The Modulation of Cerebral Pain Responses Using Desipramine in the Treatment of Irritable Bowel Syndrome (IBS)
Phase: NA
Conditions: Irritable Bowel Syndrome
Sponsor: Washington University School of Medicine

 
 
The Modulation of Cerebral Pain Responses Using Desipramine in the Treatment of Irritable 
Bowel Syndrome (IBS) 
 
Gregory Sayuk, MD 
 
 
NCT00880594 
 
Protocol Version Date:  8/15/2013

The Modulation of Cerebral Pain Responses Using Desipramine in the Treatment of Irritable 
Bowel Syndrome (IBS) 
 
 
7.5.4: Research Plan 
A. Statement of Hypotheses and Specific Aims 
 
Irritable bowel syndrome (IBS) is a common abdominal pain disorder diagnosed by symptom-based criteria 
(Rome III).  Since there are no objective measures available to establish a formal diagnosis, IBS likely 
encompasses a biologically heterogeneous group of patients with distinct pathophysiology. The majority of 
studies examining the mechanistic basis of IBS have focused on bowel-specific factors.  Recent neuroimaging 
studies using functional magnetic resonance imaging (fMRI) have demonstrated abnormal brain activation 
patterns with noxious visceral stimulation in some, but not all, IBS patients.  Regions comprising the homeostatic 
afferent processing network (HAPN), important to cognitive and affective interpretation of pain signals, 
demonstrate striking visceral pain response abnormalities in many IBS subjects. It is possible that HAPN pain 
responses have pathophysiologic relevance in a subset of IBS patients. We thus seek to characterize a putative 
IBS subgroup—those with abnormal HAPN visceral pain activations (IBS-HAPN). 
A clearer understanding the role of the HAPN in IBS pathophysiology would be of substantial clinical 
importance on two accounts.  First, centrally-acting antidepressant therapies, shown to influence the HAPN 
responses to visceral pain, are effective in only one-half of IBS subjects. The identification of objective features 
which predict IBS response to antidepressant therapy (i.e., HAPN pain responses patterns) would potentially 
allow for the development of more rational IBS treatment approaches.  Second, abnormal HAPN pain activations 
may have broader implications in IBS pain experiences.  One-third of IBS patients have comorbid somatization, a 
condition exemplified by multiple unexplained pain syndromes.  It is possible that in IBS patients with 
somatization, the abnormal HAPN activations observed with visceral stimulation may also occur with non-visceral 
(somatic) noxious stimuli. 
The overarching hypothesis of this proposal is that aberrant brain responses to visceral pain signals 
within the homeostatic afferent processing network (HAPN) represent the primary mechanism 
responsible for bowel pain experiences in a subset of IBS patients (IBS-HAPN).  Our preliminary data has 
identified a pattern of brain activation differences within the HAPN following visceral stimulation in IBS subjects 
compared to healthy controls. Using fMRI, I will further characterize the HAPN pain responses of IBS-S subjects 
to additional non-visceral stimuli and following antidepressant therapy. I predict that this protocol will reveal a 
pattern of increased HAPN pain activations to both visceral and somatic stimuli—activations that are refractory to 
antidepressant pharmacotherapy. Ultimately these findings will demonstrate the mechanistic importance of the 
HAPN to pain experiences in the IBS-S subgroup. 
 
These goals will be realized through the pursuit of the following Specific Aims:  
 
Specific Aim 1. To compare HAPN activations following noxious somatic stimulation in IBS subjects 
based on the presence of somatization. 
Hypothesis 1:  As a result of a generalized abnormal pain response within the HAPN, IBS-S subjects will 
demonstrate greater HAPN activations with noxious somatic stimuli, compared to IBS subjects with no 
somatization and healthy controls. 
   
Specific Aim 2. To compare the effects of antidepressant therapy HAPN activations following noxious 
visceral stimulation in IBS subjects based on the presence of somatization. 
Hypothesis 2: Consistent with clinical refractoriness to antidepressant treatment, IBS-S subjects will demonstrate 
a persistent pattern of abnormal HAPN visceral activations following desipramine therapy, whereas significant 
improvements in HAPN visceral activations will be observed in IBS subjects with no somatization following 
antidepressant therapy. 

 
 
B. Background and Significance. 
 
1. Irritable bowel syndrome (IBS) is biologically heterogeneous, symptom-based diagnosis. 
Irritable bowel syndrome (IBS) is recognized as a cluster of functional bowel symptoms, predominantly 
abdominal pain, in association with a change in bowel habits. 10-20% of US adults have IBS, and the diagnosis 
accounts for 20-50% of gastroenterology referrals.4  IBS results in significant functional impairments, imposing a 
major impact on affected patients’ assessments of their overall global health.5  Without identifiable 
pathophysiologic findings upon which a diagnosis can be 
established, IBS remains a diagnosis based on clinical symptoms 
(Rome III criteria).4 Over the past four decades, studies have 
examined multiple processes as potentially relevant to IBS 
pathophysiology, including gut inflammation, bowel motility, and 
visceral hypersensitivity. While many of these putative abnormalities 
have been found to be more prevalent in IBS compared to control 
populations, each measure likewise been insufficient in explaining 
IBS in all patients. 
When studied as a syndrome—a collection of nonspecific 
symptoms—IBS almost certainly represents a very heterogeneous 
condition 
with 
a 
multitude 
of 
underlying 
pathophysiologic 
mechanisms (Figure 1).4  It is likely that many of the previously 
proposed mechanisms underlying IBS are relevant in a subset of IBS 
patients. To date, however, the majority of clinical and physiologic 
studies of IBS have examined IBS subjects as a single group. The 
notion that important IBS subgroups likely exist has been largely 
disregarded in previous IBS investigations, and efforts to identify clinically- or physiologically-relevant subsets of 
IBS patients have been few. As a consequence, the biological basis for IBS remains largely unknown. 
Establishing mechanistically-relevant IBS subgroups will be paramount to furthering our understanding IBS 
pathogenesis. 
 
2. Somatization is an important clinical feature in IBS, and portends greater IBS morbidity. 
Somatization is a condition defined by the expression of pain syndromes across multiple organ systems (bowel, 
musculoskeletal, etc.) in the absence of any identifiable physical abnormality to explain the reported pain.  It is 
likely that the multiple pain symptoms associated with somatization are 
the result of a common abnormal CNS interpretation of peripheral pain 
signals, rather than a simultaneous sensory malfunction of several 
organ systems.6-9 We and others have found that 25-42% of IBS 
subjects attending gastroenterology referral clinics have identifiable 
somatization with standard instruments (PHS-15) and medical 
history.10, 11 Clinically, the presence of somatization in IBS patients 
(IBS-S) bears prognostic importance: compared to IBS patients without 
somatization (IBS-No S), IBS-S subjects report significantly greater 
numbers of: a) GI and non-GI symptoms, b) missed work days, and c) 
urgent care visits and physician consultations.12-17  Recently we 
demonstrated somatization in IBS predicts both more intense 
abdominal pain experiences, and poor bowel symptom responses to 
centrally-active pharmacotherapies (e.g., tricyclic antidepressants). 
We found that IBS-S subjects were 4.7 times more likely to report 
unsatisfactory treatment responses using antidepressant therapy, 
resulting in premature medication discontinuation (Figure 2).1, 16-18  
Figure 1. The biological heterogeneity of irritable bowel 
syndrome (IBS). Several unique mechanisms have been 
proposed as relevant to IBS pathophysiology.  However, no 
single mechanism has been sufficient to explain IBS 
symptoms in all patients.  Given that IBS is a symptom-
based diagnosis, it is likely the result of a multiple of 
underlying mechanisms, emphasizing the importance of 
identifying pathophysiologically relevant IBS subgroups. 
Figure 
2. 
 
The 
importance 
of 
somatization 
to 
antidepressant 
treatment 
discontinuation 
in 
IBS 
subjects.  IBS subjects with greater somatization using two 
different somatization measures (somatization state and trait) 
were significantly more likely to experience premature 
antidepressant treatment discontinuation due to inefficacy 
and side-effect experiences. From Sayuk G, et al.1 
IBS subjects

Figure 4. Functional neuroimaging differences in irritable 
bowel subjects (IBS) and controls following rectal balloon 
distention.  IBS subjects demonstrate greater activity within the 
homeostatic afferent processing network (HAPN) in response to 
visceral distention compared to controls. (from Mertz, et al.2)
Thus, it is known that somatization is common in IBS, and IBS-S patients experience more severe clinical courses 
which are less responsive to centrally-active IBS treatments. 
 
3. Abnormal pain processing within the homeostatic afferent processing network (HAPN) is relevant to 
IBS pathophysiology in the IBS-S subset of patients.   
Visceral pain is, by definition, the symptomatic hallmark of IBS.  Visceral pain in the absence of an identifiable 
cause (as in the case of IBS) theoretically could originate as a consequence of pain processing abnormalities at 
two distinct levels: 1) abnormal perception of chemo-mechanical 
stimuli by the enteric nervous system within the wall of the gut,19 or 
2) abnormal central nervous system (CNS) interpretation or 
modulation of the afferent sensory signals derived from the gut. 
The use of functional neuroimaging techniques (position emission 
tomography [PET] and functional magnetic resonance imaging 
[fMRI]) have resulted in recent advances in our understanding of 
the brain regions responsible for visceral and somatic pain 
processing. Over the past decade, several brain regions important 
to the affective and cognitive interpretation of visceral pain have 
been identified in animal models and healthy controls. 2, 20-26  
These areas, including subregions of the anterior cingulate cortex 
(ACC), the insula, thalamus, orbitofrontal, and prefrontal cortices, 
collectively have been described by Craig and others as 
comprising the “homeostatic afferent processing network” (HAPN) 
(Figure 3).22, 27, 28 An early study conducted by Mertz et al. found 
activations of several HAPN regions, including the ACC, prefrontal 
cortex, insula, and thalamus in both IBS subjects and controls, 
supporting the physiologic relevance of the HAPN to visceral 
sensation (Figure 4). In comparing HAPN activations with noxious 
visceral stimulation in IBS versus healthy controls, greater HAPN 
activations, 
particularly 
within 
the 
perigenual 
ACC, 
were 
appreciated in the IBS population. Several studies have replicated 
these findings, though the magnitudes of these IBS-healthy control differences in HAPN activation are less 
consistent across studies.26  In part, these observations may reflect significant protocol differences, including the 
use of different imaging modalities (PET and fMRI), study designs (noxious stimulation type, intensity, and 
duration) and data analysis methodologies. However, another likely explanation is the mechanistic heterogeneity 
that exists within IBS patients when examined as a single population.  It is likely that only a subset of IBS patients 
experience significant HAPN overactivation with noxious visceral stimulation.  Thus, when HAPN pain responses 
are examined in all IBS subjects, the inclusion of IBS subgroups 
in which HAPN overactivation is not mechanistically-relevant 
may bias findings toward the null.   
The HAPN is not only important to the emotional and 
cognitive responses to visceral pain, but likely is also relevant to 
somatic pain experiences in somatization syndromes.27  A study 
by Chang et. al. found that in IBS subjects, a greater HAPN 
response within ACC was seen in response to somatic 
stimulation, but only in those with comorbid fibromyalgia (a 
somatization syndrome).22 This finding supports my hypothesis 
that IBS subjects with somatization may experience activations 
of the HAPN with a broader spectrum of noxious stimuli, 
including painful somatic stimuli. 
  The recognition of specific a IBS population in which CNS 
pain processing abnormalities have mechanistic relevance would represent an important step in advancing our 
Figure 3. The Homeostatic Afferent Processing Network 
(HAPN).  Regions comprising the homeostatic afferent 
processing network (HAPN) are responsible for the affective and 
cognitive modulation of pain signals, and are highlighted in 
yellow.  PF, prefrontal cortex; OFC, orbitofrontal cortex; ACC, 
anterior cingulate cortex; PCC, posterior cingulate cortex; VMpo, 
MDvc and  VPL, thalamic nuclei (ventromedial portion of the 
posterior nuclear complex, ventrocaudal portion of the medial 
dorsal nucleus, and ventroposterior lateral nucleus, respectively) 
Adapted from Price, DD. Science 2000.3

understanding of IBS pathophysiology. Recently, I completed a preliminary study to examine the influence of 
somatization on cerebral activations within the HAPN in an IBS population based on the degree of somatization 
present (see Preliminary Studies, Section C).   This work successfully demonstrated significant differences in 
HAPN activations in IBS-S compared to IBS-no S subjects, strongly suggesting that abdominal pain experiences 
in IBS-S subjects are a consequence of abnormal HAPN responses to afferent intestinal sensory input. 
 
4. Antidepressant therapy and homeostatic afferent processing network (HAPN) pain responses in IBS.  
Clinical experience and controlled studies have shown that centrally-active therapies, such as the tricyclic 
antidepressants (TCAs), are of symptomatic benefit in the management of IBS.18, 29 In a recent randomized, 
double-blinded study by Drossman et al., desipramine therapy was shown to result in superior global IBS 
symptom responses compared to placebo. The post hoc analyses from that study found that somatization scores 
predicted treatment non-response using desipramine.18  It is believed that TCAs (e.g., desipramine) modify CNS 
responses to noxious visceral stimulation within the HAPN and related regions in IBS.31, 32 The effect of 
antidepressant pharmacotherapy on similar brain regions have been demonstrated in the treatment of mood 
disorders.  For example, decreases in activations within ventral regions of the prefrontal cortex have been 
previously associated with response to selective serotonin reuptake inhibitors (SSRIs) when used in mood 
disorders. 30, 31,32 The influence of another TCA, amitriptyline, on cerebral pain responses has been examined in 
IBS in a single study. This investigation demonstrated that in an IBS population, compared to placebo 
amitriptyline therapy was associated with reduced visceral pain-related activations within the HAPN, specifically 
within the perigenual anterior cingulate cortex. 33  Based on previous these observations, and our earlier cited 
work suggesting that IBS symptom response to TCA therapies is less robust in IBS-S subjects, I hypothesize that 
somatization in IBS results in less robust responses due to a refractoriness to antidepressant medications within 
the brain’s homeostatic afferent processing network. 
 
5 . Study significance. 
IBS is a common gastrointestinal diagnosis, imparting significant morbidity and 
effects on quality of life, and in turn substantial costs to society.  Despite recognizing 
IBS for decades, the likely heterogeneity inherent in the condition has continued to 
hamper our understanding of the mechanistic basis of this condition.  IBS patients 
with somatization (IBS-S) have more severe symptoms and more refractory treatment 
courses.  It is anticipated that IBS-S subjects possess a generalized central pain 
processing defect within the HAPN as the primary mechanism underlying their pain 
symptom experiences.  Consistent with the diffuse pain symptoms experienced in IBS 
subjects with somatization, it is anticipated that this abnormal HAPN activation can be 
evoked by both visceral and somatic stimulation.  IBS-No S subjects, by comparison, 
may have a primary visceral hypersensitivity which originates at the intestinal level.  In 
IBS-No S subjects, this would be expected to lead to greater afferent signaling and 
activation of the HAPN in response to noxious visceral stimulation, but not somatic 
stimuli (Figure 5).  Additionally, observations of poorer treatment responses with 
centrally-acting antidepressants in IBS-S subjects raise the possibility that this 
abnormal HAPN pain response in IBS-S subjects accounts for the refractoriness to 
centrally-active IBS therapies. These objective responses of the HAPN to noxious stimulation and pharmacologic 
intervention will allow for a more objective definition of IBS-S as an important, physiologically-relevant IBS 
subgroup. As such, this study has the potential to highlight somatization 
as an important feature to measure in future IBS studies, and moreover 
to provide mechanistic insights into a specific IBS subgroup. The broad 
scientific and clinical implication of this work lies in its emphasis of IBS 
as a heterogeneous condition, deriving from a multitude of unique 
pathophysiologic pathways, necessitating the detailed characterization 
of relevant IBS subgroups in order to move forward in our understanding 
of IBS pathogenesis and treatment. 
Figure 5.  Proposed brain-gut interactions of IBS based 
on the presence of somatization.  It is hypothesized that 
IBS subjects with somatization, IBS-S (left panel) experience 
abnormal activations of the brain’s homeostatic afferent 
processing network (HAPN) in response to visceral- and 
somatic afferent inputs.  Thus, greater HAPN activations 
would be expected with both visceral and somatic stimuli in 
IBS-S. In contrast, IBS subjects without somatization, IBS-No 
S (right panel) likely have abnormal afferent signaling from 
the bowel as their primary neurosensory abnormality.  IBS-No 
S subjects thus would be expected experience greater HAPN 
activations with visceral stimulation, but not with somatic 
stimulation.  These hypotheses will be assessed in the Aims 
of this proposal. 

Figure 6. Study design, preliminary data.  Functional MRI (fMRI) in IBS subjects with somatization (IBS-S)
compared to those without somatization (IBS-No S)  and healthy controls. Subjects with intermediate degrees of 
somatization were excluded from participation. 
 
 
C. Preliminary study: The Importance of Somatization to Central Pain Responses in IBS 
Rationale  
fMRI studies demonstrate consistent differences in CNS homeostatic afferent processing network (HAPN) 
activations in IBS patients compared to healthy controls. However, within IBS populations substantial variation in 
HAPN responses to visceral pain is observed.20, 34 Somatization is common in IBS, and results in worse clinical 
outcomes when present. This study tested the hypothesis that within an IBS population, the presence of 
somatization is associated with measurable differences in HAPN regional activations as measured by fMRI. We 
thus sought to demonstrate somatization as a clinical feature responsible for a portion of observed heterogeneity 
in CNS pain responses in IBS patients.  
Specific Aim 
To determine whether IBS subjects with somatization (IBS-S) demonstrate different patterns of central 
homeostatic afferent (HAPN) activations following noxious visceral stimuli compared to IBS-No S subjects and 
healthy controls.  It was hypothesized that IBS-S patients would exhibit significantly greater HAPN activations with 
noxious visceral stimulation compared to the IBS-No S, and healthy control groups.  
Methods 
Female IBS subjects meeting inclusion and exclusion criteria (Rome III criteria)35 and somatization (IBS-S) as 
defined in the Methods section (3.B.1, below) were eligible for study participation;  female IBS patients with no 
somatization (IBS-No S) and healthy controls served as comparator groups.  Baseline depression measures, 
including the Beck Depression Inventory (BDI), the Hamilton Depression Rating Scale (HAM-D) and anxiety 
scores (Beck Anxiety Inventory, BAI) were assessed as potential confounders.  Subjects also reported on global 
bowel symptom burden and IBS symptom severity using visual analog ratings.  The study design is illustrated in 
figure 6. 
Irritable bowel 
syndrome (IBS) 
patients
N≈24*
PHQ-15 
Somatization 
Instrument
Somatization (IBS-HDS)
N=8
Low-degree 
somatization (IBS-LDS)
N=8
Healthy controls
N=8
FUNCTIONAL MRI (fMRI)
STUDY GROUPS
Between-group 
comparisons of 
central homeostatic 
afferent  activity in 
response to visceral 
stimuli
 
 
 
 
Intervention 
Functional MRI studies were performed while applying simultaneous visceral stimulation (rectal balloon 
distension) at low- (25-mmHg) and higher-pressure (50-mmHg) levels in random order following established 
protocols.2   Subjective ratings for distention discomfort were assessed for each distention using a 10-point Likert 
scale at the end of each distention sequence. 
Data interpretation 
Cerebral activations included any statistically significant changes in fMRI activity (voxels) in a priori regions of 
interest (ROIs) within the HAPN following visceral stimulation compared to rest. These ROIs included the 
thalamus, bilateral prefrontal, orbitofrontal, anterior cingulate, and insular cortices.  Between-group differences in 
cerebral activations to noxious visceral stimuli were evaluated with 2-way ANOVA analyses. Preliminary analyses 
were conducted using data collapsed across the two visceral stimuli (“visceral stimulus”) versus “rest periods”.  
Corrections were made for multiple comparisons in regions of interest. 

Findings 
Clinical features and subjective pain ratings: 24 right-handed female subjects (8 in each study group) 
underwent fMRI during the rectal balloon distention protocol from April 2007 to June 2008. A summary of the 
clinical features and subjective pain ratings in response to the balloon distentions are reported in Table xx. 
Subject ages were not significantly different across study groups.  However, there were significant differences in 
IBS symptom ratings between the IBS-S and IBS-No S groups, with greater symptom severity, frequency, and 
global symptom burdens reported by the IBS-S group.  Depression and anxiety scores on the Beck Inventories 
also were significantly higher in the IBS-S group compared to IBS-No S and healthy controls.  Subjective 
experiences with the low-pressure (25 mm-Hg) rectal balloon distentions trended toward significantly greater 
discomfort reporting in the IBS-S group (p=0.3), but no differences were appreciated across groups with the 
higher pressure (50-mm Hg) distentions. (Table 1). 
 
Table 1.  Clinical features and subjective distention pain ratings in preliminary study. 
 
IBS-S subjects 
(N=8) 
IBS-No S subjects (N=8) 
Healthy Controls 
(n=8) 
P value* 
Age 
47.2 ±8.6 
45.0 ±9.7 
36.5 ±9.2 
0.07 
 
 
 
 
 
PHQ-15 total 
18.1 ±3.8a,e 
5.6 ±2.6 
3.7 ±1.6 
0.001 
Non-GI Somatization Diagnoses 
2.9 ±1.9a,d 
0.2 ±0.4 
0.5 ±0.5 
0.001 
 
 
 
 
 
Beck Depression Inventory 
Score 
19.1 ±10.9a,d 
3.5 ±3.3 
3.6 ±3.8 
0.001 
Beck Anxiety Inventory Score 
19.9 ±6.1a,e 
3.1 ±2.9 
1.5 ±5.4 
0.001 
 
 
 
 
 
IBS Symptom Severity Rating 
6.9 ±1.9c 
4.2± 2.8 
-- 
0.04 
IBS Symptom Frequency  
9.1 ±4.8b 
2.4 ±2.1 
 -- 
0.006 
IBS Global Burden Rating 
7.5 ±1.9b 
3.2 ±3.0 
-- 
0.005 
 
 
 
 
 
Subjective pain rating, 50-mm 
Hg  
5.1 ±2.0 
4.5 ±2.2 
4.6 ±2.2 
0.8 
Subjective pain rating, 25-mm 
Hg 
3.0 ±1.4 
2.8 ±1.4 
2.0 ±0.9 
0.3 
*  Reported p value is reflective of an ANOVA where all three study groups compared; t-tests performed to assess between-group statistical 
significances; a = p <0.001 between IBS-S and IBS-No S groups; b = p <0.01 between IBS-S and IBS-No S groups; c = p <0.05 between IBS-S and 
IBS-No S groups; d = p <0.001 between IBS-S and Healthy controls; b = p <0.01 between IBS-S and IBS-No S groups. 
 
Regional activations within the HAPN by study group 
Consistent with findings previously reported in the literature, during noxious visceral stimulation a significant 
difference in a HAPN brain region activations was seen in comparing IBS subjects and healthy controls (Table 
2 and Figure 7). Between-comparison of HAPN activations in the IBS-S and IBS-No S groups revealed 
significantly greater activation of the right prefrontal cortex, insula, and thalamus in the IBS-S subjects.  The 

IBS-S subjects also were found to have greater activation of the bilateral anterior cingulate and orbitofrontal 
cortices compared to their IBS-No S counterparts.  
 
Table 2: Regional differences in cerebral activations within the homeostatic afferent processing network (HAPN) in response 
to visceral stimulation in IBS-S subjects compared to controls, and IBS-S compared to IBS-No S subjects. 
All IBS vs. Controls
IBS-S vs. IBS-No S
Region 
Hemispher
e 
Coordinates 
(x,y,z)* 
Z 
p 
 
Z 
p 
Anterior cingulate cortex 
Left 
-8, 38, 16 
2.91 
0.004 
 
2.67 
7.3 x10-3 
Right 
9, 34, 11 
2.19 
0.02 
 
4.15 
7.6 x10-4 
Insula 
Left 
-39, 2, 7 
5.20 
9.7 x10-8 
 
1.09 
0.27 
Right 
 32, 23, 10 
5.47 
2.8 x10-8 
 
2.33 
0.02 
Orbitofrontal cortex 
Left 
-25, 13,-10 
4.27 
1.9 x10-5 
 
3.13 
0.001 
Right 
34,22,-3 
4.82 
1.4 x10-6 
 
4.84 
6.5 x10-7 
Prefrontal cortex 
Left 
-41, 40, 11 
4.87 
5.4 x10-7 
 
1.37 
0.16 
Right 
41, 50, 5 
5.78 
3.5 x10-8 
 
4.17 
2.4 x10-5 
Thalamus 
Left 
-19, -22, 11 
5.58 
1.2 x10-8 
 
1.51 
0.12 
 
Right 
12, -8, 11 
4.02 
5.8 x10-5 
 
2.09 
0.003 
 
 
 
 
All IBS vs. controls
IBS-S vs. IBS-No S
L ACC
R ACC
L OFC
R OFC
B PFC
B Insula
B Thalamus
Figure 7. Brain activations following noxious visceral stimulation. Colored pixels indicate brain regions with significant activations (Z score) in response 
to rectal balloon distention.  The regions comprising the homeostatic afferent processing network (HAPN) are circled and labeled.  HAPN regions were 
activated to significantly greater degrees in the IBS subjects compared to healthy controls (top row). Comparing IBS-S and IBS-No S subjects (bottom row), 
many of the same HAPN regions demonstrated greater activation in the IBS-S group. L=left; R=right; B=bilateral; ACC=anterior cingulate cortex; 
OFC=orbitofrontal cortex; PFC=prefrontal cortex. 

Significance of findings 
In this study, IBS subjects clearly differed in their HAPN brain responses to visceral stimulation compared to 
healthy controls. A similar pattern of HAPN activation in IBS has been observed in several other studies, 
suggesting that dysfunctional affective and cognitive processing of visceral pain signals within the HAPN may be 
of mechanistic relevance to IBS symptom experiences. 34, 36  Hypothesizing that these HAPN pain responses are 
of particular importance to IBS subjects with comorbid somatization, a similar comparison of HAPN visceral 
activations was performed between IBS-S and IBS-No S subjects. In this small study sample, significantly greater 
activations of many of these same HAPN regions were observed in the IBS-S subjects compared to their IBS-No 
S counterparts.  This finding supports my hypothesis that abnormal HAPN processing of afferent visceral signals 
may be of greater pathophysiologic relevance to the subset of IBS patients that have comorbid somatization. 
 
An important HAPN region that demonstrated greater activation in the IBS-S subgroup was the perigenual 
portion of the anterior cingulate cortex (ACC).  The ACC is the region most consistently activated by visceral 
stimulation, and thus is regarded by some as “the visceral pain center”.25  Electrical stimulation of this region 
results in a potent fear response, suggesting the key role of the ACC in emotional responses to aversive stimuli.37 
This region additionally has prominent descending connections to the dorsal vagal complex and periaqueductal 
gray, and as such is believed to play a central role in the endogenous modulation of pain and autonomic tone.  
The ACC influence on autonomic function could explain the alterations in bowel patterns often accompanying IBS 
pain experiences. Similarly, the right insular cortex (IC) demonstrated significantly greater activation in the IBS-S 
subgroup.  Studies have shown that lesions within the IC result in modulation of the affective pain response, but 
not visceral sensory responses.38  This region, along with the ACC, also has been shown to activate in response 
to viewed fearful faces (negative emotional condition).  Together, these findings suggest that abnormal right IC 
and ACC affective responses to noxious visceral stimuli may be important to IBS-S symptom experiences.40   
Greater activations of the bilateral orbitofrontal cortices (OFC), and right prefrontal cortex (PFC) were seen in 
IBS-S subjects.  These regions play a central role in the higher-order processing and integration of the cognitive 
and affective responses to visceral sensory signals, resulting experience-related behaviors and thoughts. The 
dorsolateral subdivision of the PFC is involved in the cognitive processing (attention, memory, and executive 
functions) as related to visceral sensory signaling.41  The PFC has direct reciprocal connections with brain areas 
known to be involved in mood and emotional processing, including the ACC.42-44 Via inputs from the regions 
involved in emotional regulation, and different sensory afferents (visceral, gustatory, visual), the orbitofrontal 
cortex, in conjunction with the IC, is important to the integration of emotions and visceral sensation.41  The 
preferential right sided frontal activation pattern seen in IBS-S subjects has been described previously.  Right 
sided laterality has been reported in both humans and animals to be associated with higher cortisol levels and 
autonomic responses, and greater negative affective experiences (fear, disgust).45 A similar pattern of right frontal 
activation previously was reported in one IBS study.  The composition of this IBS group, in terms of comorbid 
somatization, was detailed by the investigators.   
Thalamic activations were significantly greater bilaterally in IBS subjects compared to controls.  As a key 
center in the initial processing and subsequent relay of visceral sensory information to multiple cerebral cortical 
regions for higher-order processing, these thalamic activations could be deduced as reflecting greater afferent 
signaling in response to noxious visceral stimulation.  The thalamic nuclei are also the recipients of substantial 
descending signals from other HAPN regions, however, thus complicating this interpretation of activations in this 
region.  IBS-S subjects did also have significantly greater activations than IBS-No S subjects, though the 
magnitude of difference between groups was small by comparison to the other HAPN regions. 
Finally, these observed differences in HAPN activations between the two IBS subgroups must be considered in 
the context of the greater levels of bowel and mood symptom reporting in IBS-S subjects.  These factors 
potentially could serve as confounders in attributing the HAPN activation differences to the presence of 
somatization alone, and additional analyses will thus need to be performed on this preliminary data to establish 
the independent importance of somatization to HAPN visceral activations in the IBS-S subgroup. 
 
 
D. Research Design and Methods 
 
1. Overview 

It is likely that irritable bowel syndrome (IBS) is the result of multiple disease mechanisms which lead to the 
expression of a common set of symptoms.  The goal of this proposal is to identify and characterize a more 
homogeneous subset of IBS patients—specifically, IBS patients with comorbid somatization (IBS-S). I will define 
the IBS-S subgroup by their CNS responses within the homeostatic afferent processing network (HAPN), regions 
important to the higher level (cognitive and affective) responses to pain.  The overarching hypothesis of these 
investigations is that aberrant central pain responses within the HAPN result in somatization pain symptoms, and 
as such may have pathophyiologic relevance to the bowel pain experiences of the IBS-S subgroup.  Building 
upon my initial observations that IBS-S subjects demonstrate greater HAPN activations to visceral stimuli, 
Specific Aim 1 will examine whether these responses generalize to non-visceral (somatic) stimuli.  Given 
treatment refractoriness to antidepressants in IBS-S subjects, and the known influence of these medications on 
the HAPN, Specific Aim 2 will assess the effect antidepressant therapies on IBS-S central pain activation 
patterns. Ultimately, this work will provide greater insight into the neuropathophysiologic basis of IBS-S, allowing 
for the objectively definition of IBS-S as an important irritable bowel subgroup. 
 
 
Specific Aim 1. To compare HAPN activations following noxious somatic stimulation in IBS subjects 
based on the presence of somatization. 
 
Rationale: It has been demonstrated that CNS responses to noxious visceral stimulation differ within the HAPN 
from healthy controls subjects in some, but not all, IBS subjects.  My preliminary data supports the hypothesis 
that IBS subjects with comorbid somatization (IBS-S) demonstrate a pattern of greater CNS activation within the 
homeostatic processing afferent network (HAPN) following visceral stimulation compared to those with no 
somatization (IBS-No S).  Specifically, the regions responsible for higher-level cognitive (bilateral orbitofrontal, 
right prefrontal cortex) and affective (right insula, bilateral anterior cingulate cortex) pain processing had greater 
activations in IBS-S subjects.  Given that IBS-S subjects experience multiple somatic pain symptoms, in this Aim I 
will determine whether the previous difference in HAPN pain responses in the two IBS populations (IBS-S and 
IBS-No S) is specific to visceral stimuli, or is a more generalized pain response which can also be elicited by 
somatic stimulation. 
 
Design and Methods: HAPN responses to both visceral and somatic stimuli will be evaluated in 40 female IBS 
patients stratified by the presence of somatization (20 IBS-S and 20 age-matched IBS-No S subjects) and 20 
healthy controls. Measures of current IBS symptoms and concurrent mood symptoms will be assessed as 
potential confounders using self-report instruments and structured psychiatric interviews (see “Assessment 
measur
es”, 
below). 
Figure 8. Overview of study design for Specific Aim 1. Female IBS subjects with somatization (IBS-S) and without somatization (IBS-No S) and 
healthy controls will undergo a series of four functional magnetic resonance (fMRI) blocks, each assessing brain activations in response to 
randomized visceral and somatic stimuli. Between-group (IBS-S vs IBS-No S) differences in brain activations within the homeostatic afferent 
processing network (HAPN) in response to these stimuli will then be evaluated.

All three subject groups will undergo a baseline functional MRI study of 4 functional imaging blocks obtained 
during a protocol of randomized visceral (25- and 50-mm Hg rectal balloon distentions) and somatic stimuli (1.4 
kg-cm2 nailbed pressure) (Figure 8, above).  The primary endpoint of interest is differences in CNS activations of 
a priori regions of interest comprising the HAPN following somatic stimulation.  
 
Analysis/interpretation: As a result of a more generalized abnormal pain response within the HAPN, it is 
hypothesized that IBS-S subjects will demonstrate greater activations of the HAPN with noxious somatic stimuli, 
compared to IBS no-S subjects and healthy controls. This hypothesis thus predicts a “IBS Group x Somatic 
stimulus” effect on two-way ANOVA analysis of activations within a priori HAPN regions.  Specifically, it is 
hypothesized that the significantly increased HAPN activation pattern observed in IBS-S subjects compared to 
IBS-No S subjects that was observed following noxious visceral stimulation (preliminary data) will be present 
following noxious somatic stimulation in the IBS-S subjects, but not in the IBS-No S or healthy control groups 
(Figure 10, below).  If this HAPN activation response to somatic stimuli is indeed observed in IBS-S subjects, it 
would support my hypothesis that in IBS-S patients, the presence of somatization reflects a less discriminate 
HAPN response to noxious stimuli, which includes not only visceral, but also somatic stimuli. This global 
overactivation of the HAPN in IBS-S subjects thus could have mechanistic relevance to the multiple pain 
experiences in affected individuals. 
Alternately, if HAPN activation differences in IBS-S subjects are found only to be specific to visceral 
stimulation, the possibility that the HAPN is relevant to visceral, but not somatic, pain experiences in IBS-S 
subjects would be considered.  This would imply that several distinct, system-specific sensory defects (i.e., gut, 
musculoskeletal, etc.) may be present in somatization, rather than the global pain processing abnormality within 
the HAPN as is hypothesized to be relevant to the multiple pain experiences of IBS-S subjects.  
If greater HAPN activations are detected in both IBS-S and IBS-No S subgroups, this would be interpreted 
as possibly indicating that somatic afferent sensory signals are more potent activators of the HAPN than are 
visceral afferent signals, resulting in HAPN activation patterns with noxious somatic stimulation which are 
indistinguishable in the two IBS groups. This conclusion might be valid particularly if the difference in visceral pain 
responses seen in the preliminary data are again observed in this IBS cohort.  It would thus still be conceivable 
that differences in HAPN somatic responses could be detected in future studies which implement either less 
intense somatic stimuli (i.e., lower level thumb pressures) or alternative somatic stimuli (thermal stimuli). 
 
Pitfalls/Limitations: Critical to the study is an accurate determination of study group assignment (IBS-S or IBS-No 
S) and the limitation of potential confounders.  Thus, substantial effort will go into the determination of 
somatization status, including the use of validated self-report instruments (PHQ-15), medical chart review and 
history by the investigator, and a formal structured interview (C-DIS).  Other potentially important confounders will 
also be considered. To address differences in brain activations due to handedness, gender, and age, subjects will 
be age-matched within 5 years, and only will be right-handed females will be studied. Subjects with major 
depression, anxiety, or current antidepressant use will also be excluded.  Current depression, anxiety, and 
duration and severity of bowel symptoms will also be assessed using both validated self-report and standardized 
interviews. Additional ANOVA models will be run to confirm that HAPN activation differences attributed to 
somatization are not accounted for by other IBS disease factors (duration, symptom severity) or psychological 
features are also relevant to observed differences in brain activation. Subject movement during the fMRI study is 
also a consideration given the noxious nature of the study stimulus.  Preliminary data acquired using a smiliar 
protocol did not suggest this to be a major issue, either in terms of subjective reports of discomfort or on 
assessment of movement during quality-control assessments.  No subjects withdrew from the preliminary study 
due to protocol intolerance.  However, if movement is introduced as a problem in this investigation, it is 
anticipated that it will be increasingly present in the latter blocks of the protocol (i.e., fMRI blocks 3 and 4). The 
proposed block design of the proposed fMRI study allows for the exclusion of study blocks demonstrating 
excessive subject movement.  Study recruitment may present a potential pitfall, though in the past I have been 
successful in meeting recruitment goals through my outpatient practice given my clinical emphasis on IBS and 
related functional GI disorders.  If recruitment does prove to be an issue, recruitment efforts will be expanded to 

Figure 9. Overview of study design for Specific Aim 2. IBS patients with somatization (IBS-S) and without somatization 
(IBS-No S) will be identified.  A baseline fMRI protocol (as in Aim 1) will be obtained at baseline, followed by a 4-week, open-
label treatment with desipramine. A second fMRI protocol will be performed post-desipramine therapy at week 4.  The primary 
endpoint of interest is post-treatment differences in visceral HAPN activations between IBS-S and IBS-No S subjects.   
include the Washington University Volunteers for Health database and the use of recruitment advertisements on 
the WU Medical Center campus.   
 
Specific Aim 2. To compare the effects of antidepressant therapy HAPN activations following noxious 
visceral stimulation in IBS subjects based on the presence of somatization. 
 
Rationale: Case-control and randomized clinical trial data have established that somatization predicts symptom 
responses to IBS therapy with the tricyclic antidepressant desipramine.  Specifically, IBS-S subjects are more 
refractory to antidepressant therapies, while IBS-No S subjects have better overall responses to the same 
treatment.  A recent study has demonstrated improvements in HAPN visceral activations (anterior cingulate 
cortex) following desipramine therapy in an IBS cohort.  Given these observations, the goal of this Aim is to 
assess whether differences in visceral HAPN activation patterns following despiramine treatment can be detected 
in IBS-S and IBS-No S subjects. 
 
Design and Methods: The proposed study implements open-label desipramine therapy in 40 IBS (20 IBS-S and 
20 age-matched IBS-No S) subjects.  Participants from Aim 1 will be invited to continue on to open-label therapy 
in Aim 2.  IBS-S and IBS-No S subjects will receive a 4-week treatment course with the tricyclic antidepressant 
desipramine (DES), as detailed in section 2.D., below.  Subjects will undergo a fMRI protocol with noxious 
visceral and somatic stimuli as in Aim 1 at baseline, and then immediately following the 4-week desipramine 
therapy. Subjects will undergo baseline, and post-therapy assessment of psychiatric comorbidity 
(depression/anxiety) and IBS symptom severity (as described in 3.A. and 3.B., below). The primary endpoint is 
differences in visceral stimulation-induced brain activations of the HAPN on fMRI, ascertained between-groups 
following desipramine therapy. The proposed study design is outlined in Figure 9. 
 
 
 
Analysis/interpretation: Consistent with the clinical refractoriness to antidepressant therapy observed in IBS-S 
subjects, it is hypothesized that IBS-S subjects will exhibit a persistent pattern of abnormal HAPN activations in 
response to visceral stimulation following desipramine therapy.  By comparison, it is hypothesized that the IBS-No 
S group will exhibit decreases in HAPN activations in the post-treatment fMRI protocol (Figure 10, below).  
Region of interest two-way ANOVA models will be developed in comparing Δbaseline-DES activations, with IBS 
Group (IBS-S and IBS-No S) as a between-subject factor. If the stated hypothesis is supported by the fMRI 
findings, it would suggest that the persistent HAPN activations in response to visceral afferent signals may result 
in the less robust clinical responses to desipramine therapy in the IBS-S. This finding would be an important 
addition to the existing literature, providing both insight into the central mechanisms involved in IBS 
antidepressant response, and also into the specific relevance of HAPN pain responses to the IBS-S subgroup.   

Conversely, should HAPN activations be found to improve in both IBS groups following desipramine therapy, 
the possibility that HAPN visceral activations are indeed partially refractory to desipramine in IBS-S, but the 
detected between-group differences (IBS-S vs. IBS-
No S) in post-desipramine changes in HAPN 
activation were smaller than expected, and thus not 
statistically significant.  If this were the case, the 
ANOVA analyses would be likely demonstrate 
statistical trends between the IBS groups, and 
enrollment 
of 
additional 
subjects 
would 
be 
considered if feasible following power calculations. 
This additional step might lead to findings which 
allow for rejection of the null hypothesis.   
  
Pitfalls/Limitations: 
Pitfalls 
and 
limitations 
as 
described in Aim 1 are also applicable to this Aim, 
and will be addressed as outlined previously.  Also a 
consideration is the use of repeated fMRI measures 
within the same individual, and the influence that 
previous experiences will have on subsequent fMRI 
studies (e.g., anticipation).  Theoretically this effect 
should be distributed across treatments and IBS 
study groups equally, such that the anticipated 
effect, if any, bias towards the null.  Existing 
literature discussing this phenomenon suggests that 
the proposed regionwise analyses based in a priori hypotheses are more immune to this phenomenon.  This 
study is proposed without a placebo control arm.  This design was decided upon after discussion with my mentors 
in order to facilitate subject recruitment and protocol adherence. While an obvious limitation of the study design, 
given previous data that demonstrates the effects of tricyclic medications over placebo within the HAPN, 
foregoing a placebo-controlled arm was felt to be a reasonable approach. 
The observed changes HAPN activation seen following desipramine therapy in this Aim likely represent the 
direct effects of desipramine on these brain regions.  This conclusion can be deduced from previous pharmaco-
radiologic and animal model studies demonstating the actions of tricyclic antidepressants in these regions.  The 
possibility remains, however, the desipramine is influencing the CNS proximal to the HAPN (i.e, at the level of the 
viscera, spinal cord, or brainstem), in turn decreasing HAPN activations via a reduction in visceral afferent 
signals.  This possibility can be reconciled in future analyses which examine the data generated by this Aim, 
utilizing sophisticated functional connectivity models to establish the temporal relationships of these regional 
activations.  
 
Figure 10. Summary of the anticipated findings in Specific Aims. It is 
hypothesized that IBS-S subjects will demonstrate greater HAPN activations with 
noxious somatic stimuli, compared to IBS subjects with no somatization (IBS-S) and 
healthy controls (Specific Aim 1). It is also anticipated that IBS-S subjects will 
demonstrate a persistent pattern of abnormal HAPN visceral activations following 
desipramine therapy, whereas significant improvements in HAPN visceral activations 
will be observed in IBS subjects with no somatization following antidepressant therapy 
(Specific Aim 2). n/a=not assessed--control subjects do not participate in treatment 
arm of Specific Aim 2.  

Future directions. Recent epidemiologic studies have 
demonstrated greater prevalence of functional genetic 
polymorphisms 
of 
monoaminergic 
neurotransmission, 
including the serotonin transport protein (SERT), in IBS 
compared to healthy controls. Neurophysiologic studies in 
animal models and mood disorders have identified serotonin 
as important to nerve signaling within the HAPN.  In future 
studies, I will attempt to link established serotonergic genetic 
polymorphisms (such as SERT) to the presence of CNS 
activation patterns in IBS subjects.  Studies to-date 
evaluating the genetic basis of IBS have evaluated IBS 
subjects as a single group.  The presumed heterogeneity in 
IBS pathophysiology has driven the need for large samples 
of several hundred individuals to ascertain even weak 
associations in previous IBS genetic studies (Figure 11A).  These observations emphasize the need for the 
rational dissection of IBS into more discrete, homogeneous “endophenotypes”—intermediate phenotypes that 
help establish the causal link between genes and overt symptom expression.46  The assumption of the 
endophenotype is that it represents a more homogeneous population, and thus one which is more likely to be 
impacted by a particular system defect, and in turn a more specific set of genes (Figure 11B).47-49 Identification of 
mechanistically relevant IBS endophenotypes has been endorsed as a way forward in understanding the genetic 
basis of IBS and related somatization disorders.50-53 The IBS-somatization (IBS-S) endophenotype is an example 
of such an IBS subset.  IBS-S subjects can be identified on the basis of patient history and questionnaires, but 
also have a distinct clinical phenotype (e.g., more severe symptoms, multiple pain syndromes, treatment 
refractoriness) and are also associated with an objective measure (brain HAPN responses to noxious 
stimulation). Applying these objective filters over subjective symptom reporting alone, described functional genetic 
polymorphisms have the potential to yield much more robust impact on the syndrome.   
Functional neuroimaging techniques, in which substantial data sets are acquired from a single subjects from 
hundreds of repeated measures of brain function, is a powerful tool when coupled with genetics.  This is the 
concept of “imaging genomics”, in which signal detection power 
allows for the study of tens, rather than hundreds of subjects in 
making significant gene-brain activation associations. This 
technique has been applied successfully in other similar 
conditions, including a recent National Institute of Mental Health 
study of 14 depressed subjects found that those carrying the 
SERT promoter s allele were found to have hyper-reactive responses of their amygdala to stressful environmental 
stimuli.55-58  Genetic factors, particularly relating to candidate neurotransmitter systems, present an appealing 
area for future pursuit of a biological basis for irritable bowel in the IBS-S endophenotype.  In preparing for the 
application of these techniques in future investigations, I have enlisted Dr. Alison Goate as a consultant and 
advisor, and will integrate formal and practical genetics training opportunities into my career development plan. 
 
2. Source of subjects/calculation of estimated subject pool. 
A. Irritable bowel syndrome and somatization.  Based on published reports, it is conservatively estimated that 
approximately 15% of the US population is affected by irritable bowel syndrome.59, 60  It has been observed by our 
group and others that somatization is present in 25-42% of IBS patients.10, 11 Similarly, we have previously 
identified that within our gastroenterology referral clinic population that approximately 30% of IBS patients have 
no somatization features (IBS-No S).61  
B. Subject pool. Patients undergoing evaluation for, or presently under medical management for, a diagnosis of 
IBS at the Washington University Gastroenterology clinic by from the period of Jan 2009 until the completion of 
the study enrollment period, if interested, will be assessed for participation eligibility. Given my clinical focus on 
IBS and related functional GI disorders, it is estimated that approximately two-thirds of the patients seen in my 
Figure 11. Strategies for the future study of genetics in irritable 
bowel syndrome. Gene-to-disorder approaches (A) have relied on self-
report of symptoms in identifying study subjects with IBS, and assume 
direct linear relationships between genes and IBS. (B) By interposing 
objectives measures (e.g., more specific symptom subsets, differences in 
fMRI findings), an intermediate IBS “endophenotype” may be identified, 
potentially bridging the gap between single gene effects and the clinical 
phenotype. 

outpatient office meet criteria for IBS.  I see an estimated 400 new patients a year in the office, of whom around 
260 would be expected to have IBS.  Primary recruitment efforts for the study population thus will be based in this 
setting.  This strategy was successful in recruiting subjects to generate the primary data for this proposal.  
C. Screening, eligibility, and patient enrollment.  IBS subjects with- and without somatization features will be 
recruited.  To be eligible, subjects will have to be between 18 and 90 years of age (inclusive) and qualify for a 
diagnosis of irritable bowel syndrome according to the criteria set forth in the Rome III criteria.4  Comorbid 
somatization in these IBS subjects will be determined using the Patient Health Questionnaire-15 (PHQ-15).62  
Only subjects with high somatization (PHQ-15 ≥ 10), or low somatization (PHQ-15 ≤5), excluding GI symptoms, 
will be considered for enrollment. Medical history and chart review will be used corroborate the PHQ-15 score, 
requiring that IBS-S subject ≥2 additional somatization symdromes, while IBS-No S subjects have a history of ≤ 1 
non-GI somatization syndrome.1, 61  Persons are eligible to participate without regard to race or ethnicity.  Given 
sex differences in cerebral responses to noxious stimuli and the greater prevalence of IBS in women, only female 
participants will be sought in this study.63, 64 In view of brain hemispheric differences between left- and right-hand 
dominant individuals, all participants must be right-handed. 
 
Persons are excluded from participation for having various psychiatric, medical, and other characteristics.   
Psychiatric/cognitive exclusions include any of the following: active suicidal or homicidal ideation or a history 
of attempted suicide, current excessive alcohol use or other substance abuse disorders, active major depression, 
anxiety disorder, bipolar depression or any psychotic disorder, unwillingness to be randomized or provide 
informed consent, inability to communicate with staff or significant cognitive impairment.    
 
Medical and other exclusions include any of the following: renal or hepatic disease or impairment, diabetes, 
cardiovascular disease, cardiac arrythmia, cerebrovascular disease, or breastfeeding, pregnant, or imminent 
intention of pregnancy, history of seizures or primary neurological disorder, head trauma, brain damage, hyper- or 
hypothyroidism, history or abdominal surgery (other than cholecystectomy/appendectomy), or known structural GI 
disorder (Crohn’s disease, etc.), contraindication to MRI (metallic implant, pacemaker), or rectal balloon distention 
(e.g., proctitis/colitis).  
Exclusions related to medications:  1. Analgesics (narcotics, NSAIDs; acetaminophen OK), 2. Muscle 
relaxants 3. Psychoactive agents (antidepressants, antipsychotics) 4. Other medications (phenytoin; 
amphetamines, prescription weight-loss drugs, or bezodiazepines); 5. Thyroid medication; 6. Anticholinergic 
medications or other IBS medications (hyoscyamine, dicyclomine), 7. Cytochrome p450 substrates, 8. 
Participation in any clinical trial using any other drug.  
D. Desipramine therapy (Aim 2).  Aim 2 utilizes desipramine, a standard-of-care option in the clinical 
management of IBS.65, 66  Desipramine is a tricyclic antidepressant that has considerable clinical experience, and 
controlled-trial evidence supporting its use in the management of IBS symptoms.67-69  Dosing of desipramine is as 
follows:  Starting dose:  Desipramine 25 mg/day administered in the evening. Dosing may be increased 
dependent upon side-effects and clinical response to a maximum of 100 mg/day.  Absent significant side-effects, 
all patients are increased at the one week visit to 50 mg/day at bedtime if they have not achieved a report of 
“Adequate relief” (Assessment measures, B.3., below). Thereafter, up to week 4, the daily desipramine dose may 
be increased weekly by 25 mg up to the 100 mg/d maximum. All doses will be dispensed by the Barnes-Jewish 
Hospital (BJH) study pharmacy, and supervised by the investigator. 
 
3. Assessment measures 
Comprehensive assessment of psychiatric comorbidities (e.g., depression, anxiety) are critical for testing study 
hypotheses.  A semi-structured psychiatric diagnostic interview and DSM-IV criteria will be used to make the 
diagnosis of any co-morbid psychiatric conditions.  
 
(1) The Research Diagnostic Questions for Adult Functional GI Disorders in order to satisfy the 
presence of FGID(s) based on established diagnostic criteria (Rome III).1  
(2) The Gastrointestinal Symptom Questionnaire (GISQ) which contains the following questionnaires:   

a) GI symptom assessment (GISQ Part A): 
1. two 100-mm visual analog scales assessing recent bowel symptom burden 
and severity, and 
2. a quantification of bowel symptom frequency 
b) Somatization state and trait measures (GISQ Part B):  
 
1. the PHQ-15 somatization form,12 a validated self-report of recent somatic 
symptoms (somatization state measure), and 
 
2. a somatization diagnosis checklist (somatization trait measure). 
c) Mood disorder measures (GISQ Part C): 
 
1. the Beck Depression Inventory 
 
2. the Beck Anxiety Inventory  
d) Health-related quality of life measures (GISQ Part D & E): 
1. the Short-Form General Health Survey-36 (SF-36),13 an instrument applied 
previously in the assessment of health influence on global well-being in FGID 
and other somatic disorders. 
2. a visual analog scale assessment of  global symptom response to therapy. 
e) FGID treatment measures (GISQ Part D): 
 
1. a visual analog scale assessments of GI symptom response to therapy. 
 
2. a self-report of subject compliance with medical therapy. 
 
3. a validated questionnaire measuring beliefs about medications. 
(3) The Gastrointestinal Symptoms Questionnaire Supplement (GISQ-S) which contains the following 
questionnaires: 
a)  State-Trait Anxiety Inventory for Adults (STAI) (Part A).  The State-Trait Anxiety 
Inventory Form Y (STAI) is an instrument for measuring anxiety in adults. The STAI 
differentiates between the temporary condition of "state anxiety" and "trait anxiety." 
The qualities evaluated by the STAIS-Anxiety scale are feelings of apprehension, 
tension, nervousness, and worry. 
b) Pain Catastrophizing Scale (PCS) (Part B) is widely used to assess cognitive and 
affective responses to pain and to evaluate pain management program outcomes. 
c) Somatic Symptoms Inventory (SSI) (Part C): The SSI is a self-report questionnaire 
composed of 26 bodily complaints drawn from the hypochondriasis scale of the 
Minnesota Multiphasic Personality Inventory and the Hopkins Symptom Checklist 
somatization scale. 
d) SOMS-7 (Part D) is a 7-item instrument for the evaluation of treatment effects in 
somatoform disorders. It covers all somatic symptoms mentioned as occurring in 
somatization disorder, according to DSM-IV and ICD-10. 
e) Gastrointestinal Symptom Rating Scale for IBS (GSRS-IBS) (Part E):  The 
Gastrointestinal Symptom Rating Scale is a disease-specific instrument that includes 
15 items combined into five-symptom clusters addressing different gastrointestinal 
symptoms. The five-symptom clusters depict reflux, abdominal pain, indigestion, 
diarrhea and constipation. The GSRS has a seven-graded Likert type scale where 1 
represents absence of bothersome symptoms and 7 very bothersome symptoms. 
The GSRS is well documented to be reliable and valid and norm values for a general 
population are available.  
f)  
Life-Stress Questionnaire.  (4) Additional questionnaire measures 
a)  SES Ladder (SES-L) 
b)  Brief Pain Inventory (BPI) 
c)  Hospital Anxiety and Depression Scales (HADS) 
d)  Coping Strategies Questionnaire-Catastrophizing scale (CSQ-C) 
e)  IPIP Neuroticism and Extroversion scales (IPIP-NEO) 
f)  Early Trauma Inventory-Self Report (ETISR) 

g)  Positive and Negative Affect Scales (PANAS-SF) 
(5) Diet Assessment 
a)  Harvard School of Public Health Food Frequency Questionnaire (FFQ): 
https://regepi.bwh.harvard.edu/health/FFQ/files/2007%20BOOKLET%20FFQ.pdf 
 
 
C. Functional neuroimaging testing. 
1. Rectal balloon placement/distention. A rectal latex balloon with an external diameter of 5 cm and a length 
of 9 cm (Mui Scientific, Canada) will be placed with its distal end 4 cm from the anal verge.  A 20 min rest period 
will elapse to allow the patient to become acclimated to the presence of the rectal balloon before initiation of the 
protocol.  The rectal balloon catheter will be attached to a one end of silastic tubing, and the remaining end of the 
tubing attached to the rectal balloon will be connected to a barostat, a device designed to safely deliver controlled 
inflation of a rectal balloon at pre-determined pressures following a programmed protocol.   
2. Dolorimetry methodology.  A pre-MRI baseline session will be performed to determine a tolerance for 
pressure pain sensitivity thresholds.  A discrete pressure stimulus of 40 seconds will be applied to the left 
thumbnail using a 1-cm2 hard rubber probe attached to a hydraulic piston as described previously.27, 81-83  
Dolorimetry using standard thumb pressure has been shown to accurately and reproducibly reflect an individual’s 
overall pressure-pain sensitivity.83  A pressure of 1.4 kg-cm2 will be applied during the stimulus period, as this is 
the threshold previously established as “moderate” on a pain scale used in a fibromyalgia population.27  
3. Structural MRI scan.  All scanning will be performed on a 3.0T Siemens Allegra MR scanner at the WU 
Center for Clinical Imaging Studies (CCIR).  Acquisition of a scout scan with three orthogonal slices, followed by a 
coarse 3D sagittal T1-weighted MP-RAGE (magnetization prepared rapidly acquired gradient echo)84 will be used 
in slice registration.  The coarse MP-RAGE will be used to automatically compute fMRI slice tilts and offsets that 
optimize whole brain.  High-resolution structural images will be acquired using a 3D sagittal T1-weighted MP-
RAGE acquisition optimized for contrast-to-noise and resolution.85 High-resolution multislice oblique axial spin 
density/T2-weighted fast spin echo (FSE) structural images will be acquired using slice tilts and positions 
computed by slice registration.  The acquisition of T2-weighted data with slice pre-registration provides very 
similar slice positions in serial studies. The T2-weighted FSE data will be used in the fMRI atlas registration 
procedure. 
4. Functional MRI (fMRI) scanning.  Functional images will be collected in runs using asymmetric spin-echo 
echo-planar sequence (same as blood oxygen level-dependent, “BOLD”, contrast (T2*)). Each functional run will 
consist of sets of 39 contiguous 3.2 mm thick axial images acquired parallel to the anterior-posterior commissure 
plane, allowing complete brain coverage at high signal-to-noise ratio.86  MRI data will be reconstructed into 
images, then normalized across runs by scaling whole-brain intensity signal to a fixed value and, removing the 
linear slope on a voxel-by-voxel basis to counteract effects of drift.87 MR data will be aligned to correct for head 
motion using a six-parameter rigid body rotation and translation correction which mutually registers all frames in 
all runs for each subject.88  Between-subjects analyses will be conducted after transforming data to a common 
atlas space and then blurring images with an optimal Gaussian filter89, 90 To transform MR data into standard 
atlas, T1-weighted MP-RAGE and T2-weigted FSE anatomic images will be obtained (see above).  This overall 
strategy has been described previously.91  A sequence of linear (affine) transformations will be computed 
involving a combination of intra- and cross-modal registration procedures (e.g., T2- or T2*-weighted EPI to FSE to 
MP-RAGE to an atlas-representative MP-RAGE target).  The transforms are combined by matrix multiplication 
such that reslicing of data in conformity with the atlas then will involve only one interpolation.  The cross-modality 
registration algorithm used for this procedure is related to methods described previously.92  The intra-modality 
registration algorithm is equivalent to minimization of difference image variance.93 This is identical to the method 
used for MR-based co-registration of PET data88 This method provides a straightforward means of conducting 
quantitative group comparisons while preserving the enhanced temporal resolution of fMRI.  This analysis 
technique has been validated against single-subject analyses conducted with the N-back test.94 

Figure 12. Rectal balloon distention/dolorimetry protocol. 
6. Rectal balloon distention/dolorimetry protocol. All balloon distensions and dolorimeter activations will be 
performed with the participant in the supine position with eyes closed to minimize extraneous visual stimuli  
(potentially confounding fMRI data).  Participants will be informed that a series of stimuli are about to be delivered 
prior to a series of balloon distensions/thumb pressure 
deliveries, but will be given no insight into the degree of the 
impending distension or pressure.  The balloon distension 
protocol will consist of a series of rectal balloon distensions and 
intermixed thumb pressure deliveries performed using a block 
design as reported previously.95   Each “fMRI block” will 
constitute a total of 6 (six) 40-second “Control periods” 
alternated with 6 (six) 40-second “Stimulus periods”.  A 
continuous fMRI scan will be obtained for each of the fMRI 
blocks (Figure 11).  Each 480-second fMRI block will be 
followed by a resting period of two (2) minutes between each 
functional scan for recovery of baseline MRI signal between 
scans.  The individual fMRI blocks will randomly alternate two 
(2) rectal balloon distensions of 25 mm Hg (sensation of stool or mild pressure),2 two (2) distensions of 50 mm Hg 
(sensation of moderate pressure or discomfort),2 and two (2) thumb pressure applications of 1.4 kg/m2 
(“Moderate pain”),  with 40 second periods of rest interposed between these stimuli.  Following fMRI rectal 
distention/dolorimeter protocol sequence, the participant will be 
removed from the MRI scanner using routine protocol.   
 
Obtaining samples for genetic analysis. 
We will obtain 5 ml of saliva using an established collection technique.32, 33  All DNA samples will be collected by 
the principal investigator.  Samples will be de-identified and delivered to the lab of Dr. Rodney Newberry for 
single nucleotide polymorphism (SNP) genotyping.  Dr. Newberry is a molecular geneticist with more than 20 
years’ experience studying the genetic basis of neuropsychiatric disease.  His laboratory will employ a 
Pyrosequencer or Sequenom mass spectrometer for SNP genotyping.  Using these technologies, to date Dr. 
Newberry’s lab has genotyped several hundred SNPs in several thousand study subjects.   
 
Stool microbiome assay 
Recent work has confirmed that, independent of bowel pattern, IBS patients possess different patterns of 
intestinal flora than control subjects107 with reduced microbial diversity108 and via detailed microbiotal 
examination using high-throughput pyrosequencing, the existence of IBS subgroups, with one IBS subgroup 
resembling controls, and the other with portportional differences in Bacteroidetes and Firmicutes.109   Further, the 
sensitivity to colonic distention was successfully transferred to germfree rats inoculated with the fecal microbiota 
from IBS patients.18 
 
Sample collection (stool). Patients will be provided a sterile stool sample collection container and sterile 
tongue depressors for sample acquisition. Stool samples will be collected as soon as possible, placed within 
sterile CryoVial tubes, snap-frozen in pre-cooled methyl-butane in dry ice, and stored at -80°C until use. 
Data Analysis and Sample Size Calculations 
The proposed experimental include visceral and somatic stimuli which will be studied in a block design, and the 
obtained functional MRI activations will analyzed using the general linear model, combined with appropriate 
corrections for multiple comparisons.90, 93, 96, 97 These methods are implemented within a set of propietary display 
and analysis tools developed by Washington University investigators within the Mallinckrodt Institute of Radiology 
(MIR). The general linear model will be applied to produce statistical and magnitude maps. All analyses will be 
done assuming a standard Boynton hemodynamic response curve; this assumption has been valid in previous 
experiments conducted by Dr. Sheline which have examined similar brain regions of interest (ROIs) in this 
protocol. The statistical maps are then used to define activated regions within subjects. Random effects models 
for group comparisons will be performed in several ways. The increases or decreases in strength of response 

(Delta S/S) will the metric that is used. To statistically evaluate this, ROI analysis will be conducted in 
anatomically constrained areas along with voxel-by-voxel analysis. Both ROI-directed and voxel-by-voxel 
repeated measures ANOVAs (using parameters described below) are done across subjects using model 
estimates as inputs.  Interaction effects and other activations resulting from specific comparisons of a subset of 
the events will be reported at an uncorrected threshold of p < 0.001 for descriptive purposes. For the insular 
cortex, prefrontal cortex, orbitofrontal cortex, anterior cingulate cortex, and thalamic nuclei, predetermined ROIs 
will be used and will include any activations within these regions.  A small volume correction98 will be applied, in 
keeping with the a priori hypotheses regarding these regions. Predetermined ROIs from the HAPN in Talairach 
space will be drawn using the Talairach atlas coordinate system99 and the software ANALYZETM will then be 
applied to all the participants. Regions which would not be expected to differ between the study populations will 
also be selected for analysis, and will provide internal activation standards to provide for the specificity of the 
findings (i.e., control regions).  All selected ROIs will be used as a mask to constrain the regions in which test for 
differences are conducted. These regions will be used as a mask in an overlay on functional datasets to test the  

0.0
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0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Sample Size
Alpha = 0.050, Tails = 2
Effect
Group
Group x Treatment
Treatment
Figure 13: Study power as a function of sample size and effect.
hypotheses.  Only areas with significant F-values on the ANOVA and falling within these regions will be used in 
statistical analyses. There will be 3 event types included in the model, resulting from the combination of all 
possible stimulus and response conditions:  Visceral-Low distention (25-mm Hg), Visceral-High distention (50-mm 
Hg), and the Somatic stimulus.  Voxel-by-voxel analyses will be conducted primarily to ensure that unpredicted 
differences a priori ROIs which are significant are not overlooked.  
 
 
 
 

B.  Sample size calculations.  The power calculations performed to assess the adequacy of the proposed 
sample size are based on the preliminary data, and are driven by the hypotheses proposed in Aim 2. These 
calculations assumed accrual of all subjects prior to the 10-week interval, a 10% attrition rate in each arm (2 
subjects per group) over the course of the trial, observed group mean differences in blood flow within the anterior 
cingulate cortex from the IBS subjects studied in the Preliminary Studies section, and the previously reported 
post-desipramine reductions in ACC activations (effect size = 0.33).33 With the proposed sample size (n=20 per 
group), the anticipated 2-way ANOVA design examining the fMRI data with one between-subjects factor (Group) 
would be powered at 0.95 for detecting the anticipated difference using a conservative treatment effect size of 
0.33, while assessment of Group x Treatment effect would be powered at 0.82 (figure 12).  This sample size 
would allow the detection of a desipramine effect size of 0.25 at p<0.01, and also permit the interpretation of 
predicted non-significant interactions as consistent with the stated hypotheses.  These power calculations were 
conducted using SamplePower v2.0 (SPSS, Inc). 

 
 
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