Transcranial Direct Current Stimulation to Reduce Asymmetric Glucose Uptake in Leg Muscles of Persons With Multiple Sclerosis
Phase: NA
Conditions: Multiple Sclerosis
Sponsor: University of Iowa

Study Protocol   June 20, 2022 
NCT 04016844 tDCS and Glucose Uptake in Leg Muscles 
This study utilized a single-blind, randomized, SHAM-controlled, cross-over design. Each 
participant attended three experimental sessions, with sessions 1 (strength testing and treadmill 
familiarization) and 2 (first tDCS/PET Scan session) separated by at least 3 days to allow for 
ample recovery after strength testing and sessions 2 and 3 (first and second tDCS/PET 
sessions) separated by at least 7 days to allow the tDCS effects to subside. The experimental 
protocol is shown in Figure 1. During Session 1, subjects were consented and then filled out the 
Patient Determined Disease Scale (PDDS), which is strongly correlated with and is considered 
an alternate assessment to the Expanded Disability Disease Status Scale (EDSS) and the 
fatigue severity scale (FSS) questionnaires. The subjects then completed isokinetic strength 
testing to objectively determine their more-affected (weaker) leg. Subsequently, the subjects 
walked on a treadmill to self-select a comfortable walking pace, which was utilized in 
experimental sessions 2 and 3. At the beginning of Session 2 and 3, blood glucose, height, and 
weight were measured, and an IV catheter was inserted to facilitate FDG administration. Prior to 
Sessions 2 and 3, the subjects fasted for a minimum of 6 h and blood glucose was required to 
be ≤ 200 mg/dL to proceed with FDG administration and the PET scanning. The subjects then 
sat comfortably in a chair and received 20 min of SHAM or tDCS (3 mA; stimulation condition 
was randomized) targeting the motor cortex corresponding to their more-affected leg, as 
determined in the strength testing (see below). The subjects then rested for 10 min to allow for 
optimal stimulation effects. After this rest period, the subjects walked on a treadmill for 20 min at 
the speed determined in Session 1. Two minutes into the walking, ~10 ± 10% mCi of FDG was 
injected via IV injection. Immediately after the 20-min walking task was completed, the subjects 
underwent positron emission tomography/computed tomography (PET/CT) imaging. 
Data Analysis and Statistics 
Twenty regions of interest (ROIs) were drawn on the CT scan from each session (SHAM and 
active) by the same investigator to locate the lower limb skeletal muscles. The muscles that 
comprise the knee extensors (rectus femoris, vastus medialis, vastus intermedius, and vastus 
lateralis) and knee flexors (semimembranosus, semitendinosus, long head of the biceps 
femoris, short head of the biceps femoris, gracilis, and sartorius) were identified via visual 
inspection in the upper leg, and the plantar flexors (gastrocnemius, soleus, peroneus longus, 
peroneus brevis, flexor digitorum longus, flexor hallucis longus, and tibialis posterior) and 
dorsiflexors (tibialis anterior, extensor digitorum longus, and extensor hallucis longus) were 
distinguished in the lower leg. Figure 2 displays a representative upper leg CT image with ROIs 
identified, a corresponding PET image, and the CT and PET images co-registered. As a result 
of FDG uptake occurring during the treadmill task, glucose uptake (GU) values closely reflect 
FDG uptake during the task. For each ROI, standardized uptake values (SUVs) were calculated 
based on the injected FDG dose and subjects’ body weight. Despite the fasted state of the 
subjects, SUVs may be affected by varying insulin levels during Sessions 2 and 3. Therefore, 
SUV data were analyzed without normalization and as values normalized to the liver activity as 
a reference tissue. Moreover, SUV asymmetry indices (AIs) were calculated to determine the 
magnitude of asymmetry between the more- and less-affected legs with a previously used 
equation: ((less-affected side − more-affected side)/((0.5) × (less-affected side + more-affected 
Last approved by IRB on 06/22/20

side)) × 100). An AI value ≥ 10% was considered asymmetric. The relative distribution 
((standard deviation / mean) × 100) of GU values in PET image voxels within each muscular 
ROI was calculated as an index of spatial glucose uptake heterogeneity (GUh). The data were 
analyzed using PMOD Version 4.001 (PMD Technologies LLC, Zurich, Switzerland).  
Statistical Analysis  
Mean ± standard deviation SUVs and GUh for each muscle group (i.e., knee extensors, knee 
flexors, plantar flexors, and dorsiflexors) were calculated for each subject, and statistical 
analyses were performed for each muscle group. Normality assumptions were evaluated by way 
of histograms, Q-Q plots, and the Shapiro–Wilk test. Because these assumptions were met, 
paired t-tests were performed for each participant to compare the muscle groups of each leg 
between conditions (e.g., left knee extensors during SHAM vs. left knee extensors during 
tDCS). Significance was accepted at p < 0.05, and Cohen’s d effect size was calculated for all 
significant results. Analyses were performed using GraphPad Prism 8.1.2 (GraphPad Software, 
San Diego, CA, USA).