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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2008 Dec;89(6):476–489. doi: 10.1111/j.1365-2613.2008.00623.x

Acute colonic ischaemia in rats results in long-term structural changes without alterations of colonic sensitivity

Anna Ravnefjord *, Madeleine Pettersson *, Erika Rehnström *, Vicente Martinez *,†
PMCID: PMC2669609  PMID: 19134057

Abstract

Colonic ischaemia and mast cells have been involved in the pathophysiology of the functional gastrointestinal disorder irritable bowel syndrome, although the cause–effect relationships remain unknown. We assessed long-term histopathological and functional changes associated to an acute ischaemic episode (1 h) of the colon, followed by 8-week recovery, in rats. Functional colonic alterations [sensitivity during colorectal distension (CRD), compliance and propulsive motility] were assessed regularly during the recovery. Colonic histopathology (presence of inflammation, morphometric alterations and variations in neuronal density in the enteric nervous system) 8-week postischaemia was assessed. Following ischaemia, none of the functional parameters tested (motility, sensitivity and compliance) were affected. At necropsy, the colon presented an overall normal appearance with an increase in weight of the ischaemic area (mg/cm: 99 ± 6; P < 0.05 vs. control: 81 ± 4 or sham ischaemia: 81 ± 3). Histopathological evaluations revealed the presence of a local infiltrate of mast cells in the area of ischaemia (nb of mast cells: 142 ± 50; P < 0.05 vs. control, 31 ± 14 or sham ischaemia: 40 ± 16), without other significant alterations. Animals subjected to colonic ischaemia and treated 8 weeks later with the mast cell degranulator, compound 48/80, showed no changes in CRD-related pain responses. These studies show that acute colonic ischaemia is associated with the presence of a long-term local infiltration of mast cells, located within the serosa and muscle layers, despite the absence of functional changes, including colonic sensitivity. Considering the important pathophysiological functions of mast cells, the observed mast cell infiltration may be involved in ischaemia-induced functional changes yet to be characterized.

Keywords: colon, colonic compliance, colorectal distension, irritable bowel syndrome, ischaemia, mast cells, visceral pain

Intestinal ischaemia, either acute or chronic, is a clinical entity associated with a variety of conditions including acute mesenteric thrombosis, embolism, intestinal obstruction, inflammatory bowel disease and, probably, also functional gastrointestinal (GI) disorders. In this sense, patients diagnosed with the functional GI disorder irritable bowel syndrome (IBS) appear to be predisposed to the development of ischaemic colitis at a later stage (Cole et al. 2004; Walker et al. 2004; Suh et al. 2007). Nevertheless, a clear correlation between IBS and colonic ischaemia has not been established and it remains unclear if ischaemia is a cause or a consequence of the disease or only a manifestation of side effects associated to specific pharmacological treatments in predisposed individuals (Cole et al. 2004; Walker et al. 2004; Suh et al. 2007). Interestingly, patients with chronic heart failure, which might be in an estate of chronic partial ischaemia of the gut, have significant GI functional and histopathological alterations (Sandek et al. 2007).

In rats, acute ischaemia with reperfusion of the small intestine leads to long-term structural changes, including the development of tissue hypertrophy, neuronal cell death within the myenteric plexus and the presence of a significant inflammatory infiltrate, including mast cells (Takada et al. 1998; Lindestrom & Ekblad 2004; Chang et al. 2006). These changes appear between hours and days after an acute episode of ischaemia and persist for long time, up to 10 weeks after the initial insult (Lindestrom & Ekblad 2004). Similarly, a recent report by Sand et al. (2008) shows that acute ischaemia of the colon attracts mast cells and causes only minor loss of enteric neurons, without other significant structural alterations. Interestingly, some of these histopathological changes are similar to those observed in IBS patients (i.e. a moderate inflammatory response with an increase in the number of mast cells) (O’Sullivan et al. 2000; Barbara et al. 2004a,b; Chey & Cash 2006). In particular, mast cell infiltration in proximity to mucosal nerves has been suggested as one possible contributor to the state of colonic hypersensitivity present in a significant proportion of IBS patients (Barbara et al. 2004a,b, 2007).

The functional changes associated with intestinal ischaemia have not been fully elucidated. It is known that during acute ischaemia/reperfusion there are significant alterations of epithelial function leading to increased permeability (Murthy et al. 1997). Similarly, motor alterations, with the lost of peristaltic activity, have been associated to ischaemia-induced neuronal damage within the gut (Yano et al. 1997). To the best of our knowledge, long-term functional alterations associated to acute ischaemic events have not been characterized.

The general aim of the present study was to characterize long-term histopathological changes within the colon associated to an acute episode of colonic ischaemia as well as the potential functional alterations related to the process. Taking into consideration the potential link between ischaemia and IBS and the similarities between the histopathological changes observed in the intestine after ischaemia and in IBS patients, the possibility that acute colonic ischaemia might be a useful animal model mimicking some of the functional alterations observed in IBS patients was addressed in more detail. As altered colonic sensitivity is a hallmark of IBS (Ritchie 1973; Stacher & Christensen 2006; Azpiroz et al. 2007), we focused on alterations on colonic sensitivity in a rat model of colorectal distension (CRD), assessing visceromotor responses to CRD as a surrogate marker for visceral pain (Ness & Gebhart 1988; Tammpere et al. 2005). In addition, we also assessed changes in colonic propulsive motor activity, using faecal pellet output as a surrogate marker for motility (Martinez & Taché 2001), and changes in compliance of the colonic wall during CRD (Käll et al. 2007; Martínez et al. 2007).

Materials and methods

Animals

Adult female Sprague–Dawley rats (Harlan, The Netherlands, weighing 250–300 g) were used. Animals were housed in group cages (enriched rat cage system, 4–5 animals per cage) with free access to food (R3 pellets; Lactamin AB, Stockholm, Sweden) and water, except otherwise stated, under controlled environmental conditions (50% humidity, 21 °C) and a 12 h light:dark cycle. All experimental procedures were approved by the local Animal Ethics Review Committee of Gothenburg.

Surgical procedures

Rats were anaesthetized with isoflurane (Forene®; Abbott Scandinavia AB, Solna Sweden) and were kept on a heating pad during surgery to maintain body temperature. The peritoneal cavity was exposed with a midline incision and the colon localized. A 3–4 cm long segment of the distal colon (3–4 cm proximal to the anus) was identified, clamped proximally and distally, to avoid collateral blood flow between adjacent intestinal segments (Yano et al. 1997), and all blood supply from the mesenteric vessels interrupted with vascular clamps (Vascu-Statt®plus, soft 50–60 g; Scanlan International, St Paul, MN, USA). The restriction in blood supply (ischaemia) was maintained for 1 h. During this time, the exposed tissues were covered with gauze and constantly soaked with saline. During the time of ischaemia, the tissue became purple, indicating proper blood restriction. Thereafter, the clamps were removed; the reperfusion visually examined to secure restoration of blood flow and the abdominal cavity sutured (Dexon 4-0 and Dermalon 5-0). This region of the colon was selected because it corresponds to the area distended during CRD experiments (see below). Animals subjected to sham colonic ischaemia underwent the same procedures except for the interruption of the blood supply to the colon. After surgery, animals were given analgesics (0.015 mg/kg sc, Temgesic®, Buprenorfin, Shering-Plough, NJ, USA) and were allowed to recover in a quiet and dim room for at least 24 h. Animals were kept single-housed for 5 days after surgery and thereafter returned to the general housing conditions described above.

Colorectal distension

Rats were habituated to Bollmann cages (Plexi-glass tubes, length 18 cm, diameter 6 cm, AstraZeneca, Mölndal, Sweden) 30 min per day for three consecutive days prior to any experimental procedure to reduce motion artefacts because of restraint stress. A 3-cm polyethylene balloon (made in-house) with connecting catheter was inserted in the distal colon, 2 cm from the base of the balloon to the anus, during light isoflurane anaesthesia (Forene®; Abbott Scandinavia AB). The catheter was fixed to the tail with tape. The balloons were connected to pressure transducers (P-602, CFM-k33, 100 mmHg, Bronkhorst HI-TEC, Veenendal, The Netherlands). Rats were allowed to recover from sedation in the Bollmann cages for at least 15 min before starting the CRD procedure.

A customized barostat (AstraZeneca) was used to manage air inflation and balloon pressure control. A customized computer software (PharmLab on-line 4.0, AstraZeneca) running on a standard computer was used to control the barostat and to perform data collection. The distension paradigm generated by the barostat was achieved by generating pulse patterns on an analogue output channel. Two different CRD paradigms were used. To assess pain-related visceromotor responses and thresholds for pain, increasing phasic isobaric distensions from 10 to 80 mmHg, with a pulse duration of 30 s at 2.5-min intervals, were used. For compliance measurements, phasic isobaric distensions from 2 to 20 mmHg, with a pulse duration of 1 min at 5-min intervals, were used. In this case, pressure dependent changes in volume during isobaric CRD were taken as a measure of colorectal compliance. When assessing compliance, low distension pressures (within a range considered non-noxious) were chosen in order to avoid pain-related visceromotor responses that might interfere with the measurements of the intraballoon volume. Similar protocols have been used before to assess responses to CRD in rats (Tammpere et al. 2005; Käll et al. 2007; Martínez et al. 2007).

Data collection and analysis

When evaluating colorectal distension-induced visceral pain (10–80 mmHg), rapid pressure changes in the distending balloon, reflecting contractions of the abdominal muscle, were used to assess pain-related visceromotor responses, as previously characterized by us (Tammpere et al. 2005; Arvidsson et al. 2006). The balloon pressure signals were sampled at 50 Hz. A high-pass filter at 1 Hz was used to separate the contraction-induced pressure changes from the slow varying pressure generated by the barostat. A customized computer software (PharmLab off-line 4.0, AstraZeneca) was used to quantify the magnitude of the high-pass-filtered balloon pressure signals. Hence, manual analysis and potential bias by the investigator were avoided. The average magnitude of the high-pass-filtered balloon pressure signals was calculated for 30 s before the pulse (i.e. baseline response) and for the duration of the pulse. When calculating the magnitude of the high-pass-filtered balloon pressure signals, the first and the last 2 s of each pulse were excluded as these reflect artefact signals produced by the barostat during inflation and deflation and do not originate from the animal. The overall response to the distension procedure was calculated as the cumulative value of the high-pass-filtered balloon pressure signals for all the distension pressures used. Threshold pressures for pain were also determined during phasic, 10–80 mmHg, colorectal distension. For every animal, the pressure threshold was defined as the pressure of the distending pulse at which the response evoked exceeded the main baseline activity plus two times the standard deviation (Tammpere et al. 2005; Arvidsson et al. 2006; Käll et al. 2007; Martínez et al. 2007).

For the determination of compliance, the maximal intracolonic volume achieved during each distension (2–20 mmHg) was determined and pressure–volume curves were constructed as a measure of colonic compliance (Käll et al. 2007; Martínez et al. 2007).

Tissue sampling, histological procedures and morphometric analysis

Animals were killed 8 weeks after the induction of ischaemia, after the last CRD procedure. The colon (excluding the cecum) was carefully dissected, measured and weighed. Thereafter two 3- to 4-cm segments of colonic tissue were collected from the area where the ischaemia was induced and from the proximal colon (2–3 cm distal from the ileocaecocolic junction) respectively. In addition, a 3–4 cm segment of the distal ileum was also taken. Tissue samples were weighed and divided in two longitudinal sections. One of the sections was fixed in zinc–formalin solution (pH 7.4; Histolab Products, Göteborg, Sweden) for histological studies. The other tissue piece was weighed, frozen in liquid nitrogen and stored at −80 °C until analysis. In addition, a group of animals were killed right after the ischaemia or sham-ischaemia procedure and tissue samples were obtained from the same areas.

Paraffin sections (5 μm) of tissue samples were stained following standard histological procedures. Four different staining methods were applied for different histological measurements. Standard haematoxylin–eosin staining was used for general histological assessment and morphometric analysis (see later for details). Masson’s trichrome staining was used to visualize the presence of connective tissue, as an indicator of fibrosis. The van Gieson picric acid–acid fuchsine stain was used when assessing the total number of neurons in the enteric nervous system. Finally, a standard toluidine blue staining was used for the detection of mast cells. Mast cells were identified by their granules, which exhibit metachromatic purple staining (Galli 1990). Coded sections were analysed in a light microscope (Axioskop, Zeiss, Germany) in a blinded manner by a person unaware of the experimental procedures.

Morphometric characteristics (total wall thickness, thickness of the mucosa/submucosa and thickness of the muscle layers) were assessed in five randomly selected areas for each section, at 10× magnification, using an image analysis software (Image Access Analysis; EuroMed Networks, Stockholm, Sweden).

The density of neurons within the enteric nervous system was assessed in van Gieson picric acid–acid fuchsine-stained sections by counting the number of neuronal cell bodies in 20 fields, at 40× magnification, in three different sections for every tissue sample (i.e. a total of 60 fields per sample).

For counting of mast cells, toluidine blue-stained sections were used. The total number of mast cells in the serosa, muscle and mucosa/submucosa areas was determined in a complete tissue section of the distal ileum and the proximal and distal colon for each animal.

Analysis of markers in colonic homogenates and plasma

The content of serotonin, histamine and local inflammatory markers (IL-6, TNF-α and MIP-1α) was determined in whole colonic tissue homogenates. Tissue samples were homogenized in a buffer containing phosphate sodium saline (PBS; GIBCO, Invitrogen, Paisley, UK), 10% foetal calf serum (GIBCO) and protease inhibitor cocktail tablets (Complete Mini; Roche Diagnostics GmbH, Mannheim, Germany), as recently described (Melgar et al. 2005, 2008). Levels of IL-6, TNF-α and MIP-1α were determined using the xMAP® technology developed by Luminex® (Luminex Corporation, Austin, TX, USA) as previously described (Melgar et al. 2005). Serotonin and histamine levels were determined by using commercial RIA-kits (BA5900 and BA1000 respectively; LDN Labor Diagnostika Nord GmbH & Co., Nordhorn, Germany). In all cases, levels are expressed as picograms per 100 mg of colonic tissue.

Plasma metabolic parameters (lactate, triglycerides, free fatty acids, cholesterol and glucose) were determined by using the following commercial kits: Randox Laboratories Ltd (Crumlin, UK) kit nr. LC2389 (lactate); Roche Diagnostics kit nr. 12146029216 (triglycerides); Wako chemicals inc. (Richmond, VA, USA) kits nrs. 999–75406 (free fatty acids) and 2016630 (cholesterol); and ABX Diagnostics-Parch Euromedicine (Montpellier, France) kit nr. HK-125 for glucose.

Experimental protocols

Animals were divided in three experimental groups: ischaemia, sham ischaemia and untreated controls. Body weights were controlled daily during the first week after surgery and weekly between experimental weeks 2 and 8 (at the day of the CRD procedure). All animals were killed on week 8, immediately after the last CRD procedure. In addition, two groups of animals were used to assess the efficacy of the ischaemic procedure. In this case, at the end of the ischaemia or sham ischaemia procedure, animals were killed and tissue and plasma samples were obtained for analysis.

Faecal pellet output

Colonic motor function was assessed once every week during the first 3 weeks after ischaemia (weeks 1, 2 and 3). Animals were placed overnight in individual cages with a bottom grid and an absorbent paper disposed for urine absorption. The number and weight of faecal pellets excreted during an overnight dark period (12 h) were taken as a measure of colonic motility.

Colonic sensitivity during CRD

All animals were subjected to a CRD (10–80 mmHg) procedure before any other treatment (week 0). Thereafter, they underwent the appropriate surgical treatment and during the following 8 weeks (weeks 1 to 8) were subjected to a weekly CRD procedure.

Colonic compliance

On weeks 7 and 8, an additional CRD procedure (2–20 mmHg) was performed to assess the pressure–volume relationship during distension as a measure of colonic compliance.

Effects of compound 48/80

In a separate experiment, the effects of the mast cell degranulator, compound 48/80 (Sigma-Aldrich, St. Louis, MO, USA), on visceral pain responses to CRD (10–80 mmHg) were determined. In a group of animals (n = 7), colonic ischaemia was induced as described above. An 8-week period was allowed between the ischaemic procedure and the CRD experiments. All animals underwent two consecutive CRD procedures with an interval of 24 h. The first CRD was taken as a control. For the second CRD procedure, the animals were divided into two groups and received vehicle (saline, 1 ml/kg, i.p.; n = 3) or compound 48/80 (0.5 mg/kg, i.p.; n = 4) 30 min before starting the CRD protocol. Immediately thereafter, the animals were killed and samples from the distal colon (ischaemic area) were obtained for histological evaluation and determination of tissue levels of inflammatory-related markers (see above).

Statistical analysis

Data are expressed as mean ± SEM. Paired or unpaired Student’s t-test, as appropriate, was used for comparisons between two groups. Differences between multiple groups were determined by repeated or non-repeated measures one-way anova, as appropriate, and followed, when necessary, by a Student–Newman–Keuls multiple comparisons test. Data were considered statistically significant when P was <0.05. Correlation between parameters was assessed by linear regression analysis (GraphPad Prism®; version 4.0.3, GraphPad Software, Inc. San Diego, CA, USA).

Results

During the time of ischaemia, the colonic segment subjected to the procedure became dark reddish-blue with dilatation of the superficial blood vessels, indicative of congestion. The normal appearance of the tissue was recovered upon restoration of the blood supply (reperfusion). Histological evaluation of tissue samples obtained within 15–20 min after the ischaemia/reperfusion procedure showed moderate histopathological alterations in the area of ischaemia with oedema of the submucosa, dilatation of blood vessels and a diffuse haemorrhage extending to some areas of the mucosa, where epithelial destruction could also be observed (Figure 1a,b). In some cases, red blood cells were also observed in the smooth muscle layers. In the same animals, tissue samples obtained from the proximal colon or the distal ileum showed a normal histological structure. Detection of local levels of inflammatory-related mediators in colonic homogenates obtained from the area of ischaemia revealed a significant increase in the levels of IL-6 and slightly increased levels of histamine, when compared with samples obtained from sham ischaemia animals (Table 1). In the proximal colon and the distal ileum, IL-6 and histamine levels were unaffected, regardless of treatment (data not shown). Plasma metabolic markers (lactate, triglycerides, free fatty acids, cholesterol and glucose), determined at the end of the ischaemic procedure, had similar values in the sham ischaemia and ischaemia groups (data not shown).

Figure 1.

Figure 1

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Representative microphotographs of H&E sections showing the overall structure of the distal colon (area of ischaemia) in different experimental conditions. (a, b) Colon from animals killed right after a 1-h sham ischaemia (a) or a 1-h ischaemia procedure (b). (c–e) Colon from a control untreated rat (c) or from rats killed 8 weeks after sham ischaemia (d) or 1-h ischaemia (e). Note the presence of moderate histopathological changes after 1-h of ischaemia (b) while there was an overall absence of morphological changes associated to the previous ischaemic event at week 8 (e). Magnification: 10×.

Table 1.

Inflammation-related markers in the distal colon at the end of a 1-h ischaemia period†

TNF-α IL-6 MIP-1α Serotonin Histamine
Sham ischaemia 8.8 ± 1.8 23.0 ± 5.0 2.9 ± 0.5 46.1 ± 6.3 53.5 ± 35.0
Ischaemia 12.9 ± 2.4 42.5 ± 3.4* 2.8 ± 0.1 58.2 ± 18.9 137.0 ± 90
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*

P < 0.05 vs. sham ischaemia.

†

All values are pg/100 mg of colonic tissue. Data are mean ± SEM of 4 animals per group.

All animals recovered well from the surgical procedure. A slight body weight loss (2–3%) was observed during the 2- to 3-day period following surgery (irrespective of the induction or not of colonic ischaemia). Thereafter, all animals showed a similar increase in body weight during the course of the study (Figure 2).

Figure 2.

Figure 2

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Evolution of body weight in the different experimental groups along the experimental time. Data are mean ± SEM of 7–10 animals per group and represent the % change in body weight from day 1 (day of surgery), taken as 100%.

Effects of colonic ischaemia on faecal pellet output

Faecal pellet output (number and weight of faecal pellets excreted) was similar in the control, sham ischaemia and ischaemia groups, as determined overnight (dark phase, 12 h) once a week, during the 3 weeks after the induction of the colonic ischaemia (data not shown).

Effects of colonic ischaemia on visceral pain responses to CRD

In control animals, ascending-phasic CRD (10–80 mmHg) evoked a pressure-related visceromotor response observed as significant pressure-dependent oscillations in the intraballoon manometric recordings, as compared with baseline activity. Similar responses were observed in animals with colonic ischaemia or sham ischaemia (Figure 3a).

Figure 3.

Figure 3

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Responses to colorectal distension (CRD)-induced visceral pain in the different experimental groups. (a) Pain responses to the ascending phasic CRD (10–80 mmHg) paradigm at week 8 after colonic ischaemia or sham ischaemia. Graphs with open symbols and dashed lines represent the basal activity between distension. (b) Overall response to CRD (0–80 mmHg) throughout the experimental time. Week 0 corresponds to the assessment of pain responses before the ischaemic procedure. (c) Pain threshold during CRD throughout the experimental time. Week 0 corresponds to the pain thresholds determined before the ischaemic procedure. Data are mean ± SEM of 7–10 animals per group.

The overall response to CRD was of similar magnitude in the three experimental groups and through all distension sessions, i.e. both before ischaemia (week 0) and during the 8 weeks after ischaemia (weeks 1 to 8) (Figure 3b). Similarly, pain thresholds were also similar in the three experimental groups and relatively stable (in most cases between 30 and 40 mmHg) along the experimental time (Figure 3c).

Effects of colonic ischaemia on pressure–volume relationships during CRD

In all groups, a positive pressure–volume relationship was observed during increasing phasic colorectal distension (2–20 mmHg × 1 min). At weeks 7 and 8 after ischaemia, no significant differences in the pressure–volume responses were observed among the control, sham ischaemia and ischaemia groups (Figure 4).

Figure 4.

Figure 4

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Pressure–volume curves during phasic CRD (2–20 mmHg) in the different experimental groups at weeks 7 (a) and 8 (b) after colonic ischaemia. Data are mean ± SEM of 7–10 animals per group.

Macroscopical and microscopical analysis

Macroscopical appearance of the colon at the time of necropsy (8 weeks after ischaemia) was similar in all experimental groups. Only occasionally, a slight thickening/stiffness, approximately in the area of ischaemia, was observed in the distal colon of the ischaemic animals. Nevertheless, the colon of animals with ischaemia was slightly shortened and the relative colonic weight (mg/cm) was increased when compared with the control or sham ischaemia groups (Table 2). The change in relative colon weight was more evident when only a segment of the distal colon (3–4 cm), corresponding approximately to the area of ischaemia, was considered [control: 81 ± 4 mg/cm; sham ischaemia: 81 ± 3 mg/cm; ischaemia: 99 ± 6 mg/cm; F(2,21) = 5.639, P = 0.011]. Histological assessment of haematoxylin–eosin-stained sections revealed no evidence of lesions or structural changes associated to the ischaemic procedure in the distal colon (Figure 1c–e). In addition, Masson’s trichrome staining revealed similar collagen content, considered within a normal range, in the three experimental groups. The proximal colon and ileum showed also normal general histological features. More detailed morphometric analysis of the distal colon showed that the whole wall thickness was similar in the three experimental groups, although a slight increase, not reaching statistical significance, was observed in the ischaemia group compared with control. However, a significant thickening of the muscle layers was observed in the sham ischaemia as well as the ischaemia group (Figure 5a).

Table 2.

Length and weight of the colon 8 weeks after ischaemia‡

Body weight (g) Colon length (cm) Colon weight (mg) Relative colon weight (mg/cm)
Control 300 ± 7 17.2 ± 1.6 1755 ± 40 108 ± 11
Sham ischaemia 303 ± 6 16.9 ± 1.3 1916 ± 78 119 ± 13
Ischaemia 297 ± 4 13.5 ± 0.7*† 1974 ± 74 151 ± 11**††
F(2,21) 0.333 (P = 0.721) 3.486 (P = 0.046) 2.635 (P = 0.095) 3.957 (P = 0.035)
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*

P < 0.05 vs. sham ischaemia

**

P < 0.05 vs. control.

†

P = 0.077 vs. control

††

P = 0.067 vs. sham ischaemia.

‡

Data are mean ± SEM of 7–10 animals per group.

Figure 5.

Figure 5

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(a) Morphometry of the distal colon in the different experimental groups (see Methods for details). (b) Mast cell density in the distal ileum and proximal and distal colon in the different experimental groups. (c) Distribution of the mast cells infiltrate within the distal colon in the different experimental groups. In all cases, data are mean ± SEM of 7–10 animals per group. #P = 0.06 vs. control; *P < 0.05 vs. control (anova).

Mast cells and neuronal counting

Mast cells were occasionally observed in the mucosa and submucosa of toluidine blue-stained tissue sections from control animals. Mast cells were predominantly located around blood vessels and were more abundant in the colon than in the ileum. Similar mast cell density and distribution pattern were observed in tissue sections from animals with sham ischaemia. In animals with colonic ischaemia, there was a marked increase in the number of mast cells in the distal colon and a moderate, but still significant, increase in the proximal colon. In contrast, the density of mast cells in the ileum was only slightly increased (Figures 5b and 6). The mast cell infiltrate in the ischaemia group was very noticeable in the serosa of all animals and, in some cases, also in the muscle layer (five out of 10 animals), whereas the cell density in the mucosa/submucosa was similar as that in the control and sham ischaemia groups (Figures 5c and 6c,e). Mast cells were frequently observed in the region between the longitudinal and the circular muscle layers, i.e. in the area corresponding to the myenteric plexus (Figure 6d,f). Within the same preparation, at the points with more abundant infiltration of mast cells, the muscular layers were slightly thicker, which might be reflected in the slight thickening of the muscle layers observed overall in the ischaemia group (see Morphometric analysis). Irrespective of the treatment considered, there was a positive correlation between the relative weight of the colon and the density of the mast cells infiltrate (r = 0.615, P = 0.001). However, no correlation between the density of mast cells and the overall response to CRD was observed (r = 0.168, P = 0.434).

Figure 6.

Figure 6

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Representative microphotographs of toluidine blue-stained sections from the distal colon (area of ischaemia) 8 weeks after sham ischaemia (a) or 1-h ischaemia (b–f). (a, b) Overall view of the distal colon (10×). (c) Magnification showing the presence of numerous mast cells in the serosal side of the colon in an animal that underwent 1 h ischaemia 8 weeks before (10×). In the same image, mast cells can also be observed within the smooth muscle and in the area between the longitudinal and circular muscle layers. (d) Magnification showing the presence of numerous mast cells within the smooth muscle in an animal that underwent 1 h ischaemia 8 weeks before (20×). (e) Detail of mast cells in the serosal side of the distal colon 8 weeks after ischaemia (40×). (f) Detail of mast cells in the region of the myenteric plexus of the colon (40×). Note the localization of mast cells in close proximity on within the myenteric ganglia.

At the time of necropsy, 8 weeks after ischaemia or sham ischaemia, the number of myenteric neurons was similar among the different experimental groups, as assessed in van Gieson picric acid–acid fuchsine-stained sections (data not shown).

Inflammatory-related markers in tissue homogenates

Levels of inflammatory-related markers in tissue samples from ileum, proximal colon and distal colon (ischaemia site) were similar, irrespective of the treatment (Table 3 and data not shown). Nevertheless, in the distal colon of the ischaemia group, the levels of histamine were increased by twofold when compared with controls or the sham ischaemia group; however, statistical significance was not achieved (Table 3). There was no correlation between histamine levels and mast cell density within the distal colon (r = 0.196, P = 0.359).

Table 3.

Inflammation-related markers in the distal colon 8 weeks after ischaemia*

Control Sham ischaemia Ischaemia
TNF-α 25.0 ± 4.2 13.4 ± 2.0 37.8 ± 23.7
IL-6 83.5 ± 16.7 41.5 ± 10.6 67.1 ± 7.2
MIP-1α 4.3 ± 0.9 5.5 ± 2.5 4.2 ± 0.5
Serotonin 35.1 ± 4.9 46.7 ± 10.2 45.2 ± 9.7
Histamine 169.6 ± 46.8 187.6 ± 59.4 335.6 ± 123.6
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*

All values are pg/100 mg of colonic tissue. Data are mean ± SEM of 7–10 animals per group.

Effects of compound 48/80

In rats subjected to an episode of colonic ischaemia 8 weeks before, pretreatment with the mast cell degranulator, compound 48/80, did not affect the overall response to CRD or the threshold for pain when compared with a previous control experiment in the same animals (without any treatment) or to the response to CRD in vehicle-treated rats (Figure 7a,b).

Figure 7.

Figure 7

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Effects of compound 48/80 on the pain responses to colorectal distension (CRD) and the density of mast cells in the distal colon in rats that underwent an episode of colonic ischaemia 8 weeks before. (a) Overall response to CRD (0–80 mmHg). (b) Pain thresholds during CRD. (c) Total number of mast cells in the distal ileum and the proximal and distal (area of ischaemia) colon. (d) Distribution of the mast cells infiltrate within the distal colon. In all cases, data are mean ± SEM of three (vehicle) and four (compound 48/80) animals per group. *P < 0.05 vs. the vehicle group.

The histological evaluation of the colon and distal ileum of vehicle-treated rats revealed the presence of a mast cell infiltrate restricted to the distal colon (area of ischaemia) with a cellular density in the range observed in the previous experiments (see above). A mast cell infiltrate within the distal colon was also observed in compound 48/80-treated animals. However, in these animals, mast cells were almost completely absent in the serosal compartment, whereas the total number of mast cells in the mucosa/submucosa and muscle layers was similar to that observed in vehicle-treated animals (Figure 7c,d). The content of inflammation-related markers in the distal colon (area of ischaemia) was similar in vehicle- and compound 48/80-treated animals (data not shown).

Discussion

This study shows that a single episode of acute ischaemia (1 h) in the distal colon, followed by a 8-week recovery period, results in alterations of the colonic structure, characterized mainly by the presence of an infiltrate of mast cells in the absence of other significant inflammatory or morphological changes. Despite these changes, no significant functional alterations (i.e. changes in colonic propulsive activity, colonic sensitivity or colonic compliance) were observed during the 8-week recovery period.

In agreement with previous observations in acute ischaemic models of the small intestine (Takada et al. 1998; Chan et al. 1999; Lindestrom & Ekblad 2004), the ischaemic procedure applied to the colon was likely to be effective. This is inferred by the macroscopical changes observed in the appearance of the area of ischaemia during the time of restriction of the blood supply and also by the histopathological alterations observed in the colon of animals killed right after the ischaemic procedure. In addition, a moderate increase in the tissue levels of histamine and a significant increase in the local levels of IL-6, a marker of early acute inflammatory responses (Kamimura et al. 2003), were also observed, thus suggesting the initiation of an inflammatory-like response within the colon. These observations are compatible with the histopathological changes observed in the small intestine and colon shortly after an acute ischaemia/reperfusion event (Takada et al. 1998; Chan et al. 1999; Lindestrom & Ekblad 2004; Sand et al. 2008). In this study, we did not follow the progression of these early events, but focused on morphological changes observed 8 weeks after the acute ischaemic episode. This was based on previous studies showing the presence of significant alterations in the small intestine and colon during a period up to 20 weeks after a single ischaemic episode (Lindestrom & Ekblad 2004; Sand et al. 2008). Nevertheless, functional parameters were evaluated at several time-points postischaemia.

The most significant histopathological finding at 8 weeks postischaemia was the presence of a mast cell infiltrate in the ischaemic area (distal colon), and to a much lower extent in the proximal colon. Mast cell infiltrate was conspicuous on the serosal side. In addition, in many cases, abundant mast cells could also be observed within the smooth muscle and particularly between the circular and longitudinal layers, corresponding to the area of the myenteric plexus. Indeed, many mast cells were in close proximity or within the myenteric ganglia. However, mast cell density in the submucosa and mucosa was not affected. These observations are consistent with previous data showing a significant infiltrate of mast cells within the smooth muscle and myenteric ganglia of the ileum and colon, starting 4 weeks after an acute episode of ischaemia and persisting for 6–16 weeks thereafter, without involvement of the mucosa (Lindestrom & Ekblad 2004; Sand et al. 2008). Taken together, these observations might suggest that the mast cell infiltrate originates from the abdominal cavity and might represent a subpopulation of peritoneal mast cells (connective tissue-type mast cells). Furthermore, the reduction in mast cell density in the serosa after treatment with compound 48/80 suggest that, at least a subpopulation of these mast cells are connective-tissue mast cells, which are susceptible to degranulation upon treatment with compound 48/80 (Enerbäck & Lundin 1974). However, given the different phenotypic characteristics reported for distinct subpopulations of mast cells in rodents (Nakahata et al. 1986; Kitamura et al. 1987; Lutzelschwab et al. 1997), the mast cell population identified here needs to be further characterized in subsequent studies.

Colonic ischaemia has been linked to IBS, although the mechanistic relation remains largely unknown (Cole et al. 2004; Walker et al. 2004; Chang et al. 2006). Interestingly, a marked increase in colonic mast cells has been described in IBS patients compared with healthy subjects (O’Sullivan et al. 2000; Barbara et al. 2004a,b, 2007; Park et al. 2006). These observations suggest that ischaemia and its associated mast cell infiltration might be related to the pathophysiology of IBS. However, in contrast to that observed in rats in this study and by Sand et al. (2008), the mast cell infiltrate in IBS patients was observed in the mucosa and lamina propria (Barbara et al. 2004b, 2007). Nonetheless, the presence of mast cells in other layers of the colon has not been properly evaluated in IBS patients.

Besides the aforementioned infiltrate of mast cells and a slight increase in muscle thickness, no other significant structural alterations were observed 8 weeks after ischaemia. The histopathological examination showed no other inflammatory infiltrate, suggesting the absence of active inflammation, as indicated also by the absence of changes in inflammatory-related markers. Only slightly elevated histamine levels were observed 8 weeks after ischaemia, probably because of the local infiltrate of mast cells (Galli 1990). These observations agree with recent data showing that the rat colon was relatively unaffected with respect to morphometric changes, neuronal density or inflammatory infiltrate after acute ischaemia (Sand et al. 2008).

Heightened colonic sensitivity during experimental balloon distension and/or altered colonic motility has been demonstrated in a significant proportion of IBS patients (Ritchie 1973; Stacher & Christensen 2006; Azpiroz et al. 2007). Although the causes of visceral hypersensitivity remain largely unknown, mediators from intestinal mast cells may play a crucial role. Mast cells contain numerous neuroimmune mediators (Galli 1990) that, upon release following cell activation, can stimulate sensory nerves within the gastrointestinal tract leading to visceral hyperalgesia/allodynia (Coelho et al. 1998; La et al. 2004; Park et al. 2006; Barbara et al. 2007). Accordingly, a positive correlation between the density of mucosal mast cells and abdominal pain/discomfort has been established in IBS patients (Barbara et al. 2004a,b). These observations contrast with the results obtained here, in which no changes in CRD-induced visceral pain responses were observed after the acute ischaemic event, and no correlation between pain responses and the mast cell density could be established. The fact that the density of mucosal/submucosal mast cells was not increased after ischaemia might explain these discrepancies. This might also suggest that mucosal mast cells are more important in modulating pain-related responses than other mast cell populations. In addition, it can be speculated that the CRD paradigm used, although able to elicit pain-related responses, was ineffective inducing mast cell degranulation and, as a consequence, an increased pain response. Therefore, it is feasible to assume that the pharmacological induction of mast cell degranulation might result in sensitization of pain mechanisms leading to enhanced responses during CRD. However, in animals treated with the mast cell degranulator, compound 48/80, no changes in pain responses were observed. It is difficult to assess the significance of this negative finding because, although mast cells have been implicated in visceral pain arising from the colon (Coelho et al. 1998; Barbara et al. 2004b; La et al. 2004; Róka et al. 2007), the direct effects of compound 48/80 on CRD responses have not been previously assessed. The dose of compound 48/80 used was in the range reported in previous studies showing significant effects, avoiding general mast cell activation with systemic release of mediators that might result in confounding effects and even an anaphylactic shock (Gay et al. 2003; Liu et al. 2007). Nevertheless, a reduction in mast cell density in the serosal compartment of the colon was observed after the treatment with compound 48/80, thus suggesting that the compound was effective inducing degranulation of mast cells. Therefore, lack of changes in colonic sensitivity in our study might suggest that a more extensive mast cell degranulation, in particular activation of mucosal mast cells, as previously suggested (Barbara et al. 2004b; La et al. 2004), might be necessary to elicit sensory changes. Here we only report preliminary observations with compound 48/80; further studies should be performed to address the potential functional significance of the mast cell infiltrate observed after ischaemia. In particular, these studies should address the importance of mucosal mast cell activation to induce hypersentivity.

The effects of intestinal ischaemia on the enteric nervous system remain largely unknown. Previous reports indicated that an episode of acute ischaemia of the small intestine in rats curses with a significant reduction in the total number of enteric neurons, occurring 1 to 10 weeks after the ischaemic event (Piao et al. 1999; Lindestrom & Ekblad 2004). In our study, however, we did not observe changes in the total number of enteric neurons after a similar ischaemic event in the colon. This is in agreement with recent data showing only very marginal effects of ischaemia on colonic enteric neurons in rats (Sand et al. 2008). This might reflect a lower susceptibility of the colon to ischaemia compared with the ileum, thus suggesting that a more sustained ischaemic insult is necessary to produce a similar degree of neuronal damage. Loss of intestinal neurons is likely to result in altered intestinal secretomotor and/or sensory functions. This has been suggested as the reason for the altered peristalsis observed after intestinal ischaemia (Yano et al. 1997). Interestingly, we did not observe alterations in propulsive colonic motility, as determined by assessing faecal output, taken as marker of colonic propulsive activity (Martinez & Taché 2001), up to 3 weeks after ischaemia.

Finally, persistent stiffness of the ischaemic region has been noticed in the rat small intestine (Lindestrom & Ekblad 2004), but not in the colon (Sand et al. 2008). In our study, this was only occasionally noticed and did not correlate with other morphological changes. Intestinal stiffness has been associated to an increase in the content of connective tissue, for instance during chronic colitis (Melgar et al. 2005). In our studies, the microscopical examination of Masson’s trichrome stained tissue sections did not show changes in the content of connective tissue within the ischaemic areas indicating the absence of fibrosis. A fibrotic change in the gut might lead to a reduction in tissue elasticity and therefore to a loss of compliance during distension. Results obtained show that colonic compliance at weeks 7 and 8 after the acute ischaemic event was not altered, supporting the finding that the ischaemic insult did not result in fibrosis.

The lack of positive findings, as it relates to viscerosenstivity and compliance, is unlikely to be related to technical factors or lack of sensitivity of the system used, because in similar experimental conditions we have previously been able to detect changes in colonic sensitivity or compliance both in rats and mice (Arvidsson et al. 2005; Käll et al. 2007; Martínez et al. 2007; Martinez & Melgar 2008).

In summary, we show that an acute ischaemic episode induces long-term structural changes in the colon. Main change observed was the presence of a local mast cell infiltrate in the serosal compartment and muscular layers of the ischaemic area. However, no functional changes, as it relates to colonic propulsive activity, colonic sensitivity or compliance during CRD, were observed. The present observations warrant additional studies addressing the effects of intermittent total ischaemia or chronic partial ischaemia and their relation to sensitivity as well as the potential functional role of the mast cell infiltrate observed. These studies may contribute to determine the relationship, if any, between ischaemia, mast cells and functional gastrointestinal disorders.

Acknowledgments

We gratefully acknowledge the Morphology group (AstraZeneca R&D Mölndal) for their help in the histopathological studies and the Analytical Biochemistry group (AstraZeneca R&D Mölndal) for the bioanalytical analysis.

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