Neuroimaging and biomarkers in functional gastrointestinal disorders: What the scientists and clinicians need to know about basic neuroimaging, biomarkers, microbiome, gut and brain interactions

Chapter 3

Neuroimaging and biomarkers in functional gastrointestinal disorders: What the scientists and clinicians need to know about basic neuroimaging, biomarkers, microbiome, gut and brain interactions Jennifer S. Labusa, Gustinna Tuna, Lisa A. Kilpatricka, Satish S.C. Raob, Emeran A. Mayera, Kirsten Tillischa,c a

G. Oppenheimer Center for Neurobiology of Stress and Resilience, Vatche and Tamar Manoukian Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, CA, United States, bDigestive Health Clinical Research Center, Augusta University, Augusta, GA, United States, cDepartment of Medicine, Veterans Administration Greater Los Angeles Health Care System, Los Angeles, CA, United States

Key points ●

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The “gut–brain axis” is a bidirectional neurohumoral communication system capable of changing and regulating gut function and symptoms. Several neuroimaging techniques, notably, functional magnetic resonance imaging, positron emission tomography, radioligand techniques, magnetic resonance spectroscopy, imaging genetics, pharmacological imaging have each contributed to improve understanding. Functional and structural alterations in brain networks underlie the processing and response to visceral afferent signals in disorders of gut–brain interactions (DGBIs). The gut–microbiome plays an important role in gut–brain communication. Combining neuroimaging and other biological markers is key to advancing our understanding of the gut–brain interactions.

Introduction Brain imaging research has provided critical insight into brain structure and function, and into the role of gut–brain interactions in gastrointestinal (GI) disorders [1, 2]. The primary focus of neuroimaging studies in gastroenterology has been to gain a better understanding of the pathophysiology of a group of GI disorders referred to as functional GI disorders (FGIDS). These disorders are characterized by chronic gastrointestinal pain or discomfort and lack detectable structural abnormalities of the GI tract or diagnostic laboratory biomarkers. Over the course of two decades, these studies have led to a paradigm shift that reconceptualized FGIDs from primarily peripheral disorders of the GI tract to disorders of gut-brain interactions (DGBIs) [3]. DGBIs are defined as a group of disorders classified by the presence of GI symptoms related to any combination of motility disturbance, visceral hypersensitivity, altered mucosal and immune function, altered gut microbiota, and altered central nervous system (CNS) processing in the absence of detectable organic disease, and included esophageal (e.g., function heartburn, or chest pain), gastroduodenal (e.g., functional dyspepsia), and bowel disorders (i.e., irritable bowel syndrome (IBS)) [1, 3–5]. The “gut–brain axis” is a bidirectional communication system utilizing the autonomic nervous system, immune system, and the hypothalamic-pituitary-adrenal (HPA) axis. While attention is often focused on the “top down” influence of the CNS on the gut, the brain also receives continuous and detailed “bottom up” homeostatic signaling about the physiological condition of the body primarily via vagal afferent pathways [6]. Visceral afferent signals are modulated by cognitive and affective circuits at the level of the brain and through descending modulatory pathways, creating a communication loop that is capable of changing gut physiological activity as well as impacting the interoceptive sense of wellbeing. Dysfunction of these modulatory systems might allow non-noxious physiological stimuli to be perceived as painful or unpleasant, which Clinical and Basic Neurogastroenterology and Motility. https://doi.org/10.1016/B978-0-12-813037-7.00003-0 © 2020 Elsevier Inc. All rights reserved.

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can lead to chronic visceral pain and/or discomfort, the hallmark symptoms of DGBIs, and may feedback to heighten central stress responses and/or impact mood. In this chapter we will first review common functional, structural and molecular brain imaging techniques used to assess CNS alterations in adults with IBS, the most common DGBI. Next we will summarize the results from these neuroimaging studies, describing the specific brain alterations associated with symptom presentation and highlighting studies examining the molecular mechanisms. We describe CNS alterations in IBS patients compared to healthy individuals as well as individuals with other chronic pain conditions characterized by similar brain-body interactions with an emphasis on sex-related differences. The emerging link between the brain and the gut microbiome is explored. Fig. 1 depicts a comprehensive model of the brain gut microbiome axis in IBS. Finally we discuss the limitations of the current research and provide suggestions for addressing these challenges and pose future directions for continued progress. Several excellent reviews of brain imaging in FGIDs are also available [7–10]. This chapter is not comprehensive but instead is meant to highlight the role of neuroimaging in delineating CNS mechanisms underlying symptom presentation in IBS.

The starting point Results from imaging studies are usually depicted as blobs of color on a brain template (see Fig. 2). These blobs represent clusters of neighboring voxels, which are the basic units of measurement for any brain image. The voxel is a 3 dimensional cube of brain tissue, sized in millimeter to submillimeter range, containing over a million brain cells. Brain regions are comprised of many voxels. Psychological factors Anxiety, depression GI-specific anxiety Hypervigilance/attentional bias Catastrophizing Neurotransmitters Corticotropin-releasing factor Norepinephrine Serotonin Neurokinin-1

Brain Mechanisms

HPA axis SNS

ANS

Vagal and spinal afferents

Brain Immune Loop

CNS Structure and Function Altered structure Functional/anatomical connectivity Emotional and cognitive modulation of visceral signal

Cytokines Pro-/antiinflammatory genes

PBMC

Microbiome Immune Loop

Environmental Influences Stress Early life adversity Social support Medical system Diet

Gut Microbiome GI Symptoms Pain Altered bowel movements

Gut-related Mechanisms

FIG. 1  Brain–gut–microbiome axis in irritable bowel syndrome. The “brain–gut axis” is the bidirectional neurohumoral communication system between the brain and the gut that is continuously signaling homeostatic information about the physiological condition of the body to the brain through afferent neural (spinal and vagal) and humoral “gut–brain” pathways. Visceral afferent input is processed and continuously modulated by cognitive and affective circuits at the level of the brain and through descending modulatory pathways. Dysfunction of these modulatory systems might allow physiological (nonnoxious) stimuli to be perceived as painful or unpleasant (visceral hypersensitivity), which can lead to chronic visceral pain and/or discomfort, hallmark symptoms of DBGIs. Recently this model of brain–gut interactions has been expanded to include the bidirectional signaling between the brain and the gut microbiota, which may involve multiple neural (vagal afferents, enteric nervous system), metabolic (bacterial components and their metabolites), endocrine, and immune-related signaling mechanisms. The brain can influence microbial composition and function via endocrine, immune and neural mechanisms.



Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  33

FIG. 2  Female IBS patients show significantly increased brain activity in the right anterior insula during abdominal threat compared to a safe condition. Results from imaging studies are usually depicted as blobs of color on a brain template. Here, red to orange color gradient reflects magnitude of difference in activity during the abdominal threat compared to the safe condition.

Common neuroimaging techniques and approaches for assessing brain mechanisms in IBS Human brain activity can be measured and imaged using several techniques. Each type of brain imaging technique has a particular temporal and spatial resolution and is utilized to assess different brain features (e.g., function, structure, receptor density).

Functional imaging studies Task-based or evoked-functional magnetic resonance imaging (fMRI) The first brain imaging studies in IBS measured brain activity during rectal distention using Positron Emission Tomography (PET). PET provides functional maps of cerebral blood flow by detecting radioactivity emitted after injection of a radioactive tracer. Due to its invasiveness, limited number of measurements and low temporal resolution (~30s to minutes), PET quickly fell out of favor and was replaced by functional(f) MRI as the method of choice for investigating brain activity. Taking advantage of the differing magnetic properties of molecules, fMRI measures brain activity by assessing the ratio of oxygenated versus deoxygenated hemoglobin in a particular area of the brain, often a voxel or region of interest. In task-based fMRI, change in this blood oxygen level dependent (BOLD) signal is measured by subtracting the BOLD signal between conditions (e.g., baseline, rectal balloon inflation), and provides an indirect measurement of a change in brain activity between two different experimental condition. fMRI is a non-invasive tool and provides much greater temporal resolution than PET, measuring activity every 1–2s. fMRI generally has a spatial resolution of 2–4mm3 voxels but still does not have the precision of post-mortem studies in animals and cannot specify very small brain regions, particularly in the brainstem where considerable physiological artifact occurs. fMRI has been used successfully to compare patients with IBS to healthy controls (HCs) during a variety of disease relevant tasks (e.g., balloon distention, emotional learning, threat).

Resting-state fMRI (rsMRI) An alternative approach to task-based fMRI, task-independent, spontaneous brain activity can be acquired during a resting state. During this scan, the participant lies quietly, usually with the eyes closed, while a short functional brain scan is performed. After correcting for physiological noise and motion, the spontaneous low frequency BOLD signal fluctuations measured in brain regions (or voxels) during rest can then be correlated to determine the functional connectivity between brain regions or voxels over time. Independent components analysis can also be used to detect networks by identifying distinct, independent patterns of inter-correlations between brain regions over time. Furthermore, the interaction between these functionally connected brain networks can be assessed. In addition, resting state signal amplitude can be assessed to determine the relative contribution of specific oscillations to the whole detectable frequency range [11].

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Other less frequently applied techniques used to assess brain activity during a task or at rest in IBS include electroencephalography (EEG) used to measure electrical activity in the brain. EEG has high temporal resolution on the order of milliseconds but poor spatial localization. Although a large body of research has applied EEG to study the brain in IBS the use of this technique fell out of favor with the arrival of PET/fMRI, likely due to problems in localizing measureable activity to specific brain regions [12–21]. Another technique providing similar temporal resolution as EEG but spatial resolution comparable to PET/fMRI is magnetoencephalography (MEG), which measuring the magnetic fields produced by electrical activity in the brain. Frokjaer et al. provide an excellent review on the use of EEG and MEG to study visceral pain [22].

Macro- and micro structural imaging studies Structural (s)MRI This technique provides high spatial resolution and soft tissue contrasts to measure brain morphometry. The most common MRI sequences T1- and T2-weighted scans characterize tissue by measuring the radio frequency of magnetically perturbed protons during longitudinal and transverse relaxation (i.e., return to original alignment). T1-weighted images quantify the characterized tissue using relaxation properties of excited protons. T1-weighted images are based on the gray matter, white matter and cerebral spinal fluid in the brain. T2-weighted images provide additional contrast information (i.e., separating blood vessels from dura) and provide more accurate brain segmentation and surface reconstruction. The ratio of T1weighted /T2-weighted images can be used to detect myelin related signal intensity changes [23]. Typically these methods are applied to perform morphometry studies to describe the volume, cortical thickness, surface area and mean curvature of brain structures. The neuroplasticity of gray matter has been documented across the human lifespan [24, 25]. Alterations in gray matter may involve increased or decreased glial cells, and/or changes in dendritic spines or synapses. Ultimately, sMRI only provides nonspecific assessment of underlying tissue characteristics. In addition, tissue properties (e.g., cell size, myelination) affect relaxation times, and hence voxel intensities, and may influence voxel based morphometry [26].

Diffusion MRI Diffusion MRI is a non-invasive technique that can assess the microstructural properties and organization within brain tissue based upon the dispersion of water molecules. The most conventional form of diffusion MRI is diffusion tensor imaging (DTI). To quantify the microstructural white matter integrity of a given voxel two key metrics can be produced: fractional anisotropy (FA) and mean diffusivity (MD). FA is used to estimate the degree of directional coherence of the underlying tissue structures within an image voxel, reflecting the strength of axonal or dendritic projections, while MD can be used to estimate relative tissue compactness and degree of myelination [27]. Because they are constrained to move in the direction of axons, water molecules in dense, parallel white matter tracts have high FA values. Variations in the FA of white matter tracts can occur due to changes in axonal number, myelination, or axonal cytoskeleton integrity whereas alterations in MD may reflect differences in axonal density or branching or caliber [28–30]. In addition to assessing the microstructural integrity of brain tissue, fiber tractography is performed to identify the number of fiber tracts between specific brain regions or voxels. This technique is used to determine the anatomical connectivity underlying functional networks. DTI is limited in its ability to perform tractography, specifically in areas of the brain with complex fiber crossing [31, 32]. More sophisticated methods such as diffusion spectrum imaging (DSI) [33] and high-angular resolution diffusion imaging (HARDI) [34] use increased magnetic gradient strengths and can detect the movement of water through voxel in many more directions, providing more accurate information for tractography.

Molecular studies Radioligand PET studies While the use of PET imaging to assess task-related brain activity has gone out of favor due to the ready availability, noninvasive nature, and better temporal resolution of fMRI, specific ligand studies are still an area where PET is invaluable. PET studies permit measurement of regional availability of receptor/transporter systems in the brain by injecting radioactively labeled ligands. Ligands are available for receptor systems of major neurotransmitters including endogenous opioids, dopamine, neurokinin-1, and serotonin, with ongoing development of many others. These studies have been limited in IBS given the need for a specialized radiopharmaceutical facility in close proximity to the study location, need to generate the ligand and the involvement of radiation burden for the subjects. Despite these shortcomings, PET ligand studies will likely continue to play an important role in understanding mechanisms underlying functional brain alterations in DGBIs and for development of centrally targeted therapeutic compounds.



Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  35

Pharmacological (pH) fMRI The combination of brain imaging and administration of drugs has been referred to as pharmacological imaging [35]. phfMRI is an important noninvasive tool that can be used to assess the effects of centrally acting pharmacological compounds on the activity of the brain during rest or evoked tasks.

Interventional phenotyping with fRMI In IBS, MRI has been used as a tool to uncover central mechanisms involved in pharmacological and nonpharmacological treatments in IBS including electroacupuncture [9; 80] hypnosis [36], lidocaine treatment [37], amitriptyline [38], cognitive behavioral therapy [39], mindfulness treatment [40], and placebo [41–43].

MR spectroscopy studies This technique allows quantification of regional metabolite concentrations in brain tissue, including the neurotransmitters glutamate and GABA, and the inflammatory mediator myo-inositol, based on the differential resonance frequency of protons in different molecules. This technique has much lower spatial and temporal resolution compared to MRI [44]. To date few studies have applied this technique in DGBIs.

Imaging genetics This technique utilizes brain imaging to quantify genetic and epigenetic variation to brain structure, function, and connectivity to investigate the molecular and genetic architecture of brain phenotypes and the neural mechanisms through which genetic risk for disease may emerge [45].

Two decades of brain imaging studies in IBS: What have we discovered? Imaging studies in DGBI have used a wide variety of techniques, focusing mainly on IBS. Table 1 provides detailed information on all published neuroimaging manuscripts in IBS from 1997 to 2018 (N=89). In total 94 multimodal scanning sessions have been conducted including 13 PET, 48 tasked-based fMRI, 13 rsMRI, and 11 sMRI studies. We review the results of these studies below.

Functional imaging findings Early fMRI (and PET) studies tested for activation in the whole brain or a defined set of hypothesized regions of interest (ROI) that were selected based on preclinical or other functional imaging studies. The first decade of brain imaging studies focused primarily on brain responses to the expectation and delivery of rectal balloon distention, a stimulus designed to evoke physiologically relevant gut discomfort. Meta-analysis of the studies demonstrated that patients with IBS compared to HCs have greater brain responses in regions associated with emotional arousal and endogenous pain modulation and reduced activity in cognitive modulatory regions during the expectation of rectal distention. However HC and IBS showed similar activation of regions involved in processing of visceral afferent information [128]. Since this time, subsequent rectal distention studies have validated these findings [51, 54, 62, 72, 82, 84, 90–92, 95, 98, 129–131]. These initial imaging studies in IBS were effective in localizing and mapping brain activations in the brain during specific tasks, however functional localization did not reveal any information about the communication between active regions. As such, a strong emphasis was placed on characterizing the dynamic interplay between regions of the brain, mapping brain pathways and identifying functional networks relevant to IBS. Functional networks engaged during the expectation and response to balloon distention in IBS and HCs were the first to be quantitatively delineated and included (1) the homeostatic afferent network, central to processing visceral afferent information (information about the homeostatic state of the individual, including the viscera) to the brain via the lamina I spinal pathway to distinct thalamic subnuclei that project to the posterior insula and anterior midcingulate cortex, respectively (see Fig. 3); (2) the emotional arousal network (see Fig. 4), a network involved in arousal, and emotion-related pain amplification and comprised of the amygdala and anterior cingulate cortex (ACC) subregions and locus coeruleus complex; and (3) the cortical-modulatory network representing the modulatory influence of cortical regions with interoceptive and emotional arousal circuits [107]. Network analyses also demonstrated dampening of attentional and emotional circuitry during perceptual habituation to repeated aversive visceral stimuli [104]. Several other functional networks underlying symptoms and information processing have been identified in IBS using other less invasive experimental designs to study pain, emotional and cognitive processing including learning and extinction [49, 68, 88], attention [67], prediction error [93], emotional processing of faces, and contextual threat [60, 99] protocols.

Publication

Population

IBS subtype

Imaging modality

Group difference results

Brain-symptom correlations in IBS

Gupta et al. [46]

All F: IBS = 29 HC = 29 PV = 29

IBS-C = 11 IBS-D = 6 IBS-U = 5 IBS-M = 7

DTI

IBS vs. PV: Lower mean diffusivity in IBS.

Nan et al. [47]

All F: FC = 18 IBS = 20

IBS-C = 20

DTI

IBS-C vs. HCs: Alteration in the fractional anisotropy and radial diffusivity of the corpus callosum. IBS-C vs. FC: Differences in radial diffusivity in the corona radiata and superior longitudinal fasciculus.

Fractional anisotropy and radial diffusivity in the corpus callosum were associated with abdominal pain in all patients.

Chua et al. [48]

All F: IBS = 29 HC = 39

Not reported

sMRI

IBS vs. HC: Lower cortical thickness for L cuneus, L rostral middle frontal cortex, L supramarginal cortex, R caudal ACC, L INS, and R INS.

(−) association between duration of IBS and thickness of the L cuneus, L rostral middle frontal cortex, L supramarginal, and L INS. (−) association between severity of abdominal pain and thickness of the L cuneus, L rostral middle frontal cortex, L supramarginal, L INS, R anterior midcingulate, and R INS.

Claassen et al. [49]

IBS, F/M = 15/2 HC, F/M = 10/11

IBS-C = 2 IBS-D = 8 IBS-A = 7

fMRI

IBS vs. HC: Greater activation during acquisition phase in IBS.

Fang et al. [50]

IBS, F/M = 7/14 HC, F/M = 10/11

Not reported

DTI

IBS vs. HC: Lower fractional anisotropy and higher mean diffusivity values in IBS and decreased the apparent diffusion coefficient in IBS.

Guleria et al. [51]

M, IBS = 20 M, HC = 10

IBS-C = 10 IBS-D = 10

fMRI

IBS vs. HC: Greater activity in L anterior INS, middle temporal gyrus and cerebellum in IBS. Less activity in bilateral precuneus in IBS. IBS-C vs. IBS-D: Greater activity in R supplementary motor cortex and posterior mid-cingulate in IBS-C. Lower activity in L calcarine sulci, bilateral fusiform gyri, R middle temporal gyrus, and orbital frontal cortex in IBS-C.

Gupta et al. [52]

IBS, F/M = 8/8 HC, F/M = 9/7

IBS-C = 7 IBS-D = 4 IBS-U = 1 IBS-M = 4

rsMRI

IBS vs. HC: Greater salience network connectivity in IBS.

(+) correlation between IL6 and APOL2 genes and connectivity of temporal and cingulate cortex with salience network in IBS.

Icenhour et al. [53]

F, IBS = 44 F, HC = 20

Not reported

rsMRI

IBS-N vs. IBS-H: Increased (+) intrinsic connectivity within the salience network & sensorimotor network in IBS-H. IBS subgroups vs. HCs: Decreased (+) intrinsic connectivity in amygdala & decreased (−) intrinsic connectivity in the dorsal anterior INS in IBShypersensitive subtype.

Intrinsic connectivity of regions comprising the derived default mode network was associated with rectal perception thresholds. Intrinsic connectivity in posterior INS with sensorimotor network was correlated with reported symptom severity in IBS.

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TABLE 1  MRI studies in IBS



Kano et al. [54]

M/F IBS = 12/14 HC F/M = 15/14

IBS-D = 24 IBS-M = 2

fMRI

IBS vs. HC: IBS had greater activation of anterior midcingulate cortex, thalamus, and visual processing areas during uncertain anticipation compared with certain anticipation. Absence of bilateral INS activation during non-distention period with uncertain compared to safe cue.

Kano et al. [55]

IBS, F/M = 14/14 HC, F/M = 16/18

IBS-D = 24 IBS-M = 4

fMRI

Labus et al. [56]

IBS, F/M = 22/7 HC, F/M = 14/9

IBS-C = 11 IBS-D = 10 IBS-M = 6 IBS-U = 2

sMRI

IBS vs. HC: Distinct microbial profile in IBS was associated with morphometric alterations in sensorimotor and salience regions.

In IBS, the relative abundance of Clostridia and Bacteroidia had moderate size correlations with brain regions comprising sensorimotor and salience network regions in IBS.

Longarzo et al. [57] IBS F/M = 13/6 HC F/M = 16/10

Not reported

rsMRI sMRI

IBS vs. HC: No differences in regional gray matter volumes or functional connectivity.

In IBS, (+) correlation between Self-Awareness scores & increased connectivity between L anterior ventral INS and supramarginal gyrus. In IBS, (−) correlation between Illness Attitude Scales and connectivity between PCC and L supramarginal gyrus.

Pinto-Sachez et al. [58]

IBS F/M = 22/22

IBS-D = 27 IBS-M = 17

fMRI

IBS patients showed reduced brain activity while viewing emotional stimuli in the amygdala, ventral lateral PFC, dorsal medial frontal cortex, middle temporal gyrus, middle occipital gyrus, cuneus, and cerebellum while taking a probiotic vs. placebo.

At 6 weeks, the amygdala activation correlated with the depression scores. Greater adequate relief of IBS symptoms was associated with less activity in the amygdala.

Weng et al. [59]

IBS F/M = 6/25 HC F/M = 7/25

IBS-D = 63

rsMRI

IBS vs. HC: Decreased long and short range functional connectivity density were observed in emotional arousal, salience, default mode, and sensorimotor cortices.

(+) correlation between IBS symptom severity scores and long-range functional connectivity density values in R anterior INS. (+) correlation between disease duration and short-range functional connectivity density values in L caudate.

Hong et al. [60]

IBS F/M = 21/16 HC F/M = 158/19

IBS-C = 13 IBS-D = 11 IBS-U = 4 IBS-M = 9

fMRI

IBS vs. HC: In IBS, salience, attention, default mode, and emotional arousal regions were more activated by cue abdominal threat. IBS showed greater brain activations in the affective (amygdala, anterior INS) and attentional (middle frontal gyrus) regions, and in the thalamus and precuneus during uncued abdominal threat.

Huang et al. [61]

UCPP, F/M = 23/29 IBS, F/M = 24/15 HC, F/M = 32/29

Not reported

DTI

Lower fractional anisotropy in corticospinal tract in IBS compared to UCPPS. IBS had greater radial diffusivity in anterior thalamic radiation compared to UCPPS but not HCs. IBS had greater radial diffusivity in anterior thalamic radiation & greater fractional anisotropy in anterior thalamic radiation vs. UCPPS and HCs.

Tanaka et al. [62]

IBS, M = 16 HC, M = 16

IBS-D = 13 IBS-C = 1 IBS-M = 2

fMRI

IBS vs. HC: At baseline, corticotropin releasing hormone without colorectal distention induced more activation in the R amygdala in IBS. During rectal distention after corticotropin releasing hormone injection, R amygdala activity was greater in IBS.

HCs with higher responses to CRF showed lower brain signal and responses to rectal distension in emotional arousal and cortical inhibitory regions.

Continued

Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  37

F IBS with greater radial diffusivity had greater pain intensity.

Publication

Population

IBS subtype

Imaging modality

Group difference results

Brain-symptom correlations in IBS

Qi et al. [63]

IBS F/M = 16/49 HC F/M = 16/51

IBS-D = 132

rsMRI DTI

IBS vs. HC: IBS had higher interhemispheric functional connectivity between bilateral thalami, cuneus, PCC, lingual gyri and inferior occipital/cerebellum lobes, and lower connectivity between bilateral subgenual ACC and inferior parietal lobules.

Anxiety and depression accounted for subgenual ACC differences.

Qi et al. [64]

IBS F/M = 6/25 HC F/M = 7/25

IBS-D = 63

rsMRI

IBS vs. HC: IBS had greater (+) L amygdala connectivity with the R INS, midbrain, L pre-/postcentral gyri, R precentral gyrus, L parahippocampal gyrus, and bilateral supplementary motor area, and greater (+) R amygdala connectivity with R INS, midbrain, L parahippocampal gyrus, bilateral precentral gyri, and R supplementary motor area.

(+) correlation between: (a) IBS pain intensity & connectivity of amygdala with bilateral supplementary motor area, pre & postcentral gyri, INS, R precentral gyrus and R INS. (b) IBS-Symptom Severity Score and connectivity between L amygdala with bilateral INS, midbrain, and connectivity between R amygdala and R INS.

Qi et al. [65]

IBS F/M = 6/24 HC F/M = 7/24

IBS-D = 63

rsMRI

IBS vs. HC: IBS patients showed decreased ALFF in several core default mode network regions, while bilateral posterior INS and cuneus showed increased ALFF. IBS patients had alterations in functional connectivity of the medial PFC with orbital frontal cortex and posterior INS as well as the subgenual ACC with the PCC.

For IBS, the connectivity between the medial PFC and cuneus had a (−) correlation with pain intensities.

Gupta et al. [66]

All F: LPDV = 29 IBS = 29 HC = 29

IBS-C = 11 IBS-D = 6 IBS-U = 5 IBS-M = 7

rsMRI

PVD vs. IBS: IBS had alterations. IBS had greater connectivity in the salience network in the bilateral dorsal medial PFC. IBS had decreased connectivity of bilateral angular gyrus, and bilateral precuneus within the default mode network, but increased connectivity of R precuneus and R dorsal/ventral PCC.

Hubbard et al. [67]

All F: IBS = 15 HC = 14

IBS-C = 7 IBS-D = 4 IBS-M = 1 IBS-U = 3

fMRI

IBS vs. HC: IBS had greater activations during alerting in L anterior mid-cingulate, bilateral anterior INS, and R posterior INS and greater deactivation in L precentral gyrus during orienting. IBS had greater suppression of activity in L supplementary motor area and greater deactivation in R thalamus and activation in the R presupplementary motor area, during the executive control task.

For IBS, activity in the anterior mid-cingulate during alerting was associated with duration of GI-symptoms and overall symptom severity.

Icenhour et al. [68]

IBS F/M = 15/2 HC F/M = 11/10

IBS-D = 8 IBS-C = 2 IBS-A = 7

fMRI

IBS vs. HC: IBS showed enhanced condition stimulus induced differential activation of PFC and amygdala. IBS had greater differential cingulate activation during extinction and greater differential hippocampal activation during reinstatement.

Anxiety was associated with brain responses during memory formation and reinstatement.

Irimia et al. [69]

IBS F/M = 14/19 HC F/M = 33/23

Not reported

DTI

IBS vs. HC: IBS had greater mean fractional anisotropy. Differ within both L and R viscerotopic portions of the primary somatosensory cortex.

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TABLE 1  MRI studies in IBS—cont’d



IBS F/M = 6/25 HC F/M = 7/25

Labus et al. [71]

All F: IBS = 108 HC = 108

Lowén et al. [72]

All F: IBS = 33 HC = 18

Ma et al. [73]

IBS-D = 63

rsMRI

IBS vs. HC: IBS had increased regional homogeneity in the bilateral postcentral gyri, R thalamus, and L superior parietal lobule. IBS had decreased regional homogeneity in bilateral anterior mid-cingulate cortex/perigenual ACC, ventral medial PFC, dorsal lateral PFC and ventral lateral PFC, R caudate and angular gyrus.

Regional homogeneity values in certain brain regions correlated with disease duration, IBS symptom severity, and pain intensity levels.

sMRI

IBS vs. HC: Brain signature 1 comprised morphometric alterations of several somatosensory and motor regions, interceptive integration regions, and cognitive modulatory regions. Brain signature 2 comprised of fronto-INS, emotional modulation, and dorsal attentional regions, and visual and auditory gyri.

For IBS and HC, (−) correlation between extragastrointestinal somatic symptoms and the somatosensory and motor brain signature.

Not reported

fMRI

IBS-Hypersensitive vs. IBS-Normosensitive vs. HC: IBSHypersensitive had greater BOLD response during late phase of distention series and to the anticipation and delivery of low intensity rectal distention in INS, anterior and mid cingulate cortex. IBS-Normosensitive had greater BOLD response to repeated rectal distention in INS, PFC and amygdala.

IBS F/M = 7/14 HC F/M = 10/11

Not reported

rsMRI

IBS vs. HC: IBS had greater ALFF in L superior frontal gyrus, R hippocampus, R dorsal lateral PFC, bilateral postcentral gyrus, and R superior temporal pole, and decreased ALFF in L posterior mid cingulate. IBS had altered functional connectivity of the R dorsal lateral PFC with L posterior midcingulate, supplemental motor area, R middle frontal gyrus and L precentral gyrus and L gyrus rectus.

For IBS, (+) correlation between the R dorsal lateral frontal cortex ALFF and the duration of disease. For IBS, (−) correlation between the L posterior mid-cingulate cortex ALFF and the IBS duration.

Orand et al. [74]

IBS F/M = 85/30 HC F/M = 165/40

IBS-C = 21 IBS-D = 24 IBS-M = 55 IBS-U = 5

sMRI

In IBS, the homozygous genotype of the major ADRA1D allele was associated with gray matter increases in sensorimotor regions and the hippocampus.

In IBS patients only, the homozygous ADRA1D SNP rs1556832 major allele genotype was associated with increases in the volume of the sensorimotor regions and the R hippocampus.

Schmid et al. [41]

IBS F/M = 15/2 UC F/M = 9/6 HC F/M = 15/2

Not reported

fMRI

IBS vs. UC vs. HC: Reduced modulation during placebo analgesia compared to control within the PCC in IBS. IBS showed greater modulation in secondary somatosensory and parietal cortex vs. HC and in dorsal lateral PFC vs. UC. IBS had reduced placebo-induced modulation in mid cingulate cortex vs. HC. IBS had reduced placeboinduced modulation in hippocampus during cued-pain anticipation and in PCC vs. HC.

Woodworth et al. [75]

IBS F/M = 23/16 UCPPS F/M = 19/26 HC F/M = 26/30

Not reported

DTI

IBS vs. UCPPS: IBS had lower mean diffusivity in areas. IBS had lower fractional anisotropy in some regions. IBS had higher track density in some regions. IBS M vs. F: M had less mean diffusivity in basal ganglia, thalamus, internal capsule, brainstem, corpus callosum and corona radiata and greater mean diffusivity in a few regions involving the primary motor and sensory cortices. M had greater fractional anisotropy in most regions. Continued

Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  39

Ke et al. [70]

Publication

Population

IBS subtype

Imaging modality

Group difference results

Brain-symptom correlations in IBS

Zhao et al. [76]

IBS = 60

IBS-D

fMRI

Decreased activated voxel values were observed in bilateral INS and PFC of patients in the moxibustion group during rectal distention after treatment. Patients in electroacupuncture group showed reduced PFC activation.

Gupta et al. [77]

IBS F/M = 28/30 HC F/M = 72/38

Not reported

rsMRI

IBS vs. HC: IBS had greater intrinsic connectivity intrinsic connectivity of the salience, the left frontal parietal, and the default mode networks.

Salience and cerebellar networks, displayed significant correlations between EAL measures and within-network intrinsic connectivity.

Hong et al. [78]

IBS F/M = 9/2 UC F/M = 6/10 HC F/M = 25/16

IBS-D = 11

sMRI

IBS vs. UC: Lower cortical thickness in some brain regions. IBS vs. HC: Decreased cortical thickness in R lateral and medial orbital frontal gyri, R and the anterior INS in IBS.

(−) correlation between symptom duration and cortical thickness in L orbital frontal gyrus. (+) correlation between symptom duration and cortical thickness in L postcentral gyrus.

Hong et al. [79]

IBS F/M = 24/24 HC F/M = 24/24

Not reported

rsMRI

IBS M vs. F: Fs had decreased (+) connectivity of bilateral dorsal anterior INS with medial PFC and dorsal posterior INS. F had greater (−) connectivity of L dorsal anterior INS with L precuneus. IBS F vs. HC F: IBS had connectivity between bilateral dorsal anterior INS and dorsal medial PFC.

For M IBS, (+) correlation between bilateral dorsal anterior INS-dorsal medial PFC connectivity and the Visceral Sensitivity Index. For F IBS dorsal anterior INS with the precuneus and the medial PFC correlated with 24 hour symptom intensity.

Labus et al. [80]

All F: IBS = 82 HC = 119

IBS-C = 31 IBS-D = 22 IBS-A = 16 IBS-U = 6 IBS-M = 5

sMRI

IBS vs. HC: IBS had lower gray matter volume in superior frontal gyri, amygdala, INS, middle orbital frontal gyrus, hippocampus and, brainstem, bilaterally and the putamen, cingulate, and gyrus rectus on the L. IBS had greater gray matter volume in the L postcentral gyrus.

(−) correlations between overall IBS symptoms and gray matter volume changes in L inferior frontal gyrus, L middle orbital frontal gyrus, L lateral orbital frontal gyrus and L INS. (+) correlation between abdominal pain and gray matter in L superior frontal gyri. (−) correlation between R inferior frontal gyri and disease duration.

Zhu et al. [81]

IBS F/M = 6/9 HC F/M = 6/7

fMRI

IBS vs. HC: Moxibustion dampened activity in the PFC and ACC in IBS.

Bouhassira et al. [82]

All F: IBS = 20 HC = 11

IBS-C = 20

fMRI

IBS vs. HC: IBS had greater activation in the anterior and midcingulate, anterior INS and thalamus during painful rectal distention.

Ellingson et al. [83]

IBS F/M = 21/12 HC F/M = 72/21

Not reported

DTI

IBS vs. HC: IBS had decreased fractional anisotropy in some regions while greater fractional anisotropy was seen in sensorimotor and default mode regions. IBS M vs. F: F had decreased fractional anisotropy in the thalamus and primary sensory and motor regions. F had greater mean diffusivity in the coronal radiata, thalamic regions, and cingulate white matter bundles while decreased mean diffusivity in the globus pallidus.

(−) correlation between symptom severity and average fractional anisotropy in the ACC, basal ganglia and white matter areas near the INS.

40  SECTION | A  Foundations of neurogastroenterology and motility

TABLE 1  MRI studies in IBS—cont’d



IBS F/M = 31/29 HC F/M = 76/42

Not reported

rsMRI

F IBS vs. F HC: IBS showed greater frequency power distribution toward high frequency in the anterior INS and amygdala and low frequency in sensorimotor regions. M IBS vs. M HC: IBS showed decreased frequency power distribution toward high frequency in the INS. IBS F vs. M: F had greater frequency power distribution toward high frequency in IINS, amygdala and hippocampus and low frequency in precentral gyrus, primary somatosensory, and supplementary motor area.

For F IBS, (+) correlation between abdominal discomfort and high frequency power distribution in the L anterior INS.

Jarcho et al. [85]

F, IBS = 9 F, HC = 9 IBD F/M = 8/1

Not reported

PET with [18F]SPA-RQ

Relative to HCs, IBS tended to exhibit lower Neurokinin-1 receptor binding potential putamen, nucleus accumbens, globus pallidus, hippocampus, and amygdala, as well as cortical regions including perigenual ACC and anterior mid-cingulate cortex.

Duration of IBS symptoms was (−)ly correlated with average Neurokinin-1 receptor binding potential in caudate nucleus, putamen, each aspect of INS, and the weighted average for cortical regions of interest.

Jiang et al. [86]

IBS F/M = 70/20 HC F/M = 155/21

IBS-C F/M = 26/3 IBS-D F/M = 19/5 IBS-M F/M = 20/4 IBS-U F/M = 5/8

sMRI

F IBS vs. F HC: IBS had greater cortical thickness in preand postcentral gyrus and smaller cortical thickness in bilateral subgenual ACC, bilateral anterior INS middle INS, posterior INS and L subgenual ACC. IBS M vs. HC M: IBS trend for increased cortical thickness of the precentral gyrus and decreased cortical thickness for anterior mid-cingulate and the subgenual ACC and bilateral anterior INS.

In F IBS, (−) correlation between duration of disease and cortical thickness in R middle INS and anxiety and cortical thickness in R anterior & middle INS. In M IBS, (+) correlation between symptom severity with thickness in L middle INS, trait anxiety with thickness in subgenual ACC, R posterior INS and early adverse life events with L middle INS thickness.

Labus et al. [87]

IBS F/M = 27/20 HC F/M = 38/29

Not reported

fMRI

IBS vs. HC: All M had stronger connectivity between ACC subregions, amygdala, and INS subregions. F IBS had stronger connectivity to and from the prefrontal modulatory regions. M IBS demonstrate greater engagement of cortical and emotional arousal brain circuitry.

Labus et al. [88]

All F: IBS = 14 HC = 17

IBS-C = 6 IBS-D = 3 IBS-A = 5

fMRI

IBS vs. HC: CRF-1 reduced activity in the thalamus in IBS and HC during acquisition, the drug produced greater suppression of activity in a wide range of brain regions in IBS during extinction.

Letzen et al. [37]

All F: IBS = 11

Not reported

fMRI

Lidocaine treatment resulted in greater intrinsic connectivity of regions comprising the default mode network.

Lowén et al. [36]

All F IBS = 47

IBS-C = 5 IBS-D = 10 IBS-M = 29

fMRI

Responders in both treatments showed decreased activity in the dorsal and ventral anterior INS during high intensity distension. During post treatment rectal distension, responders to hypnotherapy showed reduced activity in the posterior INS, while responders to education showed reduced PFC activity.

Piché et al. [89]

All F: IBS = 14 HC = 14

sMRI

IBS vs. HC: IBS had decreased pain inhibition and greater shock anxiety, pain catastrophizing, depressive symptoms, and trait anxiety. IBS had thicker R posterior INS.

Association between greater suppression of activity produced by the CRF-R1 antagonist and reduction of SCR. An up-regulation of the CRF/ CRF-R1 signaling system in IBS.

(+) correlation between thickness of posterior INS and IBS duration. Continued

Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  41

Hong et al. [84]

Publication

Population

IBS subtype

Imaging modality

Group difference results

Brain-symptom correlations in IBS

Rosenberger et al. [90]

All F: IBS = 15 HC = 12

IBS-C = 2 IBS-D = 6 IBS-A = 6

fMRI

No activity in the cerebellum found in patients.

Aizawa et al. [93]

IBS F/M = 15/15 HC F/M = 15/15

IBS-C = 13 IBS-D = 13 IBS-M = 4

fMRI

IBS vs. HC: IBS had decreased activity of R dorsal lateral PFC and R hippocampus, and greater activity of L posterior INS at error feedback during set-shifting. IBS had decreased connectivity from the dorsal lateral PFC to presupplementary motor area.

In IBS, (+) correlation between coupling parameters of the modulatory input from R dorsal lateral PFC to pre-supplementary motor area and those from the ACC to pre-supplementary motor area. Latent impairment in cognitive flexibility in IBS associated with altered activity of dorsolateral PFC, INS, hippo-campus, and impaired connectivity between dorsolateral PFC & pre-supplementary motor area.

Chu et al. [94]

IBS F/M = 15/15

IBS-D = 30

fMRI

Electroacupuncture compared to sham resulted in greater activity in the R anterior INS and the R thalamus during distention.

Across all groups rectal pain ratings to balloon distension were associated with bilateral anterior INS and thalamus activity.

Larsson et al. [95]

All F: IBS = 44 HC = 20

Not reported

fMRI

IBS vs. HC: IBS had greater activation in L ventral lateral PFC during high rectal distention and in R anterior INS, R middle INS and R hippocampus during expectation of high distention. IBS-Hypersensitive vs. IBS-Normosensitive: Hypersensitive had larger BOLD signals in regions including several INS, cingulate subregions and lateral PFC regions during expected and delivered distention. Normosensitive IBS vs. HC: Normosensitive had greater activation in R hippocampus during expectation of high stimulus distention. Hypersensitive IBS vs. HC: Hypersensitive had greater activation in L posterior INS, L thalamus, and L perigenual ACC.

Lee et al. [42]

IBS F/M = 11/6 HC F/M = 11/6

Not reported

fMRI

IBS vs. HC: IBS receiving placebo treatment had greater activity in some brain regions during rectal distention including R thalamus, inferior frontal gyrus, posterior INS, and posterior midcingulate. During anticipation of rectal distention, IBS receiving placebo has greater activity in L ventral lateral PFC.

In IBS, L ventral lateral PFC activity was (−)ly correlated with the ventral lateral PFCl activity during anticipation of rectal distention.

Tillisch et al. [96]

All Fs: IBS = 11

IBS-C = 4 IBS-D = 3 IBS-A = 4

fMRI

NK-1 antagonist vs. Placebo: During painful rectal inflation, NK-1 antagonist dampened activity in some brain areas. During the nonpainful rectal inflation, the NK-1 antagonist less activity in some brain areas.

During pain condition, pain ratings and (−) affect scores correlated with NK-1 antagonist activity in the subgenual ACC, & amygdala; anxiety correlated with anterior INS, hippocampus, and amygdala activity.

42  SECTION | A  Foundations of neurogastroenterology and motility

TABLE 1  MRI studies in IBS—cont’d



All F: IBS = 10 HC = 16

IBS-C = 8 IBS-M = 2

DTI

IBS vs. HC: IBS had greater fractional anisotropy in the fornix and in the external capsule bordering the R posterior INS.

(+) correlation between pain severity, unpleasantness and anisotropy in bilateral anterior INS and & pain only in R central posterior lateral nucleus of the thalamus. (+) correlation between duration and fractional anisotropy in L posterior INS. (+) correlation neuroticism and anisotropy in L medial dorsal nucleus of thalamus. (−) correlation between PCS scores and anisotropy in Rt ACC.

Kilpatrick et al. [98]

All F: IBS = 26 HC = 29

IBS-C = 8 IBS-D = 11 IBS-A = 7

fMRI

IBS vs. HC: IBS had greater activity in L amygdala and L hippocampus during neutral visual task, as well as greater L hippocampus activity during emotional task.

Association of greater anxiety & greater amygdala response to neutral and emotional stimuli in both IBS and HC with same genotype.

Hubbard et al. [99]

All F: IBS = 14 HC = 17

IBS-C = 6 IBS-D = 3 IBS-A = 5

fMRI

IBS vs. HS: During pain expectation, CRF-1 antagonist produced dampened activity in parts of the brain in both groups. IBS showed greater BOLD responses in L locus coeruleus and hypothalamus after placebo and decreased activity in L hypothalamus after drug.

State anxiety was associated with the inhibitory effects of CRF-1 antagonist on the hypothalamus in IBS.

Labus et al. [100]

All F: IBS = 14 HC = 12

IBS-C = 14

fMRI

IBS vs. HC: In HC, ATD led to greater response of an extensive brain network to balloon distention; effect was greater during high inflation. In IBS identified near-identical pattern of loss of (−) feedback inhibition of the amygdala because of ATD on coupling between emotional arousal network nodes.

Blankstein et al. [101]

All F: IBS = 11 HC = 16

IBS-C = 2 IBS-M = 9

sMRI

IBS vs. HC: IBS had greater gray matter in the hypothalamus and decreased cortical thickness to the anterior mid-cingulate cortex.

(−) correlation between cortical thickness & descending pain modulation & PCS. (+) correlation between thickness of the anterior INS and duration of pain.

Elsenbruch et al. [92]

All F: IBS = 15 HC = 12

IBS-C = 2 IBS-D = 6 IBS-A = 6

fMRI (BOLD)

IBS vs. HC: IBS had greater pain and discomfort upon rectal distention in the scanner. IBS had greater activation in the anterior INS cortex and PFC (difference disappeared when controlling for anxiety and depression scores).

For IBS, association between anxiety symptoms and pain-induced activation of the anterior midcingulate cortex and perigenual ACC. For IBS, correlation between depression scores and activation of the PFC and cerebellar areas.

Elsenbruch [91]

All F: IBS = 15 HC = 12

IBS-C = 2 IBS-D = 6 IBS-A = 6

fMRI (BOLD)

IBS vs. HC: Stress resulted in greater increases in the ventral lateral PFC, INS, and anterior midcingulate cortex during nonpainful distensions. During painful distensions, IBS ad greater stress-induced increases in the INS and the ventral lateral PFC, but decreased activity in the dorsal lateral PFC. Relaxation-induced activity in the INS was greater in IBS.

INS activity was associated with state anxiety.

Hall et al. [102]

All F: IBS = 7 HC = 6

IBS-C = 6 IBS-D = 1

fMRI

IBS vs. HC: IBS had greater activation of the ACC, INS and ventral medial prefrontal regions. IBS failed to downregulate activity within ventral medial PFC and the PCC/ precuneus regions. HC had greater activation of the thalamus, striatal regions and dorsolateral PFC during the tonic phase (constant state) of distension protocol.

(+) correlation between greater activation of ACC, INS and ventral medial PFC and greater affective responses to painful visceral stimuli in IBS. (+) correlation between greater activation of thalamus, striatal regions and dorsolateral PFC and greater arousal of thalamus and salience-driven sustained attention & affective responses to pain. Continued

Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  43

Chen et al. [97]

Publication

Population

IBS subtype

Imaging modality

Group difference results

Brain-symptom correlations in IBS

Seminowicz et al. [103]

All F: IBS = 55 HC = 48

IBS-C = 15 IBS-D = 17 IBS-A = 19 IBS-U = 5

sMRI

IBS vs. HC: IBS had decreased gray matter density (gray matte density) in L posterior parietal cortex, L MFG, and bilateral temporal cortices.

(−) correlation between IBS duration and gray matter density in dorsolateral PFC in non-pain predominant IBS group.

Labus et al. [104]

IBS F/M = 8/4

Not reported

[15O]H2O PET

During expectation and distention conditions, perigenual ACC, mid-cingulate, PCC and dorsal medial prefrontal cortices were (+)ly correlated with the stimulus ratings during expectation and distension; while ventral medial prefrontal, dorsal lateral PFC, and parietal cortices, precuneus, and hippocampus were (−)ly correlated with the stimulus ratings. Habituation to the rectal stimuli was associated with changes in the coupling between regions of the attention network and the amygdala.

Berman et al. [105]

All F: IBS = 14 HC = 12

IBS-C = 14

fMRI

IBS vs. HC: HC had decreased activity in the INS, subgenual ACC, amygdala, and dorsal brainstem (DBS) during cued anticipation of distention. IBS had decreased anticipatory inactivation during cued anticipation of distention. IBS had greater self-rated measures of (−) affect during scanner. IBS had extensive greater activity in INS, ACC, and DBS, and decreased in the subgenual ACC during subsequent distention.

Jarcho et al. [106]

IBS F/M = 9/8

IBS-D = 11 IBS-A = 6

fMRI

Labus et al. [107]

IBS F/M = 24/ 22

Not reported

[15O]H2O PET

(−) correlation between anticipatory BOLD decreases in DBS and self-rated measures of (−) affect. Association between amplitude of the anticipatory decrease in the pontine portion of DBS and greater activation during distention in R orbitofrontal cortex and bilateral subgenual ACC

Agonist-induced symptom improvement was associated with lower distension-induced activity in bilateral orbital frontal cortex and the L middle temporal gyrus at baseline. IBS patients with lower levels of self-reported interpersonal sensitivity were also more likely to improve with treatment, and to have less activity in the L orbital frontal cortex during rectal distension. M IBS vs. F IBS: Emotional-arousal network: During expectation (EXP) of rectal balloon inflation(INF), the amygdala → subgenual ACC and amygdala → pons circuits showed greater (+) connectivity for IBS Fs and (−) connectivity in M IBS. Homeostatic-afferent network: During EXP, INS connectivity to medial orbital frontal cortex was consistently (−) in IBS M and more (+) in IBS Fs. Cortical-modulatory network: During baseline and EXP, IBS M showed greater (+) connectivity between the posterior INS → amygdala and IBS Fs shower decreased connectivity of this circuit. During INF and EXP, IBS Fs had strong (+) connectivity between medial orbital frontal cortex → amygdala, whereas IBS Ms had weak (−) connectivity in this circuitry.

44  SECTION | A  Foundations of neurogastroenterology and motility

TABLE 1  MRI studies in IBS—cont’d



All F: IBS = 10 HC = 10

Not reported

fMRI

IBS vs. HC: No group differences, but IBS had trends for greater PCC activation during painful distention in the L and R hemisphere. Abuse vs. Nonabuse: Subjects with history of abuse had greater activation in the L midcingulate cortex and the L PCC during painful distention. IBS with Abuse vs. All Others: IBS with history of abuse had greater activation in the L mid-cingulate cortex and the L PCC, with a trend in the R PCC during painful distention. IBS with history of abuse had decreased activation in the subgenual ACC.

(+) correlation between pain reports during 50mm Hg rectal distention and activation of the L mid-cingulate cortex (r = 0.75).

Price et al. [109]

All Fs IBS = 9

IBS-D = 6 IBS-C = 3

fMRI

Large reductions in pain and in brain activity within the thalamus, somatosensory cortices, INS, and ACC during the placebo conditions.

(+) correlation between brain responses and pain ratings in the secondary somatosensory, INS, and ACC.

Craggs et al. [110]

All Fs: IBS = 9

IBS-D = 6 IBS-C = 3

fMRI

Functional interaction with a “cognitive-affective” network comprising of anterior mid cingulate cortex, supplementary motor area, dorsal lateral PFC, and anterior and posterior INS change with placebo analgesia.

Lawal et al. [111]

All Fs: IBS = 10 HC = 10

IBS-D = 10

fMRI

Cerebral activity during subliminal distention observed in sensory/motor, the parietal/occipital, the cingulate gyrus, the PFC, and the INS cortex.

Naliboff et al. [112]

IBS F/M = 14/6

IBS-C = 4 IBS-D = 6 IBS-A = 10

[15O]H2O PET

After repeated visceral distention IBS (Ms + Fs) showed decreased activity in subgenual and midcingulate cortex, precuneus, and PCC. Decreased activity in amygdala, mid-cingulate cortex, perigenual cingulate, and dorsal brainstem during anticipation condition at 12 months.

Song et al. [113]

All F: IBS = 12 HC = 12

IBS-C = 6 IBS-D = 6

fMRI

IBS vs. HC: HC had decreased rectal pain scores during heterotopic stimulation. HC had greater activation bilaterally in the anterior INS, SII and putamen during rectal stimulation compared to rectal plus heterotopic stimulation. HC had greater activation in primary sensory cortex and the R superior temporal gyrus and IBC had greater activation in the R inferior lobule and bilaterally in the superior temporal gyrus during rectal plus heterotopic.

Andresen et al. [114]

IBS F/M = 5/3 HC F/M = 3/5

IBS-D = 5 IBS-A = 3

fMRI

IBS vs. HC: IBS patients had lower activity in the PFC and ACC to both subliminal and supraliminal stimulation and higher activity in the hippocampus to supraliminal stimulation. In IBS patients, decreased ACC and PFC activation with subliminal and supraliminal rectal stimuli and increased hippocampus activation with supraliminal stimuli. In IBS patients, not in controls, ACC and hippocampus were also activated by auditory stimulation.

Kwan et al. [115]

IBS F/M = 6/3 HC F/M = 7/ 4

Not reported

fMRI

IBS vs. HC: IBS had urge-related responses in the primary sensory cortex. IBS had pain-related responses in the medial thalamus and hippocampus. HC had urge- and pain-related activations in the R anterior INS and the R anterior.

Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  45

Ringel et al. [108]

Continued

Publication

Population

IBS subtype

Imaging modality

Group difference results

Morgan et al. [38]

F IBS = 19

IBS-C = 7 IBS-D = 11 IBS-A = 4

fMRI

Activation of perigenual ACC, R INS and R PFC during rectal pain. Low dose tricyclic antidepressant decreased pain related cerebral activations in the perigenual ACC and L posterior parietal cortex during stress.

Nakia et al. [116]

IBS F/M = 4/6

[α‐[11C] methyl‐l‐ tryptophan‐ PET

5-HT antagonist vs. Placebo: M IBS had greater 5-HT synthesis in parahippocampal gyrus and cerebellum. F IBS had greater 5-HT synthesis in parahippocampal gyrus. 5-HT antagonist: IBS M > F L: medial frontal gyrus, superior frontal gyrus, inferior temporal gyrus, medial occipital gyrus, PCC; R: precuneus, fusiform gyrus, cerebellum. Placebo: M > F L: precentral gyrus, middle frontal gyrus, superior frontal gyrus, inferior occipital, superior and medial temporal gyrus; R: PCC, cerebellum. F > M L: medial frontal gyrus, inferior frontal gyrus; R: anterior mid-cingulate cortex.

Lieberman et al. [43]

IBS F/M = 13/10

[15O]H2O PET

Greater placebo induced reductions in bilateral anterior mid-cingulate and increases in R ventral lateral PFC during distention were associated with greater selfreported symptom improvement. Relationship between R ventral medial PFC and symptom improvement was mediated by bilateral ACC activity.

Nakia et al. [117]

IBS F/M = 6/6 HC F/M = 6/6

α-[11C] methyl-ltryptophan PET

F IBS compared to F HCs had higher rates of serotonin synthesis in the R medial temporal gyrus.

Naliboff et al. [118]

IBS F/M = 23/19

Not reported

[15O]H2O PET

M IBS vs. F IBS: F patients had greater activation in amygdala and ACC. M patients had greater activation in dorsal lateral PFC, dorsal pons/PAG and mid posterior INS.

Ringel et al. [119]

All F: IBS = 6 HC = 6

Not reported

[15O]H2O PET

IBS vs. HC: IBS had lowered greater in L ACC but higher thalamic activity during distention. Nonabused vs. Abused: Nonabused (N = 7) had greater activity in the L ACC during distention. Abused (N = 4) had no increase in ACC during distention.

IBS-D = 23

Brain-symptom correlations in IBS

For IBS, (+) correlation between thalamic activity and stimulus intensity.

46  SECTION | A  Foundations of neurogastroenterology and motility

TABLE 1  MRI studies in IBS—cont’d



Verne et al. [120]

IBS F/M = 6/3 HC F/M = 6/3

IBS-C = 3 UBS-D = 6

fMRI

IBS vs. HC: IBS had greater activation in the INS, cingulate cortex, and PFC for the 35 mmHg rectal distention. IBS had greater activation in the ventral posterior lateral and dorsomedial thalamus, INS, somatosensory cortex, cingulate cortex, and PFC for the 55 mmHg rectal distention. IBS had greater activation in the INS, somatosensory cortex, and the cingulate cortex during immersion of the R foot in 45°C water, and greater activation in the dorsomedial lateral thalamus, INS, somatosensory cortex, cingulate cortex, and the PFC during immersion of the R foot in 47°C water.

Berman et al. [121]

IBS F/M = 26/23

IBS-D = 30 IBS-A = 19

[15O]H2O PET

Serotonin antagonist dampened activity in emotional arousal and autonomic, reward and pain during baseline and the expectation condition only. No differences observed during rectal distention.

Bernstein et al. [122]

IBS F/M = 4/10

Not reported

fMRI

IBS vs. HC: IBS had lower activity in ACC during distention. IBS vs. IBD: IBS had greater percentage of pixels activated in the ACC over pain and stool conditions. IBS had greater deactivation of L somatosensory cortex.

Bonaz et al. [123]

IBS F/M = 10/1

IBS-C = 6 IBS-D = 3 IBS-A = 2

fMRI

Significant deactivation in the R hemisphere, within posterior INS cortex, amygdala, and striatum.

Mayer et al. [121]

IBS F/M = 26/23

IBS-D = 30 IBS-A = 19

[15O]H2O PET

Compared to placebo, Alosetron, a serotonin antagonist dampened activity in amygdala, ventral striatum, subgenual ACC, hypothalamus, dorsal pons/periaqueductal gray, midcingulate cortex, anterior INS, and thalamus.

Naliboff et al. [124]

IBS F/M = 2/10 HC F/M = 2/10

IBS-D = 7 IBS-A = 5

[15O]H2O PET

IBS vs. HC: IBS showed lateralized activation of R PFC. IBS had decreased activation of perigenual cortex, temporal lobe, and brain stem. IBS had greater activation of rostral ACC and PCC.

Berman et al. [125]

IBS F/M = 13/17

[15O]H2O PET

M IBS vs. F IBS: Ms had greater regional activations. Rectal pressure activated the INS bilaterally in the Ms but not in Fs.

Correlation between INS activation & objective visceral pressure. Correlation between ACC activation and ratings of subjective discomfort.

Mertz et al. [126]

IBS F/M = 16/2 HC F/M = 14/2

Not reported

fMRI

IBS vs. HC: IBS had greater number of pixels activated in the ACC and greater intensity of pain at 55 mmHg distention.

Subjective pain intensity ratings correlated with and ACC activation in HCs but not IBS.

Silverman et al. [127]

IBS = 6 HC = 6

IBS-C = 3 IBS-D = 2 IBS-A = 1

[15O]H2O PET

IBS vs. HC: HC had activation of ACC during actual delivery of painful pressure and during simulated delivery of painful stimuli. IBS had no ACC activation, but L dorsolateral PFC activation.

Reduced activity in amygdala ventral striatum, and dorsal pons were correlated with IBS symptom reduction.

Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  47

ACC, anterior cingulate cortex; ALFF, amplitude low frequency fluctuations; CRF, corticotropin releasing factor; FC, functional constipation; HC, healthy control; IBS, irritable bowel syndrome; INS, insula; L, left; R, right; PCC, posterior cingulate cortex; PFC, prefrontal cortex; PV, provoked vestibulodynia; UCPP, ulcerative colitive/pelvic pain. IBS subtypes: D, diarrhea; C, constipation; M, mixed; A, alternating; U, unspecified. This table comprises all published IBS neuroimaging manuscripts in adults diagnosed with irritable bowel syndrome (IBS) from 1997 to 2018. Magnetic Resonance Imaging modalities include functional (fMRI), resting state (rsMRI), structural (sMRI), and Diffusion Tensor Imaging (DTI).

48  SECTION | A  Foundations of neurogastroenterology and motility

FIG. 3  Sensorimotor network. The homeostatic afferent network considered part of the sensorimotor network responsible for central processing and modulation of viscerosensory and somatosensory information.

FIG. 4  Emotional arousal network. This network acts as an important link between stimulus appraisal and autonomic nervous system activity with the gut, and is key for determining the magnitude and duration of autonomic modulation of various gut functions. ACC, anterior cingulate cortex; MCC, ­mid-cingulate cortex.

Using rsMRI, a set of interacting canonical brain networks associated with specific behavioral domains (i.e., emotion, executive control, attention, sensorimotor processes) have been identified [132, 133]. Importantly, resting state and task-evoked networks closely correspond suggesting that these networks are continuously at work even in the absence of a specific task [133]. Across evoked and resting state studies, IBS patients compared to HCs demonstrate functional alterations in regions comprising default mode (DMN) [53, 57, 65, 134–136], emotional arousal (closely related to the central autonomic network) [59, 60, 64, 70, 73, 84, 107, 128], central executive [53, 60, 67, 93], sensorimotor processing (including the homeostatic afferent network) [53, 59, 70, 73, 84], and salience [59, 60, 79, 84] networks. Mayer et al. provide a complete review of these networks and their relevance in IBS [6]. Activity in these networks has shown small to moderate correlations with self-reported symptoms, cognitive functioning and mood measures. In addition, the intrinsic connectivity of regions compromising sensorimotor and salience networks have been associated with rectal balloon distention perception thresholds [53], while the intrinsic connectivity of the default mode network during rest has been associated with gut permeability [136]. These networks are all involved in the processing and response to visceral afferent signals and may underlie the disordered information processing reported in patients with pain disorders like IBS, such as biased threat appraisal (catastrophizing) and expectancy of outcomes (e.g., salience network), autonomic hyper-arousal (emotional arousal and central autonomic networks), and symptom focused attention (central executive network) [137].

Structural imaging findings Studies of gray matter morphometry in IBS compared to HCs have reported both decreases and increases in the morphometry of regions comprising brain networks reported as altered in functional imaging studies [48, 78, 80, 86, 89, 101, 103]. These alterations have been shown to correlate with associated pain inhibition, disease duration, symptom severity, early adverse life events and mood, each highlighting potentially different pathophysiological mechanisms. In addition to



Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  49

differences in regional morphometry, alterations in the architecture of structural networks have also been shown to differ between HC and IBS, supporting structural reorganization of cortical and subcortical regions [80]. Data-driven analyses using supervised learning indicates that scores on two brain signatures comprising the morphometry of (1) primary somato-sensory and motor regions, and (2) multisensory, emotional arousal and salience regions can discriminate IBS versus HC with a predictive accuracy of 70%. Although not sufficient for diagnosis, these finding underscore the existence of structural brain alterations in IBS, the role of the sensory and motor brain regions, and the utility of data-driven analyses.

White matter findings Generally speaking, studies have reported microstructural alterations consistent with increased strength of axonal or dendritic projections and increased myelination in sensorimotor, corticothalamic, and basal ganglia circuits involved in sensorimotor integration and pain processing. Patients with IBS compared to HCs show 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. Patients with IBS also demonstrate reduced MD within the globus pallidus and higher MD in the thalamus, internal capsule, and the part of the coronal radiata projecting to sensory/motor regions, suggestive of differential changes in axon/dendritic density in these regions [83]. Differences in the mean FA of the left and right viscerotopic portions of the primary somatosensory cortex (S1) have also been reported [69]. Only studies with small samples (IBS <20) have reported alterations in white matter of tracts associated with emotional processing [50, 97]. Together, these results support the hypothesis that patients with chronically recurring visceral pain from IBS have long-term microstructural changes within the brain, particularly in regions associated with pain processing and integration of sensorimotor information.

Sex-specific brain alterations in IBS Brain imaging studies provide evidence for sex differences within brain structure and connectivity, as well as responses to evoked stimuli in IBS. sMRI studies in female or female-predominant samples have found small to moderate (d=0.30 to 0.60) effect size differences in regional gray matter morphometry between IBS and HCs that parallel the alterations reported in the aforementioned disease-relevant networks, providing some insight into brain structure-function relationships in IBS [48, 57, 78, 80, 86, 89, 101, 103]. Sex differences in IBS-related structural brain alterations have been observed, largely involving salience and sensorimotor regions. Female, but not male, patients with IBS demonstrate increased sensorimotor and decreased insular and subgenual ACC cortical thickness compared to same-sex HCs [86]. Female patients demonstrate microstructural alteration in sensorimotor-related tracts involving the thalamus, primary sensory and motor regions, and the corona radiata (associated with the corticospinal, corticopontine, and corticobulbar tracts) compared to that in male patients, while these sex differences are not seen in HCs [83]. rsMRI studies further demonstrate sex differences in sensorimotor, salience and emotional-arousal alterations in IBS. Female patients show greater lower frequency power in the sensorimotor cortex and higher frequency power in the insula and amygdala compared to that in male patients and same-sex controls [138]. Furthermore, sex influences the nature of insula intrinsic connectivity changes in IBS [77, 79, 84]. In particular, altered connectivity between the insula and default mode network may be more relevant to female patients than male patients, suggesting sex differences in internally-directed resources in response to stressful and salient events [79]. Moreover, sex differences in ACC functional connectivity have been reported, with enhanced ACC connectivity with emotional-arousal regions such as the amygdala and hippocampus in female patients compared to that in male patients [77, 87, 107]. In contrast to non-evoked studies, evoked-pain studies do not show sex differences in sensorimotor reactivity. However, sex differences in the reactivity of salience regions have been observed. Insula pain-related responses appear to be enhanced to a greater extent in male patients compared to that in female patients [87, 125]. In contrast, ACC pain-related responses may be enhanced to a greater extent in female patients compared to male patients [118]. However, male patients with IBS have demonstrated greater ACC reactivity to emotional stimuli previously shown to elicit greater behavioral and brain responses in healthy male subjects compared to healthy female subjects [87]; thus, male and female patients may have similar or analogous changes in emotional-arousal reactivity, with sex-specific triggers. Overall, these studies show that sex influences IBS-related alterations in the structure and functional organization of salience, sensorimotor, and emotionalarousal regions, as well as the reactivity of salience and emotional-arousal regions.

Comparison with other chronic pain disorders To determine shared and distinct mechanisms in IBS, recent studies have added chronic pain control comparison groups. For example, in IBS compared to provoked vestibulodynia, distinct alterations have been reported in (1) the intrinsic connectivity of default mode, salience, and sensorimotor network [66]; (2) gray matter volume of sensorimotor cortices,

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posterior insula and thalamus [139] and (3) in the microstructural properties of basal ganglia and the internal capsule in a region adjacent to the pallidum, which contains cortico-spinal and thalamo-cortical fibers [46]. Compared to patients with urologic chronic pelvic pain syndrome, IBS patients show distinct differences in the white matter properties of the anterior thalamic radiation, the pallidum in the basal ganglia, and the corona radiata, which project to and from the cortex, along with projections to the internal capsule [61, 75]. These results suggest IBS-specific sensorimotor-thalamic-brainstem connectivity and perturbations.

Brain imaging, genetics and signaling systems underlying symptoms and pathophysiology in IBS The research on the influence of neurotransmitters in modulating brain networks in IBS is still in its infancy. Much work has focused on chronic stress system activation in IBS capitalizing on the fact that the brain communicates through the gut via the autonomic nervous system and the HPA axis, mediating behavioral, autonomic, and neurochemical responses to stress [140]. Using imaging genetics, the interactions of early adversity with stress-related gene polymorphisms and regional brain structure in HC and IBS females have been documented [141]. In this study, the combined genetic ­variation in the glucocorticoid receptor NR3C1 gene and the interleukin 1 beta (IL-1β) gene interacted with early adverse life events to influence the thickness of subgenual ACC. Also, the greater prevalence of early adverse life events in the IBS group was thought to play a role in the reduced thickness of subgenual ACC in IBS patients, who had less common NR3C1 haplotypes, and were homozygous for the major IL-1β allele. These findings support an interaction between genetic polymorphisms related to stress and inflammation with early adverse life events and brain structure. Distinct gene expression profiles in peripheral blood mononuclear cells (PBMCs) associated with increased sympathetic nervous system activity during chronic stress [142–145] have also been investigated in IBS compared to HCs. These gene expression profiles involve pro-inflammatory and anti-inflammatory genes which have been found to have IBS-specific associations with the intrinsic connectivity of the salience network. Based on these results, the authors suggest that IBS patients may have chronically activated stress signaling pathways that maintain a pro-inflammatory state in the periphery. Specifically, primary alterations in the brain’s salience network may underlie chronically increased sympathetic nervous system outflow in IBS, which in turn contributes to the generation of a conserved transcriptional response to an adversity gene expression pattern [146] in peripheral immune cells [52]. MR spectroscopy has shown reductions in hippocampal glutamate–glutamine in IBS compared to HCs and these reductions are associated with emotional stress indicators in IBS patients. The authors interpreted these findings as suggesting dysregulation of inhibitory hippocampal feedback on the HPA axis [147].

Corticotropin-releasing factor signaling Many studies have looked at corticotropin-releasing factor (CRF), which has a central role in the stress response regulating the HPA axis [148], and mediates behavioral, autonomic, and neurochemical responses to stress [140]. For example, consistent with a prior EEG study [20], a corticotropin releasing hormone (CRH) receptor agonist was shown to increase colorectal distention-induced activity in the amygdala, a key emotional-arousal region in HCs. In IBS compared to HCs, CRH administration was reported to be associated with increased activation of the amygdala at rest and less response in the amygdala during colorectal distension. The authors suggested this might be due to greater receptor density or an increased receptor affinity for CRH in this brain region [62]. Using this same experimental design with fMRI, a negative association between ACTH response to CRH and activity in the perigenual anterior cingulate cortex during rectal distention was reported in HCs but not in IBS patients and interpreted as suggesting an impaired top-down inhibitory input from the perigenual ACC to the HPA axis. This could potentially lead to altered neuroendocrine and gastrointestinal responses to corticotropin releasing hormone [55]. Using an orally administered CRH antagonist and fMRI, it has also been shown that CRF signaling via CRF receptor 1 is involved in fear acquisition. Also extinction learning is upregulated in patients with IBS [88]. These mechanisms have a central role during pain expectation, and a CRF1 antagonist dampens regional activity and engagement of the emotional-arousal network in IBS [99].

Noradrenergic signaling pathways A single study has used pharmacological manipulation of central α2A receptors to study the effects of the central noradrenergic signaling on the brain in IBS [149]. This study reported alterations in emotional arousal, as well as attention and sympathetic nervous system activity between patients with IBS and HCs, and highlighted the complex interaction between norepinephrine, CRF, and serotonin. Related to noradrenergic signaling, catechol-o-methyltransferase [COMT] is involved in the metabolism of both catecholamines (epinephrine, norepinephrine, and dopamine) and enkephalins and has been



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implicated in dysregulation of pain modulatory systems in chronic GI pain [149, 150]. A single imaging genetics study has examined single nucleotide polymorphisms (SNPs) of the catecholaminergic signaling systems, specifically genes encoding COMT in IBS [74]. In this study, catecholaminergic SNPs were associated with IBS symptom severity and gray matter alterations in specific brain regions involved in sensory processing and emotional arousal. In addition, this study reported a complex effect between catecholaminergic SNPs, early adverse life events and diagnosis on brain morphometry.

Neurokinin-1 receptor signaling Two studies in patients with IBS have investigated substance P, a neuropeptide that modulates effects of acute or chronically recurring physical and emotional distress and acts centrally and peripherally through neurokinin-1 receptors (NK-1Rs). Tillisch et al. [96] reported that inhibition of the NK1R decreased anxiety and dampened activity in regions comprising the emotional arousal network in response to noxious visceral distension in women with IBS. In a related PET ligand study quantifying the availability of NK-1Rs, Jarcho et al. [85] found that IBS patients had diminished NK-1R availability relative to HCs, but greater availability than IBD patients. Patients with longer duration of IBS had the lowest NK-1R availability in putamen, caudate nucleus, and insula, regions involved in sensory integration and pain processing.

Serotonin signaling system Serotonin (5-hydroxytryptamine; 5-HT) is a key neurotransmitter in the enteric nervous system and the CNS and is thought to be critical for gut–brain communication. Importantly, treatments targeting 5-HT receptors, have been found to be beneficial in IBS. The first study to examine the central role of the serotonin system in IBS utilized PET to measuring brain activity during baseline, rectal distention, and anticipation of undelivered rectal distention before and after a randomized, placebo-controlled, 3-week trial of a 5-HT3 receptor antagonist. This antagonist had previously been shown to improve diarrhea, pain, and global symptoms in women with IBS. Compared with placebo, the 5-HT3 antagonist dampened activity in emotional arousal (ventromedial prefrontal cortex, subgenual cingulate, amygdala) and autonomic (hypothalamus), reward (ventral striatum) and pain (dorsal pons/periaqueductal gray) regions, both during baseline and during expectation of painful stimulus [121, 151]. Reduced activity in amygdala, ventral striatum, and dorsal pons was correlated with IBS symptom reduction. These finding suggested that the symptom improvements may be due, in part, to the antagonists inhibitory effects on brain regions associated with emotional arousal and expectation. Using α-[11C]methyl-l-tryptophan PET imaging, studies have reported that women with IBS compared to HCs have greater 5-HT synthesis rates in the medial temporal gyrus [117]. Furthermore, after a randomized, placebo-controlled 2-week trial of a 5-HT3 receptor antagonist, a significant sex and treatment interaction was observed in 5-HT synthesis rates during rectal distension in the basal ganglia, fusiform gyrus and the posterior and subgenual cingulate cortices. Furthermore, regardless of treatment, sex differences in 5-HT synthesis during rectal distension were observed in regions associated with the default mode, visual processing, and cognitive control networks [116]. This observation provides further support for the role of the 5-HT signaling system in IBS. Labus et al. [152] demonstrated that lowering 5-HT levels via acute tryptophan depletion led to a normalization of the effective connectivity pattern for the emotional arousal circuitry in patients with constipation predominant IBS. A handful of imaging genetics studies has examined the role of 5-HT gene polymorphisms in IBS. Fukudo et al. [153] studied the effect of 5-HT transporter gene-linked polymorphic region (5-HTTLPR) in HCs undergoing a PET study assessing brain response to rectal distention. Individuals with the s allele of 5-HTTLPR had lower transcriptional efficiency of the promoter than those with the l allele, resulting in lower 5-HTT expression and lower cellular uptake of 5-HT to presynaptic nerve terminals in serotonergic neurons. This study indicated that patients with s allele compared to those with the l allele had greater activity in regions comprising the emotional-arousal network. A follow-up study in males only indicated that that males with weaker 5-HT transporter functioning showed alterations in the effective connectivity of the emotional arousal network during aversive visceral stimuli compared to those with l carriers [154]. Kilpatrick et al. [154] examined the effect of a polymorphism of relatively common variant c.−42C>T, a region of the 5-HT3 receptor type gene (HTR3A), on brain response during the matching of emotional faces and the matching of forms in HCs and IBS. Regardless of diagnosis, the C/C genotype of the c.−42C>T polymorphism, compared with T carrier status, was associated with increased anxiety and amygdala responsiveness during emotional and non-emotional tasks. In IBS, the C/C genotype was associated with severity of symptoms. This study supports the notion that 5-HT signaling modulates the responsivity of the amygdala in IBS, a key region of the emotional arousal network. Together these studies highlight the critical role of neurotransmitters in the functioning of emotional arousal, cortical inhibition, salience, and sensorimotor networks involved in processing signals from visceral afferents in IBS. Further s­ tudies

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are needed to determine the effect of alterations in signaling system on brain morphometry and white matter properties. Overall, brain imaging provides a powerful tool for examining molecular mechanisms underlying the gut–brain axis.

Brain-gut-microbiome axis Brain imaging has emerged as a valuable tool to study the brain-gut-microbiome axis. A growing body of preclinical literature has demonstrated bidirectional signaling between the brain and the gut microbiota, which may involve multiple neural (vagal afferents, enteric nervous system), metabolic (bacterial components and their metabolites), endocrine, and immunerelated signaling mechanisms. In turn, the brain can influence microbial composition and function via endocrine, immune and neural mechanisms [155]. Functional brain changes in response to a probiotic intervention were first studied in healthy women [156]. Women ingesting the probiotic twice daily for 4weeks showed altered engagement in a network comprised of sensorimotor regions (including the posterior insula), the basal ganglia, periaqueductal gray, and inhibitory cortical regions (ventral lateral prefrontal and dorsal frontal cortex). Recently, in response to a 6 week probiotic treatment that reduced depression scores, IBS patients showed reduced brain activity while viewing emotional stimuli in the amygdala, a key emotional arousal region, similar to the inhibitory cortical regions reported previously, including temporal and occipital regions [157–158]. Examining gut microbial profiles in healthy women indicated that 2 microbial-based clusters of women could be identified, a Bacteroides-dominant cluster and a less common Prevotella-dominant cluster. Compared to the Bacteroidesdominant group, the women with a Prevotella-dominant gut profile showed greater behavioral responses to negative emotional stimuli, and this response was associated with both functional and structural differences in the hippocampus [157]. In IBS, distinct microbial profiles have been demonstrated and found to be associated with morphometric alterations in sensorimotor cortices including posterior and anterior insula and the basal ganglia [56]. In addition, specific microbial taxa, Clostridia belonging to phylum Firmicutes and Bacteroidia belonging to phylum Bacteroidetes, show moderate-sized associations with several sensorimotor integration regions including the thalamus, basal ganglia (caudate nucleus, putamen, pallidum, nucleus accumbens), motor cortex, anterior insula and ventral prefrontal regions. Metagenes enriched in the IBS-specific microbial profile were also found to be associated with the morphometry of the posterior insula, the core viscerosensory/interoceptive region. Furthermore, abundance of Clostridia from the gut microbiome in IBS has been shown to correlate with functional and structural organization in sensorimotor regions including the posterior insula and basal ganglia, as well as evoked symptoms (e.g., transit time, rectal perception threshold) [158]. In sum, these studies support the importance of investigating the role of the microbiome in gut-brain communication.

Current limitations Although much progress has been made over the course of two decades of brain imaging research in IBS, there remains room for improvement. First and foremost, despite the known issues with reliability of small sample research, most brain imaging studies in IBS remain underpowered (Table 1). As can be seen in Fig. 5, 10–20 subjects per group does not provide the power necessary to detect moderate Cohen's d >= 0.50 effect size differences commonly observed in brain imaging studies. Furthermore, negative findings from underpowered small sample studies are uninformative and the reliability of significant findings are questionable at best. Large samples should be the rule not the exception. As the financial burden of imaging research remains quite high, multi-site collaborations as well as increased contributions toward and use of brain imaging data repositories such the Pain and Interoception Imaging Network (PAIN) repository [152] is crucial for maximizing the benefits and minimize the costs of data sharing. The diversity of inclusion criteria, imaging protocols, and choice of outcomes across neuroimaging studies in DGBIs has also made comparisons between studies difficult. Greater international collaboration to set standards for this research could also advance the field. Furthermore, most research has been performed in all female or predominantly female samples with less attention to men with IBS, despite the high prevalence of the disorder in men (Table 1). Given initial findings regarding sex-differences it is important that analyses do not combine men and women in analyses but analyze them separately. From a statistical point of view, findings from studies using sex as a covariate in mixed uneven samples (e.g., 80% female, 20% male) do not provide an accurate assessment of brain alterations in IBS, many of which are sex-specific. Also, many studies report sex differences based on within sex comparisons between IBS and HCs and not more appropriately on direct comparisons between male and female IBS. Few studies have investigated IBS in pediatric and adolescent samples [129, 130, 159–162]. Finally, there is glaring lack of developmental and longitudinal studies that are critical for determining whether brain alterations are the result or consequence of having IBS.



Neuroimaging and biomarkers in gastrointestinal disorders Chapter | 3  53

FIG. 5  Effect size detection threshold as a function of sample size. This analysis is based on a two-tail independent t-test of group means, N1/N2 allocation=1, alpha=0.05, and 80% power.

Moving forward and the path ahead As research continues, it is critical to bear in mind that great heterogeniety exists in the clincal presentation of IBS specifically, and in DGBIs more generally. As such continued efforts at investigating differences in known patient subgroups such as those based on features such as measurable pain hypersensitivity [72], bowel habit, or microbial profiles is important. It will also be important to evaluate aspects of DGBIs which may identify common features that cross over different subtypes of DGBIs as well as non-GI pain disorders—factors such as stress sensitivity, presence or absence of wide-spread pain, or psychological symptoms. More studies are needed to investigate the role of neurotransmitters in IBS brain alterations and to validate findings from single neurotransmitter studies. Hopefully in the future molecular studies will be a powerful tool for furthering our understanding of mechanisms underlying DGBIs.

Conclusions In summary, neuroimaging research has advanced our understanding of IBS and DGBIs by delineating symptom-associated brain alterations along with their molecular, genetic, and microbial associations. This research has ushered in a paradigm shift with regard to the way we conceptualize the disorder, from a peripheral condition with no organic markers to a ­condition characterized by CNS dysfunction. This research not only provides information on underlying pathophysiological mechanisms but provides objective targets for non-pharmacological and pharmacological treatment studies. Ultimately combining the brain imaging and other biological markers will prove most successful in advancing our understanding of the DGBIs.

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