The aim of the present study was to characterize a possible inflammatory response of the jejunum and colon to mechanical manipulation similar to what would be applied during colic surgery in horses. A focus was placed on neutrophilic and eosinophilic granulocytes.
The hypothesis of the study was that mechanical manipulations of the equine intestine would result in a measurable inflammatory reaction of all layers of the intestinal wall.
We also hypothesized that laparotomy and intestinal manipulation would result in an inflammatory reaction of distant non-manipulated intestinal segments.
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47 5 Manuscript A
Influence of mechanical manipulations on the local inflammatory reaction in the equine colon
HOPSTER-IVERSEN, C., HOPSTER, K., STASZYK, C., ROHN, K., FREEMAN, D.
and RÖTTING, A. K.
Equine Veterinary Journal 2011, 43: 1–7. doi: 10.1111/j.2042-3306.2011.00378.x
Abstract:
Reasons for performing study: Large intestinal diseases in horses are characterized by inflammation, which could arise from the disease process with some contribution from intestinal manipulation. The effects of the latter are unknown but important to surgeons and could contribute to post-operative complications.
Objectives: To characterise type and degree of intestinal inflammation induced by various mechanical stimuli in the equine ascending colon.
Methods: Laparotomy was performed in 12 horses, the left dorsal colon was exteriorised and 3 segments were randomly exposed to one of the following mechanical manipulations: 1) enterotomy, 2) enterotomy and 10 minutes of mechanical mucosal irritation, and 3) 10 minutes of mechanical serosal irritation.
Intestinal biopsies were harvested before, immediately after and 30 minutes after each manipulation for histological evaluation. Eosinophils were detected with Luna's stain and neutrophils were identified by immunohistochemical staining for calprotectin. Additionally, left dorsal colon samples from 14 horses from a jejunal ischaemia-reperfusion study were collected immediately after laparotomy (7 horses) and at the end of the experiment without previous manipulation of the colon (7 horses). Horses were euthanized at the end of both studies.
48 Results:
Redistribution of mucosal neutrophils and eosinophils towards the luminal surface and increased neutrophilic infiltration of the submucosa were demonstrated after serosal and mucosal irritation. All manipulations resulted in serosal infiltration with neutrophils. Laparotomy and small intestinal manipulation increased mucosal eosinophilic infiltration
Conclusions and potential relevance: Mechanical intestinal manipulation caused a rapid local inflammatory reaction in the mucosa, submucosa and serosa including a mucosal eosinophilic response. These changes could exacerbate existing inflammation in horses with large colon disease. Colic surgery can lead to intestinal inflammation in non-manipulated intestine and this could contribute to a higher morbidity rate in horses after prolonged colic surgery. An intestinal biopsy should be collected at the beginning of surgery to avoid false interpretations.
Introduction:
During colic surgery, which is the most frequent emergency surgery in horses, manipulation of the intestine is inevitable. In humans and in rodents intestinal manipulations resulted in a local inflammatory reaction with subsequent decreased intestinal motility post-operatively (Kalff et al. 2003, Schwarz et al. 2004). In horses inflammatory intestinal reactions have been described after ischaemia-reperfusion injury (Moore et al. 1994, Little et al. 2005, Grosche et al. 2008), parasitic infection (Collobert-Laugier et al. 2001, Rötting et al. 2008) and after experimentally induced colitis (McConnico et al. 1999). Inflammatory cells involved include eosinophilic and neutrophilic granulocytes. Eosinophilic granulocytes are part of the resident cell population of the intestinal submucosa and mucosa. Within the intestinal mucosa of healthy horses a higher number of eosinophilic granulocytes is present in caecum, colon ascendens, and colon transversum than in stomach, duodenum, jejunum, and colon descendens (Rötting et al. 2008). Most of the mucosal eosinophilic granulocytes are located close to the lamina muscularis mucosae. The intestinal submucosa also contains larger numbers of resident eosinophilic granulocytes. The
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functions of eosinophilic granulocytes in the intestine are not completely understood, but they play an important role in inflammatory bowel disease in several species including horses (Rothenberg 2004, Makinen et al. 2008). Previous studies have shown a response of local intestinal eosinophilic granulocytes to several stimuli in horses. Parasite infection resulted in a focal accumulation of eosinophilic granulocytes (Collobert-Laugier et al. 2001 and Rötting et al. 2008) and to date an intestinal eosinophilic response most often is attributed to parasitic or immune-mediated disease. However, accumulation of eosinophilic granulocytes in the intestinal mucosa has also been observed after experimental ischaemia and reperfusion (Moore et al. 1994 a,b, Rötting et al. 2005), after in vivo intestinal strangulation (Rötting et al. 2005), and after experimentally induced colitis (McConnico et al. 1999). In addition to eosinophilic accumulation, the eosinophilic granulocytes migrated within the intestinal mucosa from the area close to the lamina muscularis mucosae towards and into the intestinal lumen after experimental ischaemia and reperfusion and after in vivo large and small intestinal strangulation (Rötting et al. 2005). The eosinophilic granulocyte is well equipped to initiate and maintain a local inflammatory reaction and therefore is of particular interest for a better understanding of the pathogenesis and clinical consequences of intestinal inflammation in horses.
The neutrophilic granulocytes is the predominant inflammatory cell type commonly associated with mucosal damage caused by ischaemia-reperfusion injury in human medicine and other species (Grisham and Granger 1988, Grisham et al. 1990, Schoenberg et al. 1991, Kubes et al. 1992, Moore et al. 1994, Gayle et al. 2002). In equine medicine an increased neutrophilic infiltration has been described after ischaemia and reperfusion injury in the small intestine (Little et al. 2005) and the colon (Grosche et al. 2008). The neutrophilic infiltration was time-dependant and activation of neutrophils resulted in the release of inflammatory mediators including cytokines, reactive oxygen metabolites, proteases and other regulatory proteins (McMicheal and Moore 1994). Intestinal inflammation can increase the morbidity of post-operative equine colic patients. The detrimental effect may differ depending on the intestinal segment and intestinal wall layer involved and can include
post-50
operative adhesion formation, post-operative ileus, enteritis, exacerbation of existing inflammation and endotoxaemia. The length of colic surgery and the types and severity of mechanical manipulations performed may influence the intestinal inflammatory reaction.
In this study we aimed to characterise the local colonic inflammatory reaction after mechanical manipulations as they may occur during colic surgery and after laparotomy and small intestinal manipulations only. In addition to a neutrophilic inflammatory reaction we evaluated the potential role of local eosinophilic granulocytes in the inflammatory reaction to these stimuli.
Material and methods Horses
This study was a terminal experiment approved by the Ethical Commission of the Veterinary University of Hannover, Germany. Twenty-six adult horses of various breeds with a mean age of 13.01 years (range 3-25 years) without gastro-intestinal disorders were studied. Before study begin, the horses were treated with a Moxidectin formulation (Equest)1 according to manufacturers recommendations and the faeces were evaluated for intestinal parasitism using a faecal flotation test. After a negative result (no parasitic eggs in the faeces) was obtained, horses were then kept in stall with paddock for two weeks and were allowed unlimited access to hay and water.
Study design:
Horses were premedicated with 0.8 – 1.1 mg/kg bwt xylazine (Xylapan)2 i.v. and general anaesthesia was induced with 0.05 mg/kg bwt diazepam (DiazepamAbZ) 3 i.v. and 2.2 mg/kg bwt ketamine (Narketan)1 i.v. Following induction and tracheal intubation, horses were positioned in dorsal recumbency. Anaesthesia was maintained by balanced anaesthesia with isoflurane (Isofluran CP)2 in 100% oxygen and continuous rate infusion (CRI) of xylazine (Xylapan)2 at 0.7 mg/kg bwt/hour.
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Mean arterial blood pressure, heart rate, respiratory rate, respiratory pressures, inspiratory oxygen fraction, expiratory isoflurane concentration were monitored continuously with the “Kardiocap 5”-monitor4 and arterial blood gases were measured every twenty minutes. Dobutamine (Dobutamin-ratiopharm 250mg)5, intravenous fluids (lactated Ringers solution (Ringer-Laktat-Lösung)6 ) and hydroxyethylstarch (hetastarch) (Hemohes 10 %ig)6, were administered to effect to maintain a mean arterial blood pressure above 60 mmHg. Horses did not receive lidocaine infusions during surgery.
Study 1:
In this study 12 horses were used. A median laparotomy was performed, the left dorsal colon was exteriorised and divided into 3 adjoining segments of a length of approx. 30 cm with penrose drains without compromising vascular supply and randomly exposed to one of the following mechanical manipulations:
Group 1:
An antimesenterial enterotomy was performed without further manipulation.
Additionally a control sample was collected from the enterotomy site (Group 1:
enterotomy).
Group 2:
An enterotomy was carried out followed by 10 minutes of mechanical irritation of the mucosa with sponges (Group 2: mucosal irritation).
Group 3:
The last segment was exposed to 10 minutes of mechanical irritation of the serosa with sponges. No control sample was harvested from this segment to avoid any influence the required enterotomy could have on the inflammatory reaction in this model of serosal irritation (Group 3: serosal irritation).
All manipulations were performed by the same person (A.K.R.).
Full thickness colon wall samples (2 x 2 cm) approx. 15 cm apart were harvested at three time points from the anti-mesenterial side of the intestine:
Control: before begin of the manipulations
t0: immediately after the end of each manipulation.
t1: 30 minutes after t0.
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After final sampling, horses were euthanatized with an overdose of sodium pentobarbital without regaining consciousness.
Study 2:
14 horses from an ischaemia-reperfusion injury study of the jejunum were also included in the sample collections. In 7 horses, full thickness samples of the left dorsal colon were collected immediately after opening the abdomen before commencing with intestinal manipulations. In the remaining 7 horses analogous samples were obtained at the end of the experiment (approx. 3 hours surgery time) without any previous manipulation of the colon. Time of surgery was not significant different in both groups. After final sampling, horses were euthanatized with an overdose of sodium pentobarbital without regaining consciousness.
Histological evaluation:
The samples were fixed in either formalin (10%) or in Bouins solution (formaldehyde, picric acid and acetic acid). Subsequently, samples were embedded in paraffin and cut into 5-μm-thick sections. All histological examinations were performed by the same investigator (C.H-I.) blinded to treatment group.
Slides were stained with Luna’s eosinophil stain to determine eosinophil accumulation and distribution. To perform Luna’s eosinophil stain, slides fixed in Bouin were desiccated in xylene, stained with Biebrich scarlet-hematoxylin, differentiated in 1% acid alcohol and subsequently treated with lithium carbonate to achieve a transition of colour to blue. Eosinophil granules and Charcot-Leyden crystals stain red, erythrocytes orange and all nuclear elements are blue (Luna 1992).
Activated neutrophilic granulocytes were identified using calprotectin immunohistochemistry staining (Little et al. 2005, Grosche et al. 2008). Calprotectin is a protein expressed in neutrophils, blood monocytes and tissue macrophages during acute inflammation (Nacken et al. 2003; Yui et al. 2003).
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Formalin-fixed sections were dewaxed in xylole and treated with hydrogen peroxide (3%) in methanol for 10 min. to block the endogenous peroxidase. Non-specific binding of antibodies was inhibited by incubating sections in normal goat serum (BioGenex)7 for 30 min. Afterwards sections were incubated with primary antibodies (anti-calprotectin, clone MAC387, monoclonal, mouse IgG1)8 at a dilution of 1:4000, over night at 4 °C. Immunoreactions were visualized by use of a immunohistochemical detection system employing an indirect biotin-streptavidin amplified staining system (Super sensitive IM Link-Label Detection)7. Therefore sections were incubated with a biotinylated immunoglobulin for 20 minutes. This was followed by incubation with horseradish peroxidase-labeled streptavidin for 20 minutes. The entire antibody-enzyme complex was then visualized by adding a chromogenic system (Liquid DAB)7 until adequate colour development was seen.
Sections were counterstained with toluidin blue and covered with DePex9.
A computer-based program (Image Pro Express 6.3)10 was used for histomorphometric analysis of the images obtained by light microscopy. The histomorphometric measurements were performed as described in previous studies published by Rötting et al (2005, 2008). The length of mucosal surface epithelium was measured and the percentage of denuded epithelium calculated. The mucosa was divided into five sections as follows: the mean distance from the muscularis mucosae to the luminal surface was divided into successive quarters for sections M1-M4, and the luminal surface of epithelial cells was section M5 (Fig. A.1). Absolute numbers of eosinophils and neutrophils were counted in each section and the mean number of eosinophils or neutrophils per section was calculated. These mean values were then used to calculate the percentage of eosinophils or neutrophils in each of the five sections relative to the total eosinophil or neutrophil count in all sections for each horse.
54 Figure A. 1
Fig. A.1: Division of mucosa into four equal sections with M1 bordering the Lamina muscularis mucosae and M4 next to the luminal surface. M5 is the lumen
A: control samples from study 1
B: sample 30 min. after mucosal irritation (group 2) with a redistribution of eosinophilic granulocytes towards the luminal surface. Arrows point at eosinophilic granulocytes.
The absolute number of eosinophils and neutrophils per square millimetre of mucosa was also calculated as a measure of the eosinophil and neutrophil density.
In addition, the other histological layers of colon wall (serosa, circular muscle layer, intermuscular layer, longitudinal muscle layer and submucosa) were evaluated for neutrophilic and eosinophilic infiltration by counting the number of neutrophils or eosinophils present in each of 3 random areas of each histological layer using a
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calibrated grid with a length of 250 x 390 μm in 100fold magnification for the circular and longitudinal muscular layers. The serosa, intermuscular layer and submucosa was evaluated in 200fold magnification with a grid length of 250 x 390 μm. For more detailed information about distribution of neutrophilic and eosinophilic granulocytes within the submucosa, the later was further divided into three sections (SM 1, 2, 3) with section SM1 next to the Lamina muscularis mucosae, section SM3 adjacent to the circular muscular layer and SM2 in the middle between SM1 and SM3.
Three randomly selected areas were analysed in each slide for each variable in each layer and the mean values calculated and used for statistical analysis.
Statistical analysis:
Goodness of fit for normal distribution of model residuals of all parameters was rejected by visual assessment of normal probability plots and the Kolmogorov-Smirnov test, i.e. data was neither normally nor log-normally distributed, and for that nonparametric methods were used for examination of data.
For description median as well as min and max were calculated for all variables.
Independent samples were compared by the Wilcoxon Two-Sample Test, in case of repeated measurements pair wise comparisons was calculated with the signed rank test. Results were considered significant if p < 0.05. Analyses were carried out with the statistical software SAS, version 9.2 11.
Results Study 1:
Macroscopically, all mechanical manipulations of the colon resulted in marked oedema formation and haemorrhage into all layers of the intestinal wall. The histomorphometric measurements showed a significant increase of denuded mucosal surface epithelium after mucosal (group 2, p < 0.00488) and serosal irritation (group 3, p < 0.0211).
56 Group 1:
After enterotomy alone the only significant change was a neutrophilic infiltration of the serosa 30 minutes after the end of manipulation (p < 0.0391, table A.1).
Group 2:
Mucosal irritation reduced the percentage of eosinophilic granulocytes in sections M2 (p < 0.00342) and M3 (p < 0.02441) after the end of manipulation when compared to control samples (table A.2).
The absolute number of neutrophilic granulocytes increased in the mucosa (M1: p <
0.03125, M2 p < 0.00098, M3 p < 0.00391, M4: p < 0.01172), the submucosa (SM1:
p < 0.00732, SM2: p < 0.04688) and the serosa (p < 0.00977) after the end of mucosal irritation when comparing to the control samples and to samples at t0.
Group 3:
Serosal irritation alone resulted in a significant redistribution of eosinophilic granulocytes towards the luminal surface (M4: p < 0.00977, table A.2) after the end of manipulation when compared to control samples and to samples at t0. Additionally, an increased infiltration of the submucosa (p < 0.01953), circular muscle layer (p <
0.01074) and serosa (p < 0.00439) with neutrophilic granulocytes was observed after the end of serosal irritation when compared to control samples (table A.1)
Study 2:
Laparotomy and small intestinal manipulation without manipulation of the colon increased mucosal infiltration and numbers of eosinophilic granulocytes (p < 0.036, table A.3). There were no significant changes in neutrophilic infiltration.
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ControlEnterotomy Mucosal Irritation Serosal Irritation Laparotomy (Study 1b) 0 min 30 min 0 min 30 min 0 min 30 min 0 min 3 h M10 a 0 – 2.33 0.67 0 – 2.33 0.33 0 – 3.33 0 0 – 0.67 1 A 0.33 – 10.33
0.33 0 – 3.33 0.67 0 – 4.33 1.33 0 – 5
0.67 0.33 – 46.33 M20.67 0 - 11 1.33 0 – 6 0.67 0 – 9 0.5 b 0 – 1.67
3 B 0.33 – 14.33 0.67 0 – 4.33 2.33 0.33 – 11.67
3.67 0.33 – 20.67
1 0.33 - 36.67 M30.83 0 – 8 1.33 0 - 3 0.67 0 – 5.67 0.17 b 0 – 2 2 B 0.67 – 5.330.83 0 – 6.67 3 0 - 18 3.33 1.33 – 11
0.67 0.67 – 15.67 M40.67 0 – 8.67 0.33 0 - 2 0.5 0 - 4 0.5 b 0 – 3 2.33 B 0 – 6.33 0.83 c 0 – 6.67 2.67 C 0 – 23.33
2.33 1.67 – 2.67
0.67 0 – 6.67 M50 0 – 0.67 0 0 - 0.67 0 0 – 2 0 0 – 1.67 0 0 – 14.67 0.83 0 – 3.67 0 0 – 1 0.33 0 – 1.67 0 0 - 0 SM10.17 a 0 – 1.33 0.33 0 – 1 0.5 0 – 2 0.33 b 0 – 1.67 1 A,B 0 – 36.33 0.33 c 0 – 0.33 0.5 C 0 – 14 0.33 0 – 1.67 0.67 0 – 7.333 SM20.17 a 0 – 1 0.33 0 – 1.67 0.67 0 – 1.33 0.5 A 0 – 1 0.33 0 – 2.33 0.33 0 – 1.33 0.67 0 – 2.33
0.67 0.33 – 3.33
0 0 – 1.67 SM30 0 – 0.67 0 0 – 0.33 0.33 0 - 1.67 0.33 0 – 2.67 0.33 0 – 10.67 0.33 0 – 1.5 0.17 0 – 1.33 0.33 0 – 0.67 0 0 - 0.33 CM 0.33 a 0 – 1.67 0 0 – 1.67 0.75 0 – 2.33 1 0.33 – 2 0.67 0 – 3 0.83 A 0 – 2 0.17 0 – 2.33 0.67 0 – 2 0.67 0 – 1 IM 0.17 0 – 4 0 0 – 1 0.25 0 – 2.33 0 0 – 2.33 0 0 – 2.33 0 0 – 25 1 0 – 2.33 0 0 - 0.33 0 0 - 0.33 LM 0.33 0 - 7 0.17 0 – 1.33 0.42 0 - 1.5 0 0 – 4.67 0 0 – 1.67 0.42 0 – 4.5 0 0 – 4.33 0.17 0 - 2.32 1 1 – 1.33 Serosa 0.67 a 0 – 88.33 0 b 0 - 7 10.5 B 0 - 43 2.08 c 0 – 49.33 6.83 A,C 0.67 - 85 1 0 - 5 3 A 0.33 - 28 0.33 0 - 1 0.33 0 - 1
Table A 1 Absolute numbers of neutrophilic granulocytes in the colon
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Table A.1: Absolute number of neutrophilic granulocytes in the mucosain different localisations compared between control samples and different manipulations types. Values are expressed as median values; small numbers are range of minimum and maximum values. Values with capital letters are significantly different from values with the same small letter within the identified histological layer. Neutros: neutrophilic granulocytes, SM: submucosa, CM: circular muscle layer, IM: intermuscular layer, LM: longitudinal muscle layer, Table A.2: Percentage of eosinophilic and neutrophilic granulocytes in the colonic mucosal sections. Values are expressed as median values; small numbers are range of minimum and maximum values. Values with capital letters are significantly different from values with the same small letter within the identified histological layer.
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Table A 2 Percent distribution of eosinophilic and neutrophilic granulocytes in the colonic mucosa ControlEnterotomy Mucosal Irritation Serosal Irritation Laparotomy (Study 1b) t0 t1 t0 t1 t0 t10 min 3 h Eosinophils M1 (%)
49.86 17.95 – 84.14
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Table A 3: Absolute numbers of eosinophilic granulocytes in the colon Control Enterotomy Mucosal Irritation Serosal IrritationLaparotomy (study 1b) t0 t1 t0 t1 t0 t10 min 3 h M1 11 a 2.33 - 2817.5 A 3 – 5110.5 1 – 56.675.33 A 0 – 12.337.67 0.33– 19.67 17 0.67 – 50.339.33 1 – 28.6712.33 0 – 25.3315.67 5.33 – 37 M2 5 a 1 - 29.6710.67 0.67 – 196 0.33 – 7.330 A 0 – 111.67 0 – 11.679.5 0.33 – 338.33 0.33 - 196 b 0 – 9.6715 B 5.67 – 19 M3 1.67 0 – 18.673.33 0 – 19.332.83 0 – 16.670 0 – 12.670.33 0 – 4.673.33 0 – 15.673 0.33 – 111.33 0 – 11.334.33 1.33 – 8 M4 0 0 – 11.330.33 0 – 4.330.17 0 – 33.330 0 – 4.330 0 – 1 0.33 0 – 61 0 – 91.67 10 – 33.330.33 0.33 – 1.67 M5 0 0 – 4.330 0 – 0.330 0 – 0.330 0 – 0.670 0 – 0.670 0 – 3.330 0 0.24 SM1 2 0 – 8.333 0 – 9.334.33 0 – 9.673 0 – 6 3.33 0 – 112 0 – 6 2.5 0 – 4.336.67 0.33 – 2015.67 0.67 – 36 SM2 0 0 – 0.330 0 – 1 0.33 0 – 1.330 0.17 0 – 1 0 0 – 0.330 0 – 0.330.33 0 – 2 2 0 – 7.33 SM3 0 0 – 0.330 0 – 0.670 0 - 10 0 – 0.330 0 – 0.670 0 – 1 0 0 – 0.670.33 0 – 2.330.33 0 – 2 CM 0 0 0 0 0 0 0 0 0 0 – 0.33 IM0 0 – 0.330 0 0 0 0 – 0.330 0 0 – 0.330 0 – 0.330 LM 0 0 0 0 0 0 0 0 – 0.670 0 Serosa0 0 – 0.670 0 0 0 0 – 0.330 0 – 0.670 0 0 – 0.330 0 – 0.33
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Table A.3: Absolute number of eosinophilic granulocytes in the mucosa in different localisations compared between control samples and different manipulations types. Values are expressed as median values; small numbers are range of minimum and maximum values. Values with capital letters are significantly different from values with the same small letter within the identified histological layer. Eos: eosinophilic granulocytes, SM: submucosa, CM: circular muscle layer, IM: intermuscular layer, LM: longitudinal muscle layer.
62 Discussion
In the present study we observed a local inflammatory response in the colon layers to mechanical manipulations of the colon. The least response was in enterotomy alone with only an increased serosal infiltration with neutrophilic granulocytes. Both mucosal and serosal irritation caused a measurable inflammatory reaction.
Laparotomy and small intestinal manipulation without any colon manipulation also resulted in a measurable local inflammatory reaction in the colon.
The inflammatory reactions observed in the present study included a redistribution of mucosal eosinophilic granulocytes towards the luminal surface as observed in previous studies in horses. Redistribution of eosinophils has previously been described as a response to parasitic infections (COLLOBERT-LAUGIER et al. 2001, RÖTTING et al. 2008), experimental ischaemia and reperfusion injury (MOORE et al.
1994, 1995b, RÖTTING et al. 2005), in vivo intestinal strangulation (RÖTTING et al.
2005), and experimentally induced colitis (McCONNICO et al. 1999). We conclude that eosinophilic granulocytes are part of many inflammatory responses of the equine colon and their role is not limited to parasitic infections and immune-mediated diseases. The change of distribution of mucosal eosinophilic granulocytes appears to be histologically more obvious than an increase of absolute number of eosinophilic granulocytes. A time point of 10 minutes was chosen for the duration of mechanical manipulations in this study. We expected this time frame to provide a measurable inflammatory reaction of the intestine. Previous studies have found adhesion formation after short (15 min.) intestinal manipulations (SCHIPPERS et al. 1998). A time frame of 30 minutes was chosen for the collection of the second sample after the end of intestinal manipulation. This time frame was based on previous results in a study from RÖTTING et al. (2003), were a significant redistribution of mucosal eosinophilic granulocytes was seen after 30 minutes. Similarly, 30 minutes should be long enough to identify a neutrophilic infiltration (GROSCHE et al. 2008).
Neutrophilic granulocytes also were involved in the inflammatory response as seen as an increased infiltration of the mucosa, the submucosa and the serosa with neutrophilic granulocytes. A previous study by LITTLE et al. (2005) reports significant
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neutrophilic inflammation of the jejunum in horses with clinical ischaemia injury. The infiltration was identifiable in all intestinal layers, particularly in the serosa, fascial planes around circular and longitudinal muscle fibres, and myenteric plexus. Another study by GROSCHE et al. (2008) described an increase in calprotectin-stained cells within the submucosal venules and a migration of neutrophils into the mucosa towards the epithelium in equine colon after ischaemia and reperfusion injury. In the latter study the number and distribution of calprotectin positive cells in the equine colon corresponded to the number of histologically identified neutrophilic granulocytes.
The two colon layers with the most obvious inflammatory response in this study were the serosa and the mucosa. Possible consequences of an inflammatory reaction in the colonic serosa can include post-operative adhesion formation. A recent study by GORVY et al. (2008) reported that adhesions were neither associated with the
The two colon layers with the most obvious inflammatory response in this study were the serosa and the mucosa. Possible consequences of an inflammatory reaction in the colonic serosa can include post-operative adhesion formation. A recent study by GORVY et al. (2008) reported that adhesions were neither associated with the