Clinical trial NCT04263454
Conditions: Fibromyalgia
Keywords: neuroinflammation neuroimaging fibromyalgia endotoxin chronic pain
Sponsor: University of Alabama at Birmingham
Start date: 2020-03-11; Completion date: 2021-12-22
Enrollment: 36 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The major goal of this study is to determine if the innate immune response is dysregulated in people with fibromyalgia, compared to healthy controls. Magnetic resonance spectroscopy will be used to measure changes in the brain's metabolic profile following an experimental immune stimulus, to test whether individuals with fibromyalgia show a heightened immune response in the brain. The ultimate goal of this research is to better understand the role of brain inflammation in the pathophysiology of chronic pain and fatigue, which will guide the development of more effective therapies for these conditions.

Detailed description:
The study consists of two sessions: the screening session and the experimental session. During the initial phone screening, individuals who contact the study team will be given information about the study, including a description of all study-related procedures. If interested, participants will then be taken through a phone screen interview that will ask them to report demographic information (date of birth, race, ethnicity), height and weight, and list of medical conditions and current medications, surgical history within the past year, amount of weekly consumption of alcohol, tobacco products, and illicit substances, viral or bacterial infections within the past 3 months, recent vaccines received, and current participation in other research. The standard MRI Safety form will be administered to ensure participants do not have ferromagnetic implants or other contraindications to MRI (e.g. claustrophobia).A self-report will also be administered to ascertain that potential fibromyalgia participants meet 2016 American College of Rheumatology (ACR) criteria for fibromyalgia. The phone screen is intended to reduce participant burden by identifying exclusion criteria without the need to attend at UAB. Participants who meet initial screening criteria will be invited to attend an in-person screening visit.

During the screening visit, participants will go to the Clinical Research Unit (CRU) on the 15th floor of Jefferson Tower. Participants will be asked to fast prior to this visit. Participants will provide written informed consent before any study procedures are initiated. Participants will provide a urine sample for pregnancy testing. Vital signs (blood pressure, heart rate), aural temperature, height and weight will be measured and recorded. Participants will undergo 12-lead electrocardiogram and provide a maximum of 60cc of blood into labeled blood collection tubes for screening tests. A tender point exam will be administered to assess fibromyalgia symptomatology. The procedure uses a pressure algometer, a hand-held instrument with a small rubber tip that is applied to the participant's skin over standardized regions of the body (occipital, low cervical, upper trapezius, supraspinatus, anterior second rib, lateral epicondyle, gluteal, greater trochanteric, or medial knee). Pressure is slowly increased at a constant rate until the participant reports the sensation changing from one of pressure to one of pain or discomfort. Participants will complete a demographics form , Brief Pain Inventory, Fibromyalgia Impact Questionnaire, Multidimensional Fatigue Inventory, and Multiple Abilities Symptom Questionnaire. The blood will be tested by the UAB Hospital Labs to ensure that the participant meets the study criteria. The ECG will be forwarded to the study physician who will assess for evidence of conduction abnormalities or of ischemia. If the blood tests or ECG are abnormal, the study physician will determine if the participant should be advised to see their PCP. Following availability of all results from the screening visit, participants will be contacted via phone and informed of their eligibility to attend the experimental session.

During the experimental session, the administration of endotoxin, brain imaging, and related procedures will be performed at the CRU. No fasting is required prior to this visit, and participants will be asked to eat breakfast prior to arrival and will be provided with meal suggestions. Baseline vital sign measurements consisting of resting blood pressure and pulse rate will be measured and recorded within 60 minutes prior to the start of imaging. ECG and pregnancy tests will be repeated and participants will be excluded from participation in the session if conduction abnormalities are found (same criteria as used during screening) or if participants are found to be pregnant. Participants will undergo MRI imaging for approximately 1 hour. Participants will leave the scanner and complete questionnaire measures assessing participant's current level of fatigue, pain, and mood symptoms. Grip strength will also be assessed. A low dose of endotoxin (0.3ng/kg) will then be administered by the study nurse via intravenous infusion. Vital signs will be periodically monitored for the rest of the session. Participants will remain in the laboratory for three hours before being placed into the MRI scanner to complete a second, identical, imaging session. The self-report questionnaire measures and grip strength will be repeated every hour following endotoxin administration in order to quantify changes in symptoms due to LPS. To avoid participant discomfort and to optimize study integrity, a standardized meal of approximately 600-kcal will be provided to each participant between the two MRI sessions, as well as a later snack of approximately 200-kcal. The meal is standardized to caloric quantity and timing to offset any effects of food intake on the immune response.

Arms: arm 1: All healthy control participants will undergo the same screening and experimental procedures. Healthy controls must report no other medical conditions ( including fibromyalgia) to be considered for inclusion. All healthy controls will have an MRI scan performed both before and 3 hours post 0.4ng/kg endotoxin injection. arm 2: All fibromyalgia participants will undergo the same screening and experimental procedures. However, fibromyalgia participants will need to meet 2010 American College of Rheumatology diagnostic criteria for fibromyalgia. All fibromyalgia participants will have an MRI scan performed both before and 3 hours post 0.4ng/kg endotoxin injection.
Interventions: intervention 1: Endotoxin, Escherichia Coli intervention 2: Magnetic Resonance Spectroscopy
Intervention descriptions: intervention 1: Endotoxin is a purified lipopolysaccharide prepared from E. coli O:113 and vialed under good manufacturing practice guidelines. intervention 2: MRI is a non-invasive imaging technique and does not utilize ionizing radiation.

Protocol documents:
Prot_002.pdf: 
The Innate Central Nervous System Immune Response to an 
Experimental Immune Challenge in People With Fibromyalgia 
 
 
Protocol 
 
 
NCT04263454 
 
 
Version Date: 01/06/2021 
 
 
Jarred Younger, Professor, Principal Investigator 
University of Alabama at Birmingham 
Birmingham, AL 35294 
 


Methods 
Design  
This study utilized a mixed (group-by-time) design to compare changes in brain 
metabolites before and after an experimental endotoxin challenge between FM and HC groups. 
Participants underwent an MRI 1.5 hours before receiving the endotoxin infusion, and 4 hours 
after. The following were measured with EPSI in 47 brain regions as primary outcomes: brain 
temperature, CHO, MI, and NAA.  
Procedures  
Study procedures were approved by the University of Alabama at Birmingham (UAB) 
Institutional Review Board and conducted in accordance with the Declaration of Helsinki. 
Participants provided written informed consent prior to any procedures. A payment of $50 was 
provided following completion of the screening visit, and $600 was provided for the 
experimental session. Screening and experimental sessions were performed at the Clinical 
Research Unit (CRU) located at UAB Hospital. Brain imaging was conducted at the UAB 
Civitan International Imaging Laboratory (CINL). During the experimental session, participants 
were transported between locations via rideshare transportation.  
Participants  
Women with FM aged 18-55 and healthy age-matched women were recruited from the 
Birmingham community via online advertisements on the UAB Clinical Trials website. Our 
laboratory maintains a database of individuals who have indicated an interest in research, from 
which additional participants were contacted. Participants in the FM group met the revised 
diagnostic criteria by the American College of Rheumatology,1,39,40 endorsed at least eleven out 
of nineteen positive tender points on a physical exam, and reported an average daily widespread 


pain severity of at least 6 on a 0-10 scale. HCs reported average daily pain of 1 out of 10 or less. 
The following exclusion criteria were applied to both groups: i) MRI contraindications; ii) ECG 
abnormalities (QTc>450ms, QRS>120ms); iii) fever >99.0 °F; iv) hypotension or hypertension 
(systolic blood pressure outside of 100-140 range, diastolic blood pressure outside of 60-90 
range); v) resting heart rate <55bpm; vi) body mass index (BMI) outside of 19.0-39.9 range; vii) 
current smoker (within 12 months of study); viii) pregnant, planning to become pregnant, or 
breastfeeding; ix) high alcohol consumption (>1 drink/day or >4 drinks on a single occasion per 
week); x) use of illicit substances within six months of participation; xi) vaccination within four 
weeks of participation; xii) viral or bacterial illness requiring medical attention or antibiotics 
within three months of participation; xiii) concurrent participation in another research study; xiv) 
surgical procedure within three months of participating; xv) history of rheumatologic or 
autoimmune disease (e.g., multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, 
Hashimoto’s thyroiditis); xvi) other major medical conditions (e.g., cancer, blood or clotting 
disorder, HIV, Hepatitis C infection, diabetes); xvii) regular use of anti-inflammatory or 
immunomodulatory drugs, xviii); current use of opioid analgesics; xix) Hospital Anxiety and 
Depression Scale (HADS)41 depression total score ≥13; xx) values outside clinical reference 
ranges on tests of renal function, tri-iodothyronine, thyroxine, thyroid stimulating hormone, 
complete blood count with differential, and 25-hydroxy vitamin; xxi) detectable rheumatoid 
factor or antinuclear antibody, xxii) erythrocyte sedimentation rate >60 mm/h, xxiii) high 
sensitivity C-reactive protein (hsCRP) >10 mg/L, and xiv) ongoing litigation or worker’s 
compensation claim.  


Screening visit 
Initial inclusion and exclusion criteria, including medical history, ACR criteria, HADS 
score, and MRI safety, were established via brief phone calls. Interested individuals presented in 
person to the CRU to complete the remaining screening procedures. Participants underwent a 
urine pregnancy test and ECG. A tender point exam was administered by applying up to 4kg 
pressure at a rate of 1kg per second to the nineteen tender points established by the ACR39 with 
an FPK20 manual algometer (1cm diameter; Wagner Instruments, Greenwich, CT). Thirty-three 
cc blood was collected by research nurses and transported to the UAB Hospital laboratory for 
processing and confirmation of inclusion/exclusion criteria. Participants also completed the Brief 
Pain Inventory (BPI)42 and Fibromyalgia Impact Questionnaire43 (FIQ, FM participants only).  
Experimental Session  
Participants presented to the UAB CRU and underwent a repeat ECG for safety and were 
then transported to the CINL for brain imaging. Participants returned to the CRU, where 
endotoxin from E. coli (0.4ng/kg body weight, N=14, or 0.3ng/kg body weight, N=11) was 
administered via infusion over 30 minutes. The endotoxin dose was reduced to 0.3ng/kg after 
several participants experienced adverse events following 0.4ng/kg infusions. Two standardized 
meals of 834 kcal and 508 kcal were provided to participants prior to the first MRI session and 1 
hour after the endotoxin infusion, respectively.  
Image Acquisition 
MRI data were collected on a Siemens Magnetom Prisma 3-Tesla scanner with 20-
channel head coil. Participants laid supine, were provided with hearing protection, and were 
instructed to keep their eyes closed during the acquisition. A T1-weighted structural brain scan 


was collected with a magnetization-prepared rapid gradient echo (MPRAGE) sequence with the 
following parameters: TR: 2000 ms, TE: 2.51 ms, TI: 901ms, flip angle: 8°, field-of-view 
(FoV): 230 × 230 mm, matrix: 256 × 256, 176 slices, 0.9 mm isotropic voxel resolution. Fast 3-
dimensional echoplanar spectroscopic imaging (EPSI) with spin-echo excitation was performed 
following automatic and manual iterative shimming, with the following parameters: TR: 
1550 ms, TE: 17.6 ms, inversion time (TI) for lipid inversion nulling: 198 ms, flip angle: 71°, 
FoV: 280 × 280 mm, matrix: 50 × 50, 18 slices, 5.6 × 5.6 × 10 mm voxel resolution. The 
acquisition time for EPSI was 17:48 min. Participants had their body temperature measured in 
both ears with a Braun Pro 4000 ThermoScan thermometer before and after the MRI.  
Image processing  
The Metabolite Imaging Data Analysis System (MIDAS)44,45 was used to process the 
whole-brain spectroscopy data as previously described.46-48 Preprocessing is fully automated and 
includes reconstruction of the spectroscopic images, lipid extrapolation and Gibbs ringing 
correction, corrections for b0 shifts, Eddy current correction, interpolation to 64×64×32 voxels, 
smoothing with 5×7mm Gaussian kernel (final voxel size: 5.6×5.6×5 mm), and automated fitting 
of the CHO, creatine (CR), MI, and NAA peaks with Gaussian line shapes. Apparent brain 
temperature was calculated with the following formula: TCR = −102.61(ΔH20−CR) + 206.1°C, 
where ΔH20−CR is the difference in resonance frequencies between the water and CR preaks.49,50 
The metabolite and temperature maps were co-registered with the structural images and the 
structural images were then non-linearly transformed to Montreal Neurological Institute (MNI) 
space. Finally, the inverse spatial transform was applied to a modified Automated Anatomical 
Labeling (AAL) atlas51 with the PRANA module in MIDAS to obtain atlas regions in subject 


space. Mean metabolite levels and temperature were extracted from each ROI for statistical 
analyses.  

SAP_003.pdf: 
The Innate Central Nervous System Immune Response to an 
Experimental Immune Challenge in People With Fibromyalgia 
 
 
Statistical Analysis Plan 
 
 
NCT04263454 
 
 
Version Date: 02/16/2023 
 
 
Jarred Younger, Professor, Principal Investigator 
University of Alabama at Birmingham 
Birmingham, AL 35294 
 


Statistical Analyses  
Statistical analyses were conducted with IBM SPSS for Mac version 27. Body 
temperatures measured in the left and right ear were averaged for each participant. Baseline data 
were compared between groups using independent-samples t-tests.  
Brain metabolite levels were expressed as ratios over the local CR concentration. Brain 
temperatures below 35.0°C and above 42.0°C were excluded as an additional data quality control 
measure, as temperatures outside of this range are not physiological. Mixed analyses of variance 
(ANOVA) were conducted with time as the within-subjects factor (levels: pre-endotoxin, post-
endotoxin) and study group as the between-subject factor (levels: FM, healthy control). 
Endotoxin dose (0.4ng/kg vs 0.3ng/kg) and its interaction with time was added as an additional 
between-subjects factor, and age and the age-by-time interaction was entered as a covariate of no 
interest. The effects of interest were the time-by-group interaction (to test whether FM and HC 
participants exhibited different responses to endotoxin) and the dose-by-time interaction (to test 
whether participants receiving 0.3ng/kg versus 0.4ng/kg endotoxin exhibited different 
responses). Significant interactions were followed up with repeated-measures t-tests assessing 
the effect of time separately for FM patients and HCs. The dependent variables were brain 
temperature, CHO/CR, NAA/CR, and MI/CR in the 47 brain regions. Because of the small 
sample size and lack of statistical power, all tests were considered significant at p < 0.05. 
However, all tests meeting a more stringent false discovery rate threshold are highlighted. The 
false discovery rate threshold of 0.05 yielded a corrected p-value threshold of 0.0113.