Results

Eosinophils are part of the normal cell population of the mucosa in the colon and small intestine (RÖTTING et al. 2008). In contrast, neutrophils are not part of the resident cell population of the intestine and accumulation of neutrophils in the intestinal layers may reflect tissue injury. In the present study, there were only sporadic neutrophils in the control samples of jejunum and colon. Eosinophils on the other hand were present in the mucosa of the control samples.

7.3.1 Eosinophils

The histological evaluation of the mucosa was performed to identify a redistribution of the eosinophils as identified in previous studies as a result of tissue injury (RÖTTING et al. 2003, 2005, GROSCHE et al. 2012).

The mechanical manipulations performed in the present study resulted in a redistribution of mucosal eosinophils towards the luminal surface of the mucosa. The eosinophilic response to mechanical manipulations was most pronounced in the colon, with redistribution towards the luminal side of the mucosa after serosal irritation. Previous studies observed a mucosal redistribution and infiltration of eosinophils after direct colonic injury through HOCl (RÖTTING et al. 2003),

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experimentally induced colitis (McCONNICO et al. 2005), strangulation, and I/R (MOORE et al. 1994, RÖTTING 2005, GROSCHE et al. 2011). In addition to the direct effects of colonic injury on eosinophilic accumulation, jejunal manipulation also resulted in a colonic mucosal accumulation of eosinophils in unmanipulated colon.

Previous studies in rodents also describe a panenteritis after jejunal manipulation (SCHWARZ et al. 2004). This should be kept in mind when harvesting intestinal biopsies during laparotomy or laparoscopy as intestinal biopsies are frequently collected at the end of surgery after manipulation of the intestine. A possible mucosal inflammatory reaction with eosinophilic participation observed in such samples could potentially be the result of surgical manipulation and not a pre-existing inflammatory condition. Also, rectum biopsies should be interpreted with care if collected after several transrectal examinations as a possible identified inflammatory reaction could be the result of the mechanical manipulation of the rectum. When an eosinophilic infiltration of intestine is observed, the infiltrated layer should be identified to better interpret these findings. In cases of eosinophilic infiltration of the muscular layers as has been observed in idiopathic focal eosinophilic enteritis (IFEE) and diffuse eosinophilic enteritis (DEE) (MAKINEN et al. 2008) the eosinophilic infiltration is more likely to be of clinical consequence than a mucosal eosinophilic accumulation without redistribution of eosinophils.

In contrast to the colon, the jejunum demonstrated a less pronounced change in eosinophils in response to mechanical manipulations. This may be due to the anatomical localization as eosinophil numbers have been demonstrated to be less in the jejunum than in the large intestine. Therefore a significant change in eosinophilic accumulation or distribution would be harder to identify.

In the present study and in previous studies describing a mucosal eosinophilic response after oxidative injury in-vitro (RÖTTING et al. 2003) and ischaemia-reperfusion injury (RÖTTING 2005, RÖTTING et al. 2008) there was a lack of factors normally associated with eosinophilic accumulation in the intestine, such as parasitic disease or allergic response. As the eosinophilic granulocyte is part of the resident cell population of the intestinal tract it can quickly respond to stimuli. Eosinophils may be involved in any rapid local inflammatory response because they are well equipped

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to initiate and maintain an inflammatory reaction through secretion of pro-inflammatory cytokines, chemokines, and lipid mediators.

7.3.2 Neutrophils

In contrast to eosinophils, neutrophils are predominantly located in the circulation and need to be recruited to the site of injury by inflammatory mediators as leukotriene B4, complement factor C5a, and platelet-activating factor (WITKO-SARSAT et al. 2000).

These cytokines promote chemotaxis, adherence and extravasation of the neutrophils. Despite this recruitment pathway, a neutrophilic response was already observed immediately after the end of manipulations in the present study. This is similar to previous studies after I/R injury of the jejunum, where a neutrophilic infiltration was observed as early as 5 min. after beginning of reperfusion (VENTE 2011). These findings indicate that mechanical manipulations initiate an inflammatory reaction with expression of inflammatory mediators very rapidly which subsequently leads to rapid chemotaxis and activation of neutrophils.

The inflammatory reaction of the mucosa seen in the present study after removal of the Doyen forceps, mucosal irritation in the jejunum, or mucosal irritation in the colon could result in a transposition of bacterial toxins through the intestinal layers into the blood or lymphatic circulation. Previous studies in rodents (TÜRLER et al. 2007) and humans (REDDY et al. 2006) observed a translocation of bacterial toxins to the regional lymph nodes or distant intestinal segments after intestinal manipulations. A possible clinical consequence of the impairment of the mucosal integrity is endotoxaemia, frequently observed in horses after injury of the large intestine (MORRIS 1991). This may lead to a further exacerbation of a systemic inflammatory response after tissue injury of the intestine as observed in some horses after colic surgery.

In the submucosa, an accumulation of neutrophils was observed after mucosal irritation of the mucosa, of the serosa of the colon, after removal of Doyen forceps, and after mucosal irritation in the jejunum. In inflammatory bowel disease (IBD) neutrophils and monocytes leave the blood stream and migrate into the inflamed submucosa and mucosa through the intestinal wall into the lumen. Similar to the

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results in the present study, intestinal manipulation in the rat also resulted in marked inflammatory changes in the submucosa (KALFF et al. 1998).

In the present study, a neutrophilic infiltration of the circular muscle layer was observed in the colon after serosal irritation and in the jejunum after manual decompression. Similar to those results, previous animal studies (KALFF et al. 1999, TÜRLER et al. 2007) and clinical studies in humans (KALFF et al. 2003) observed a neutrophilic inflammation after surgical manipulation. In these studies this was associated with a decrease in gastrointestinal motility. Therefore, surgical manipulation and the associated inflammatory reaction of the circular muscle layer have been proposed to contribute to the development of postoperative ileus in surgical patients. The effects of the neutrophilic infiltration of the circular muscle layer were not examined in the present study. However, a tendency towards a neutrophilic infiltration of the circular muscle layer was also observed in a study by RATHMANNER et al. (2011) in the proximal resection margin of horses which subsequently developed POI.

No significant infiltration of the intermuscular layer and longitudinal muscle layer with neutrophils or eosinophils was observed in the present study. This may be due to the sample collection time, as an inflammatory reaction in those layers could become evident at a later time. Previous studies in horses after I/R injury of the jejunum identified a neutrophilic infiltration of the longitudinal layer after reperfusion (LITTLE et al. 2005, VENTE 2011). The type of injury could have an influence on the localization of the inflammatory reaction in the individual muscle layer. Most histological studies on equine jejunum after I/R injury identified a more prominent neutrophilic infiltration of the longitudinal muscle layer compared to the circular muscle layer in the ischaemic intestine (LITTLE et al. 2005, VENTE 2011). The more prominent affection of the longitudinal muscle layer in I/R injury of the jejunum has also been described in mice, where PONTELL et al. (2011) identified prominent degenerative changes in the longitudinal muscle of areas subjected to I/R, in contrast to the largely morphological unaffected circular muscle layer. The authors observed a neutrophilic infiltration of both muscle layers after I/R, but the neutrophilic infiltration

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in the longitudinal layer was double that of the circular muscle layer. In addition, a minor infiltration of the circular and longitudinal muscle layers occurred in non-injured regions and in intestine from sham-operated mice. The authors concluded that I/R injury caused an inflammatory response that exceeds the response to laparotomy.

However, in most of naturally occurring cases of I/R injury in equine medicine, the affected intestinal segments are resected and so the segment at greatest risk of reperfusion injury is usually removed. Although ischaemic segments are not resected if judged to be viable, these segments do not seem to develop an obvious reperfusion injury. Important factors affecting the development of POI may be intestinal handling, intestinal distension, duration of the surgery, or the use of drugs.

For instance in non-colic patients, duration of general anaesthesia and surgery has been positively associated with the duration of reduced postoperative faecal output (LITTLE et al. 2001). Differences in the inflammatory response of intestinal muscle layers to different injury models should be kept in mind when evaluating the motility effects of pro-kinetic drugs. To the authors knowledge there is no information about the relative importance of the longitudinal versus the circular muscle layer to the development of POI. Drugs may also affect both muscle layers differently.

The most obvious inflammatory reaction was observed in the serosa in the present study with all manipulation types resulting in a neutrophilic infiltration of the serosa. A reaction of the jejunal serosa to serosal rubbing with dry sponges has been observed in horses (GERRING and HUNT 1986). Additionally, serosal infiltration with neutrophils was observed after I/R injury of the jejunum (LITTLE et al. 2005). A possible consequence of serosal inflammation may be adhesion formation, a complication observed after colic surgery in horses and in more than 90% of human patients after major abdominal surgery (LIAKAKOS et al. 2001). Factors associated with the formation of postsurgical adhesions include trauma, thermal injury, infection, ischaemia, and foreign bodies leading to injury of the serosa and peritoneum with subsequent adhesion formation. Neutrophils have been proposed to participate in the adhesion formation through accumulation in the peritoneal fluid and secretion of inflammatory mediators (VURAL et al. 1999). The significance of surgical trauma of a

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laparotomy wound versus laparoscopy was evaluated in a canine model and demonstrated a significant higher rate of adhesions after laparotomy (SCHIPPERS et al. 1998). The surgical lesion may not be the main reason for adhesion formation as has been demonstrated in the study by GORVY et al. (2008), where the site of adhesion was not related to the site of the primary lesion. A more likely reason for adhesion formation may be a panenteritic inflammatory response initiated by manipulations of the intestine, the injury of the intestine, or peritonitis. Additionally, the type of injury is probably also important for the risk of adhesion formation with strangulating lesions possibly having a higher risk than simple displacements or impactions, as the latter would likely cause as less severe inflammatory response.