The Large Intestine Rectum and Anus

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Colorectal distension (CRD) is the most widely used model of organ distension, and has been characterized in both the rat (6) and the mouse (42,43). This method reproducibly generates painful responses in both animals and humans as the result of a natural visceral stimulus, and is minimally invasive: a balloon or similar device can be inserted anally. Such techniques produce acute pain and can be combined with intracolonic treatment with chemicals that produce insult or inflammation such as acetic acid. Normally, the responses to CRD are recorded using electrodes that have been implanted in the abdominal muscle a week or so before measuring responses to distension. These electrodes allow the study of the visceromotor response in the absence of anesthesia: the electrical contractile activity of the muscles in response to a painful (or painless) distension.

Figure 3 TNBS-induced pancreatitis produces referred muscle hypersensitivity. These panels show the response frequency (percentage of positive responses) to mechanical stimulation of the abdomen with von Frey-like monofilaments. Panel A reports the responses to a 40.7 mN filament before (BL), and up to six weeks following induction of pancreatitis (*p< 0.001), while panel B shows the responses to all filaments tested three weeks after induction of pancreatitis (*p< 0.05). Although omitted here for clarity of presentation, baseline response frequencies were similar to those recorded for the vehicle and were significantly different from the TNBS treatment data for responses to filaments of 6.76 mN and greater. Abbreviation: TNBS, 2, 4, 6-trinitrobenzenesulfonic acid. Source: Redrawn from Ref. 39.

One of the postulated risk factors for irritable bowel syndrome (IBS), a key symptom of which is visceral pain, is acute transient infection, and this has been applied to animal models. Such infectious stimuli as that produced by the nematode Nippostrongylus brasiliensis or Trichinella spiralis result in hypermotility, hypersecretion, and intestinal inflammation. This is accompanied by increased visceral sensitivity and is used as a chronic model, because these effects can be seen after the inflammation has resolved, although effects may be related more directly to the jejunum rather than the colon (44). If an insult such as an infection is experienced by neonatal animals, it may induce a sensitization that can significantly affect nociceptive processing in adults, and this is the theoretical basis of animal models involving developmental stimuli. Indeed, both mechanical and chemical neonatal colonic irritation in rats have been reported to produce visceral hypersensitivity in adults (45). Furthermore, environmental changes during neonatal life can have similar effects. For example, separation of rat pups from their mothers for 180 minutes on each of postnatal days 2 to 14 results in increased EMG output in response to CRD compared to nonhandled litters (46). Similar studies show exaggeration of the immune response and long-term changes in the colonic epithelial barrier of these animals (47). These effects may be the result of increased anxiety, another stimulus that can reproduce some IBS symptoms in adult rats following partial restraint (48) and other experimental stressors (49).

Genetic models have been used with some success to examine visceral pain. For example, the Wistar Kyoto rat (a high-anxiety strain) exhibits significantly more colonic hypersensitivity than other strains, including the commonly used Sprague Dawley rat strain (50). Specific transgenic models have been produced with intestinal inflammation, results that have been used to propose the pathophysiology of inflammatory bowel disease (IBD). Such genetic models include the knockout of genes such as that for the anti-inflammatory cytokine interleu-kin-10, which results in increased cytokine production by TH1 lymphocytes and chronic enterocolitis with some IBD-like symptoms (51).

Finally, chemical stimuli are perhaps the most diverse group of stimuli. Most of these will generate a state of colonic hypersensitivity (e.g., to balloon distension) by inducing inflammation. While intraperitoneal injection of substances is unreliable (and questionably ethical) for visceral pain studies (52,53), localized intracolonic application of compounds such as acetic acid (54), antibiotics (55), butyrate (56), capsaicin (57), DSS (58), glycerol (59), mustard oil (60), TNBS (61), turpentine (62), or zymosan (63) will either directly produce pain-like behaviors (e.g., capsaicin and mustard oil) or induce inflammation. These compounds are either used to study their acute effects (recording within minutes or hours of instillation) or to produce long-lasting, persistent, visceral hypersensitivity (days or even months after instillation), depending upon the agent.

It is beyond the scope of this review to give a detailed account of each of these models; however, we shall discuss two: one inflammatory (TNBS) and one without colonic inflammation (butyrate). TNBS is used routinely in many laboratories for the study of behavioral, biochemical, pathological, and electrophysiological changes in rodents. Initially developed in the rat, the TNBS model of colitis (in which colon inflammation and symptoms develop that are frequently associated with human IBD) has since been documented in the mouse (64,65). TNBS (also known as picrylsulfonic acid), as a hapten, is not itself immunogenic, but can become so when combined with larger carrier molecules. It is therefore incapable of producing any immune reaction alone and so is coadministered with a "barrier-breaking" compound, ethanol, to produce acute colonic mucosal damage (66) and increased MPO activity (42). The MPO activity in a given tissue is a measure of neutrophil infiltration, and therefore an index of inflammation, supported by both macro- and microscopic histology. This combination therefore enables both entry and immunogenicity of TNBS instilled into the colon, leading to the introduction of intestinal bacterial flora into the colon wall and macrophage activation. These macrophages will secrete a number of cytokines that result in a TH1-dominant cascade. Alterations in the mucosal serotonergic system are also present. For example, downregulation of the 5-HT reuptake transporter increases the amount of 5-HT available to depolarize the peripheral terminals of both intrinsic and extrinsic primary afferent neurons, probably by activation of 5-HT3 receptors. This is by no means an exhaustive account of proposed mechanisms; further details can be found in the literature (67,68).

Previous studies document that TNBS produces colon inflammation and colon hyper-sensitivity in rats (69-71), and we are now assessing the behavioral characteristics of this model in mice (Robinson and Gebhart, unpublished). TNBS colitis in mice is normally initiated by the intracolonic instillation, via the rectum, of the hapten (in our experience, mortality is negligible at doses up to 10 mg mL_1) dissolved in ethanol (typically 25-50%) under light anesthesia. Alternatively, the mixture has been injected into the colon during laparotomy (72). Typically, colonic inflammation and elevated MPO activity are seen a few days following instillation of TNBS. Animals can, however, exhibit colonic hypersensitivity after the resolution of this inflammation, as measured by visceromotor response to distension (Fig. 4). This may therefore be an interesting model for the study of postinflammatory hyperalgesia.

A new, noninflammatory model (no mucosal damage or detectable MPO activity was observed) of visceral hypersensitivity was recently described by Bourdu et al. (56). This model uses twice-daily intracolonic instillation of butyrate solution (1 mL of 8-1000 mM; 200 mM was selected as the most appropriate concentration) over three days. Treated rats show a significantly decreased threshold for CRD-evoked pain behaviors and greater pain score (rated 0-5) than rats treated intracolonically with saline. Referred cutaneous hyperalgesia, measured using von Frey filaments to probe the lumbar abdomen, was present up to 12 days after

Figure 4 TNBS can produce colonic hypersensitivity in the mouse. The upper two panels show electromyographic recordings [visceromotor response (VMR)] in response to a 20-second colorectal distension (black bar) before (A) and seven days after (B) intracolonic installation of TNBS. Panel C shows the VMR recorded at four different distending pressures (15,30,45, and 60 mmHg for 10 seconds) before (o) and after (•) TNBS, revealing increased responses after colon inflammation (i.e., colonic hypersensitivity). Data are standardized to the 60 mmHg distending pressure measured before TNBS instillation. Data in all panels are taken from the same animal. Source: D.R. Robinson and G.F. Gebhart, unpublished data.

Figure 4 TNBS can produce colonic hypersensitivity in the mouse. The upper two panels show electromyographic recordings [visceromotor response (VMR)] in response to a 20-second colorectal distension (black bar) before (A) and seven days after (B) intracolonic installation of TNBS. Panel C shows the VMR recorded at four different distending pressures (15,30,45, and 60 mmHg for 10 seconds) before (o) and after (•) TNBS, revealing increased responses after colon inflammation (i.e., colonic hypersensitivity). Data are standardized to the 60 mmHg distending pressure measured before TNBS instillation. Data in all panels are taken from the same animal. Source: D.R. Robinson and G.F. Gebhart, unpublished data.

butyrate treatment (200 mM concentration). All these effects were significantly more pronounced in female rats (Fig. 5), a feature of this model that, in conjunction with the lack of visible intestinal inflammation, referred nonvisceral hypersensitivity, and colorectal hypersen-sitivity, may make it particularly relevant in the study of IBS. Intracolonic administration of butyrate has also been reported to prolong TNBS-induced hypersensitivity in rats (73).

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