Influence of mechanical manipulations on the local inflammatory reaction in the equine colon




Reasons for performing study: Large intestinal diseases in horses are characterised 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 exteriorised and 3 segments randomly exposed to different mechanical manipulations: 1) enterotomy, 2) enterotomy and mucosal irritation and 3) serosal irritation. Intestinal biopsies were harvested before, immediately after and 30 min after each manipulation for histological evaluation. Eosinophils were detected with Luna's stain and neutrophils 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 subjected to euthanasia at the end of both studies.

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 nonmanipulated 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.


During colic surgery, which is the most frequent emergency surgery in horses, manipulation of the intestine is inevitable. In man and rodents, intestinal manipulations result 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. 1994a; Little et al. 2005; Grosche et al. 2008), parasitic infection (Collobert-Laugier et al. 2001; Rötting et al. 2008b) 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. 2008a). Most of the mucosal eosinophilic granulocytes are located close to the lamina muscularis mucosae. The intestinal submucosa also contains more resident eosinophilic granulocytes, whose functions in the intestine are not completely understood, although it is known that 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 result in a focal accumulation of eosinophilic granulocytes (Collobert-Laugier et al. 2001; Rötting et al. 2008b) and an intestinal eosinophilic response is most often 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. 1994a,b; Rötting 2005), after in vivo intestinal strangulation (Rötting 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 2005). The eosinophilic granulocyte is well equipped to initiate and maintain a local inflammatory reaction and is,therefore, of particular interest for a better understanding of the pathogenesis and clinical consequences of intestinal inflammation in horses.

The neutrophilic granulocyte is the predominant inflammatory cell type commonly associated with mucosal damage caused by ischaemia-reperfusion injury in humans and in other species (Grisham and Granger 1988; Grisham et al. 1990; Schoenberg et al. 1991; Kubes et al. 1992; Moore et al. 1994a; Gayle et al. 2002). In horses, 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 (McMichael 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 may include post 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.

Materials and methods


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 gastrointestinal disorders were studied. Before the study began, the horses were treated with a Moxidectin formulation (Equest)1 according to the manufacturer's recommendations and faeces evaluated for intestinal parasitism using a faecal flotation test. After a negative result (no parasitic eggs in the faeces) was obtained, horses were kept in a stall with paddock for 2 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. Balanced anaesthesia was maintained by with isoflurane (Isofluran CP)2 in 100% oxygen and continuous rate infusion (CRI) of xylazine (Xylapan)2 at 0.7 mg/kg bwt/h.

Mean arterial blood pressure, heart rate, respiratory rate, respiratory pressures, inspiratory oxygen fraction and expiratory isoflurane concentration were monitored continuously with the Kardiocap 5 monitor4 and arterial blood gases measured every 20 min. Dobutamine (Dobutamin-ratiopharm 250 mg)5, i.v. fluids (lactated Ringer's 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: A median laparotomy was performed in 12 horses. The left dorsal colon was exteriorised and divided into 3 adjoining segments of a length of approximately 30 cm with penrose drains without compromising vascular supply. An antimesenterial enterotomy was performed on one random segment without further manipulation (Group 1: enterotomy); additionally, a control sample was collected from the enterotomy site. An enterotomy was carried out in a second random segment followed by 10 min of mechanical irritation of the mucosa with sponges (Group 2: mucosal irritation). The last segment was exposed to 10 min of mechanical irritation of the serosa with sponges (Group 3: serosal irritation). No control sample was harvested from this segment to avoid any influence that the required enterotomy could have on the inflammatory reaction in this model of serosal irritation. All manipulations were performed by the same person (A.K.R.). Full thickness colon wall samples approximately 15 cm apart were harvested before the manipulations (control) from the enterotomy site, immediately after the end of each manipulation (time-point 0) or 10 min after placement of the enterotomy for Group 1 and 30 min after the end of each manipulation (time-point 30). After final sampling, horses were subjected to euthanasia with an overdose of sodium pentobarbital without regaining consciousness.

Study 2: Fourteen 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 (approximately 3 h surgery time) without any previous manipulation of the colon. Time of surgery was not significantly different between groups. After final sampling, horses were subjected to euthanasia with an overdose of sodium pentobarbital without regaining consciousness.

Histological evaluation

The samples were fixed in either formalin (4%) or in Bouin's solution (formaldehyde, picric acid and acetic acid) and were subsequently 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's solution were desiccated in xylene, stained with Biebrich scarlet-haematoxylin, 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).

Formalin-fixed sections were dewaxed in xylole and treated with hydrogen peroxide (3%) in methanol for 10 min to block the endogenous peroxidase. Nonspecific binding of antibodies was inhibited by incubating sections in normal goat serum (BioGenex)7 for 30 min. Sections were then incubated with primary antibodies (anti-calprotectin, clone MAC387, monoclonal, mouse IgG1)8 at a dilution of 1:4000, overnight at 4°C. Immunoreactions were visualised 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 min. This was followed by incubation with horseradish peroxidase-labelled streptavidin for 20 min. The entire antibody-enzyme complex was then visualised by adding a chromogenic system (Liquid DAB)7 until adequate colour development was seen. Sections were counterstained with toluidine blue and covered with DePex9.

Image Pro Express 6.310 was used for histomorphometric analysis of the images obtained by light microscopy. The histomorphometric measurements were performed as described in previous studies (Rötting 2005;Rötting et al. 2008a). The length of mucosal surface epithelium was measured and the percentage of denuded epithelium calculated. The mucosa was divided into 5 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 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 5 sections relative to the total eosinophil or neutrophil count in all sections for each horse. The density of eosinophils and neutrophils/mm2 of mucosa was also calculated.

Figure 1.

Division of mucosa into 4 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 indicate eosinophilic granulocytes in Luna's eosinophil stain, in which eosinophils are stained red and nuclear elements are blue; bar = 200 μm.

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 in each of 3 random areas of each layer using a calibrated grid of 250 × 390 µm in 10× magnification for the circular and longitudinal muscular layers. The serosa, intermuscular layer and submucosa were evaluated in 20× magnification with a grid of 250 × 390 µm. For more detailed information about distribution of neutrophilic and eosinophilic granulocytes within the submucosa, the later was further divided into 3 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 were neither normally nor log-normally distributed, and thus 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 were calculated with the signed rank test. Results were considered significant if P<0.05. Analyses were carried out with the statistical software SAS, version 9.2911.


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).

After enterotomy alone (Group 1) the only significant change was a neutrophilic infiltration of the serosa 30 min after the end of manipulation (P<0.0391, Table 1).

Table 1. Absolute numbers of neutrophilic granulocytes in the mucosa in different localisations compared between control samples and different manipulations types. Values are expressed as median and range
 ControlEnterotomyMucosal irritationSerosal irritationLaparotomy
0 min30 min0 min30 min0 min30 min0 min3 h
  1. Values with capital letters are significantly different from values with the same lower case letter within the identified histological layer. SM: submucosa; CM: circular muscle layer; IM: intermuscular layer; LM: longitudinal muscle layer.

M10 a0.670.3301 A0.330.671.330.67
M20.671.330.670.5 b3 B0.67 c2.33 C3.671
M30.831.330.670.17 b2 B0.83 c3 C3.330.67
M40.670.330.50.5 b2.33 B0.83 c2.67 C2.330.67
SM10.17 a0.330.50.33 b1 A,B0.33 c0.5 C0.330.67
SM20.17 a0.330.670.5 A0.330.330.670.670
CM0.33 a00.7510.670.83 A0.170.670.67
Serosa0.67 a0 b10.5 B2.08 c6.83 A,C13 A0.330.33

Mucosal irritation (Group 2) 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 2).

Table 2. Percentage of eosinophilic and neutrophilic granulocytes in the colonic mucosal sections. Values are expressed as median and range
 ControlEnterotomyMucosal irritationSerosal irritationLaparotomy
0 min30 min0 min30 min0 min30 min0 min3 h
  1. Values with capital letters are significantly different from values with the same lower case letter within the identified histological layer.

Eosinophils M1 (%)49.8663.7757.9952.2476.4150.2940.8148.9933.98
Eosinophils M2 (%)29.31 a25.2226.351.28 A15 A32.6829.8916.5822.19
Eosinophils M3 (%)9.61 a7.539.260 A2.5610.215.98.3319.07
Eosinophils M4 (%)0 a0.480.572.4900.92 b6.19 A,B16.670.83
Eosinophils M5 (%)000000000
0–5.90–69 0–0.780–2.560–1.140–7.01
Neutrophils M1 (%)016.677.940 b16.67 B13.8911.1110.1833.33
Neutrophils M2 (%)31.2826.9832.1116.67 b34.15 B16.6732.9428.9527.78
Neutrophils M3 (%)28.9725.3221.76.6719.72319.9328.3616.87
Neutrophils M4 (%)14.66 a6.088.1226.3913.78 A19.0520.8122.596.85
Neutrophils M5 (%)00000004.760

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 time point 0.

Serosal irritation alone (Group 3) resulted in a significant redistribution of eosinophilic granulocytes towards the luminal surface (M4: P<0.00977, Table 2) after the end of manipulation when compared to control samples and to samples at time point 0. 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 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 3). There were no significant changes in neutrophilic infiltration.

Table 3. Absolute numbers of eosinophilic granulocytes in the mucosa in different localisations compared between control samples and different manipulation types. Values are expressed as median and range
 ControlEnterotomyMucosal irritationSerosal irritationLaparotomy
0 min30 min0 min30 min0 min30 min0 min3 h
  1. Values with capital letters are significantly different from values with the same lower case letter within the identified histological layer. SM: submucosa; CM: circular muscle layer; IM: intermuscular layer; LM: longitudinal muscle layer.

M111 a17.5 A10.55.33 A7.67179.3312.3315.67
M25 a10.6760 A1.679.58.336 b15 B
M5000 00000


In the present study, a local inflammatory response to mechanical manipulations of the colon was observed in the colon layers. 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. 2008b), experimental ischaemia-reperfusion injury (Moore et al. 1994a,b; Rötting 2005), in vivo intestinal strangulation (Rötting 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 min 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 min 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), where a significant redistribution of mucosal eosinophilic granulocytes was seen after 30 min. Similarly, 30 min should be long enough to identify a neutrophilic infiltration (Grosche et al. 2008).

Neutrophilic granulocytes were also involved in the inflammatory response as seen as an increased infiltration of the mucosa, the submucosa and the serosa with neutrophilic granulocytes. Little et al. (2005) reported significant 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. 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 2 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 might include post operative adhesion formation. Gorvy et al. (2008) reported that adhesions were neither associated with the primary lesion site, nor with small intestinal resection or endotoxaemia, the authors concluding that surgical trauma was the most important stimulus in adhesion formation.

The mucosal inflammatory reaction observed in this study was associated with loss of mucosal epithelial cells and included a neutrophilic and eosinophilic response. A possible consequence of the mucosal inflammation is an impairment of the local gut-blood barrier with the result of leakage of endotoxins into the circulation. Clinical signs of this inflammation might include post operative endotoxaemia and colitis.

The local inflammatory reaction observed in our study after mechanical manipulation of the intestine conforms to studies in laboratory animals where a panenteritis has been reported after laparotomy and local intestinal manipulation (Kalff et al. 2003). A local enteric inflammatory reaction has been considered to play a potential role in the pathogenesis of post operative ileus. Several studies in rats and man have shown that intestinal manipulation during surgery induces a local inflammatory reaction in the intestinal muscularis with infiltration of macrophages, monocytes and neutrophilic granulocytes. Local inflammatory cells are activated and release inflammatory mediators (IL-1, IL-6) and motor-active substances (Wehner et al. 2007). As a result, the motility of the intestine can be decreased for several hours after abdominal surgery (Kalff et al. 2003). Strong infiltration of leucocytes and an increase in inflammatory mediators (IL-6, IL-1β, TNF-α) in the tunica muscularis mucosae, along with a decrease in the measured intestinal motility, has been described in several in vitro studies (Kalff et al. 1998, 1999, 2003; Türler et al. 2002; Schwarz et al. 2004). In our study, the only change within muscular layers was an increased neutrophilic granulocyte infiltration in the circular muscle layer after serosal irritation. Other factors discussed as possible aetiologies for the development of post operative ileus are an increase in activity of the sympathetic system, the activation of inhibitory adrenergic and nicotinergic nerves (Bredtmann et al. 1990; De Winter et al. 1997; Kalff et al. 1998; Kehlet and Holte 2001) and effects of anaesthetics (Bueno et al. 1978; Ogilvy and Smith 1995).

Laparotomy and small intestinal manipulation alone resulted in colonic mucosal inflammation in this study. In this part of the study, colon samples were collected from 7 horses immediately after laparotomy without any manipulation of the intestine. In the remaining 7 horses, the colon was left in situ without any manipulation while the small intestinal ischaemia and reperfusion study was performed. Only after the small intestinal manipulation was the colon exteriorised and a sample collected. This approach was chosen to eliminate any effect of colon manipulation or colon biopsies on the inflammatory reaction in the second group in this study. This approach does have a limitation in that it is possible that the second group could have had an infiltration of the intestinal wall prior to surgery. However, horses were randomly assigned to one sample time point and the initial enterotomy and colon manipulation to gain a colon sample at the beginning were considered more severe.

The effect of small intestinal manipulation alone without any manipulation of the colon has also been studied in rodents. Schwarz et al. (2004) demonstrated a molecular, cellular and functional measurable inflammatory panenteric reaction in the unmanipulated gastric and colonic muscularis after small intestinal manipulations with activation of IL-6 and infiltration of neutrophilic granulocytes in the unmanipulated colonic region and associated with suppressed colonic circular muscle contractility.

The intestinal inflammatory response and especially the eosinophilic inflammatory response observed in this study occurred very quickly after laparotomy with and without direct mechanical irritation of the colon. Intestinal biopsies obtained during surgery should therefore be harvested at the beginning of surgery to avoid misinterpretation of the histological findings, especially when a diagnosis of mucosal eosinophilic enteritis is considered.

In conclusion, our findings demonstrate a rapid colonic inflammatory response after direct colonic mechanical irritation as well as after laparotomy and manipulation of small intestinal segments. This response could account for some of the post operative complications encountered in surgical colic patients and should be considered when harvesting intestinal biopsies and interpreting the results of histological evaluation of biopsies.

Conflicts of interest

No conflicts of interest have been declared.


We would like to thank Mrs Gudrun Wirth for her histological preparations.

Manufacturers' addresses

1 Vétoquinol GmbH, Ravensburg, Germany.

2 CP-Pharma Handelsgesellschaft mbH, Burgdorf, Germany.

3 AbZ Pharma GmbH, Blaubeuren, Germany.

4 Datex-Ohmeda GmbH, Duisburg, Germany.

5 Ratiopharm GmbH, Ulm, Germany.

6 B. Braun Melsungen AG, Melsungen, Germany.

7 Firma BioGenex, San Ramon, California, USA.

8 DCS Diagnostik Systeme, Hamburg, Germany.

9 Serva Electrophoresis GmbH, Heidelberg, Germany.

10 Media Cybernetics, Bethesda, Maryland, USA.

11 SAS Institute Inc, Cary, North Carolina, USA.