Non-invasive Approaches to Identify the Cause of Fatigue in Inflammatory Bowel Disease Patients.
Non-invasive Approaches to Identify the Cause of Fatigue in Inflammatory Bowel Disease Patients.
Crohn's disease (CD) presents with severe symptoms, but fatigue is a very predominant symptom
 that negatively impacts upon quality of life. Fatigue affects ~40% of patients when well and
 80% of patients when the disease is active. It is the second commonest symptom that an IBD
 patient gets throughout their life-time. The IBD priority-setting partnership between the
 James Lind Alliance and the British Society of Gastroenterology has recently identified
 fatigue as an area of unmet clinical need and a priority research field, in which diagnosis
 and therapeutic intervention are lacking.

 Based on other diseases that present with fatigue, the cause of fatigue may be divided into
 peripheral fatigue, mainly driven by anomalies in muscle mass and function and central
 fatigue, mainly driven through decreased blood supply to the brain during exercise probably
 due to decreased heart and lung fitness.

 Research in IBD fatigue until now has been patchy with no convincing evidence that any
 treatment helps. There has been no research aimed at studying whole body function. It is
 imperative to have a better understanding of the alterations in muscle, brain, heart and lung
 function seen in these patients before specific treatments are researched.

 In this study, the investigators aim to recruit 32 CD patients, half with fatigue and half
 without. Subjects with active disease or with other known reasons of fatigue will be
 excluded. Findings in this group will be compared to 16 other healthy control volunteers of a
 similar age, gender and Body Mass Index. The study aims to recruit all participants over 36
 months, and will target people aged from 16 to 60 years of age.

 Once recruited, the participants will be asked to provide their consent to take-part in 3
 experiments on two separate days. These experiments have been designed to carefully consider
 potential fatigue burden, experimental practicality, and participant availability.

 Objective 1: The investigators aim to measure muscle fitness and strength by asking subjects
 to exercise using a stepper, whilst body mass and composition will be measured using an
 X-ray. This session will take 2 hours and be undertaken on one day.

 Objective 2: Peripheral fatigue: The investigators aim to non-invasively measure the recovery
 of muscle physiology after exercise by using magnetic resonance imaging after 5 min of
 exercise undertaken with a limb cuff. This will take ~1 hour.

 Objective 3: Central fatigue: while in the scanner and performing exercise, the investigators
 aim to non-invasively measure heart and brain blood flow before and after a few minutes of
 exercise using magnetic resonance imaging. This will take 2 hours.

 Experimental work for Objectives 2 and 3 will be undertaken on the same day. There will be
 ample time for recovery in between and during the different studies. There will be no further
 commitment from the participants required after these 2 study visits.

 IBD fatigue has never been studied in such detail. This unique work will allow identification
 of fatigue mechanisms, which can then be targeted with exercise, nutritional, or medical
 treatments.
Background information Inflammatory bowel disease (IBD) fatigue is, with diarrhoea, the most
 common medical symptom in patients with active Crohn's disease (CD) and the second commonest
 complaint after arthralgia in CD patients in remission. The IBD priority-setting partnership
 between the James Lind Alliance and the British Society of Gastroenterology has highlighted
 that IBD fatigue is a top 10 UK research priority.

 The prevalence of fatigue ranges from 41-48% when in remission, increasing up to 86% when the
 disease is active. Chronic fatigue is associated with an impaired health-related quality of
 life in IBD and a reduced physical activity, which in paediatric cohorts plays a major role
 in normal growth development. In the absence of reversible clinical causes such as anaemia,
 inflammatory burden or nutrient deficiency, it is as yet unclear what causes fatigue in these
 patients. Present approaches are limited in offering clues to potential therapeutic
 approaches or accurate clinical evaluation for CD patients. This is likely due to a lack of a
 multicomponent approach and poor methodological sensitivity and reliability when assessing
 fatigue. A multidimensional concept of physical, cognitive and affective components have been
 suggested for IBD fatigue. Yet, it remains unclear how to treat IBD fatigue. Exercise
 therapy, psychosocial interventions and solution-focused therapy, are all suggested as
 treatment options but none have been shown to be convincingly effective.

 Fatigue arises from peripheral and central processes, which are both equally important. While
 peripheral fatigue is ascribed to muscle aetiology, central fatigue is a progressive
 exercise-induced reduction in voluntary activation to the muscle due to failure of the
 central nervous system (CNS). Present approaches have been limited in failing to adequately
 quantify peripheral fatigue at a muscle level and central fatigue at the level of the CNS.

 Peripheral fatigue: the need of muscle activity

 Physical fatigue is linked to reduced muscle mass and quality (sarcopenia) and consequently
 reduced muscle function. Drivers of muscle mass loss include physical inactivity and anabolic
 resistance, which to a large extent blunt the stimulatory effect of protein nutrition on
 muscle protein synthesis. In keeping with this, patients with active CD show a significant
 decrease in expression of hypertrophy skeletal muscle signalling pathways, but no changes in
 the expression of atrophy signals.

 Poor muscle quality is an important driver of metabolic dysfunction that is often overlooked
 as a driver of peripheral fatigue. In preliminary unpublished data, the investigators have
 shown for the first time that skeletal muscle of patients with active CD exhibits blunted
 glucose uptake in response to glucose feeding, suggesting diminished muscle metabolic quality
 in CD patients.

 In fatigued IBD subjects, medium-to-large effect sizes for impaired aerobic fitness (VO2max),
 muscle strength and physical activity are seen in subjects with fatigue when compared to
 subjects with no reported history of fatigue and the normal population. Physical inactivity
 in quiescent CD disease could be an explanation underpinning these observations, pointing to
 inactivity secondary to disease burden being the aetiology of fatigue in CD, much as in
 chronic obstructive pulmonary disease (COPD). Physical inactivity has already been shown in
 CD in both paediatric and adult cohorts and has been significantly correlated to disease
 activity but is still prevalent in remission. Although this might imply circularity in the
 aetiology of fatigue in the context of physical inactivity, it is unlikely that these two
 factors have a simple direct cause and effect relationship. Indeed, when habitual physical
 activity is matched between volunteers, skeletal muscle fatigue is still most prevalent in
 CD, and predominantly in those subjects who complain of fatigue. Collectively, these data are
 indicative that both reduced cardio-respiratory fitness and peripheral muscle deconditioning
 (possibly linked to reduced muscle mitochondrial density) are contributing to early fatigue
 development in IBD patients who report symptoms of fatigue.

 Central fatigue: the need of cardio-respiratory regulation

 Skeletal muscle, the cardio-respiratory system and the CNS are interlinked. In preliminary
 data, it has been shown for the first time that it is possible to assess change of brain
 perfusion (or cerebral blood flow; CBF), an indirect measurement of haemodynamic measurement
 of brain neuronal activity, using functional MRI techniques, in relation to decreased
 cardiovascular fitness with age. These data have demonstrated that CBF measured directly
 using arterial spin labelling (ASL) or indirectly using blood-oxygen-level-dependent (BOLD)
 was reduced in older people with reduced cardiorespiratory fitness, and who are likely more
 fatigable than young volunteers. This suggests that the coupling between CBF and brain
 activity can be modulated by cardiac output, and thus be manipulated to improve brain
 function linked to central fatigue in CD patients.

 This highlights the potential of non-invasive functional brain MRI techniques to determine
 the effect of brain perfusion during exercise on central fatigue in CD patients. Furthermore,
 these quantitative approaches could be used to determine whether improvements in
 cardiovascular fitness and appropriate downstream exercise or pharmacological interventions
 can alter central fatigue.

 1.Aims and purpose of the proposed investigation

 Hypothesis: The investigators hypothesize that the aetiology of IBD fatigue lies collectively
 with peripheral muscle deconditioning and brain central fatigue, the latter being secondary
 to a reduction in cardio-respiratory fitness and/or cardiac output, such that both central
 and peripheral fatigue contribute to fatigue development in IBD.

 Aims:

 To address this hypothesis, the investigators aim to recruit a cohort of fatigued and
 non-fatigued CD patients in remission and age-, gender- muscle mass-, and physical
 activity-matched healthy volunteers. Fatigue symptoms will be defined through relevant
 questionnaires at screening. The investigators will subsequently objectively quantify muscle
 strength of the knee extensors during a maximal voluntary isometric contraction, and fatigue
 development in the quadriceps muscle group. The latter will be defined as the loss in muscle
 peak-torque development over 20 consecutive isokinetic knee-extensions. Peripheral (muscle)
 and central (CNS) roles in fatigue aetiology will be investigated. Peripheral muscle
 deconditioning will be investigated by quantifying muscle phosphocreatine (PCr) re-synthesis
 during recovery from exercise using 31phosphorous MRS, while the potential of central fatigue
 will be examined through various measures of CNS perfusion and fractional brain oxygen
 extraction using MRI.

 Detailed plan of investigation and scientific procedures/methodology

 The investigators aim to recruit 32 patients with CD in remission, with and without a history
 of IBD fatigue (n=16 for IBD fatigue cohort and n=16 for no IBD fatigue cohort) within 36
 months. These cohorts will be compared to 16 age-, gender- muscle mass-, and physical
 activity-matched healthy controls (from our existing Nottingham Digestive Diseases Biomedical
 Research Unit and MRC/ARUK databases) to undergo the experimental protocol outlined below.
 The investigators will specifically match for physical activity to investigate its potential
 primary effect on fatigue.

 Objective 1 (Day 1): To determine muscle strength, fatigue and cardiorespiratory fitness.

 Following consent, CD and healthy matched controls volunteers will perform 3 maximal
 isometric knee extensions of the quadriceps muscle group (each separated by 30s) to determine
 muscle strength. Following this, volunteers will perform 20 maximal single leg knee
 extensions on a Cybex isokinetic dynomometer (Cybex Norm, USA) at a constant angular velocity
 of 90 degrees per second to determine muscle torque development. Each maximal voluntary
 contraction will be initiated from a position of 90 degrees knee flexion and continued
 through to the point of full knee extension. After each contraction, the leg will be returned
 passively to the start position (approximately 1.1s) from which the next contraction will be
 immediately initiated. The outcome measure and the gold-standard definition of fatigue in
 this objective will be defined as the decline in peak torque generation for each contraction
 over the 20 knee extensions. Maximal voluntary contractions at this velocity has previously
 been shown to result in recruitment of both type I and type II muscle fibres of the
 quadriceps muscle group. Thirty minutes later, volunteers will undergo a continuous,
 incremental, supine exercise test using an MR compatible step ergometer (Ergospect diagnostic
 pedal). Exercise will commence at a low workload, which will increase every 3 min to
 determine peak VO2 during supine exercise using on-line expired gas analysis (Cosmed, Rome,
 Italy). Carbon dioxide (CO2) production and oxygen (O2) consumption will be measured
 throughout the test and used to measure peak oxygen uptake and respiratory exchange ratio
 (RER). These data will be normalized to body composition as measured by dual-energy X-ray
 absorptiometry (DEXA; Lunar Prodigy, GE Medical Systems, Bucks, UK) and will provide a
 gold-standard measure of cardiorespiratory (or aerobic) fitness.

 Objective 2 (Day 2): To determine brain perfusion and central fatigue.

 Recently, in collaboration with researchers at the Sir Peter Mansfield Imaging Centre at the
 University of Nottingham, the investigators have developed a protocol to quantify the
 cortical vascular response to exercise in volunteers and its modulation in ageing and
 cardiorespiratory fitness. Here, the same approach will be used to assess cortical vascular
 response to exercise in the brain regions associated with central fatigue of CD patients.

 To study this mechanism in CD patients, the investigators propose to measure cardiac output
 and, cerebral and regional perfusion using Blood-Oxygen-level-dependent (BOLD) measures and
 arterial spin-labelling (ASL)-MRI as well as fractional oxygen extraction using TRUST-MRI
 respectively during supine exercise of the quadriceps with the MR compatible ergometer
 (Ergospect diagnostic pedal) at given values of relative VO2max (as measured in Objective 1).

 This multimodal approach will determine a potential uncoupling between brain perfusion and
 neuronal activity and the role of central hypoxemia in central fatigue of IBD patients
 compared to healthy controls. Furthermore, these data will be correlated to cardiorespiratory
 fitness, muscle strength and fatigue, and will determine the predominant role of central
 processes in fatigue of IBD patients. If these brain vascular measures are associated with
 fatigue development, then this index of neurovascular coupling could benefit from exercise
 training in a similar way to muscle tissue.

 Objective 3 (Day 2): To determine muscle deconditioning.

 To maintain energy (ATP) during exercise, skeletal muscle regulates oxidative (mitochondrial)
 and non-mitochondrial production of ATP. In healthy individuals, the relative contribution of
 non-mitochondrial ATP production to total energy production is generally dictated by the
 exercise intensity, which determines the magnitude of accumulation of metabolic by-products
 that are directly linked to muscle discomfort and fatigue. In disease states, exacerbated
 fatigue and muscle pain has been attributed to reduced muscle blood flow and/or reduced
 mitochondrial mass.

 To separate these two processes in CD patients, limb blood flow will be occluded with a cuff,
 whilst participants perform repeated plantar flexion exercise in a MRI scanner. This will
 drive PCr, (the available reserve of ATP) to zero. Specifically, participants with a lower
 limb cuff will perform 30 contractions over a 5-minute period using a MR compatible
 ergometer. Once the cuff is removed, they will undergo non-invasive and well-established in
 vivo 31P magnetic resonance (31P MRS) measurements on the gastrocnemius to determine PCr
 re-synthesis rate during resting recovery. 31P MRS data will be averaged, processed and
 quantified using in-house methods.

 Since the rate of PCr re-synthesis is directly proportional to mitochondrial mass and oxygen
 delivery is not limiting during exercise recovery, this will allow us to measure muscle
 metabolic deconditioning in vivo.

 Sample size

 1. Cardiorespiratory fitness: The investigators have previously shown that the VO2max of a
 healthy volunteer is 3.65 (0.5) L/min, whilst the VO2max of a fatigued IBD patient is
 1.99 (0.44) L/min and a non-fatigued patient is 2.43 (0.75) L/min. Assuming a power of
 80% and α=0.05, 6 subjects in each group are needed to show a significant difference
 between the healthy volunteers and the non-fatigued cohort, and 13 patients in each
 group are needed to show a difference between the 2 CD cohorts.

 2. Muscle Fatigue: Peak muscle Torque extension 180⁰/s (Nm) is lower in fatigued IBD vs
 non-fatigued IBD patients (60.73 vs 73.5, SD 12.3 respectively). Assuming a power of 90%
 and α=0.05, 16 CD subjects in each group are needed to show a difference between
 fatigued and non-fatigued CD cohorts. Importantly, the rate of muscle fatigue (defined
 as the gradient of force decrease from repetition 2 to 30 of an exercise protocol) is
 significantly greater in CD than healthy volunteers (5.2 Nm/min vs 1.3 Nm/min, SD 3. In
 this instance, assuming a power of 90% and α=0.05, 11 subjects in each group are needed
 to show a difference between CD and healthy volunteers.

 3. Muscle conditioning: recent studies have shown that Pcr re-synthesis rate is 27.9 ± 0.6
 s in lean sedentary subjects, whilst being 36.5 ± 2.4 s in Type 2 diabetes mellitus
 patients (T2DM). Thus, assuming a power of 100% and α=0.05, 3 subjects in each group are
 needed to show a difference between healthy volunteers and potentially fatigued cohort
 such as T2DM patients.

 4. Anabolic resistance: Furthermore, our recent work on anabolic resistance has already
 shown significant differences in muscle quality between CD and healthy volunteers
 (significant difference observed n=5 patients). Percentage forearm fatigue in the
 dominant arm is 26.75%±11.1 and 18.3±7.8 in CD and controls respectively. Assuming a
 power of 80% and α=0.05, 11 subjects are needed in each arm.

 5. MRI: It is not possible to power this exact MRI study as pilot data in these IBD cohorts
 has not been collected. However, the investigators have previously shown that brain
 perfusion in the young increases by 18±6% for 30% VO2max during a cardiorespiratory
 fitness test. Whilst in the elderly, it increases by only 3±4 %. Assuming a power of
 100% and α=0.05, 10 subjects in each group are needed to show a significant difference
 between healthy volunteers (young) and a potentially fatigued cohort (elderly). Further,
 for fMRI BOLD analysis it is generally considered that a minimum of 12 subjects is
 needed in each cohort, with improved sensitivity of MR scanners (and thus higher
 temporal signal-to-noise ratio (SNR) now allowing improved individual subject analyses.

 For the above reasons, the investigators feel that 16 subjects in each group (fatigued CD,
 non-fatigued CD and healthy volunteers) will suffice to show a difference in our chosen
 objective endpoints.

 Data analysis Fatigue will be measured as the decline in peak torque development over 20
 isokinetic knee extensions as described above. PCr re-synthesis rate and fractional brain
 oxygen extraction will be the primary endpoints in objectives 2 and 3.

 31P MRS data will be analysis using standard NMR software (jMRUI Software for the clinical
 and biomedical MRS). Precisely, all spectra from the time series will be apodized (15-20 Hz
 Lorentzian shape). From these spectra, the signal intensities of 5 peaks will be determined
 after lineshaping. The pH will be determined prior to and at the end of the exercise protocol
 by measuring the frequency shift between the PCr and Pi, whilst in-house written code in
 MATLAB (MathWorks Inc., Matrix House, Cambridge, UK) will be used for further calculations of
 the PCr recovery rate. In this case, peak area intensities of the PCr peak from jMRUI will be
 used as input in the script to fit the data time courses using a mono-exponential function:
 PCr (t) = PCr (t0) + C (1 - e-t/τ); with PCr being the intensity level of the PCr peak, C a
 constant, τ [s] the rate constant for PCr recovery, and t [s] time, starting at the end of
 the exercise protocol (t0). The Reproducibility of 31P MRS measures of muscle energetics has
 been recently evaluated in trained human and show that coefficient of variation during
 exercise (27%) and at rest (8%) was relatively low, and difference were detectable with 15
 volunteers. This confirms that 31P MRS approach provides reproducible and experimentally
 useful measures of PCr re-synthesis rate in human skeletal muscle.

 BOLD MRI data will be analysed using standard neuroimaging packages (Statistical Parametric
 Mapping (SPM); Wellcome Department of Imaging Neuroscience and FMRIB Software Library
 software. Images will be motion-corrected, normalized to a standard Montreal Neurological
 Institute template to facilitate inter-participants averaging. For all data acquisitions, the
 investigators will record the vectocardiogram (VCG) and respiratory motion to allow data to
 be corrected for physiological noise. Contrast vectors will be formed to identify the CNS
 response to the exercise stimulus. The investigators will form statistical maps of those
 brain regions which show a decrease/increase in BOLD signal following the exercise
 intervention. These statistic maps will be used in contrasts by intervention and between
 participant groups. Results will be pooled in a second level, random effects group analysis
 (RFX) and active brain areas displayed at a false discovery rate corrected probability P <
 0.05, k > 5. These analyses have been previously carried out and published by our group. ASL
 data will be motion corrected and modelled to quantify perfusion in ml/100g/min, and global
 and regional perfusion measures compared before and during the exercise intervention. TRUST
 data will be used to compute fractional oxygen extraction before and during exercise
 intervention. Data will be presented as mean +/- standard error of the mean (SEM). Binary
 comparisons will be carried out by t-tests while multiple comparisons will be carried out
 with an analysis of variance with a post-hoc Bonferroni correction in between the 3 groups.
 The effects of cytokines, vitamin D and disease duration will be investigated through a
 correlation co-efficient and post-hoc multivariate analysis.

 The investigators will stratify subjects based on the quantitative outcome measures: PCr
 re-synthesis rate, cardiorespiratory fitness as measured through VO2max, decline in muscle
 output and change in regional perfusion and fractional brain oxygen extraction irrespective
 of their fatigue status. This will aid our understanding regarding which alterations in
 physiology and metabolism are best associated with specific fatigue symptoms. Doing this will
 allow objective quantification of a subjective cut-off for the IBD Fatigue scale, thereby
 better defining fatigue.
Inclusion Criteria:

 Non-Fatigued CD Patients:

 -  Harvey Bradshaw index <4

 -  CRP<5mg/dl or

 -  Faecal calprotectin <50ug/g or,

 -  As evidenced by recent endoscopy or cross-sectional imaging,

 -  General fatigue and physical fatigue score on the Multiple Fatigue Inventory-20 <13

 -  Score of <3 in the Fatigue questionnaire.

 Fatigued CD Patients:

 -  Harvey Bradshaw index <4 and,

 -  CRP<5mg/dl or,

 -  Faecal calprotectin <50ug/g or,

 -  As evidenced by recent endoscopy or cross-sectional imagining and,

 -  General fatigue and physical fatigue score on the Multiple Fatigue Inventory-20 >14

 -  Score of >4 in the Fatigue questionnaire.

 Inclusion criteria (Healthy volunteer participants)

 -  26 Healthy Volunteers 16-75 years old matched for:

 -  Gender

 -  Muscle mass

 -  Physical activity

 -  General fatigue and physical fatigue score on the Multiple Fatigue Inventory-20 <7.

 All participants should have a good command of both verbal and written English and be able
 to give informed consent.

 Exclusion Criteria:

 Exclusion criteria (CD and Healthy volunteer participants)

 Potential participants with any of the following criteria will be excluded:

 -  >8 on the Hospital Anxiety and Depression score

 -  Haematological or biochemical abnormalities (e.g. anaemia (haemoglobin <13g/dl in a
 male and 12g/dl in a female)

 -  Renal failure

 -  Hypokalaemia

 -  Pregnancy or childbearing in the last 6 months

 -  Vitamin B complex deficiencies)

 -  Active or previous prescriptions of corticosteroids in the last 12 weeks

 -  Overt muscle wasting (defined as 2 standard deviations outside the age-related norm as
 measured by DEXA)

 -  Fatigue starting after the onset of thiopurine therapy

 -  Present arthritis or arthralgia

 -  Surgical intervention in the last 12 weeks

 -  Other aetiologies of chronic liver disease, specifically alcohol or drug induced liver
 disease, autoimmune or viral hepatitis, cholestatic or metabolic/genetic liver disease
 by specific clinical, biochemical, radiographic and /or histological criteria.

 -  Significant cardiovascular or respiratory disease

 -  Thyroid disease

 -  Current Infection

 -  Neurological or cognitive impairment

 -  Significant physical disability

 -  Pregnancy or breastfeeding. Participants will be informed before the DEXA scan that
 pregnancy is an exclusion and standard NHS procedures will be followed- pregnancy
 tests will be available in the female toilets of the Physiology Unit for self-testing)

 -  Any other conditions in addition to the above that the investigators consider may
 affect study measurements or safety

 -  Inability to understand verbal and/or written explanation of the study requirements

 -  >5mSv ionizing radiation exposure in the past 12 months