Anthony J. Bauer

Role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents

BACKGROUND & AIMS We have shown that intestinal manipulation leads to a significant inhibition of circular muscle contraction. We hypothesized that the inflammatory mediator inducible nitric oxide (NO) plays a role in surgically induced ileus. METHODS Rats and inducible NO synthase (iNOS) knockout and wild-type mice underwent a simple intestinal manipulation. Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize iNOS expression. Nitrite and NO production were measured in muscularis cultures. Spontaneous and bethanechol-stimulated jejunal circular muscle contractions were measured in an organ bath. RESULTS Intestinal manipulation resulted in significant iNOS messenger RNA induction in mucosa and muscularis. Immunohistochemistry localized iNOS in phagocytes within the muscularis. Nitrite and NO production increased 59.8-fold 24 hours after manipulation. L-n(6)-(1-iminoethyl) lysine (L-NIL) inhibited this response. In control rats, selective iNOS inhibition did not increase spontaneous muscle activity, but after manipulation L-NIL significantly improved spontaneous activity. iNOS knockout mice showed a significant 81% decrease in neutrophil infiltration into the muscularis after intestinal manipulation compared with wild-types. Contractile activity was normal in knockout mice after intestinal manipulation. CONCLUSIONS These results show that leukocyte-derived inducible NO inhibits gastrointestinal motility after manipulation and plays an essential role in the initiation of intestinal inflammation.

Mechanisms of postoperative ileus

Postoperative ileus is an iatrogenic condition that follows abdominal surgery. Three main mechanisms are involved in its causation, namely neurogenic, inflammatory and pharmacological mechanisms. In the acute postoperative phase, mainly spinal and supraspinal adrenergic and non‐adrenergic pathways are activated. Recent studies, however, show that the prolonged phase of postoperative ileus is caused by an enteric molecular inflammatory response and the subsequent recruitment of leucocytes into the muscularis of the intestinal segments manipulated during surgery. This inflammation impairs local neuromuscular function and activates neurogenic inhibitory pathways, inhibiting motility of the entire gastrointestinal tract. The mechanisms underlying the recruitment of the inflammatory cells, and their interaction with the intestinal afferent innervation, are discussed. Finally, opioids administered for postoperative pain control also contribute to a large extent to the reduction in propulsive gastrointestinal motility observed after abdominal surgery.

Hydrogen inhalation ameliorates oxidative stress in transplantation induced intestinal graft injury.

Ischemia/reperfusion (I/R) injury during small intestinal transplantation (SITx) frequently causes complications including dysmotility, inflammation and organ failure. Recent evidence indicates hydrogen inhalation eliminates toxic hydroxyl radicals. Syngeneic, orthotopic SITx was performed in Lewis rats with 3 h of cold ischemic time. Both donor and recipient received perioperative air or 2% hydrogen inhalation. SITx caused a delay in gastrointestinal transit and decreased jejunal circular muscle contractile activity 24 h after surgery. Hydrogen treatment resulted in significantly improved gastrointestinal transit, as well as jejunal smooth muscle contractility in response to bethanechol. The transplant induced upregulation in the inflammatory mediators CCL2, IL‐1β, IL‐6 and TNF‐α were mitigated by hydrogen. Hydrogen significantly diminished lipid peroxidation compared to elevated tissue malondialdehyde levels in air‐treated grafts demonstrating an antioxidant effect. Histopathological mucosal erosion and increased gut permeability indicated a breakdown in posttransplant mucosal barrier function which was significantly attenuated by hydrogen treatment. In recipient lung, hydrogen treatment also resulted in a significant abatement in inflammatory mRNA induction and reduced neutrophil recruitment. Hydrogen inhalation significantly ameliorates intestinal transplant injury and prevents remote organ inflammation via its antioxidant effects. Administration of perioperative hydrogen gas may be a potent and clinically applicable therapeutic strategy for intestinal I/R injury.

Intra-Abdominal Activation of a Local Inflammatory Response Within the Human Muscularis Externa During Laparotomy

ObjectiveTo investigate the initiation of a complex inflammatory response within the human intestinal muscularis intraoperatively so as to determine the clinical applicability of the inflammatory hypothesis of postoperative ileus. Summary Background DataMild intestinal manipulation in rodents initiates the activation of transcription factors, upregulates proinflammatory cytokines, and increases the release of kinetically active mediators (nitric oxide and prostaglandins), all of which results in the recruitment of leukocytes and a suppression in motility (i.e., postoperative ileus). MethodsHuman small bowel specimens were harvested during abdominal procedures at various times after laparotomy. Histochemical and immunohistochemical techniques were applied to intestinal muscularis whole-mounts. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed for interleukin (IL)-6, IL-1&bgr;, tumor necrosis factor (TNF)-&agr;, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Signal transducers and activators of transcription (STAT) protein phosphorylation was determined by electromobility shift assay. Organ bath experiments were performed on jejunal circular smooth muscle strips. GW274150C and DFU were used in vitro as iNOS and COX-2 inhibitors. ResultsNormal human muscularis externa contained numerous macrophages that expressed increased lymphocyte function associated antigen-1 (LFA-1) immunoreactivity as a function of intraoperative time. RT-PCR demonstrated a time-dependent induction of IL-6, IL-1&bgr;, TNF-&agr;, iNOS, and COX-2 mRNAs within muscularis extracts after incision. Mediators were localized to macrophages with STAT protein activation in protein extracts demonstrating local IL-6 functional activity. DFU alone or in combination with GW274150C increased circular muscle contractility. Specimens harvested after reoperation developed leukocytic infiltrates and displayed diminished in vitro muscle contractility. ConclusionsThese human data demonstrate that surgical trauma is followed by resident muscularis macrophage activation and the upregulation, release, and functional activity of proinflammatory cytokines and kinetically active mediators.

Biphasic response to gut manipulation and temporal correlation of cellular infiltrates and muscle dysfunction in rat

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.

Inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents

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.

LPS-induced muscularis macrophage nitric oxide suppresses rat jejunal circular muscle activity.

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 inhibitorl- 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.

CD206-Positive M2 Macrophages That Express Heme Oxygenase-1 Protect Against Diabetic Gastroparesis in Mice

BACKGROUND & AIMS Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay. METHODS Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction. RESULTS Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages. CONCLUSIONS CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.

Leukocytes of the intestinal muscularis: their phenotype and isolation.

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. J. Leukoc. Biol. 63: 683–691; 1998.

Colonic Postoperative Inflammatory Ileus in the Rat

ObjectiveTo investigate local inflammatory events within the colonic muscularis as a causative factor of postoperative ileus. Summary Background DataSurgically induced intestinal muscularis inflammation has been hypothesized as a mechanism for postoperative ileus. The colon is a crucial component for recovery of gastrointestinal motor function after surgery but remains unaddressed. The authors hypothesize that colonic manipulation initiates inflammatory events that directly mediate postoperative smooth muscle dysfunction. MethodsRats underwent colonic manipulation. In vivo transit and colonic motility was estimated using geometric center analysis and intraluminal pressure monitoring. Leukocyte extravasation was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase–polymerase chain reaction. In vitro circular muscle contractility was assessed in a standard organ bath. The relevance of iNOS and COX-2 inhibition was determined using DFU or L-NIL perfusion. ResultsColonic manipulation resulted in a massive leukocyte recruitment and an increase in inflammatory mRNA expression. This inflammatory response was associated with an impairment of in vivo motor function and an inhibition of in vitro smooth muscle contractility (56%). L-NIL but not DFU significantly ameliorated smooth muscle dysfunction. ConclusionsThe results provide evidence for a surgically initiated local inflammatory cascade within the colonic muscularis that mediates smooth muscle dysfunction, which contributes to postoperative ileus.