A five year, two-site, randomized controlled study is proposed to investigate the efficacy of NMES +FES versus Passive + FES (control group) on health-related secondary complications after SCI (Table 1). A third site will be used for protein and RNA analysis that will generated from muscle biopsy samples. Patches of muscle biopsy samples will be shipped overnight at the completion of the trial for analysis purpose (see below). All training aspects will be conducted at the SCI Exercise Body Composition Laboratory and MRI Research center located at the McGuire VA Medical Center (VAMC) and the Clinical Research Center (CRC) located at Virginia Commonwealth University (VCU). All participants will undergo metabolic studies, body composition assessments using anthropometry, dual x-ray absorptiometry and magnetic resonance imaging (MRI) and muscle biopsies. After informed consent, each subject will undergo a complete physical examination, including neurological assessment, and International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI), formerly known as the “ASIA” exam.
Additionally, MRI scans will be obtained for trunk VAT, lower extremity skeletal muscles and IMF cross sectional areas (CSA).2,3 Participants will then be escorted to the VCU CRC and will remain in the CRC overnight for metabolic testing. This procedure will be used for the three study visits (baseline, post-intervention 1 and 2). Resting blood pressure and fasting metabolic labs will be obtained including HbA1c, as well as lipid panels, C-Reactive Protein (CRP), Interleukin–6 (IL–6), Tumor Necrosis Factor alpha (TNF-α), and free fatty acids (FFA). This will be followed by an intravenous glucose tolerance test (IVGTT). The vastus lateralis muscle will be biopsied to measure proteins expression, myosin heavy chain expression and to determine mitochondrial ETC and enzymatic activities. The design of the study is outlined in Figure 2.
The recruitment process started in October 2015 and will end in September 2020. Forty-eight participants will be randomly assigned into either NMES+FES or control + FES groups for 24 weeks. The participants will be matched based on level of injury (tetraplegia vs. paraplegia) and time since injury (less versus more than 10 years) using block randomization using a 2 x2 design developed by the study statistician. Randomization was conducted using n-query computer program at the baseline prior enrollment in the trial (Table 1). Current recruitment status is outlined in Figure 3.
Participant Inclusion and Exclusion Criteria
All participants will be between 18–65 years old, men/women24, greater than one-year post SCI, with BMI < 30 Kg/m2.23 Participants with traumatic motor complete or incomplete C5-L2 level of injury, the American Spinal Injury Association (ASIA) Impairment Scale (AIS) classification A, B, or C will be considered for the trial. Participants with any of the following pre-existing medical conditions will be excluded: cardiovascular disease, uncontrolled type II DM, uncontrolled hypertension, insulin dependence, pressures sores stage 3 or greater, hematocrit above 50%, symptomatic urinary tract infection, or participants with neck of femur or total body osteoporosis (T-score equal or worse than –2.5 according to the World Health Organization guidelines.)26
A recent video publication provided full details on the NMES-RT protocol.27 Briefly, NMES will be applied to the knee extensor muscles via surface electrodes to induce concentric-eccentric actions. Two 8 X10 cm2 adhesive carbon electrodes will be placed on the skin over the knee extensor muscle group. Current from the stimulator will be manually increased in 5-second intervals to evoke full knee extension against gravity followed by gradual reduction of current to induce eccentric action during relaxation. Training will be performed twice weekly for 12 weeks. Each training session will consist of 4 sets of 10 repetitions of NMES with 30 Hz, 450µs pulses and a current sufficient to evoke full knee extension.28–30 The first week of the RT will be conducted with no ankle weights to ensure that the knee extensor muscles can extend the weight of the lower leg against gravity.3,23 Once full knee extension is achieved in a sitting position, an increment of 2lbs will be used on a weekly basis with the criteria that full knee extension should be achieved before further increases in load.21–24,31
Passive Range of Motion for Control
A member of the research team supports the leg proximal to the ankle joints and moves it from 90° knee flexion close to full knee extension. The leg will be maintained up for 5 seconds and returned down for 5 seconds. The passive movements will be repeated in the same fashion described in RT protocol: 10 reps for the right leg followed by 10 reps for the left leg for total of 4 sets x 10 reps.
Functional Electrical Stimulation-Lower Extremity Cycling (FES-LEC)
A recent video publication provided full details on the FES-LEC protocol 27. Briefly, rides will be performed with each subject using conductive adhesive gel electrodes.19, 32 Rectangular adhesive electrodes are placed on the skin of the knee extensor, hamstrings, and gluteus maximus muscle groups. Pulse frequency will be set at 33.3 Hz, pulse duration at350 µs and resistance will be adjusted every 10 minutes to maintain a speed of 40–45 revolutions per minute (RPM). Table 2 highlights the progression of resistance in 0.5 Nm increments per 10-minute stage over the course of 12 weeks. The progression in resistance was customized based on the subject’s performance riding the FES-LEC over 12 weeks. Initially, the servo-motor of the bike is permitted to assist during the warm-up phase (5 minutes) and during training to reduce muscle spasms. Participants are then allowed 2–3 minutes of rest between each 10-minute stages. Time to maximum (100%) current amplitude, power, and distance covered will be recorded at the end of each stage to determine whether these parameters improved in both groups (NMES+FES vs. control). The fatigue threshold will be set at 18 RPM; if RPM falls below 18 RPM; the bike will automatically shift from active to passive cycling (cool-down). During the three-minute cool-down period, participants will be passively cycled with no electrical stimulation. The cool down period will then be followed by 5 minutes of recovery, during which the participant will be still connected to the bike but in a complete resting position. Heart rate will be monitored every 30 seconds and blood pressure every 2 minutes.
The use of NMES-RT or FES-LEC may result in the following potential harms and complications
- Light-headedness, shortness of breath and altered heart rate & blood pressure leading to autonomic dysreflexia. Muscle soreness of the neck, upper back, shoulders, arms & hands.
- Fracture of the exercising bones.
- Autonomic dysreflexia (slow heart rate, high blood pressure, headache flushing &sweating) which may be life threatening
- Skin irritations or pressure injuries from shear stress that may lead to breaking the skin.
- Fainting or heart attacks.
Blood pressure will be continuously monitored during exercising sessions to ensure no signs of orthostatic hypotension or autonomic dysreflexia and participants will be regularly checked for any changes in their physical status or health compared to their initial admission in the study and the SCI provider will be notified immediately. Prior admission to the study, participants will undergo DXA scans (see below) to ensure adequate bone health especially at the hip joints (T-scores < –2.5 SD), distal femur and proximal tibia (bone mineral density should be greater than 0.6 gm/cm2) to reduce the likelihood of fracture during exercising. The skin will continuously be checked on a regular basis prior to and following removal of the electrodes to ensure no redness or tendency to develop skin irritations pressure injuries.
Other unanticipated adverse events will be monitored throughout the study via exams, vital signs, laboratory tests, review of medical charts, and verbal concerns voiced by the participant or an associated caregiver. Finally, data safety monitoring board of independent research investigators will annually review the study related adverse events, adequacy of subjects’ compliance and adherence to the protocol.
1.1 Dietary records and basal metabolic rate (BMR)
Each participant will follow a standard diet pattern during the entire period of the study (45% carbohydrate, 35% fat and 25% protein) to balance dietary habits between both groups.23 All participants will be asked to complete a 3-day food record monitoring their energy intake each week. Previous work showed that 3-day records are equivalent to 5-day records in reporting caloric intake and percentage macronutrients.28 Caloric needs will be determined using subjects measured BMR. The diaries will be evaluated weekly by the dietitian and monthly feedback will be provided via phone interview or secure email communication to ensure adherence to the recommended dietary plan throughout the study. All participants will meet with the dietitian three times: at baseline, P1, P2.
After an overnight fast for 10–12 hours, participants will be kept in a dark room for 20–30 minutes to attain a resting state, during which basal metabolic rate will be measured by using a canopy that covers the whole head and a portable COSMED K4b2 (COSMED USA, Chicago, IL). BMR and respiratory exchange ratio (RER) will be recorded by indirect calorimetry.3,33,34
1.2 Serum total, free testosterone, IGF, FFA
Total Testosterone measurements will be performed by radioimmunoassay after sample extraction and column chromatography. The interassay coefficient of variation (CV) is 12.5% or less for all quality control samples analyzed. Free testosterone concentrations will be calculated by measuring sex hormone binding globulin (SHBG) and albumin (www.issam.ch/freetesto.htm).35 Plasma IGF-I and IGFBP–3 concentrations will be measured byimmunoluminometric assay (Quest Diagnostics, Madison,NJ) and RIA (Diagnostics Systems Laboratories Inc., Webster,TX), respectively. 10 ml of blood will be collected from the indwelling venous catheter and lipid profile (HDL-C, LDL-C, total cholesterol, and TG) will be determined using standard analyses procedures.
1.4 Inflammatory biomarkers
Before starting the intravenous glucose tolerance test (IVGTT) and following a 12-hour fast, blood will be collected from the indwelling venous catheter and CRP, IL–6, TNF-, and free-fatty acids (FFA) will be determined by the VCU-CRC using available enzyme-linked immunosorbent assay (ELISA) assay kits.36,37
1.5 Intravenous Glucose Tolerance Test (IVGTT; Primary outcome variables)38.39
An IVGTT will be used to determine insulin sensitivity and glucose effectiveness. Each subject will undergo an IVGTT before (baseline), and 12 weeks after interventions (Post 1 and Post 2). After a 10 to 12-hour fast, an indwelling catheter with an intravenous saline drip (0.9% NaCl) will be placed, following 20 minutes of glucose injection, a bolus of insulin (0.02 U/kg) will be injected to determine insulin sensitivity. Plasma glucose will be measured by the Autoanalyzer glucose oxidase method and plasma insulin concentrations will be determined by commercial radioimmunoassay. The SI (glucose disposal rate per unit of secreted insulin per unit time; i.e. insulin sensitivity) and SG (glucose mediated glucose disposal rate) will be calculated from a least-squares fitting of the temporal pattern of glucose and insulin throughout the IVGTT using the MINMOD program.38,39
1.6 Oxygen uptake and energy expenditure during FES-LEC
One week prior to intervention (week 1), post-interventions 1 (week 14) and 2 (week 27), peak oxygen uptake (VO2) will be measured using a COSMED K4b2 (COSMED USA, Chicago, IL) portable metabolic unit.19 After calibration, subjects will be asked to place the mask on their face to monitor oxygen (VO2) and carbon dioxide (VCO2) production. A three-minute resting phase allows the subject to get used to breathing with the mask on while they are attached to the RT–300 bike. After the resting phase, VO2 will be measured during the three-minute warm-up phase, the resistance of the bike will be gradually increased by 2 Nm every 2 minutes until fatigue. During testing, the servo motor will be tuned off, and the cool-down phase will then be followed by the recovery phase.19
VO2 and VCO2 will be monitored throughout exercise to determine total energy expenditure using the Weir equation. Five minutes of recovery will be recorded to determine the efficacy of each intervention on energy expenditure and substrate utilization. Heart rate (via polar HR monitor) will be recorded every 30 seconds and blood pressure (COSMED 740) will be recorded before, every 2 minutes during cycling, and for another 5 minutes after cycling to ensure full recovery to baseline.
2.1 Body mass index (BMI) and Anthropometrics
Each participant will be asked to empty their bladder and then will propel onto a wheelchair weighing scale to evaluate weight in Kg. The wheelchair will be measured separately, and the difference will be taken for the final weight. The height of each participant will be determined with the subject on his/her right side in the supine position. Two smooth wooden boards will be placed at the participant’s head and heels and the distance between them corresponds to the height in nearest cm. The BMI (Kg/m2) will be calculated as weight (Kg)/height2 (m2). Anthropometrics will be determined in duplicate by identifying the narrowest region of the trunk from sitting and lying positions. After normal expiration, a tape measure will be used around the participant’s trunk to measure waist circumference (WC).33,40,41
2.2 Dual energy x-ray absorptiometry (DXA)
DXA will be used to measure body composition in SCI individuals, specifically regional and total fat mass (FM) and FFM. Total body and regional (lumbar spine, proximal femur, and forearm) DXA scans will be performed using a GE Lunar iDXA (Lunar Inc., Madison, WI) bone densitometer at the Hunter Holmes VAMC hospital. All scans will be performed and analyzed using Lunar software version 10.5. After scanning, total and regional % FM and FFM will be determined using DXA software. The longitudinal precision of total and regional body composition using DXA was recently determined in persons with SCI 42
2.3 Magnetic resonance imaging (MRI)
Skeletal muscle CSAs will be determined before (baseline), and twice after 12-week interventions (post-intervention 1 and post-intervention 2) using a 1.5 Tesla GE magnet. Transaxial images, 0.8 cm thick and 1.6 cm apart, will be taken from the hip joint to the knee joint (thigh) and from knee to the ankle (leg) using the whole-body coil.19,20,43–46, T1-weighted imaging will be performed using a fast spin-echo sequence to capture visceral fat images. To measure visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), transverse slices (0.8 cm thickness) are acquired every 0.4 cm gap from the xyphoid process to the femoral heads. Images will be acquired in a series of two stacks with L4-L5 used as a separating point. Participants will be asked to take a deep breath in and hold their breath for 10–15 seconds to reduce the respiratory-motion artifact associated with MRI for the abdominal region.19,29,47
2.4 Skeletal muscle torque and fatigue
Skeletal muscle torque and fatigue of the knee extensor muscle groups (left and right) will be evaluated at baseline, following 12 weeks at P1 and then 12 weeks following FES-LEC for both groups at P2 using a Biodex isokinetic dynamometer (Biodex Medical Systems, Shirley, NY). Participants will be in a seated posture with the trunk-thigh angle and the knee flexed at 90º (where 0 corresponds to full knee extension). Isometric torque will be measured after adjusting the amplitude of the current to 50 and 100 mA (frequency 30 Hz and pulse duration 450 µs). Fatigue will be assessed by measuring the torque elicited at 10, 20, 30, 40, 50 60, 80 and 100 Hz before and immediately after intervention Isokinetic torque will be measured at 60, 90 and 180 degrees/sec using the same electrical stimulation protocol.19
3.1 Skeletal muscle biopsy
Three biopsy samples of the vastus lateralis muscle (100mg wet wt.) will be obtained by a 14-gauge Tru-Cut needle to compare the effects of evoked NMES+FES vs. control on protein levels of IRS–1, GLUT–4, IGF–1 and PGC–1 α, AMPK, Akt and mTOR as well as the phosphorylation forms of AMPK, AKT and mTOR. We will also test whether NMES+FES enhanced mitochondrial enzymatic activities and ETC compared to control. Skeletal muscle biopsies of the right vastus lateralis muscle will be obtained prior to training and after 12 and 24 weeks of interventions.48,49
Western blot analysis will be performed to determine the protein concentrations as previously described.50 Briefly, proteins will be resolved by SDS-PAGE then transferred to two supported nitrocellulose membranes by wet electromembrane transfer at 110 V for 2 hours, and then blocked in 6% bovine serum albumin (BSA)/Tween-Tris-buffered saline (TTBS) for 1 hour. Membranes will be incubated separately with the appropriate primary antibody overnight. The membranes will be washed separately (three times for 20 minutes) in 6% BSA/TTBS and incubated with appropriate secondary antibody in 4 mL 3% BSA/TTBS at 4°C for 1 hour. The membranes will be washed (four times for 45 minutes) in 6% BSA/TTBS. Proteins will be visualized using an enhanced chemiluminescence detection system (GE Lifesciences) according to the manufacturer’s instructions. Western blots will be quantified by scanning with A GS800 densitometer. Optical densities of the Western blots will be measured using image-analysis software (Molecular Analyst; Bio-Rad).
3.2 Mitochondrial electron transport chain (ETC) activities
Samples of skeletal muscle are studied for assessment of electron transport chain activity. Measurement of maximal enzyme activity is performed in freshly-isolated detergent solubilized mitochondria using cholate as detergent. Analysis of freshly isolated samples are initially performed to assure that maximal activity of electron transport complex I is obtained. Skeletal muscle is isolated, homogenized and a potion subjected to detergent solubilization in cholate. The enzyme activity of electron transport chain complexes is obtained by using specific substrates and inhibitors to isolate segments of the ETC. Assays are performed in 0.1M phosphate buffer using spectrophotometric endpoints. Complex I activity (NADH coenzyme Q reductase) is measured following the oxidation of substrate NADH to decylubiquinol, a synthetic coenzyme Q, as electron acceptor. Activity is measured in the presence and absence of the specific complex I inhibitor rotenone. In a similar manner, the activity of other complexes is measured using specific substrates, acceptors and inhibitors. Complex II is measured using succinate as substrate, and the reduction of 2,6 dichlorophenolindophenol as the electron acceptor followed as spectrophotometric indicator, with and without thenoyltrifluoroacetone as the inhibitor of complex II. Complex III activity is measured using the reduced synthetic coenzyme Q molecule decylubiquinol as substrate, oxidized cytochrome c as acceptor and antimycin-A as the complex III specific inhibitor. The reduction of cytochrome c as acceptor is followed as the spectrophotometric endpoint. Complex IV is measured by following the first-order oxidation of added exogenous reduced cytochrome c using azide as the specific inhibitor.51 Citrate synthase, a tricarboxylic acid cycle component enzyme, is measured as an indicator of mitochondrial mass. 51–55
T-tests and Pearson chi-square tests will be used to compare characteristics that may act as confounding variables, including age, level of injury, time since injury, %FM, as well as the three outcome variables (Si, Sg, VO2) to ensure that each group is not different with respect to each variable at baseline. A repeated-measures ANOVA will be performed with glucose effectiveness as the primary study outcome and effects for treatment group (NMES+FES vs. control), time (baseline, post-intervention, and post-intervention 1), and the interaction between the two factors included in the model. Similar repeated measured ANOVA models will be fit with each of the body composition, metabolic profile, protein expression, and ETC measures (Aim 3). All statistical analysis will be performed at the 0.05 level with SAS V9.4 (Cary, NC).
We have acquired pilot data for all outcomes of this project. Glucose effectiveness possesses the smallest effect size from baseline to follow up with NMES-RT showing a mean baseline effectiveness of.01716 and post-treatment mean of.02258. Cohen’s f was found to be 0.271 using a sample variance of 0.0001.25 Assuming the correlation between the pre- and post-treatment measurements is0.3735 and 3 total measurements are acquired,38 subjects will need to complete the study at 90% power using a Type–1 error rate of 0.05. At least 48 subjects will need to be recruited, adjusting for a 20% dropout rate. All sample sizes were calculated using the software G*Power (V18.104.22.168). Sample size of 24 was calculated using insulin sensitivity data without regard to dropout rate.
Other factors, such as insulin sensitivity and VO2, are considered; each representing larger effect sizes than glucose effectiveness (f = 0.313 and f = 0.432, respectively). Our study would result in a power of 97.51% and 99.82% for observing the baseline to follow-up difference between the NMES+FES and control + FES groups, provided the correlation between baseline and follow-up measurements are 0.4 for the outcomes.