Method

In the present study, all manipulations were performed on horses under general anaesthesia after sedation and induction with xylazine, ketamine and diazepam. The anaesthesia protocol was the same in all horses. NSAIDs were not administered before anaesthesia to avoid any influence on the local inflammatory reaction. To the author’s knowledge, there are no studies on the possible influence of xylazine, diazepam or ketamine on neutrophils or eosinophils in the intestinal tract. In animal models, isoflurane as well as sevoflurane and halothane has been reported to have protective effects in myocardial models of ischaemia-reperfusion injury (KOWALSKI et al. 1997). De ROSSI et al. (2002) demonstrated an inhibition of the activation of L-selectine and an attenuation of integrine by isoflurane. Nevertheless, all horses acted as control and manipulation groups so that a possible effect of isoflurane should not have influenced the results.

The manipulations performed in the present study are in part more severe than often performed during colic surgery. Less aggressive manipulations would have likely caused an inflammatory reaction less easily identifiable by the histological analysis used in the present study. The severity of intestinal manipulation performed in this study is discussed below.

Enterotomy is routinely performed in cases of primary obstipation of the jejunum, ileum, colon, or caecum or secondary impaction of the large intestine following small intestinal ileus to empty the colon or caecum of ingesta. The enterotomy in this study was performed the same as it would happen during colic surgery.

Mucosal irritation after enterotomy may be caused by emptying of the intestine.

Additionally, a mucosal irritation is already present in cases of sand impaction and less so with other impactions, depending on the composition of the impaction.

Mucosal irritation in this study was severe but could reflect the degree of manipulation necessary in severe impactions, especially sand impactions.

Mechanical manipulation of the serosa is inevitable during handling of the intestine.

The amount of manipulation of the intestine is depending on the existing condition of the intestine and the experience of the surgeon. A more experienced surgeon usually needs a shorter time to identify and correct a displacement or volvulus, and uses a less invasive technique, thereby minimizing the degree of tissue trauma compared to a less experienced surgeon. Serosal irritation in this study is severe compared to irritation during a routine colic surgery. However, a serosal irritation mimicking the severity of damage used in this study could occur adjacent to an enterotomy or anastomosis site, especially if performed by a less experienced surgeon.

In cases of colic surgery involving distension of the small intestine, the jejunum is frequently emptied into the caecum by manual compression or “running” of the bowel. The manipulation is usually performed continually over the complete length of the jejunum but in some cases, several decompressions are necessary to empty the jejunum. The degree of manipulation when mimicking emptying of the small intestine in this study was severe (figure 1) and likely would only occur locally and in particularly difficult small intestinal lesions (such as epiploic foramen incarceration with a small epiploic foramen).

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Figure 1 Jejunum after manual emptying of the jejunum

Figure 1: Jejunum after 10 min. of manual emptying (arrow) compared to unmanipulated jejunum (star).

In cases of strangulating lesions, resection of devitalized intestinal segments and anastomosis is required. To avoid leakage of the intestinal contents into the surgical field during the anastomosis, the intestinal lumen is often closed, and Doyen clamps or penrose drains are commonly used for this purpose. The severity of the compression of intestine through the Doyen forceps in the present study is similar to the degree of damage produced during colic surgery by Doyen forceps.

A standardized manipulation time of 10 min. was chosen to remove the influence of manipulation time on the results. The Doyen forceps were left in place for 30 min, as this reflects the time necessary for sewing an anastomosis, depending on the

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experience of the surgeon. The control samples in the present study were taken from the enterotomy site to minimize effects of sample collecting on the inflammatory reaction. Samples were collected immediately after the end of manipulation and 30 min. later. Previous studies have demonstrated a response of eosinophils and neutrophils within this time frame (RÖTTING et al. 2003, GROSCHE et al. 2008, VENTE 2011). However, a more pronounced change especially in neutrophilic infiltration may have been observed in samples collected at a later time point, as previous studies in rodents (KALFF et al. 1998, 1999a, 1999b, mice (De JONGE et al. 2004b) and humans (KALFF et al. 2003) observed an increase in neutrophilic infiltration of the muscularis over time. For reasons of animal welfare, the horses studied in the present study were euthanized while anesthetized.

The tissue samples were fixed in Bouin´s solution and formalin. At the beginning of the study, we evaluated both fixing mediums and the staining techniques and found Bouin´s solution better suitable for the eosinophilic detection with LUNA. The immunhistochemistry staining for calprotectin was performed on formalin-fixed samples as previously reported in literature. A previous study on rectal biopsies from horses found less eosinophils in samples fixed in Bouin´s solution compared with formalin (LINDBERG et al. 1996). However, as the control samples and the samples after manipulations were all prepared in the same manner, the influence of fixing medium on the results should be negligible. Neutrophilic granulocytes were identified by calprotectin staining. A previous comparison of neutrophils directly identified in haematoxylin and eosin stain and calprotectin-positive cells by GROSCHE et al.

(2008) found a good correlation between both techniques.

The histological analysis of the mucosa was performed as already described by RÖTTING (2005) to identify a possible accumulation and redistribution of eosinophils or neutrophils towards the luminal surface of the mucosa.