The Innate Central Nervous System Immune Response to an Experimental Immune Challenge in People With Fibromyalgia
Phase: PHASE2
Conditions: Fibromyalgia
Sponsor: University of Alabama at Birmingham

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.