic SMC (LT-SMC) also showed after several months in culture the expression of α-actin whereas normal SMC cultures lost this specific marker during propagation. Furthermore, LT-SMC retained the fusiform shape that is also lost by normal SMC. In macrophage cultures, the nucleofection does not result in the cellular immortalization. Conclusion: Our results demonstrate for the first time that intestinal SMC can be immortalized by large-T antigen expression. Thereby, the LT-SMC retained the phenotype of normal intestinal SMC for several months. Resident muscularis macrophages transfection by electroporation is insufficient. The macrophage approach will be repeated by microinjection of the v-myc plasmid into the nucleus of these cells. The generation of both cell lines is essential for the investigation of cell- and organ-specific intestinal dysfunctions, subsequently identifying molecular targets to prevent postoperative ileus.

Inhibition of resident muscularis macrophage function could be a promising clinical approach to prevent postoperative ileus (POI). As an indispensable condition, the aim of this study was to investigate if depletion and inactivation of these inflammatory cells influence anastomotic healing. Methods: Standardized ileal and colonic anastomoses were performed, mice were randomized into four groups: wildtype (WT); pharmacologically depleted and inactivated mice (pMD), by means of chlodronate liposomes and gadoliniumchloride; heterocygote (op+/−) littermates; genetically depleted osteopetrosis (op−/−) mutant mice. Tissues from the anastomoses were removed 2-, 5- and 14 days after surgery and used for functional (anastomotic bursting pressure), histochemical (anastomotic healing score, cross polarisation microscopy) and molecular (collagen I and III, matrix metalloproteinases II, IX and XIII) investigations. Results: Comparison of bursting pressure values and histological healing scores showed no significant differences between the macrophage depleted groups (pMD and op−/−) and the control groups (WT and op+/−). RT-PCR measurements suggest that the investigated genetic events (Col 1, Col 3 and MMP 13 gene expressions), leading to synthesis of extracellular matrix (ECM) components, passes off similar between macrophage depleted groups (pMD, op−/−) and controls (WT). Finally, crosspolarisation microscopy on pricrosirius-red stained sections revealed no obvious disturbance in production and deposition of collagen especially in the submucosal layer. Conclusions: In our current model, perioperative pharmacological and genetical macrophage depletion and inactivation did not affect anastomotic healing.

Background: Abdominal surgery results in a molecular and cellular inflammatory response in the intestine, leading to postoperative ileus. It was hypothesised that resident macrophages within the intestinal muscularis have an important role in this local inflammation. Aims: To investigate whether chemical or genetic depletion of resident muscularis macrophages would lead to a reduction in the local inflammation and smooth-muscle dysfunction. Methods: Two rodent models were used to deplete and inactivate macrophages: (1) a rat model in which resident macrophages were depleted by chlodronate liposomes; (2) a model of mice with osteopetrosis mice, completely lacking the resident muscularis macrophages, used as an additional genetic approach. Animals with normal or altered intestinal macrophages underwent surgical intestinal manipulation. The inflammatory response was investigated by quantitative reverse transcriptase-polymerase chain reaction for mRNA of MIP-1α, interleukin (IL)1β, IL6, intracellular adhesion molecule 1 (ICAM-1) and monocyte chemotractant protein 1 (MCP)-1 in the isolated small bowel muscularis. In addition, muscularis whole mounts were used for histochemical and immunohistochemical analysis to quantify leucocyte infiltration and detect cytokine expression. Subsequently, in vitro muscle contractility and in vivo gastrointestinal transit were measured. Results: Both models resulted in markedly decreased expression of MIP-1α, IL1β, IL6, ICAM-1 and MCP-1 after manipulation compared with controls. In addition to this decrease in inflammatory mediators, recruitment of leucocytes into the muscularis was also diminished. Macrophage-altered animals had near normal in vitro jejunal circular muscle function and gastrointestinal transit despite surgical manipulation. Conclusions: Resident intestinal muscularis macrophages are initially involved in inflammatory responses resulting in postoperative ileus. Depletion and inactivation of the muscularis macrophage network prevents postoperative ileus.

Unsere Ergebnisse zeigen, dass ein mechanisches Trauma die Expression und/oder Freisetzung proinflammatorischer Mediatoren in intestinalen Makrophagen und glatten Muskelzellen induziert. Die dabei von Makrophagen oder SMC freigesetzten Mediatoren induzieren im jeweils anderen Zelltyp eine molekulare Antwort. Aufgrund der sehr fruhzeitigen, stretch-induzierten Reaktion der SMC postulieren wir eine initiale Rolle dieser Zellen beim postoperativen Ileus. Diese geht mit einer wechselseitigen Aktivierung intestinaler Makrophagen einer, die dadurch die massive Inflammation der Tunica muscularis initiiert.