Visceral and Somatic Hypersensitivity in TNBS-Induced Colitis in Rats
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- Zhou, Q., Price, D.D., Caudle, R.M. et al. Dig Dis Sci (2008) 53: 429. doi:10.1007/s10620-007-9881-6
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Inflammation of visceral structures in rats has been shown to produce visceral/somatic hyperalgesia. Our objectives were to determine if trinitrobenzene sulfonic acid (TNBS) induced colitis in rats leads to visceral/somatic hypersensitivity. Male Sprague-Dawley rats (200–250 g) were treated with 20 mg of TNBS in 50% ethanol (n = 40) or an equivalent volume of ethanol (n = 40) or saline (n = 25) via the colon. Colonic distension, Von Frey, Hargreaves, and tail reflex tests were used to evaluate for visceral, mechanical, and thermal sensitivity. The rats demonstrated visceral hypersensitivity at 2–28 days following TNBS administration (P < 0.0001). The ethanol-treated rats also demonstrated visceral hypersensitivity that resolved after day 14. TNBS-treated rats demonstrated somatic hypersensitivity at days 14–28 (P < 0.0001) in response to somatic stimuli of the hind paw. TNBS colitis is associated with visceral and somatic hypersensitivity in areas of somatotopic overlap. This model of colitis should allow further investigation into the mechanisms of visceral and somatic hypersensitivity.
KeywordsTrinitrobenzene sulfonic acid induced colitisVisceral hypersensitivitySomatic hypersensitivityViscerosomatic convergenceIrritable bowel syndrome
Visceral pain is a common and debilitating disorder. Many common gastrointestinal disorders such as irritable bowel syndrome are characterized by chronic visceral pain. Despite the fact that visceral pain is a common clinical finding, the pathophysiology is still unclear. The mechanism(s) of visceral pain are not as well studied as those which contribute to somatic pain. Patients with visceral pain often exhibit a wide variety of somatic symptoms, including back pain, migraine headaches, and muscle pain. These symptoms may be consistent with central sensitization, referral of visceral pain to somatic tissues outside the area of immediate referral [1–5], or neural cross-talk in which afferent activation of one visceral structure influences efferent output in other structures and organs and is mediated by convergence of sensory pathways in the spinal cord [6–10].
Somatic hypersensitivity may also occur in clinical conditions where visceral pain is directly referred to a corresponding area of somatic tissue. Referred visceral hypersensitivity is most often related to visceral pain being directly referred to the corresponding area of the abdominal wall [2, 11, 12]. Animal models have been developed to evaluate referred hypersensitivity following a nociceptive visceral stimulus. Uterine inflammation in rats has been shown to increase sensitivity to stimulation of flank muscles . Others have used bladder inflammation or urethral calculus to evaluate hypersensitivity of the paws and tail [11, 14, 15]. However, to date, no study has demonstrated both visceral and somatic hypersensitivity following colonic inflammation.
Our current study used an animal model of visceral pain to evaluate if trinitrobenzene sulfonic acid (TNBS) induced colitis leads to visceral and somatic hypersensitivity. We monitored the behavioral development in rats at multiple time points following the inflammatory colonic stimulus. We tested visceral and somatic hypersensitivity at 2, 7, 14, 21, and 28 days following TNBS administration. Our overall objective in this study was to determine if visceral pain induced by acute colitis leads to peripheral somatic hypersensitivity. To accomplish this, we performed colorectal distension as well as mechanical and thermal cutaneous stimulation to test visceral and somatic hypersensitivity following TNBS administration. Our hypothesis was that TNBS-induced colitis produces both visceral hypersensitivity of the colon and somatic hypersensitivity in the hind paws and tail.
Materials and methods
A total of 105 male adult Sprague-Dawley rats weighing 200–250 g were used in this experiment. The rats were housed in pairs under constant temperature and humidity with 12-h light–dark cycles, and were given free access to food and water. Administration of TNBS with 50% ethanol was used to produce colonic inflammation . Prior to administration of TNBS in the colon, the animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (50–90 mg/kg). With a 5–6-cm 24-gauge catheter, 20 mg (per rat) of TNBS (Sigma Chemical Co.) in 50% ethanol (total volume, 0.4 ml), was instilled into the lumen of the colon 3–4 cm proximal to the anus (n = 40). An equivalent volume of saline (n = 25) or ethanol (n = 40) was administered into control rats or vehicle rats, respectively. Rats were kept in a vertical position for several minutes to avoid leakage of the instilled intracolonic solutions. Rats were monitored daily for changes in body weight, body condition, physical appearance, and behavior following treatment. No adverse effects were observed in any of the rats.
Somatic and visceral pain testing were performed 2, 7, 14, 21, and 28 days following administration of TNBS, ethanol, or saline under blinded conditions and the order of testing was counterbalanced across groups. Behavioral testing was done following a 12-h fast. The rats were euthanized after all behavior tests had been completed and the colon was removed for histopathological study. All procedures were approved by the North Florida/South Georgia Veterans Health System Institutional Animal Care & Use Committee.
Visceral pain testing
A balloon (3 cm-long, 1.5-cm maximum diameter) made of polyethylene was secured to tubing attached to an automated distension device (G & J Electronic, Toronto, Canada) and was used to perform colonic distension. The balloon was lubricated and placed into the rat’s distal colon so that the tip of the balloon was 1 cm from the anus. The rats were allowed 10 min to acclimatize before behavior-testing began. With use of an an automated distension device (G & J Electronic, Toronto, Canada) the rats were restrained in a plastic containment device and received phasic distension (0–80 mmHg in 5-mmHg ascending increments) of the colon until the first contraction of the testicles, tail, or abdominal musculature occurred, which was defined as the visceral pain threshold indicative of the first nociceptive response as previously described [17, 40]. The colonic distensions were repeated four times with 5–10 min interstimulus intervals and the mean pressures at the nociceptive threshold were recorded for each rat.
Somatic pain testing
Mechanical hypersensitivity was measured using an automatic Von Frey device (dynamic plantar aesthesiometer; electronic unit/filaments and calibration weights, from Ugo Basile Biological Research Apparatus, Italy). Rats were placed on a wire mesh floor in a plastic enclosure. A computer-driven filament was then extended up through the mesh floor and exerted an increasing amount of pressure (maximum 50 g) onto the rat’s hind paw. The force in grams required until the rat withdrew its hind paw was defined as the mechanical pain threshold. Both hind paws were tested in each rat. The stimulus was repeated four times following a 5 min interstimulus interval and the mean was calculated for each rat’s hind paw.
A thermal stimulus was delivered using the Hargreave technique (7371 plantar test device from Ugo Basile Biological Research Apparatus, Italy) . Rats were placed in a plastic enclosure on a Plexiglas surface and the heat stimulus was applied underneath the plastic chamber. The time in seconds (latency) until the rat withdrew its hind paw was recorded for each rat. Both hind paws were tested in each rat. The stimulus was repeated four times following a 5 min interstimulus interval and the mean was calculated for each rat’s hind paw.
Tail flick reflex
The tail reflex test was performed by immersing the rat’s tail 6–7 cm in 50°C water. The length of time in seconds (latency) until the rat withdrew its tail was measured. The stimulus was repeated four times following a 5 min interstimulus interval and the mean was calculated for each rat.
Histopatholgical evaluation of colonic tissues
Immediately following the somatic and visceral pain testing, all rats (eight TNBS-treated, eight ethanol-treated, and five saline-treated rats per time point of 2, 7, 14, 21, and 28 days; total 40 TNBS-treated, 40 ethanol-treated, and 25 saline-treated) were euthanized using sodium pentobarbital (120 mg/kg, intraperitoneally). Following euthanasia, 3 cm of the descending colon was removed and processed for histopathological evaluation. The tissue was fixed in formalin and processed using standard techniques for hematoxylin and eosin staining. The severity of the lesions in the colon and mucosa was graded using a system previously described . The grades of colitis included the following:mild (+1) infiltration of a limited number of neutrophils in the lamina propria with minimal interstitial edema; moderate (+2) infiltration of a moderate number of neutrophils in the lamina propria with moderate interstitial edema; severe (+3) diffuse infiltration of neutrophils in the lamina propria with severe interstitial edema.
All statistics were run using Prism version 6. Two-way analysis of variance (ANOVA) following the Bonferroni posttest was used to analyze all behavioral test data. Values are expressed as means ± the standard deviation.
All rats treated with TNBS had colitis (+2 to +3) characterized by diffuse infiltration of neutrophils in the lamina propia with severe interstitial edema as previously described . The colitis was present at all of the time points: 2, 7, 14, 21, and 28 days following TNBS administration each group of rats were euthanized. The ethanol-treated colons appeared to colitis (+1 to +2) characterized by infiltration of a limited or moderate number of neutrophils in the lamina propria with minimal or moderate interstitial edema at days 2 and 7 following ethanol administration. The saline-treated colons appeared normal without any pathophysiologic changes.
Rats were observed twice daily for 30–60 min. TNBS-treated rats demonstrated abnormal behavior previously described in acute pain models, including repeated licking of the lower abdomen, testicles, and hind paw, and a hunched position in comparison with control rats [5, 13, 17]. This behavior started several hours after TNBS administration and lasted for several days. This abnormal behavior has been previously described in other experimental models of visceral inflammation [17, 40]. In comparison with saline-treated control rats, TNBS-treated rats had an intermittent runny, loose stool up to day 28 after treatment and exhibited a slight decrease in body weight in comparison with control rats. The difference in body weight was not significant. The ethanol-treated rats demonstrated minimal behavioral changes after ethanol administration, such as repeated licking of the lower abdomen and the area of the testicles. These types of behavior subsided 5–7 days later.
The Von Frey test on hind paws revealed hypersensitivity at 14, 21, and 28 days following TNBS administration (Fig. 2). Two-way ANOVA indicated P < 0.0001. Bonferroni’s posttest revealed P < 0.001 for rats 14, 21, and 28 days after administration of TNBS compared with saline-treated and ethanol-treated rats.
Thermal nociceptive stimululation was applied to hind paws using Hargreaves’ method . Hypersensitivity as indicated by a reduced latency in response to thermal stimulation was found at 14, 21, and 28 days after TNBS injection (Fig. 3). Two-way ANOVA indicated P < 0.0001. Bonferroni’s posttest revealed P < 0.001 for rats 14, 21, and 28 days after TNBS administration when compared with saline-treated controls and ethanol-treated animals.
Tail flick reflex
The tail reflex test response to 50°C water stimulus indicated hypersensitivity at 21 and 28 days following TNBS injection (Fig. 4). Two-way ANOVA indicated P < 0.0001. Bonferroni’s posttest indicated P < 0.001 for rats 21 and 28 days following TNBS administration when compared with saline-treated controls and ethanol-treated rats.
The results of our current study suggest that TNBS-induced colitis in rats produces both visceral and somatic hypersensitivity. The presence of somatic hypersensitivity is a new finding as previous studies using TNBS-induced colitis in rats have only evaluated colonic hypersensitivity [20–24]. There have been several previous studies that have used visceral cavities (i.e., bladder, uterus) other than the colon to evaluate somatic hypersensitivity [5, 14, 15, 25–30]. Our current study revealed increased somatic hypersensitivity in the TNBS-induced colitis model. This is in contrast to several other studies that have shown decreased somatic sensitivity in areas outside the region of referred pain following visceral stimulation [31–34]. However, unlike the present study that examined somatic sensitivity over 28 days, these previous studies examined somatic sensitivity for only brief periods after visceral stimulation (e.g., 4 h ). Thus, whereas the immediate effects of noxious visceral stimulation on somatic sensitivity may be inhibitory, the long-term effects are facilitatory and more relevant for persistent visceral pain conditions.
From a mechanistic perspective it is interesting that hypersensitivities to different stimuli developed at different time points. For example, visceral hypersensitivity developed at 2 days, mechanical and thermal hypersensitivity to radiant heat developed at 14 days, and hypersensitivity on the tail reflex test developed at 21 days. Interestingly, these time points of development are the same as the development of increased expression of different types of N-methyl-d-aspartate (NMDA) receptor subunits between 14 and 28 days following TNBS administration . In contrast to visceral hypersensitivity that is based on immediately inflamed tissues, delays in somatic hypersensitivity may be due to gradual spatial spread of spinal cord neuron sensitization and/or delayed upregulation of different types of glutamate receptors, including NMDA and non-NMDA receptors.
The somatic hypersensitivity present is likely to be a result of central sensitization of spinal dorsal horn neurons that receive somatovisceral convergence [6–10, 36]. An additional factor could be related to neural cross-talk in the pelvis . The persistent colitis may result in the peripheral and/or central release of some excitatory mediator that produces and maintains central sensitization, eventually leading to somatic hypersensitivity.
In our study, there was persistent colitis up to 28 days following TNBS administration. These findings differ from those of Asfaha et al.  in which the inflammation peaked at day 3 and then gradually resolved. Our findings may be different as we used Sprague-Dawley rats and not Wistar rats. The intensity and duration of inflammation may very well be strain-related. The study by Wells and Blennerhassett  used a different concentration of TNBS from our study, which makes it difficult to compare the results with our findings. Our TNBS-induced colitis data were most consistent with the findings of Ness and Gebhart . Another likely reason for the differences in the duration of inflammation is the agent used to induce colitis. Mahgoub et al.  used intracolonically administered 3% ascetic acid, which produced severe colitis 24 h after administration. Kimball et al.  used mustard oil, which caused a peak inflammation at day 3 and was resolved by day 7. Natah et al.  found that a quantitative analysis of the endothelial barrier antigen (EBA) after TNBS-induced colitis resulted in occluding expression in the frontal cortex. A significant decrease in EBA expression was shown at 2 and 7 days. However, this study was different from our current study, in which we measured somatic behavioral changes after TNBS induced colitis.
Although hypersensitivity in chronic visceral pain disorders such as irritable bowel syndrome has previously been thought to be limited to the gastrointestinal tract, these patients also exhibit a wide variety of somatic symptoms, including back pain, migraine headaches, heartburn, dyspareunia, and muscle pain, consistent with our present results. Collectively, these somatic symptoms are consistent with the possibility that patients with chronic visceral pain disorders may also suffer from central hyperalgesic dysfunction . In fact, recent investigations suggest that in both animal models and patients with chronic visceral pain there is evidence of referred somatic hypersensitivity [19, 45–48]. These findings are different from earlier studies which revealed somatic hyposensitivity in patients with chronic visceral pain disorders such as inflammatory bowel disease and irritable bowel syndrome [3, 4, 49–52]. One very plausible explanation for these different findings in humans may be due to the specific type of nociceptive stimuli used. It is possible that previous studies failed to reveal somatic hypersensitivity in outside referral areas because they did not use nociceptive stimuli that were intense or long enough to stimulate NMDA receptor mechanisms associated with sensitization and somatic hypersensitivity.
In summary, TNBS-induced colitis in the rat leads to visceral and somatic hypersensitivity. The somatic hypersensitivity present reflects tonic impulse input from the inflamed colon along with central sensitization. Further studies are needed to determine the duration of visceral and somatic hypersensitivity following resolution of TNBS-induced colitis. In addition, molecular studies of the spinal cord are needed to be done to evaluate potential mediators of colitis-induced visceral and somatic hypersensitivity.
G.N.V. is supported by a Merit Review Award (PI: GN Verne) from the Medical Research Service of the Department of Veteran Affairs and NIH Grant 1-R01-NS053090–01 (PI: GN Verne).