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- Materials and Methods
- Author Contributions
- Conflict of Interests
Background Abdominal surgery involving bowel manipulation commonly results in inflammation of the bowel wall, which leads to impaired intestinal motility and postoperative ileus (POI). Mast cells have shown to play a key role in the pathogenesis of POI in mouse models and human studies. We studied whether mast cells contribute to the pathogenesis of POI by eliciting intestinal barrier dysfunction.
Methods C57BL/6 mice, and two mast cell-deficient mutant mice KitW/W-v, and KitW-sh/W-sh underwent laparotomy (L) or manipulation of the small bowel (IM). Postoperative inflammatory infiltrates and cytokine production were assessed. Epithelial barrier function was determined in Ussing chambers, by measuring transport of luminal particles to the vena mesenterica, and by assessing bacterial translocation.
Key Results In WT mice, IM resulted in pro-inflammatory cytokine and chemokine production, and neutrophil extravasation to the manipulated bowel wall. This response to IM was reduced in mast cell-deficient mice. IM caused epithelial barrier dysfunction in WT mice, but not in the two mast cell-deficient strains. IM resulted in a decrease in mean arterial pressure in both WT and mast cell-deficient mice, indicating that impaired barrier function was not explained by tissue hypoperfusion, but involved mast cell mediators.
Conclusions & Inferences Mast cell activation during abdominal surgery causes epithelial barrier dysfunction and inflammation of the muscularis externa of the bowel. The impairment of the epithelial barrier likely contributes to the pathogenesis of POI. Our data further underscore that mast cells are bona fide cellular targets to ameliorate POI.
Postoperative ileus (POI) is characterized by a transient cessation of intestinal motor activity following abdominal surgery, and as a result, patients suffer from complications and prolonged hospital stay.1,2 The costs related to POI have been estimated to amount 1.47 billion dollars annually in the USA, illustrating its large socio-economical impact.1 With regard to the pathogenesis of POI, it has become evident from animal and human studies that postoperative intestinal hypomotility in POI is the result of an influx of leukocytes into the manipulated muscularis externa.3,4 Neutrophil infiltrates have been shown to inhibit local contractile activity, i.e., via the release of nitric oxide (NO),5–7 or general motility via the activation of sympathetic inhibitory neural reflexes.8 The importance of this inflammatory response in POI is underscored, for instance, by the success of therapeutic strategies aimed at blocking neutrophil recruitment to ameliorate POI.1,3,9 However, the pathophysiological mechanisms behind the immune response to bowel manipulation remain to be clarified. In this respect, an important factor could be the reduced epithelial barrier function resulting from bowel handling that was previously observed in rodent models of POI.5,10 This would be in line with previous observations that bowel wall mechanical stretch11 and manipulation12 augments inflammatory responses of bowel wall macrophage populations and local dys-contractility via TLR activation.
We have previously shown that mast cells are crucial players in the intestinal inflammation that mediates POI 13 and that mast cell stabilizers and histamine receptor antagonists are instrumental in reducing POI in animal models13 and human POI.4,14 Mast cells are implicated in barrier dysfunction in animal models of chronic stress,15,16 allergic inflammation,17 parasitic infection,18 and endoxemia.19 Thus, given the implication of mast cells in the pathogenesis of POI, and their potential to regulate intestinal barrier function, we assessed the role of mast cell-induced barrier dysfunction in the occurrence of POI using two mast cell-deficient mouse strains, KitW/W-v and KitW-sh/W-sh. Here, we show that intestinal manipulation (IM), during abdominal surgery, is associated with intestinal barrier dysfunction and inflammation of the manipulated bowel muscularis externa. Our data indicate that both inflammation and barrier dysfunction are mediated by mast cells and can be considered as factors contributing to POI pathogenesis.
- Top of page
- Materials and Methods
- Author Contributions
- Conflict of Interests
The prolonged impairment of gastrointestinal motility after intestinal manipulation is a significant confounding factor in postoperative recovery. Rodent models and human studies have demonstrated that surgical inspection and manipulation of the bowel leads to the activation of antigen presenting cells that reside in the intestinal muscularis layer.1,25 The general paralysis of the entire GI tract – including the unmanipulated segments – is a commonly seen characteristic of POI. This clinically important aspect of POI involves the activation of an inhibitory neural reflex arch by local inflammatory infiltrates,8 and was recently also shown to involve the production of IFN-γ by CCR9 + T-cells that are activated at the site of manipulation.25 We have shown previously that the activation of mast cells resulting from local manipulation of the bowel is a pivotal factor in the pathogenesis of POI and the inflammatory response to local manipulation.4,13,14 Hence, we questioned in the current study whether mast cell derived mediators contribute to POI either as local activators of dendritic cells recruited to the gut wall or via epithelial permeability changes.
We show here that intestinal manipulation induces barrier dysfunction via a mechanism that is crucially dependent on mast cells. In patients, barrier dysfunction frequently occurs during abdominal surgery and has been associated with increased postoperative septic morbidity in surgical patients undergoing laparotomy.31–33 In addition to this model of POI,13 as well as in human ileus,4 mast cell activation has been associated with disturbed intestinal barrier function in ulcerative colitis34 and several disease entities such as stress-induced hypersensitivity of the bowel15 and endotoxemia.19 In these models, the rapid release of serine proteases following triggering of mast cells,15 possibly via release of corticotropin releasing factor (CRF),34,35 is responsible for an increase in epithelial permeability. Likewise, the nature of the mast cell mediators that affect barrier function in our model involves similar rapid mechanisms and mediator release. This mechanism may involve the activation of protease activated receptor-2 (PAR-2) that is expressed on epithelial cells,36 although most studies have focused on PAR-2 activation by protease activity in the lumen.
Our data are in line with earlier observations in patients and rodent models implicating a disturbed intestinal barrier function after intestinal surgery involving bowel handling.37,38 It has been shown that in POI, bacterial products may reach the intestinal muscularis after IM of the small bowel,38 and that antibiotic treatment decreases muscular inflammation after colon manipulation.12 The implications of this process for the pathogenesis of POI are incompletely understood, but irrespective thereof, the clinical impact of bacterial translocation during surgery is significant. A recent study which included 927 patients over 13 years showed that bacterial translocation was associated with increased postoperative septic morbidity in surgical patients undergoing laparotomy.31
In this study, we assessed barrier integrity by measuring bacterial translocation to the MLN. Most probably this process reflects dendritic cell39 (or CX3CR1 expressing macrophages40) – mediated uptake of bacteria that are still viable once transported by dendritic cells to the MLN. Thus, the bacterial translocation is dependent on a number of immunological processes including phagocytosis, killing and bacterial cultures in the MLN may not reflect merely epithelial integrity. Therefore, we performed measurements of barrier function in the small intestine in Ussing chambers, reflecting the para- and transepithelial transport of the 40 kD HRP, as well as in vivo measurement of real time changes in epithelial leakage to the vena mesenteria. Using this combination of methods, we demonstrated that IM in our model of POI led to a mast cell dependent epithelial barrier dysfunction. This mechanism may explain the important role of mast cells in the pathogenesis of POI and validate mast cells as a bona fide drug target to shorten POI and improve postoperative recovery and barrier function. We performed our experiments in two strains of mast cell-deficient mice, KitW/W-v, and KitW-sh/W-sh mice, both of which carry mutations in the Kit gene [White spotting (W) locus]. C-kit is the receptor for stem cell factor (SCF) and involved in regulation of hematopoiesis, proliferation and migration of primordial germ cells and melanoblasts during development. The KitW/W-v mice carry the W mutation, resulting in deletion of the transmembrane domain of the c-kit protein as well as the dominant negative Wv mutation, a point mutation that affects c-kit kinase activity. On the other hand, the mast cell-deficient Kit-w-sh/w-sh mice carry a mutation that reflects an inversion in the kitlocus spanning a 2.8 mb segment. Hence, the resulting phenotypes are different: Kitw-wv mice have phenotypic abnormalities including sterility, anemia, lack of interstitial cells of Cajal (ICC), and have defects in hematopoiesis that lead to an absence of intra epithelial T-cells, neutropenia, and poor mobilization of blood neutrophils.26,41,42 In contrast, Kit-w-sh/w-sh mice, bearing the W-sash [W(sh)] inversion mutation, have mast cell deficiency, but are neither anemic nor sterile. Adult Kit-w-sh/w-sh mice have been shown to have a profound deficiency in tissue mast cells but normal levels of major classes of other differentiated hematopoietic and lymphoid cells.27
As the pathogenesis of POI is mediated by neutrophil influx, we reasoned that the protection after IM in our POI model in these KitW/W-v mice might not be selectively dependent on the lack of mast cells. Therefore, we also carried out our experiments in the KitW-sh/W-sh mice for two reasons: first, in contrast to the KitW/W-v, the KitW-sh/W-sh mice can be tested against C57BL/6 WT control mice. Second, KitW-sh/W-sh have normal neutrophil numbers, are fertile, contain normal number of intra epithelial lymphocytes (IELS), and are not anemic. The latter probably explains why the effects of IM-induced neutrophilic extravasation and inflammatory response were much less pronounced in the KitW-sh/W-sh compared with the KitW/W-v.
Interstitial cells of Cajal (ICC) require intact ckit signaling for proper development and are defective in both ckit mutant mice, although ICC subsets have been shown to develop in KitW-sh/W-sh mice.43 As KitW/W-v mice are hemizygous, we assessed basic GI motility in KitW-sh/W-sh that are on a C57BL/6 background. Indeed, we observed a disturbed motility and delayed transit under normal conditions compared with C57BL/6 mice. However, this difference did not reach significance and hence, we decided to include KitW-sh/W-sh in our motility analyses.
The observation that the bacterial translocation in KitW/W-v was almost completely abolished in both affected KitW/W-v and its control Kit+/+ may reflect a defective representation of innate immune cells in the lamina propria in these mice, given the purported role of lamina propria APCs in bacterial sampling.39,44 In addition, we observed that inflammatory parameters, bacterial translocation, and permeability to HRP differed between the control groups: C57BL/6 and Kit+/+. This is probably due to differences in immune responses that generally exist between mouse strains: for example between Balb/c and C57BL/6.45
In this study, we found that luminal bacteria were involved in IM-induced inflammation and ileus by treating mice with antibiotics, indicating that bacterial translocation contributes to the POI. It has been described that the immune response is induced soon after IM, for example ICAM-1 mRNA is expressed in the muscularis within 15 min of manipulation,3 whereas luminal products start to appear in the muscularis externa externa 6 h after intestinal manipulation,38 indicating that translocated bacterial antigens may not trigger muscularis immune responses, but may exacerbate immune responses, as shown by our data presented here. It is important to note that lamina propria intestinal macrophages that form the first line of defense generally do not produce high levels of cytokines upon bacterial challenge. Rather, dendritic cells in draining lymph are activated by luminal antigens which may not lead to mucosal inflammation but can contribute to the pathogenesis of POI in unmanipulated areas.25
We show in this study that IM is associated with a postoperative decrease in MAP. Hypoperfusion of intestinal tissue following abdominal as well as non-abdominal surgery has been associated with impaired barrier function preceded by hypotension, mesentery hypoperfusion and enterocyte damage.29,46 In addition, these studies show that aberrations in actin reorganization, cell proliferation and mitochondrial function are maximal at 60 min after mechanical bowel manipulation (i.e., the same time point at which we measured intestinal barrier function in the current study) and was partially recovered after 24 h46,47 (at which we measured inflammatory mediators). Although MAP decreases after IM, when MAP was pharmacologically lowered, the intestinal barrier function was not affected and inflammation did not occur to a similar extent. Circulating SNP-derived NO causes smooth muscle relaxation and subsequent microvascular vasodilatation. NO might affect inflammatory processes and intestinal barrier48,49 function independent of blood pressure alterations, but we show that NO has no effect on these processes. Of note, MAP measured in the carotid artery in our study probably reflects the blood pressure of the internal organs, but we cannot exclude that perfusion in the small intestine is different from the carotid artery.
Concluding, we show that IM elicits a mast cell dependent inflammatory response and intestinal barrier disturbances that may contribute to the pathogenesis of POI. Our study further underscores the potential of mast cell stabilization in ameliorating postoperative recovery, warranting that this treatment strategy should be pursued in clinical setting.