Long Duration Activity and Metabolic Control After Spinal Cord Injury
Phase: NA
Conditions: Spinal Cord Injuries
Sponsor: Richard K Shields

Study Protocol and Statistical Analysis Plan 
Study Title: Long Duration Activity and Metabolic Control After Spinal Cord Injury 
NCT03139344 
Document Version: 11/3/2022 
 
 
 

Study Objectives 
Skeletal muscle is the largest endocrine organ in the body, playing an indispensable role in glucose 
homeostasis.  Spinal cord injury (SCI) prevents skeletal muscle from carrying out this important function. 
Dysregulation of glucose metabolism precipitates high rates of metabolic syndrome, diabetes, and other 
secondary health conditions (SHCs) of SCI.  These SHCs exert a negative influence on health-related 
quality of life (HRQOL).  New discoveries support that a low level of activity throughout the day offers a 
more effective metabolic stimulus than brief, episodic exercise bouts. 
The purpose of this study is to translate this emerging concept to the population of individuals with SCI 
by using low-force, long-duration electrical muscle stimulation to subsidize daily activity levels.  The 
long-term goal of this research is to develop a rehabilitation strategy to protect the musculoskeletal 
health, metabolic function, and health-related quality of life of people living with complete SCI. 
 
Specific Aim 1: To compare acute changes in skeletal muscle gene regulation in individuals who receive 
a single session of low frequency (LF) or high frequency (HF) exercise.  
Hypothesis 1: The expression of genes regulating skeletal muscle metabolism will support that HF 
and LF both initiate a shift toward an oxidative muscle phenotype.  
Specific Aim 2: To compare changes in skeletal muscle gene regulation and systemic biomarkers of 
metabolic health in individuals with SCI who receive LF or HF for 6 months.  
Hypothesis 2: LF and HF will both yield gene expression changes indicating a shift toward a slow-
oxidative muscle phenotype. 
Hypothesis 3: LF training will yield systemic biomarker changes indicating improved glucose 
homeostasis and reduced systemic inflammation.  
Specific Aim 3: To measure subject-reported HRQOL using the PROMIS survey metric.   
Hypothesis 4: LF training will yield improved self-reported QOL via PROMIS physical and mental 
health scales.  
 
 

Design 
Population: Individuals with motor-complete spinal cord injury (SCI) (AIS A – B), 21 to 60 years of age. 
Exclusion Criteria: Pressure ulcers, chronic infection, lower extremity muscle contractures, deep vein 
thrombosis, bleeding disorder, recent limb fractures, any comorbid disease known to affect bone 
metabolism (such as parathyroid dysfunction), pregnancy, anti-osteoporosis medications, Vitamin D 
supplements, Metformin or other medications for diabetes 
Enrollment: Non-random assignment 
Study Arm 1: Acute gene regulation in response to a single session of low frequency electrically induced 
exercise 
Study Arm 2: Acute gene regulation in response to a single session of high frequency electrically induced 
exercise 
Study Arm 3: Adaptations in gene regulation, systemic metabolic markers, and patient-report metrics in 
response to training with low frequency electrically induced exercise 
Study Arm 4: Adaptations in gene regulation in response to training with high frequency electrically 
induced exercise 
Study Arm 5: Comparator participants will undergo selected outcome measures to provide comparison 
values for Experimental arms. 
 
 
Methods 
Baseline Characteristics: Participants will provide information on age, sex, race, ethnicity, and level of 
SCI (quadriplegia, paraplegia) 
Study Arms 1 and 2:  Participants will undergo unilateral vastus lateralis skeletal muscle biopsy: they will 
be positioned in supine, the skin over the biopsy site will be sterilized, up to 3 mL of 1% lidocaine will be 
injected locally, and a 1/8th inch incision will be made with a scalpel.  The biopsy needle will then be 
inserted and four cores of muscle (~ 20 mg) will be obtained from four passes of the needle through the 
same puncture site. The incision site will be closed with steri-strips or a butterfly bandage and the 
subject will be told not to perform electrical muscle stimulation or wet the area for 48 hours. Muscle 
specimens will be immediately processed for microarray analysis.  Participants will then undergo a single 
session of electrically-induced low frequency (Arm 1) or high frequency (Arm 2) exercise to the 
quadriceps and hamstrings muscles of the non-biopsied limb.  Stimulating electrodes will be placed over 
the quadriceps and hamstrings muscles, and electrical stimulation will be given to induce an isometric 
closed kinetic chain knee extension moment.  Participants will rest for 3 hours and then the vastus 
lateralis biopsy procedure will be repeated on the limb that performed electrically-induced exercise. 
Study Arm 3: Participants will complete a baseline assessment including the biopsy procedure described 
above, completion of the PROMIS self-reported quality of life survey, and venipuncture.  Venous blood 
will be collected into vacutainer tubes with anticoagulant for glucose, kept on ice in dark conditions. The 

blood will be centrifuged and analyzed with a blood auto-analyzer.  After two weeks, participants will 
begin low frequency electrically induced exercise training.  They will perform electrically induced 
exercise as described above for 6 months. At the conclusion of training, participants will undergo repeat 
biopsy, PROMIS survey, and venipuncture. 
Study Arm 4: Participants will complete a baseline muscle biopsy assessment using the procedure 
described above.  After two weeks, participants will begin high frequency electrically induced exercise 
training.  They will perform electrically induced exercise as described above for 6 months.  At the 
conclusion of training, participants will undergo repeat biopsy. 
Study Arm 5: Comparator participants will complete the PROMIS self-reported quality of life survey. 
Outcome Measures: Microarray will be used to measure mRNA transcription levels for genes that 
regulate cellular adaptation to exercise (peroxisome proliferator-activated receptor gamma coactivator 
alpha – PGC1-alpha), muscle oxidative metabolism (pyruvate dehydrogenase kinase – PDK4, actin 
binding Rho activating protein – ABRA, and nuclear receptor subfamily 4 group A member 3 – NR4A3), 
and muscle fiber type (myosin heavy chain 6 – MYH6, myosin light chain 3 – MYL3, myosin heavy chain 7 
– MYH7, actin 3 – ACTN3).   
Standard clinical assays will be used to measure levels of serum biomarkers for metabolic control 
(fasting insulin, fasting glucose, fasting glucose-insulin ratio, fasting hemoglobin A1c) and systemic 
inflammation (c-reactive protein – CRP). 
For the PROMIS participant-report metric of health-related quality of life, raw scores will be used to 
calculate the physical health t-scale and the mental health t-scale. 
 
 
 

Statistical Analysis Plan 
Gene mRNA transcript levels will be expressed as Log2 intensity (arbitrary units).  Each blood biomarker 
will be expressed as its commonly-used clinical unit of measure (eg. mg/dL).  PROMIS physical and 
mental health scores will be expressed as t-scores, which are anchored to the U.S. population mean (50) 
and standard deviation (10). 
Change over time for each gene-related outcome measure will be assessed via one-way ANOVA.  For 
study Arms 1 and 2, gene expression levels from the baseline biopsy will be contrasted to the 3 hour 
post-exercise biopsy.  For study Arms 3 and 4, gene expression levels from the pre-training biopsy will be 
contrasted to the post-training biopsy. 
For study Arm 3, change over time in pre-training and post-training values for systemic biomarkers and 
for PROMIS scores will be assessed via 2-sided t-tests. 
Pre-training values for PROMIS scores in study Arm 3 will be contrasted with the comparator cohort 
(study Arm 5) via 2-sided t-tests.