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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.

Introduction: Inflammatory events within the gut muscularis contribute to dysmotility. We hypothesized that manipulation during organ harvesting initiated an inflammatory response via muscularis macrophages and that this cascade was amplified during reperfusion., Methods: Small bowel transplantation was performed in Lewis rats. To investigate the impact of organ harvesting on muscularis inflammation, cold whole-body perfusion was performed after versus prior to organ harvesting. The role of macrophages was investigated by transplantation of the macrophage-depleted gut. Leukocyte infiltration was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase polymerase chain reaction. Contractility was assessed in a standard organ bath., Results: Organ harvesting and ischemia-reperfusion induced leukocyte recruitment and mRNA upregulation in the muscularis: interleukin-6 12217-fold, monocyte chemoattractant protein-1 62-fold, ICAM-1 12-fold, cyclooxygenase-2: 8-fold, iNOS: 150-fold. Although organ harvesting with cold ischemia prevented early gene expression, peak expression at 3-hour reperfusion was not changed by modification of the harvesting technique. Compared to controls, transplanted animals showed a 63% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted gut exhibited significantly fewer leukocytes and only a 16% decrease in contractility., Conclusions: Gut manipulation during organ harvesting initiates an inflammatory response within the muscularis that is massively intensified during reperfusion. This change contributes to muscular dysfunction. Furthermore, the results suggested that resident macrophages play a key role in initiating this process.

Introduction: Isogeneic intestinal transplantation elicits an inflammatory response within the intestinal muscularis that is associated with dysmotility. Usually the inflammation and the postoperative motor dysfunction resolve within a few days after small bowel transplantation (SBTx). However, the onset of acute rejection in allogeneic SBTx is again associated with dysmotility. We hypothesized that dysmotility during acute rejection is based on coexpression of kinetically active mediators by the alloreactive leucocyte infiltrate., Materials and Methods: Rat SBTx (BN to Lew and BN to BN) was performed without immunosuppression. Animals were sacrificed at 1, 4, and 7 days after SBTx. Leukocyte infiltration was investigated in muscularis whole mounts by immunohistochemistry. Mediator mRNA expression was determined by reverse transcriptase polymerase chain reaction. Muscle contractility was assessed in a standard organ bath., Results: Transplanted animals showed a significant inflammatory response within the muscularis at day 1 after SBTx. However, allogeneic transplanted animals exhibited a significant second inflammatory peak at day 7 (mRNA induction: iNOS 150-fold; tumor necrosis factor-alpha 18-fold; interferon-gamma 397-fold), parallel to the onset of rejection. This change was associated with a significant leukocyte infiltration. Compared to controls, allogeneic transplanted animals showed a 29% decrease in smooth muscle contractility on days 1 and 4 and a 71% decrease of contractility on postoperative day 7., Conclusions: The motor dysfunction of transplanted small bowel during acute rejection is associated with an inflammatory reaction in the intestinal muscularis. The initial unspecific inflammation process immediately after transplantation is reactivated and intensified during acute rejection.

Background: Pathophysiological states that produce intestinal ischemia/reperfusion injury (I/R) initiate an inflammatory cascade and cause ileus. The aims of this study were to investigate the local cellular responses and molecular mechanisms, which contribute to intestinal dysmotility after selective intestinal I/R injury., Methods: ACI rats were subjected to 75 min SMA clamp-induced ischemia followed by reperfusion and were killed at 0 min, 30 min, and 24 hr. Whole mounts of the jejunum were used to immunohistochemically quantify alterations in leukocytes, and circular muscle strips were used to assess organ bath muscle function. Muscularis and mucosa extracts were isolated from the intestine and used for reverse transcription assisted polymerase chain reaction mRNA measurements of granulocyte-colony stimulating factor and interleukin-6, and for determination of nuclear factor kappa B and Stat3 activation., Results: Intestinal I/R injury resulted in the significant recruitment of neutrophils and monocytes into the intestinal muscularis and a functional suppression in jejunal circular muscle contractions. These I/R injury induced cellular responses were preceded by the molecular activation of nuclear factor kappa B, up-regulation of granulocyte colony-stimulating factor and interleukin-6 mRNA and phosphorylation of the downstream signaling and transcription factor Stat3., Conclusions: I/R injury evokes a molecular and cellular inflammatory response within the intestinal muscularis that is associated with a subsequent decrease in intestinal motility.

Background: Surgical manipulation of the intestine results in the massive movement of leukocytes into the intestinal muscularis at 24 hours. This is associated with muscle inhibition. The aim of this study was to temporally associate leukocyte extravasation with ileus after surgical manipulation., Methods: Rats underwent a simple manipulation of the small bowel and were killed at various times (0, 0.25, 0.5, 1, 3, 6, 12, and 24 hours) postoperatively. Jejunal circular-muscle contractile activity was assessed in a standard organ bath. Both extravasating and resident leukocytes were immunohistochemically stained in muscularis whole mounts., Results: Contractile activity was significantly reduced immediately after surgery, but rapidly returned to control levels at 3 hours. After recovery, muscle function decreased at 12 and 24 hours (41% and 81%, respectively). The resident muscularis macrophage network demonstrated cellular activation 1 hour postoperatively. The number of leukocytes increased over time (neutrophils, 67.5-fold; monocytes, 98.2-fold; and mast cells, 47-fold at 24 hours)., Conclusions: The functional results demonstrate a biphasic response in the suppression of muscle activity after surgical manipulation. Regression analysis (r2 = 0.998) of the temporal development of leukocyte infiltration and the protracted phase of muscle inhibition provides evidence for a correlation between cellular inflammation and postoperative dysmotility.

Background & Aims: Postoperative ileus is a poorly understood and common problem. We previously demonstrated an association between a suppression in jejunal circular muscle activity and a massive extravasation of leukocytes into the muscularis after surgical manipulation of the small bowel. This study was pursued to establish a direct causal link between these events., Methods: Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize expression of adhesion molecules: P-selectin, intercellular adhesion molecule 1 (ICAM-1), and lymphocyte function-associated antigen 1 (LFA-1). Leukocyte infiltration and in vitro jejunal circular muscle function were quantified in controls and manipulated animals with and without antibody treatment (1A29, WT.1, and WT.3)., Results: Surgical manipulation caused a significant up-regulation within the muscularis of ICAM-1 and P-selectin messenger RNA. ICAM-1 and P-selectin protein expression was increased within the muscularis microvasculature, and ICAM-1 and LFA-1 were expressed on infiltrating cells. Administration of adhesion molecule antibodies prevented the recruitment of monocytes and neutrophils into the muscularis and also averted jejunal circular muscle dysfunction., Conclusions: The data demonstrate that adhesion molecule antibodies prevent surgically induced suppression of intestinal muscle contractions and therefore suggests that late postoperative ileus is mediated through a leukocytic inflammatory response within the intestinal muscularis externa.

Cellular mechanisms of sepsis-induced ileus remain an enigma. The study aim was to determine the role of nitric oxide (NO) in mediating the suppression of rat jejunal circular smooth muscle activity during endotoxemia. Isolated muscularis inducible NO synthase (iNOS) mRNA was measured by RT-PCR, immunohistochemistry was employed to localize iNOS protein, and contractile activity was measured in an organ bath. The low basal expression of muscularis iNOS mRNA expression was increased in a time-dependent fashion after lipopolysaccharide (LPS), resulting in a 20-fold increase over controls 3 h after injection. Immunohistochemistry of muscularis whole mounts and dissociated muscularis cells for iNOS revealed staining only in the muscularis macrophages 12 h after LPS. LPS caused a 68% reduction in spontaneous muscle activity 12 h after injection, which improved by 53% after the in vitro application of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine. Similar results were obtained in C57BL/6 mice but not in iNOS knockout mice. These data demonstrate that macrophage iNOS plays an important role in mediating LPS-induced intestinal circular muscle suppression.

Intestinal stasis followed by mucosal barrier breakdown and the generation of locally produced cytokines has been proposed as the cause of systemic infection and multiple organ failure following hemorrhagic shock. The aim of this study was to investigate the underlying mechanisms of impaired intestinal muscle function leading to ileus following hemorrhagic shock. Rats were subjected to severe hemorrhagic shock (mean arterial pressure 40 mm Hg) followed by resuscitation and were killed early at 4 h or late at 24 h. Other groups consisted of control and sham animals. Intercellular adhesion molecule (ICAM-1) mRNA levels were significantly elevated in the muscularis but not in the mucosa using the semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). There was a marked infiltration of neutrophils into the muscularis early and late after shock. Furthermore, smooth muscle contractility in response to bethanechol was significantly decreased, being more pronounced in the early group. Immunohistochemistry revealed signal for ICAM-1 in the muscularis microvasculature and on infiltrating cells. These results suggest that the expression of ICAM-1 within the muscularis vasculature after hemorrhagic shock promotes the local recruitment of leukocytes and that this inflammatory response is accompanied by a subsequent impairment of intestinal contractility.

Objective: To investigate the pathophysiologic mechanisms that lead to ileus after abdominal surgery., Summary Background Data: The common supposition is that more invasive operations are associated with a more extensive ileus. The cellular mechanisms of postsurgical ileus remain elusive, and few studies have addressed the mechanisms., Methods: Rats were subjected to incremental degrees of surgical manipulation: laparotomy, eventration, "running," and compression of the bowel. On postsurgical days 1 and 7, muscularis infiltrates were characterized immunohistochemically. Circular muscle activity was assessed using mechanical and intracellular recording techniques in vitro., Results: Surgical manipulation caused an increase in resident phagocytes that stained for the activation marker lymphocyte function-associated antigen (LFA-1). Incremental degrees of manipulation also caused a progressive increase in neutrophil infiltration and a decrease in bethanechol-stimulated contractions. Compression also caused an increase in other leukocytes: macrophages, monocytes, dendritic cells, T cells, natural killer cells, and mast cells., Conclusion: The data support the hypothesis that the degree of gut paralysis to cholinergic stimulation is directly proportional to the degree of trauma, the activation of resident gut muscularis phagocytes, and the extent of cellular infiltration. Therefore, postsurgical ileus may be a result of an inflammatory response to minimal trauma in which the resident macrophages, activated by physical forces, set an inflammatory response into motion, leading to muscle dysfunction.

The basal presence of immunologically potent cells within the intestinal muscularis externa and their functional significance is unclear. Our aim was to investigate the basal distribution of various leukocyte populations within the rat jejunal muscularis. In addition, we sought to immunohistochemically phenotype the muscularis macrophage in jejunal whole-mounts, isolate these cells in primary culture, and investigate their ontogenesis. Macrophages form a regularly distributed network that expresses major histocompatibility complex class II, CD14 receptors, and a low level of CD11/CD18. The macrophages are activated by dissection and are present in fetal animals. Enriched macrophage cultures show a normal resident phenotype and remain present for weeks in dissociated muscularis cultures. The results also demonstrate the presence of neutrophils, monocytes, mast cells, and lymphocytes within the muscularis and suggest that the dense network of muscularis macrophages may be a potent resident trigger for inflammation in response to tissue injury or bacterial translocation.

Bacterial lipopolysaccharide (LPS) is a causative agent of sepsis-induced ileus. Although it is known that LPS activates macrophages and initiates inflammation, the consequences of LPS on the macrophage network and a potential inflammatory response within the intestinal muscularis have not been investigated. This study was designed to identify cellular and functional changes in rat intestinal muscularis after intraperitoneal LPS. Histo- and immunohistochemistry were used to phenotype leukocytes. Functional alterations were determined using an organ bath. Compared with controls, LPS caused a 21-fold increase in staining for the lymphocyte activation marker-1 (LFA-1) localized to the ED2+ macrophage network 1 h after injection. This was followed by a significant infiltration of neutrophils, mast cells, and monocytes into the muscularis. LPS also caused a 62% reduction in spontaneous circular muscle activity and a 91% suppression of bethanechol-stimulated contractions 12 h after injection. These results demonstrate that endotoxemia 1) acutely activates the muscularis macrophage network, 2) causes the extravasation of leukocytes, and 3) results in circular muscle impairment.