Irritable bowel syndrome (IBS) is a common chronic

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1 GASTROENTEROLOGY 2012;142: Brain Responses to Visceral Stimuli Reflect Visceral Sensitivity Thresholds in Patients With Irritable Bowel Syndrome MATS B. O. LARSSON,*,, KIRSTEN TILLISCH, A. D. CRAIG,*, MARIA ENGSTRÖM,, JENNIFER LABUS, # BRUCE NALIBOFF, # PETER LUNDBERG,, **, MAGNUS STRÖM,*, EMERAN A. MAYER, and SUSANNA A. WALTER*,, *Department of Clinical and Experimental Medicine/Gastroenterology, Linköping University; Center for Medical Image Science and Visualization (CMIV) Linköping University; Department of Medical and Health Sciences/Radiology, **Radiation Physics, Linköping University, Linköping, Sweden; Department of Gastroenterology, Department of Radiation Physics, University Hospital Linköping, County Council of Östergötland, Linköping, Sweden; Division of Digestive Diseases, Department of Medicine, # Department of Psychiatry, Oppenheimer Family Center for Neurobiology of Stress, David Geffen School of Medicine at UCLA, Los Angeles, California; Barrow Neurological Institute, Phoenix, Arizona BACKGROUND & AIMS: Only a fraction of patients with irritable bowel syndrome (IBS) have d perceptual sensitivity to rectal distension, indicating differences in processing and/or modulation of visceral afferent signals. We investigated the brain mechanisms of these perceptual differences. METHODS: We analyzed data from 44 women with IBS and 20 female healthy subjects (controls). IBS symptom severity was determined by a severity scoring system. Anxiety and depression symptoms were assessed using the hospital anxiety and depression score. Blood oxygen level-dependent signals were measured by functional magnetic resonance imaging during expectation and delivery of high (45 mmhg) and low (15 mmhg) intensity rectal distensions. Perception thresholds to rectal distension were determined in the scanner. Brain imaging data were compared among 18 normosensitive and 15 hypersensitive patients with IBS and 18 controls. Results were reported significant if peak P-values were.05, with family-wise error correction in regions of interest. RESULTS: The subgroups of patients with IBS were similar in age, symptom duration, psychological symptoms, and IBS symptom severity. Although brain responses to distension were similar between normosensitive patients and controls, hypersensitive patients with IBS had greater activation of insula and reduced deactivation in pregenual anterior cingulate cortex during noxious rectal distensions, compared to controls and normosensitive patients with IBS. During expectation of rectal distension, normosensitive patients with IBS had more activation in right hippocampus than controls. CON- CLUSIONS: Despite similarities in symptoms, hyperand normosensitive patients with IBS differ in cerebral responses to standardized rectal distensions and their expectation, consistent with differences in ascending visceral afferent input. Keywords: Barostat; Visceral Sensitivity; Functional MRI; Anticipation. Irritable bowel syndrome (IBS) is a common chronic abdominal pain disorder characterized by recurrent abdominal pain associated with altered bowel habits, 1 and a high prevalence of d anxiety. 2 The pathophysiology of IBS is incompletely understood; altered brain gut interactions are thought to play an important role in the cardinal symptoms, particularly abdominal pain. 3,4 In the absence of generally agreed upon biomarkers, the diagnosis relies on symptom reports and exclusion of organic disease. 1 Chronic visceral pain can be considered as a complex experience that signals an adverse condition in the gastrointestinal (GI) tract, requiring a behavioral response. 5 However, this experience is highly subjective and is modulated by several different emotional and cognitive factors, including anxiety, attention, and expectation. 6 Chronic pain is therefore not linearly related to peripheral sensory input. 7 For example, hypervigilance toward GI sensations, anxiety, and symptomrelated fears are common in patients with functional GI disorder and play an important role in perceived symptom severity. 8,9 In IBS patients it is not known whether the reported abdominal pain reflects d afferent input from the gut to the brain, or central alterations in the signals from the gut (central pain amplification), or both. 10,11 Multiple mechanisms for central pain modulation have been described in animal models, and evidence for altered engagement of some of these, including diffuse noxious inhibitory controls, have been described in IBS patients. 12 Recently, Berman et al 13 showed that in IBS patients alterations during cued expectation of an aversive rectal stimulus correlated with subsequent brain responses to the delivered stimulus in the bilateral pregenual anterior cingulate cortex Abbreviations used in this paper: BOLD, blood oxygen level dependent; DLPFC, dorsolateral prefrontal cortex; fmri, functional magnetic resonance imaging; GI, gastrointestinal; HAD, Hospital Anxiety and Depression scale; HC, healthy control; HIPP, hippocampus; IBS, irritable bowel syndrome; INS, insula; MRI, magnetic resonance imaging; pacc, pregenual anterior cingulate cortex; ROI, region of interest by the AGA Institute /$36.00 doi: /j.gastro

2 464 LARSSON ET AL GASTROENTEROLOGY Vol. 142, No. 3 (pacc), a subregion involved in descending inhibitory pain modulation. Because IBS patients and healthy controls (HCs) generally differ in perception thresholds for reports of discomfort to controlled rectal balloon distension, this stimulus has been used for experimental pain provocation in numerous IBS studies Enhanced perception of visceral stimuli is considered to be an important component in the pathophysiology of IBS, 19 yet a large proportion of IBS patients have perception thresholds within the normal range, 16,20,21 even though all IBS patients present with a similar symptomatology. 22,23 Furthermore, there is a poor correlation between visceral hypersensitivity in IBS and symptom severity Results of brain imaging studies in IBS have varied or have been contradictory, perhaps owing to different study designs, but perhaps also owing to heterogenous study populations, including patients with and without visceral hypersensitivity A recent meta-analysis of published studies on brain responses to rectal distension supports the conclusion that brain responses to rectal distension differ between IBS patients and HCs. 31,32 IBS patients showed greater activation during rectal distension in brain regions concerned with emotional arousal, cognitive modulation, and interoceptive processing. 32 However, similar to the poor correlations between perception thresholds to rectal stimuli and symptom severity, there were poor correlations between changes in brain responses to rectal stimulation during repeated stimulation and associated changes in symptom severity. 9 Given that visceral hypersensitivity is considered an important feature in the pathophysiology of IBS, it is of interest to examine whether there are differences in the cerebral response to standardized visceral pain stimuli between the patients with normal visceral sensitivity and those with d visceral sensitivity. Here we refer to subjects with normal visceral sensitivity as normosensitive and subjects with d sensitivity as hypersensitive. Such differences have not been investigated in any of the published studies. In the current study, using a validated paradigm of rectal distension and expectation of such distension, we aimed to answer the following questions: (1) do brain responses to these stimuli differ between normosensitive and hypersensitive IBS patients, despite a similar symptom burden? (2) Are brain responses of normosensitive IBS patients similar to those of HCs? We used a combination of aversive and nonaversive rectal distension, as well as an expectation of pain paradigm, to differentiate topdown and bottom-up mechanisms that may play a role in the observed response patterns. Materials and Methods Subjects Forty-four right-handed women with IBS, fulfilling Rome III criteria, were included (mean age, 35.5 y; range, y). Patients were referred by general practitioners to the Linköping University Hospital in Sweden. They were evaluated by a trained gastroenterologist; standard clinical investigations were performed to exclude organic GI disease. The control group consisted of 20 healthy right-handed women (mean age, 32.2 y; range, y) recruited by advertisement. Exclusion criteria were organic GI disease; metabolic, neurologic, or psychiatric disorders; nicotine intake; centrally acting medication; pacemaker; implanted metal in the brain; and claustrophobia. Additional exclusion criterion for controls was a medical history of GI symptoms or complaints. All subjects completed the Hospital Anxiety and Depression (HAD) scale 33 and IBS patients additionally completed the IBS Severity Scoring System 34 and the Visceral Sensitivity Index. 35 Control subjects received a monetary compensation of 1000 Swedish kronor. Two HCs and 11 patients had to be excluded from the data analysis for the following reasons: incomplete data collection (n 2), balloon leakage (n 1), excess motion (n 4), major scanner artefacts (n 2), and intolerance to the procedure (n 4). Thus, the data from 18 control subjects and 33 IBS patients were analyzed. Ethics. Written and oral informed consent was obtained from all subjects. The study protocol was approved by the Regional Research Committee for Ethical issues at Linköping (Sweden). Questionnaires HAD scale. The HAD scale is a self-assessment scale that was developed for detecting states of depression and anxiety in medical outpatient settings. 33 The scale consists of 7 items each for anxiety and depression. Responses are graded on a 4-point scale. The summed scores for anxiety and depression, respectively, range from 0 to 21, with a higher score indicating more pronounced symptoms. IBS Severity Scoring System. The IBS Severity Scoring System was developed for rating IBS symptoms. 34 The scoring system incorporates 5 items: abdominal pain severity, pain frequency, bowel distension, bowel dysfunction, and quality of life/global well being. The items are scored on a visual analog scale ranging from 0 to 100 mm. The maximum achievable score is 500. Mild, moderate, and severe cases are indicated by scores of , , and greater than 300, respectively. Visceral Sensitivity Index. The Visceral Sensitivity Index consists of a 15-item scale to measure GI symptomspecific anxiety by assessing the cognitive, affective, and behavioral response to fear of GI sensations, symptoms, and the context in which these occur. 35 Ratings of present intensity and unpleasantness of GI symptoms. A scale ranging from 0 to 10 was used to determine the following: (1) intensity of present GI symptoms (low scores indicate faint intensity, and high scores indicate extremely intense symptoms), and (2) unpleasantness of subjects GI symptoms (low scores indicate neutral feelings, and high scores indicate intolerable unpleasantness). Study Protocol The temporal overlap of menses period with the experimental day was avoided for all subjects. Subjects were required to cease medications and avoid alcohol at least 24 hours before the experiment. Subjects came to the experimen-

3 March 2012 BRAIN RESPONSES TO VISCERAL STIMULUS IN IBS 465 tal site after 4 hours of fasting. The barostat balloon was installed into the rectum with the subjects in a left lateral position. The rectal balloon catheter consisted of a noncompliant polyethylene bag (maximal volume, 520 ml), which was attached to a polyethylene tube. The subjects then were placed in the magnetic resonance imaging (MRI) scanner and fitted with headphones (allowing 2-way communication), and high-resolution video MR goggles for visual stimulus presentation in the MR scanner (Resonance Technology, Inc, Los Angeles, CA). Determination of perception thresholds. After 5 minutes of rest in the scanner bed, a 10-minute resting scan was performed. Thereafter, subjects underwent the thresholding procedure to test visceral sensitivity. Intermittent phasic isobaric rectal distensions of 30 seconds duration were administered by an electronic barostat (Dual Drive Barostat, Distender series II; G&J Electronics, Inc, Toronto, Canada). After each distension the balloon was emptied. The time interval between the onset of each distension was 1 minute. Visceral sensitivity was tested by using the ascending method of limits with pressure increments of 5 mm Hg. Thirty seconds after onset of each distension the subjects were shown a 4-point scale to grade their sensation and report it through the microphone: 0, no sensation; 1, first sensation/some sensation; 2, urge to defecate; and 3, maximum tolerable distension. After determination of sensitivity thresholds functional MRI (fmri) data were collected. fmri Distension Paradigm Twenty high (45 mm Hg) and 18 low (15 mm Hg) rectal distensions with a duration of 15 seconds each were delivered in pseudorandom order. The distensions were preceded by a varied expectation period (5 8 s). In the beginning of each expectation period subjects were shown a red cue for the high distension and a blue cue for the low distension. Between the rectal distensions, 18 periods were inserted pseudorandomly during which subjects were shown grey cues with no subsequent distension (used as baseline). Subjects rated the present intensity and unpleasantness of GI symptoms before and after thresholding and at the end of the experiment. Directly after having completed the run subjects rated the last low and high stimulus, respectively (0, no sensation; 1, some sensation; 2, urgency; and 3, maximum tolerable pressure). In this article, we focus on the differences between hypersensitive and normosensitive IBS patients according to their response to expectation and delivery of standardized low and high rectal distensions. The pressure threshold for classifying patients as normosensitive or hypersensitive was defined as the lower range of the maximum tolerable distension pressure of HCs. As in several previous fmri studies of IBS patients, the experimental stimuli were equal for all subjects, regardless of the individual visceral sensitivity perception thresholds obtained using the ascending series of longer stimuli. 13,36 38 The intent of this design was to obtain a standardized peripheral visceral afferent input from the gut. The experimental protocol is illustrated in Supplementary Figure 1. fmri Data Acquisition fmri images were acquired by a Philips Achieva 1.5-T MR scanner (Philips, Amsterdam, Netherlands). For experimental design and presentation of information, Superlab Pro 4 software was used (Cedrus Corp, San Pedro, CA). Functional brain images were acquired by using a blood oxygen level dependent (BOLD) sensitive gradient echo sequence, using the following acquisition parameters: repetition time, 3 s; echo time, 40 ms; flip angle, 90 ; voxel size, 3 3 3mm 3 ; 35 slices were acquired in an interleaved mode with a 0.5-mm slice gap. Data Analysis Statistical Parametric Mapping 8 (Wellcome Trust Centre for Neuroimaging, available: spm/software/spm8/) was used for the analysis of the BOLD fmri data. Images were realigned to the first image of the time series, normalized to a standard brain atlas (Montreal Neurological Institute space) at 2 2 2mm 3, and smoothed using an 8-mm full-width half-maximum Gaussian kernel. Subjects were excluded from further analysis if the realignment parame- Table 1. ROI Center Coordinates (Center of Mass) and Volumes ROI x y z Volume, mm 3 PAG L R Thalamus L R DLPFC L ,984 R ,272 VLPFC L ,968 R ,344 HIPP L R AMYG L R amcc L ,208 R ,528 pacc L ,112 R ,344 sgacc L R Anterior INS, ventral L R Anterior INS, dorsal L R Mid-INS L R Posterior INS, ventral L R Posterior INS, dorsal L R amcc, anterior midcingulate cortex; AMYG, amygdala; HIPP, hippocampus; PAG, periaqueductal gray; sgacc, subgenual anterior cingulate cortex.

4 466 LARSSON ET AL GASTROENTEROLOGY Vol. 142, No. 3 ROI analyses were considered significant at a P value of.05 or less, family wise error corrected. Figure 1. Rectal distension sensitivity thresholds of hypersensitive and normosensitive IBS patients and HCs, obtained by the ascending method of limits before the imaging experiment, sorted by sensory level. Mean values and standard deviations are shown. Differences were calculated with a nonpaired t test. ters indicated excessive movement ( 3 mm) or the BOLD fmri images contained major artifacts. A first-level general linear model was specified for each subject and modeled the following conditions: (1) high distension (45 mm Hg), (2) low distension (15 mm Hg), (3) expectation of high distension, (4) expectation of low distension, and (5) baseline. In addition, motion parameters were included as regressors. or contrast images comparing the active conditions with baseline were calculated, thresholded at P.01 uncorrected, and entered into second-level regionof-interest (ROI) analyses. The general linear model and a ROI approach were applied to test for group differences in brain activity during the distension and expectation conditions. ROIs included regions associated with emotional arousal and regions associated with processing of visceral sensations including pain, that is: amygdala, hippocampus, thalamus, periaqueductal gray, anterior midcingulate cortex, subgenual anterior cingulate cortex, pacc, dorsolateral prefrontal cortex (DLPFC), ventrolateral prefrontal cortex, and 5 insula (INS) subregions: anterior ventral, anterior dorsal, posterior ventral, posterior dorsal, and midinsula. 32,39 ROI center coordinates and volumes are shown in Table 1. ROIs with labels are shown in Supplementary Figure 1. The ROI analyses were implemented using Statistical Parametric Mapping 8 small-volume correction. Results Clinical and Psychophysical Characterization of IBS Subgroups HCs (n 18) had a maximum tolerable rectal pressure of mm Hg (mean, range, mm Hg). Eighteen IBS patients had a maximum tolerable pressure of 40 mm Hg or higher (mean, mm Hg; range, mm Hg) and thus were considered normosensitive. Fifteen IBS patients had a maximum tolerable rectal pressure less than 40 mm Hg and were considered hypersensitive (mean, mm Hg; range, mm Hg). There was no overlap between the hypersensitive IBS group and HCs in terms of pressure thresholds. The hypersensitive group had significantly lower thresholds for first sensation and urgency than the normosensitive group and the HC group. The threshold for first sensation was a mean of mm Hg (5 15) for the hypersensitive group and mm Hg (5 25) and mm Hg (10 25) for the normosensitive IBS group and the HCs, respectively (Figure 1). The clinical data for the 2 IBS groups are shown in Table 2. The 2 groups were similar in terms of disease duration, IBS symptom severity score, anxiety and depression symptoms, and GI symptom related anxiety score (Visceral Sensitivity Index). Eleven hypersensitive and 10 normosensitive subjects had severe symptoms according to the Severity Scoring System. IBS patients as a group had significantly higher anxiety and depression scores than HCs. Hypersensitive IBS patients reported higher symptom intensity and unpleasantness ratings than the normosensitive patients (P.04) on the day of the distension paradigm, even though the difference reached statistical significance only before the thresholding procedure. Both IBS groups had significantly higher ratings of GI symptom intensity than HCs (Figure 2A). After the imaging experiment, both IBS subgroups rated the last low distension and the last high distension of the experiment significantly higher than the Table 2. Age, Symptom Duration, and Somatic and Psychological Evaluation in 33 Female IBS Patients With Normal and Increased Visceral Sensitivity Compared With Healthy Women Controls (n 18) Normosensitive IBS patients Hypersensitive IBS patients (n 18) P value a (n 15) P value b P value c Mean age, y (range) 32.5 (21 54) 32.5 (20 60) (21 60) Mean HAD anxiety score (range) 3.0 (0 11) 8.2 (0 17) (2 17) Mean HAD depression score (range) 1.3 (0 3) 3.7 (1 10) (0 8) Mean SSS intestinal (range) 319 ( ) ( ).099 Mean VSI (range) 44.3 (8 68) 44.7 (16 63).950 SSS, Severity Scoring System; VSI, Visceral Sensitivity Index. a Comparison between normosensitive IBS patients and healthy controls. b Comparison between hypersensitive IBS patients and normosensitive IBS patients. c Comparison between hypersensitive IBS patients and controls.

5 March 2012 BRAIN RESPONSES TO VISCERAL STIMULUS IN IBS 467 during expected and delivered distension are shown in Table 3. They include several INS and cingulate subregions and lateral PFC regions. Hypersensitive IBS compared with HCs. Hypersensitive IBS patients had larger BOLD signals during the high rectal distension in left posterior INS (cluster size, 64; [ ], P.04); left pacc (cluster size, 311; [ ]; P.01), and left thalamus (cluster size, 54; [ ]; P.04). Normosensitive IBS compared with HCs. Normosensitive IBS patients and HCs did not differ significantly in their BOLD response to either distension. During expectation of the high distension the normosensitive IBS group had more BOLD response than the HCs in the right HIPP ([ ]; P.01). HCs had more activation than the normosensitive IBS group during the low distension in the right anterior INS (P.02; [ ]). distributions of cerebral BOLD activation for IBS subgroups and HCs during expectation and delivery of the high distension are shown in Figure 3. Significant BOLD s and s during the expectation and delivery of the low and high distension are shown in Supplementary Tables 1 and 2. Figure 2. (A) Ratings of present intensity of GI symptoms during the experiment. A scale ranging from 0 to 10 was used to determine the intensity of present GI symptoms with a lower score indicating faint intensity and a higher score indicating extremely intense symptoms. Mean values and standard deviations are shown. Differences between groups were calculated with a nonpaired t test. (B) Ratings of the last low and high distension of the imaging experiment. Subjects were rated on a 4-point scale as follows: no sensation, 0; some sensation, 1; urgency, 2; and maximum tolerable pressure, 3. Mean and standard deviation are shown. Differences were calculated with a nonpaired t test. HCs. There was, however, no difference between the IBS subgroups (Figure 2B). Brain Responses to Distension and Expectation of Abdominal Pain As shown in Table 3, IBS patients as a group had greater BOLD signals than HCs in the left ventrolateral prefrontal cortex in response to the high distension and in the left middle INS in response to the low distension. During expectation of the high distension, IBS patients had more activation in the right anterior and middle INS and hippocampus (HIPP). There were no regions of significantly higher BOLD activation in HCs than in IBS patients. Hypersensitive IBS compared with normosensitive IBS. ROIs in which the hypersensitive IBS patients had larger BOLD signals than the normosensitive patients Discussion The key findings of the study were as follows: (1) despite similarities in symptoms, hypersensitive and normosensitive IBS patients differed substantially in their BOLD response to high-pressure rectal distensions, and to expectation of such distensions; (2) during rectal distension there were no significant differences in cerebral response between normosensitive IBS patients and HCs; and (3) during expectation of the high-pressure stimulus, normosensitive IBS patients had more activation in the right hippocampus than HCs. Group differences in brain responses during the highintensity distension were observed in primary interoceptive cortex (posterior INS [pins]), suggesting that this region is sensitized or it receives d ascending input from the dorsal horn during rectal distensions. The fact that d activity in the midinsular cortex and thalamus also was seen during the expectation of an aversive sensation, without any acute peripheral stimulus, suggests that differences in top-down modulatory mechanisms (eg, descending endogenous pain modulation systems), also may play an important role in the observed abnormalities. The group comparison between HCs and all IBS patients confirms previous findings by showing greater INS responses (both anterior and middle subregions) during pain expectation in IBS patients 13,30 and greater activation of the ventrolateral PFC to aversive rectal distensions. 29,30,38,40,41 The fact that no group differences were observed during distension in other regions of the homeostatic afferent network (previously referred to as pain matrix; eg, INS, MCC, and thalamus)

6 468 LARSSON ET AL GASTROENTEROLOGY Vol. 142, No. 3 Table 3. Brain Regions (ROIs) in Which the BOLD Response Was Greater in all IBS Patients (n 33) Than in HCs (n 18) and in Hypersensitive IBS Patients (n 15) Than in Normosensitive IBS Patients (n 18) ROI size Peak p(fwe) X-coordinate Y-coordinate Z-coordinate IBS controls 45-mm Hg distension VLPFC L mm Hg distension Insula, mid L Expectation of 45-mm Hg distension Insula, anterior ventral R Insula, mid R Hippocampus R Expectation of 15-mm Hg distension No significant findings Hypersensitive normosensitive 45-mm Hg distension Insula posterior L DLPFC R amcc R pacc L pacc R mm Hg distension No significant findings Expectation of 45-mm Hg distension Insula, posterior R Thalamus R Expectation of 15-mm Hg distension Insula, anterior ventral R amcc, anterior midcingulate cortex; VLPFC, ventrolateral prefrontal cortex. may be attributed to the fact that this difference was contributed only by the hypersensitive, but not by the normosensitive, group, and therefore not detectable in the combined group (Table 3 and discussion later). The findings are discussed in terms of the 2 primary hypotheses of the study. Do Brain Responses to Aversive Rectal Stimuli and Their Expectation Differ Between Normosensitive and Hypersensitive IBS Patients? Perceptual responses. The 2 IBS subgroups, defined by their subjective response to an acute rectal stimulus, did not differ in their subjective ratings of IBS symptom severity or symptoms of anxiety and depression. These results confirm previous reports showing a poor correlation between perceptual responses to controlled rectal distension and subjective IBS symptoms 27 and emphasize that factors other than perception of an acute visceral stimulus contribute to patients reports of clinical symptoms. However, the mechanisms underlying this d sensitivity in hypersensitive patients are incompletely understood. The hypersensitive group, as evaluated by the ascending method of limits, had lower perception thresholds for first sensation and for first sensation of urgency compared with either normosensitive IBS patients or HCs. This leftward shift of the stimulus response curve in hypersensitive IBS patients has been described earlier 42 and could be caused by amplification of the experimental sensory inflow somewhere along the afferent pelvic visceral pathway: (1) at the gut receptor level, (2) in the dorsal horn of the spinal cord, (3) in the thalamus, or (4) in the primary interoceptive cortex (posterior INS), the interoceptive association cortex, or the middle and

7 March 2012 BRAIN RESPONSES TO VISCERAL STIMULUS IN IBS 469 Figure 3. BOLD response ( 0.01) within the ROIs in HCs (n 18), normosensitive IBS patients (n 18), and hypersensitive IBS patients (n 15) during (a) the expectation of high rectal distension (45 mm Hg), and (b) high rectal distension (45 mm Hg). Red, d BOLD signal; blue, d BOLD signal. HYP, hypersensitive IBS; NORM, normosensitive IBS. anterior INS. Alternatively, a leftward shift (with or without a reduction in threshold) could be caused by abnormal engagement of descending inhibitory mechanisms 29,43 or by greater activation of endogenous facilitatory systems in the hypersensitive IBS group. Both of the latter mechanisms could result in d excitability of dorsal horn neurons, amplifying ascending afferent input to the brain. 44 As previously pointed out, the ascending method of limits paradigm maximizes selective attention to threat mechanisms, and it has been suggested that d ratings may be more reflective of hypervigilance and selective attention to perceived discomfort than sensory hypersensitivity. 45 During the high-intensity rectal distension the hypersensitive group had more activation in the posterior INS and anterior midcingulate cortex, key regions of a brain network consistently activated during interoceptive stimuli, including noxious stimuli (homeostatic afferent network). 46 Furthermore, they had a reduced deactivation in the pacc compared with HCs and normosensitive IBS, consistent with alterations in the engagement of descending pain modulation systems. The pacc is considered to be the primary cortical region in the descending pain inhibition system, 47 with a high concentration of opioid receptors. 48 In most reported studies evaluating the endogenous descending pain inhibition system the peripheral stimulations were calibrated individually 47,49,50 and the present results might have been different if we had individually adjusted the high stimulus level to reflect distension tolerance. However, according to the results of the pressure thresholds obtained with the ascending method of limits, the range between the ratings of first sensation of urgency and maximum tolerable pressure was proportionally much larger for the normosensitive IBS patients and HCs than for the hypersensitive IBS group. This finding could indicate that the HCs as well as to some degree the normosensitive patients may mobilize mechanisms that helped them to tolerate the increasing visceral discomfort, allowing them to accept further pressure increments, whereas the hypersensitive IBS patients may lack this ability. This concept is consistent with our observation that hypersensitive IBS patients had more DLPFC activation than normosensitive patients. It previously has been shown that the expectation of pain relief is mediated by regions within both right and left DLPFC, 47,51 and that the bilateral DLPFC was activated during the task to suppress the feeling of pain while enduring a tonic painful heat stimulation. 49 Based on these findings, one may speculate that the hypersensitive IBS patients engage this prefrontal mechanism more than normosensitive patients to reduce the aversive experience, but that the mechanism does not appear to be functioning appropriately. In the present study a visual cue predicted the experimental stimuli. It has earlier been shown that expectation of a noxious stimulus can modulate the processing of a subsequent aversive stimulus in the brain. 13,52 Engagement of ains, ACC, and thalamus have been observed during cued expectation of a somatic pain stimulus. 53 IBS patients have been shown to engage ains to a greater

8 470 LARSSON ET AL GASTROENTEROLOGY Vol. 142, No. 3 extent during pain expectation 13 and this has been interpreted as a possible prediction bias related to the expected pain intensity. Confirming this hypothesis, hypersensitive IBS patients, compared with the normosensitive IBS group, had more activation of the right ains during the expectation of a nonaversive stimulus. During the expectation of the high-pressure distension, greater activation of different INS subregions, for example, the posterior/ middle INS, as well as thalamus was observed. The greater activation of these homeostatic afferent regions is consistent with inappropriate engagement of descending pain facilitatory mechanisms triggered by the prediction of an aversive stimulus. Are Brain Responses of Normosensitive IBS Patients Similar to Those of HC Patients? No statistically significant differences between normosensitive IBS patients and HCs were observed in brain responses to rectal distension despite differences in HAD scores, suggesting that anxiety and depression symptoms were not a major modulator of brain responses to rectal distension. However, during the expectation of the high stimulus, normosensitive IBS patients had more activation in the right hippocampus than HCs. Bingel et al 54 recently showed that negative pain treatment expectancy was associated with d activity in the HIPP, a brain region implicated in the anxiety-driven exacerbation of pain. 55 In accordance with this, the present findings could be explained by enhanced negative expectations in the IBS patients compared with HCs, probably owing to their generally d anxiety levels compared with HCs but not because of a greater pain experience by the subsequent distension. The lack of influence of anxiety symptoms on visceral sensitivity is surprising in view of several reports that psychological factors have an impact on pain perception, pain reporting, and visceral sensitivity thresholds in IBS patients. 8,45,56,57 Similarly, it previously has been reported that differences in brain responses to a visceral stimulus between normosensitive IBS patients and HCs were no longer seen when controlling for anxiety and depression scores as confounding variables. 58 Conclusions Despite similarities in IBS symptoms, female IBS patients differentiated by normal or high perceptual sensitivity to acute rectal distension showed significantly different brain responses to both the actual stimulus and to its expectation. When viewed together, the findings are most consistent with the hypothesis that altered engagement of descending pain modulation systems s the excitability of the dorsal horn, resulting in d ascending input to brain regions processing interoceptive input. Brain responses were not affected significantly by general or symptomrelated anxiety, thus cognitive factors, such as prediction error, could be responsible for these differences. However, our findings cannot rule out alternative mechanisms, such as peripheral sensitization. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Longstreth GF, Thompson WG, Chey WD, et al. Functional bowel disorders. Gastroenterology 2006;130: Svedlund J, Sjodin I, Dotevall G, et al. Upper gastrointestinal and mental symptoms in the irritable bowel syndrome. Scand J Gastroenterol 1985;20: Mayer EA, Naliboff BD, Chang L, et al. V. 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10 472 LARSSON ET AL GASTROENTEROLOGY Vol. 142, No. 3 Received July 20, Accepted November 2, Reprint requests Address requests for reprints to: Susanna Walter, MD, PhD, Endokrin-Magtarmmedicinska Kliniken, Mag-tarm Mottagningen, Universitetssjukhuset Linköping, Linköping, Sweden. susanna.walter@lio.se; fax: Conflicts of interest The authors disclose no conflicts. Funding Supported by the County Council of Östergötland, Sweden; Lions Forskningsfond för Folksjukdomar; Bengt Ihresfond, Svenska Läkaresällskapet; Magnus Bergvall fond; and National Institutes of Health grants DK 64531, DK 48351, and K23 DK73451.

11 March 2012 BRAIN RESPONSES TO VISCERAL STIMULUS IN IBS 472.e1 Supplemental Figure 1. The time-course of the imaging experiment is shown in the graph. Subjects rated their sensory thresholds during phase C: 0, no sensation; 1, first sensation/some sensation; 2, urge to defecate; and 3, maximum tolerable distension.

12 Supplementary Table 1. Increase and Decrease of BOLD Activity in Hypersensitive IBS Patients, Normosensitive IBS Patients, and HCs During High and Low Rectal Distensions 45-mm Hg distension Hypersensitive patients Normosensitive patients Healthy controls size Peak p(fwe) X Y Z 45-mm Hg distension size Peak p(fwe) X Y Z 45-mmHg distension size Peak p(fwe) X Y Z Insula anterior dorsal R Insula anterior dorsal R Insula anterior dorsal L Insula anterior ventral R Insula mid-l Insula anterior dorsal R Insula mid-l Insula mid-r Insula anterior ventral R Insula mid-r Amygdala R Insula mid-l Insula post dorsal L Hippocampus L Insula mid-r Insula post dorsal R Thalamus L Insula post ventral L Insula post ventral R Thalamus L mm Hg distension 45-mm Hg distension 45-mm Hg distension Amygdala L DLPFC L Insula anterior ventral R Hippocampus R DLPFC L DLPFC L DLPFC R DLPFC R amcc L DLPFC R pacc L VLPFC R pacc L amcc L pacc R pacc L pacc R pacc R sgacc R sgacc L sgacc L sgacc R sgacc R mm Hg distension 15-mm Hg distension 15-mm Hg distension Insula anterior ventral R Insula mid-r Insula post dorsal L Insula post dorsal R Insula post ventral L Thalamus L mm Hg distension 15-mm Hg distension 15-mm Hg distension DLPFC R Insula anterior dorsal R DLPFC R Amygdala L Insula anterior ventral R DLPFC R DLPFC R amcc L amcc, anterior midcingulate cortex; sgacc, subgenual anterior cingulate cortex; VLPFC, ventrolateral prefrontal cortex. 472.e2 LARSSON ET AL GASTROENTEROLOGY Vol. 142, No. 3

13 Supplementary Table 2. Increase and Decrease of BOLD Activity in Hypersensitive IBS Patients, Normosensitive IBS Patients, and HCs During the Expectation of High and Low Rectal Distensions Expectation 45-mm Hg Hypersensitive patients Normosensitive patients Healthy controls size Peak p(fwe) X Y Z Expectation 45-mm Hg size Peak p(fwe) X Y Z Expectation 45-mm Hg size Peak p(fwe) X Y Z Insula anterior dorsal L Insula anterior dorsal L Insula anterior dorsal R Insula anterior dorsal R Insula anterior dorsal R Insula anterior ventral R Insula anterior ventral L Insula anterior ventral L Insula post dorsal L Insula anterior ventral R Insula anterior ventral R Insula post ventral L Insula mid-l Insula mid-l VLPFC R Insula mid-r Insula mid-r Insula post dorsal L Insula post dorsal L Insula post ventral L Hippocampus L Insula post dorsal R Insula post ventral R Hippocampus R Insula post ventral L VLPFC L Thalamus R Insula post ventral R amcc L Hippocampus L VLPFC L Insula ant ventral L VLPFC R Amygdala L amcc L Amygdala R amcc R Hippocampus R Amygdala L Amygdala L Hippocampus L Hippocampus L Hippocampus R Hippocampus R Thalamus L Thalamus R Expectation 45-mm Hg Expectation 45-mm Hg Expectation 45-mm Hg pacc L Expectation 15-mm Hg equivk Expectation 15-mm Hg equivk Expectation 15- mm Hg equivk Insula anterior ventral R Insula post dorsal L Insula mid-r Insula post ventral L VLPFC R Thalamus R Expectation 15-mm Hg amcc, anterior midcingulate cortex; FWE, family wise error; VLPFC, ventrolateral prefrontal cortex. Expectation 15-mm Hg Insula ant dorsal R DLPFC L DLPFC R amcc L Expectation 15-mm Hg March 2012 BRAIN RESPONSES TO VISCERAL STIMULUS IN IBS 472.e3