The study cohort included 68 IBS patients and 68 HCs; all participants were right-handed and gave written informed consent to the procedures approved by the local Medical Research Ethics Committee of Jinling Hospital in accordance with the Helsinki Declaration. All the participants were volunteers. All patients were recruited from the Digestive Disease Clinic of our hospital and were clinically diagnosed with IBS by a gastroenterologist with expertise in functional gastrointestinal disorders based on the Rome III criteria (Drossman, 2006) and the healthy controls were collected from the local communities. The diagnostic criteria included recurrent abdominal pain or discomfort associated with two or more of the following: relief/improvement by defecation, onset related to a change in stool frequency, and onset related to a change in stool appearance.

Subjects were excluded if they reported a history of gastrointestinal surgery, psychiatric illnesses or substance abuse, treatment with any centrally acting medications such as selective serotonin reuptake inhibitors, aspirin or non-steroidal anti-inflammatory drugs for over 2 weeks before enrollment, major medical or neurological conditions and head motion of more than 1.0 mm in translation or 1.0° in rotation during magnetic resonance imaging (MRI). Two IBS patients and one healthy subject were excluded for excessive head motion, and one IBS patient was excluded for falling asleep in the MR scanner during scanning according to his self-reporting after scanning. The remaining 65 IBS patients (49 men, 16 women, mean age: 34.00 ± 11.82 years) and 67 HCs (51 men, 16 women, mean age: 31.21 ± 10.70 years) were included in the final analysis and were matched for age, sex and education level.

Questionnaires were completed before MR scanning for all participants, including Mini-Mental State Examination (MMSE; Folstein et al., 1983), Montreal Cognitive Assessment (MoCA; Nasreddine et al., 2005) and Zung Self-Rating Anxiety and Depression Scales (SAS; Zung, 1971) and SDS (Zung et al., 1965), respectively). IBS patients were also assessed by the IBS-Symptom Severity Score (IBS-SSS; Francis et al., 1997), IBS-QOL score (Patrick et al., 1998) and the visual analog scale (VAS, 0–100 points with the descriptors “no pain sensation” at 0 and “the most intense pain sensation imaginable” at 100; Price et al., 1994).

Subjects were scanned using a 3 Tesla MR instrument (TIM Trio, Siemens Medical Solutions, Erlangen, Germany). Foam padding was used to minimize head motion for all subjects, who were instructed to rest with their eyes closed, not think of anything in particular, and not fall asleep. First, high-resolution three dimensional T₁-weighted structural images were acquired in sagittal orientation using a magnetization-prepared rapid acquisition gradient-echo sequence (repetition time/echo time [TE] = 2300 ms/2.98 ms, flip angle = 9°, field of view (FOV) = 256 mm² × 256 mm², matrix size = 256 × 256, slice thickness = 1 mm, 191 slices in the sagittal orientation). Second, resting-state fMRI (rs-fMRI) data were obtained using a single-shot, gradient-recalled echo-planar imaging sequence (TR/TE = 2000 ms/30 ms, FOV = 240 mm² × 240 mm², flip angle = 90°, matrix = 64 × 64, voxel size = 3.75 mm³ × 3.75 mm³ × 4 mm³, 30 axial slices aligned along the anterior-posterior commissure, 250 volumes). Then, diffusion tensor images were obtained using a spin echo-based echo planar imaging sequence in contiguous axial planes, including 20 volumes with diffusion gradients applied along 20 non-collinear directions (b = 1000 s/mm²) and one volume without diffusion weighting (b = 0 s/mm²). Each volume consisted of 30 contiguous axial slices covering the whole brain (TR/TE = 4100 ms/93 ms, FOV = 240 mm² × 240 mm², matrix = 128 × 128, voxel size = 1.8 mm³ × 1.8 mm³ × 4 mm³).

For each group, a random-effects one-sample t test was performed with SPM8⁷ for individual VMHC maps. Significant clusters were identified using the joint expected probability distribution (Poline et al., 1997) with height (P < 0.005) and extent (P < 0.05) thresholds corrected at the whole-brain level. To assess differences of VMHC between groups, a random two-sample t test was then performed, while eliminating the effects of age, sex and educational level by regression. Significance thresholds were set at a corrected P < 0.05, with the joint expected probability distribution as done in the abovementioned one-sample t test. Path length, tract count and mean FA of the fiber connecting the bilateral ROIs were compared between groups by a two-sample t test using SPSS v16.0 (SPSS Inc., Chicago, IL, USA), which was considered significant at P < 0.05. The comparison between the IBS and control groups was also performed by including anxiety and depression as covariates (Zhou et al., 2013) to evaluate the effect of anxiety and depression on interhemispheric coordination during two-sample t test. Since the SAS and SDS scores manifested a high correlation here (patient group: Spearman rho = 0.77, P < 0.001; control group: Spearman rho = 0.69, P < 0.001), they were taken together, rather than separately, as covariates to evaluate the psychosocial effect on brain connectivity as done in previous studies in IBS (Zhou et al., 2013; Qi et al., 2015).

To investigate the relationship between abnormal interhemispheric connectivity and clinical indications of IBS, any functional and structural connectivity that differed significantly between IBS patients and HCs was extracted and correlated with IBS-SSS, IBS-QOL, SAS, SDS and pain intensity scores of IBS patients using Pearson’s correlation analysis, which was considered significant at P < 0.05.

The demographic and clinical data are summarized in Table 1. There were no differences in age, sex or education level between groups (P > 0.05, all). Scores for MMSE (>26) and MoCA (≥26) for all subjects were in the normal range, while lower MMSE and higher anxiety and depression symptom scores were observed in IBS patients relative to HCs (P < 0.05; Table 1). All cases were diarrhea-predominant based on bowel habits, with pain reported as a symptom by each IBS patient.

Figure 1 shows the spatial patterns of VMHC in each group. Visual inspection indicated that the functional coordination was a global brain phenomenon, with regional differences in strength consistent with previous studies (Zuo et al., 2010; Ji et al., 2014; Li et al., 2015). The HC and IBS groups did not differ in global VMHC (IBS patients: 0.64 ± 0.08; controls: 0.63 ± 0.07; t = 0.67, P = 0.50); however, inter-group comparisons showed that IBS patients had lower VMHC in bilateral ventral anterior cingulate cortices (vACC) and inferior parietal lobules (IPL), as well as higher VMHC in bilateral thalami, cuneus, posterior cingulate cortices (PCC), lingual gyri and inferior occipital/cerebellum lobes relative to HCs (Table 2; Figure 2). Inclusion of anxiety and depression as covariates (along with age, sex and education level) eliminated the VMHC difference in the vACC (Figure 2).

Two commissural fibers connecting the left and right vACC and cuneus were detected in all HCs and patients. The commissural fibers that connected the other bilateral ROIs displaying abnormal VMHC were detected in less than half of the subjects. Therefore, we only extracted the anatomical parameters in commissural fibers connecting the bilateral vACC and cuneus. The result of a two-sample t test between groups did not show any significant differences in path length, tract count or FA of the two fibers (Figure 3).

The VMHC value in vACC showed a slight negative correlation with the QOL scores of IBS patients (r = −0.25, P = 0.04; Figure 4). The VMHC value in other regions had no significant correlation with clinical indices in IBS patients.

Our results showed that IBS patients had higher interhemispheric functional connectivity of bilateral thalami. The thalamus is a core region of the homeostatic afferent network, which encompasses the sensory input entering the thalamus from the brainstem, and then projects to the insular and anterior mid-cingulate cortex, mediating affective, motivational and motor aspects of the stimulus (Al Omran and Aziz, 2014). Convergent evidence has suggested that the thalamus was more activated in IBS patients than the HCs during visceral distension (Ringel et al., 2003; Yuan et al., 2003) and cutaneous heat stimuli (Verne et al., 2003). In a recent rs-fMRI study using regional homogeneity (ReHo), an indicator measuring the degree of regional synchronization of fMRI time series (Zang et al., 2004), Ke et al. (2015) reported higher regional synchronization in the thalamus of IBS patients relative to controls. The ReHo in that study and VMHC used in this study focus on the degree of regional- and interhemispheric-coordination, respectively. Thus, the finding of a higher VMHC of the thalamus in IBS was in accordance with previous findings and added important insights into understanding the role of thalamus in enhanced visceral information processing of IBS.

The cuneus and PCC also showed higher VMHC in IBS patients. The cuneus plays an important role in integrating somatosensory inputs with other sensory stimuli and with cognitive processes such as attention, learning and memory (Fulbright et al., 2001). It is also considered to be the center of the cortical network associated with the experience of pain (Fulbright et al., 2001). One recent rs-fMRI study also demonstrated increased intrinsic activity of the bilateral cuneus in IBS, which was regarded as partly accounting for the chronic pain of the disease (Qi et al., 2015). PCC is involved in internally directed cognition, conscious awareness and working memory (Leech and Sharp, 2014). Previous studies have reported higher PCC activation in IBS patients (Naliboff et al., 2001; Verne et al., 2003), and even higher in those with a history of sexual or physical abuse (Ringel et al., 2008). However, it is worth noting that a subsequent study showed decreased intrinsic brain activity in the PCC (Qi et al., 2015). These discrepancies may be explained by the use of different methodologies. The previous study that showed decreased PCC activity applied the amplitude of low-frequency fluctuation (ALFF) algorithm, an index evaluating the strength or intensity of spontaneous neural activity at rest (Zang et al., 2007), while the VMHC used here measures the strength of interregional temporal correlation (functional connectivity) between hemispheres.

We also found higher VMHC in the lingual gyrus and inferior occipital/cerebellum in IBS patients. Few studies have reported abnormalities in these regions in IBS patients. Ke et al. (2015) showed increased regional synchronization of cerebellum regions in IBS; however, the consistency of these findings needs to be confirmed in future studies.

Our finding that decreased interhemispheric functional connectivity in the vACC was shown for IBS patients, but the inclusion of anxiety and depression as covariates abolished this difference. The ACC is a multifunctional structure situated in the medial frontal lobe that is highly interconnected with the insular, prefrontal, limbic and other subcortical structures, thus cognitive and affective factors may exert influence on pain transmission through the ACC (Bush et al., 2000; Vogt, 2005). The vACC is the principal site of autonomic (primarily vagal) regulation in the frontal lobe and plays a key role in the visceral aspects of emotion (Vogt, 2005; Shackman et al., 2011). Recent studies have revealed that the symptoms of anxiety and depression partially mediated the central processing of visceral stimuli in patients with IBS (Elsenbruch et al., 2010), and abolished differences in multiple white matter tracts between patients with functional gastrointestinal disorders and HCs (Zhou et al., 2013). A recent fMRI study also reported that the inclusion of anxiety and depression as covariates abolished the decreased ALFF of ACC in IBS patients (Qi et al., 2015). Thus, our findings are supported by previous neuroimaging studies and indicate that the high level of depression and anxiety may lead to affective dysregulation of the ACC in IBS patients. Together with previous studies (Wilder-Smith et al., 2004; Ringel et al., 2008), we conclude that ACC might be the key area associated with affective dysregulation in IBS.

In addition to decreased VMHC in the vACC, lower VMHC in the IPL was also observed in IBS patients relative to controls. The IPL is also implicated in pain modulation (Schwedt and Chong, 2014). A previous study demonstrated lower spontaneous brain activity in bilateral inferior parietal cortices in IBS patients (Ke et al., 2015); therefore, our results are compatible with previous neuroimaging studies in IBS.

We performed an inter-group analysis for the commissural tracts connecting the bilateral ACC and cuneus, but did not observe significant differences between groups. Some DTI studies in IBS have demonstrated abnormalities of microstructural white matter integrity (Chen et al., 2011; Ellingson et al., 2013). For instance, Chen et al. (2011) reported that IBS patients had an increased FA in the fornix and the external capsule compared with HCs. Ellingson et al. (2013) also found IBS patients had lower FA in thalamic regions, the basal ganglia and sensory/motor association/integration regions as well as higher FA in frontal lobe regions and the corpus callosum. However, across these investigations that employ DTI, no consistent areas of abnormal microstructural white matter were reported. The value of directly comparing our findings with previous DTI studies was limited by the differences in study samples, analysis methodology (measurements of the microstructure of regional white matter in most previous studies and of the whole fiber tract that connects cerebral hemispheres in the current study), as well as the mixed findings within the previous DTI studies.

Taking into account that IBS patients showed abnormal interhemispheric functional connectivity but no significant changes in interhemispheric anatomic connectivity, we speculated that as for functional disorders such as chronic pain disorders including IBS, functional connectivity may be more sensitive than structural connectivity in detecting the exact mechanisms underlying the symptoms. In general, the functional connectivity which measures blood oxygenation level-dependent fluctuations is thought to be more flexible and sensitive; while the structural connectivity is relatively stable (Bullmore and Sporns, 2009; Zhang et al., 2011). The inconsistent changes of interhemispheric functional and structural coordination have also been reported in other brain disorders without significant lesions by conventional MRI. For instance, Wu et al. (2015) reported widespread impaired interhemispheric functional connectivity but no affected interhemispheric structural connectivity in benign childhood epilepsy with centrotemporal spikes.

While this study has advanced our understanding of the changes in neural networking associated with IBS, it also had some limitations. First, our study cohort consisted of Chinese patients with diarrhea-predominant IBS, and as such, the results may not be generalizable to patients of other ethnicities or with other subtypes of IBS. Second, future studies will need to address the question of whether the observed results are altered by IBS treatment. Besides, further studies with more subjects with depression or anxious symptom and with different pain degrees are needed to investigate how the psychosocial factor and pain might modulate the observed differences. Third, as a newly developed approach, the exact physiological implication of VMHC and its relationship with anatomical connectivity warrant further examination.