Effects of Activity Dependent Plasticity on Recovery of Bladder and Sexual Function After Human Spinal Cord Injury
Phase: NA
Conditions: Spinal Cord Injuries
Sponsor: University of Louisville

Study Title: Effects of Activity Dependent Plasticity on Recovery of Bladder and 
Sexual Function after Human SCI 
 
 
NCT Number: NCT03036527 
 
 
Document Date: February 9, 2017 

IRB # 14.0062, Effects of Activity Dependent Plasticity on  
Recovery of Bladder and Sexual Function after Human SCI 
 
 
 
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B. SIGNIFICANCE 
Approximately 1,275,000 people in the U.S. live with paralysis from spinal cord injury (SCI) 1. Disabling SCI 
sequelae include impairments of locomotor, sensory and autonomic functions 2, 3. The U.S. could save an 
estimated $400 billion on direct and indirect lifetime costs if we can develop therapies to treat SCI. Also, the cost 
in emotional stress and well-being to the individual and family is demanding. Bladder and sexual dysfunction 
consistently ranks as one of the top disorders drastically affecting quality of life after SCI 2, 3. Urological 
complications are responsible for most clinical conditions and hospital readmissions 4, and individuals with SCI 
are eleven times more likely to die from diseases of the urinary system than non-injured individuals.   Locomotor 
Training (LT) has shown a range of benefits on health and function in both human 5-17 and animal 18, 19 models.  
Improved bladder and sexual function has been reported by patients undergoing LT even years after injury; 
however this phenomenon has not been studied.  The mechanistic insights of how LT affects not just walking 
recovery but urological and sexual function not only will lead to imminent treatments for those suffering with SCI 
but may influence the treatment of other neurological disorders such as multiple sclerosis, Parkinson’s, and 
stroke, and stimulate investigations on other systems such as cardiovascular, pulmonary and bowel dysfunctions. 
 
1. Urological Dysfunction and SCI. Deficits in urological function after SCI manifest as: detrusor hyperreflexia 
(bladder contractions at low volumes, causing incontinence and smooth muscle hypertrophy), detrusor-sphincter 
dyssynergia (uncoordinated bladder and external urethral sphincter contractions, causing inefficient emptying and 
smooth muscle hypertrophy), decreased compliance (unable to store urine under appropriately low pressures) 
and loss of continence requiring lifelong management, maintenance, and health care visits 20. Current therapies to 
improve efficiency of bladder voiding, management and continence after SCI include catheterization, 
pharmacologic and surgical interventions, functional electrical stimulation, and urethral stents 21, 22. 
Catheterization can induce scarring, stricture formation, cystitis and frequent urinary tract infections due to 
introduction of bacteria into the urethra 23. Pharmacological agents alter internal urethral sphincter tone (α 
blockers) or relax the detrusor muscle (anticholinergics increase bladder compliance and decrease 
pressure) but do not treat voluntary voiding. These drugs have side effects like dry mouth and constipation 
further complicating the issue of fluid restriction to control timely urine evacuation 21, 24. Surgeries to reduce high 
bladder pressures and chronic urinary incontinence include urinary diversion and lower urinary tract 
reconstruction 22, 25, 26. Surgery to implant neural control devices to artificially influence voiding circuitry often 
require ablation of intact neural tracts. There is a critical need for a successful treatment that restores normal 
lower urinary tract function as the compensatory strategies continue to diminish the capacity of the bladder, 
require life-long maintenance, have deleterious side effects and lead to recurring illness . 
 
2. Locomotor Circuitry and Bladder Function. The interaction of lower limb musculature with the bladder and 
its sphincter has been observed sporadically over the years, as far back as 1933, in both humans 27, 28 and 
animals 29, 30. Flexor and extensor reflexes can be modulated by the state of bladder filling and voiding in both 
normal patients and those with CNS damage 28. In patients with spasticity, the general pattern is that detrusor 
contractions precede limb flexor spasms 31. Our experience with patients undergoing LT reveals two important 
observations: 1) catheterization is required before each session because a full bladder will inhibit stepping; and 
2) an individual who received LT in combination with epidural stimulation of the lumbosacral region regained the 
ability to routinely void without catheterization. These observations suggest an interaction of the locomotor and 
bladder circuitry that is emphasized after SCI. Afferent input from the bladder and/or the external urethral 
sphincter affects the limb musculature and repetitive activation of the locomotor circuitry seems to affect bladder 
function. We propose that this vesicosomatic relationship can be influenced by LT to enhance bladder integrity 
and function and improve the health/quality of life for those suffering from SCI. 
Recent discoveries in humans related to activity-dependent plasticity have led to a widely implemented activity-
based (generates neuromuscular activation below the level of injury) rehabilitation intervention, LT, for those with 
incomplete SCI 14, 32-35. The therapeutic intervention is usually implemented in those with incomplete injuries, 
even though the mechanistic studies have been done in clinically complete SCI, because while LT optimizes the 
spinal circuitry, remaining residual supraspinal inputs may be required to sufficiently excite these networks for 
successful walking 36. Generation of locomotion by the interaction of afferent input with central pattern generating 
networks has been shown in spinally transected animals 18, 37-46 and several of these properties exist in the 

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functionally isolated human spinal cord 10, 11, 47-52. Motor patterns observed during stepping in individuals with 
clinically complete SCI 11, 39, 53, 54 are driven by sensory information available to the spinal cord interneuronal 
networks 47, 51, 55, 56. Multiple sensory inputs from the periphery during locomotion, particularly limb loading 57 and 
stepping rate 58 provide information to these networks to improve stepping 53, 55, 
59-61. 
One individual with a clinically motor complete SCI did not produce motor activity in the legs even after 170 
sessions of LT. However, in the presence of epidural stimulation, he was able to stand independently and 
voluntarily move his legs indicating that the spinal circuitry requires a sufficient level of excitability to execute 
motor tasks.  Intense, repetitive LT with sufficient spinal network excitability (via residual supraspinal input or 
epidural stimulation) conceivably induces a net improvement in functional reorganization of the neural circuitry in 
response to afferent feedback and supraspinal commands and/or epidural stimulation 62-65. Improving these 
synaptic inputs might therefore also lead to adaptive changes to other systems such as those controlling the 
bladder and genitalia since much of the motor and autonomic output of the spinal cord is driven in large part by 
afferent input and local or propriospinal circuitry emphasized after SCI conditions 51, 66-69. 
 
3. Sexual Dysfunction and SCI  In men with SCI, the degree of sexual dysfunction depends on level of the 
lesion.  For our research, only injuries cranial to T10 are considered as the spinal reflex arcs for erection and 
ejaculation are considered to be left intact, with only the removal of supraspinal input 70.  Most individuals with 
SCI’s above T10 demonstrate reflexogenic erections of varying degrees in response to very slight stimulation of 
the penis 71, 72. Though erections are easily initiated, they are not easily sustained 73 which has been proposed to 
be a result of altered penile sensitivity 74.  In 95% of SCI men with lesions cranial to T10, normal ejaculation is 
severely impaired or impossible 72, 75 despite the intact spinal reflex arc, suggesting the ejaculatory reflex circuitry 
is more dependent on supraspinal control than is the erection circuitry.  The ejaculatory dysfunction may also 
relate to the decrease in penile sensation. Many SCI individuals who do not respond to normal tactile stimulation 
of the penis may ejaculate to intense vibratory stimulation of the ventral penile midline 76 suggesting that massive 
recruitment of all low and high threshold penile mechnoreceptive afferent neurons can provide enough input to 
the spinal ejaculatory circuit. In addition to erectile dysfunction and ejaculatory failure, abnormal sperm motility 
and viability as early as two weeks post-SCI contribute to neurogenic reproductive dysfunction post-SCI 77. 
 
4. Neurotrophins. Exercise and neuromuscular activity are highly influential on expression of neurotrophins 
(such as BDNF and NT-3 in lumbar spinal cord and soleus muscle - 78) known to modulate cellular and 
synaptic function in the adult 79-82. Among various strategies, step training after contusive SCI in animal studies 
promotes the greatest changes in neurotrophin production and functional recovery (such as the normalization 
of BDNF levels in the lumbar spinal cord and soleus muscle - 83). Other forms of exercise attempted in the 
contused rat model, such as swim training, only had a transient effect, and stand training had no effect on 
neurotrophins and functional recovery 83. 
Transplantation of NT-3/BDNF expressing fibroblasts into the injury site after contusive SCI has been 
reported to increase bladder and locomotor function 84 and viral-vector-mediated delivery of NT-3 via i.m. 
injection after SCI promotes locomotor recovery and electrophysiological changes in motoneurons akin to 
training 80. Combined step training and neurotrophin therapy results in greater locomotor gains than either alone 
85. Although increased levels of NT-3/BDNF appear to be adaptive/beneficial when expressed in the spinal cord 
or muscles (at least for locomotor functions), increased NGF in the bladder and corresponding spinal segments 
plays a significant role in dysfunction. Upregulation of neurotrophic factors post-SCI 86 is responsible for the re-
emergence of the spinal voiding reflex within 2 weeks of injury 87, but chronic changes inNGF appear to be 
responsible for bladder afferent hypersensitivity, hypertrophy, and sprouting of axons 88, all of which lead to 
bladder over-activity. NGF and BDNF have been implicated as key factors in a variety of bladder dysfunctions 89. 
NGF delivery (intrathecal infusion at L6-S1 spinal level in adult rats) causes bladder DRG afferents to become 
hyper-excited and results in detrusor hyperreflexia 90, while NGF removal, via antibody treatment (intrathecal 
infusion at L6-S1 spinal level in adult rats), has been shown to relieve detrusor hyperreflexia and detrusor-
sphincter dyssynergia 91, 92. Sequestering BDNF (daily tail vein administration of TrkB-Ig2, which specifically binds 
BDNF and neutralizes it) has been shown to improve bladder function in a chronic cystitis model 93. Exercise 
therapies also influence neurotrophin expression in visceral target organs, not just skeletal muscle 94, 95. We 

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therefore hypothesize that LT and epidural stimulation will influence neurotrophin levels within the spinal cord and 
periphery, leading to enhanced locomotor and visceral function. 
 
 
C. INNOVATION. 
 
- This proposal focuses on the effects of activity dependent plasticity induced by locomotor training following 
chronic SCI on non-locomotor systems, those involved with bladder and sexual function. 
 
- Bladder and sexual dysfunction after SCI is rarely studied in experimental animals, yet is overwhelmingly the 
most significant concern for those suffering from SCI and urological complications results in significant morbidity 
and mortality. In addition, small improvements in bladder and sexual function can have a tremendous impact on 
these individuals’ continual health and quality of life. 
 
- This proposal involves the collaboration of scientists with extensive experience in animal and human SCI 
models. In our currently funded animal study, rats undergoing step training are also being compared to a SCI 
group receiving the same intensity of exercise without weight bearing by stepping on only their forelimbs 
(equivalent to the arm crank or non-weight bearing exercise control group proposed here), as well as to an 
uninjured group. These ongoing parallel studies in animals will provide further insights to underlying 
mechanisms that can also be tested in the human model. 
 
- A novel multi-disciplinary combination of outcome measures including EMG recordings, stepping kinematic 
measures, urodynamics, International Index of Erectile Function, and molecular assessments of bladder tissue 
will also be conducted in this study. Histological, biochemical, and molecular assessments of spinal cord and 
dorsal root ganglion tissue are in the process of being conducted in rats to identify specific mechanisms not 
testable in humans. 
 
- The current proposed study will increase our understanding of human lumbosacral spinal networks and guide 
the use of innovative therapeutic strategies that would be immediately available to not only improve the motor 
output during standing and walking but ameliorate bladder and sexual dysfunction and thus improve quality of life 
in individuals after SCI. 
 
APPROACH 
1. General Design. Individuals who sustained a SCI will undergo assessments for bladder and sexual function 
who are receiving different types of physical therapy interventions by three possible avenues:  1) clinically, 2) 
as part of another research study that has an activity-based intervention (IRB 07.0066; 07.0268; 13.0625; 
14.1284) or 3) a control intervention as part of this study. This approach allows us to efficiently and cost effectively 
understand the role of specific mechanisms of activity-based interventions on devastating secondary conditions 
resulting from SCI.11 
 (Specific Aims 1 and 2). Specific Aim 1 and 2 is designed to understand the effects of weight-bearing 
activity-based therapy on bladder function and sexual function.  The weight-bearing activity-based intervention will be 
provided via a standardized locomotor training program that is provided clinically at Frazier Rehab Institute within 
the NeuroRecovery Network (NRN); or similar interventions in a research protocol of stepping (IRB 07.0066) or a 
standing program with or without neuromuscular electrical stimulation (NMES) - (07.0268, 14.1284).  The stand 
only intervention may also be provided as part of this study.  The control group intervention will be provided via a 
non-weight bearing exercise program that consists of either standardized arm crank or upper extremity 
neuromuscular electrical stimulation (UE-NMES) provided within this study.  The UE-NMES may also be provided 
as a standardized occupational therapy program that is provided clinically at Frazier Rehab Institute within the 
NeuroRecovery Network (NRN).  We may also evaluate research participants at the midpoint.  The research 
participants (n=30) selected for Aims 1 and 2 will be from a convenience population to provide a sufficient number 
of controls in the non-weight bearing group. Blocked randomization will be used and the specific intervention 
(n=10 step; n=10 stand; n=10 non-weight bearing) will be chosen based on the physical examination by the 
occupational therapist.   
 
(Specific Aim 3 ).  Specific Aim 3 is designed to understand the effects of epidural stimulation with and 

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without weight-bearing activity-based therapy on bladder and sexual function.  The up to 10 SCI individuals will 
be evaluated before and after their interventions defined from their epidural stimulation protocols (IRB 07.0066 and 
13.0625).   The interventions may include:  stand and/or step training with LT, cardiovascular (CV) training, and/or 
voluntary movement (VM) training, as provided by our research team at the University of Louisville Human 
Locomotion Research Center. We may also evaluate research participants at the midpoint of training. 
 
When a research participant is enrolled into the non-weight bearing training group, a physical 
examination may be provided (for cervical and upper thoracic SCIs – T3 and above) by an occupational 
therapist and s/he will recommend either the arm crank training or UE-NMES intervention for the control group.   
Each individual will serve as their own control reducing the variability among individuals related to the injury 
itself, time since injury, medications taken, therapies received, differences in degree of sexual dysfunction, and 
many other factors that cannot be controlled in the human experience. The inclusion criteria for both cohort 
research participants are: 1) stable medical condition, 2) Non-progressive, suprasacral SCI, 3) Bladder and 
sexual dysfunction as a result of SCI, 4) Indication that the period of spinal shock is concluded. The exclusion 
criteria for both cohort research participants are: 1)Cardiopulmonary  disease or dysautonomia that would 
contraindicate LT, stand, or non-weight bearing training; 2) Painful musculoskeletal dysfunction, untreated 
fracture, severe contractures, untreated pressure wounds or active urinary tract infection that would affect training 
and/or assessments; 3)  Untreated psychiatric disorders or ongoing drug abuse; Urodynamic parameters, 
erectile dysfunction evaluations, urine biomarker and biopsy sample retrieval, and EMG activity assessments will 
be conducted as outlined in Figure 1. 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Timeline for Aims 1 and 2 (left) and Aim 3 (right). For Aims 1 and 2 research participants with 
incomplete SCI (American Spinal Injury Association Impairment Scale (AIS) B, C or D 96-98), 
urodynamic/erectile function assessments and biopsies will be conducted prior to enrollment in the 
standardized NRN LT program and after 80 sessions of the intervention. Assessments may also be 
conducted at the midpoint of training. Training will be done in groups of three (one LT, one stand training, 
one non-weight bearing training). For Aim 3, urodynamic/erectile function assessments and EMG activity will 
be conducted on individuals with severe SCI (those who are motor complete and sensory complete [AIS A] 
or sensory incomplete [AIS B]) (i) before LT or at baseline, (ii) after 80 sessions of LT plus stand or an 
equivalent time-frame of participant usual care activity, prior to implantation, (iii) after 80 sessions of step 
plus stand training (mid training LT) or CV training in combination with epidural stimulation (ES), (iv) after 80 
sessions of LT plus stand training (post training LT) or CV, VM training in combination with epidural 
stimulation, and (v) after 6 month follow-up LT (total of 4 follow-ups or 2 years) or CV, VM, stand training 
plus follow-ups in combination with epidural stimulation (n=2 per year for a total of 10 over 5 years). 
 
 
2. Approach for Specific Aims. 
a. Specific Aim 1: To determine the effects of weight-bearing task-specific training for locomotion (stepping on 
a treadmill) after traumatic incomplete and complete SCI in humans on a) urodynamic parameters, b) 
interactions between lower limb and urinary bladder circuitries, and c) bladder neurotrophin levels. Weight- 

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bearing (stand-only) and non-weight-bearing exercise (arm crank, UE-NMES) will serve as controls. 
SCI research participants are expected to exhibit marked urological deficits (e.g., detrusor hyper-reflexia with 
detrusor-external sphincter dyssynergia). An example showing a baseline cystometrogram (to demonstrate 
feasibility) from a 32 year old male, obtained 4 yrs. 3 mo. post-SCI (C7 level; AIS B), is provided in Figure 2. 
We hypothesize that individuals with severe SCI after LT will have significant improvements in continence 
and voiding ability, as evidenced by an increase in bladder capacity, lower detrusor pressures and voiding 
pressures, the ability to resume normal voiding at least partially without catheterization, and lower post-void 
residual volumes. 
 
 
 
 
Figure 2. At the beginning of the filling curve at around 60 ml, the subject had an uninhibited bladder contraction 
that reached 40 cm H2O of pressure, demonstrating bladder overactivity and low compliance. The bladder was 
still able to be filled up to 580 ml, as after the initial bladder contraction the pressure decreased only to rise again 
to 40 cmH2O. After the end of the filling phase, the subject was asked to void, but was unable to achieve any flow 
to what appeared to be detrusor sphincter dyssynergia type 3 as the sphincter EMGactivity remained high during 
the entire voiding phase. 
 
Cystometry measures obtained from a T5 AIS Grade A subject (two years post-SCI) during preliminary 
studies of LT effects on bladder function indicates an increase in compliance and doubling of volume at first 
uninhibited bladder contraction after 80 daily sessions of LT (Figure 3). The recording in Figure 3 provides an 
example of how cystometry (see details below in General Methods) measurements of resting pressures, bladder 
capacity, and voiding volumes will be quantified. Lower resting pressures reflect the ability of the bladder to 
store more urine (indicative of a change in compliance). For the current proposed clinical study, we will also 
measure changes in voiding frequency over time using established documented voiding diaries. Therefore, 
improvements in a variety of specific quantifiable parameters related to bladder function are expected to 
contribute to better continence as well as improved voiding efficiency revealed by a decrease in residual volume 
and voiding frequency, an outcome that would likely lead to improved urological health and quality of life for 
those suffering from SCI. 
 

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Figure 3.  Example of cystometry recordings from a 29 year old male AIS Grade A (motor and sensory complete 
SCI) two years after injury (T5 neuro level). Improvement of compliance and doubling of the volume at the time of 
his first uninhibited bladder contraction was noted after 80 sessions of LT. Note that this individual’s bladder 
medications were discontinued 24 hours prior to each of the cystometry sessions. In addition, this individual 
reported having chills through both legs when his bladder was full after, but not before LT (he said he started 
noticing the sensation after several weeks of training). Note that he is sensory complete as determined by AIS 
standards and the fact that he could tell when his was full (and thus the need to catheterize) was a huge benefit 
for him (we have found similar reports for all four subjects that have been studied to date in these preliminary 
studies, although the location and type of sensation has varied – see Specific Aim 3 below). 
 
Data from our animal studies (Figure 4 below; manuscript in preparation) shows that following 80 step 
training sessions the ability of SCI rats to empty the bladder increased. The mean voiding efficiency (percent 
volume voided/volume infused) of the trained group was significantly greater than non-trained (Figure 4a, trained 
39.78% ± 17.72; non-trained 22.29% ± 11.02, p=.042). This was accompanied by a significant increase in the 
maximum amplitude of bladder contraction (MAC, pressure in mm Hg) and a significantly increased 
intercontraction interval (ICI, time in seconds) (Figure 4, MAC p=.043, trained 6.54±4.75 non-trained 
2.56±1.39; ICI p=.018, trained 25.40±12.98 non-trained 12.25±3.77). Contraction time (trained 25.23±15.35 
sec, non-trained 16.03±3.67 sec), resting pressure (trained 16.10±5.52 mmHg, non-trained 17.80±2.46 
mmHg), and bladder weight (trained 0.36±0.11 g non-trained 0.40±0.10 g) demonstrated no differences 
between groups. However, bladder weight (grams) significantly correlated with voiding efficiency in only the 
trained group (p=.004, r= -.946, r2=.895, n=6). Bladder hypertrophy post-SCI can result from detrusor sphincter 
dyssynergia in a manner similar to bladder outlet obstruction which is reversible with outlet relief 99, 100. The 
sphincter must be relaxed during a bladder contraction to allow emptying. We can infer that the sphincter of the 
trained rats was in partial coordination to allow for the flow of urine, an increase in voiding efficiency, and the 
significant relationship between bladder weight and voiding efficiency for trained rats. 
 

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Figure 4. Bladder NGF mRNA and Cystometry. a) Percent change from the non-trained mean for NGF, 
voiding efficiency (percent volume voided per volume saline infused), maximal amplitude of contraction, and 
intercontraction interval. Mean NGF significantly decreased and mean voiding efficiency, intercontraction 
interval (seconds), and maximal amplitude of contraction (mmHg) significantly increased with training. b) 3D 
relationship between cystometry measures. c & d) 3D relationship between NGF and cystometry measures. 
Significant group differences were detected by group using multiple regression analysis (MAC and ICI were 
good predictors of voiding efficiency with significant group differences (R2=.870, p<.05, N=14)). Therefore, 
training modified the relationship of voiding efficiency, MAC, and ICI. 
 
However, it is not clear whether step training induced bladder recovery is due to activation and plasticity in 
lumbosacral circuitry (bladder and locomotion). To assess potential underlying mechanisms in the current 
proposed study, bladder neurotrophin levels are also being evaluated after SCI. We expect that LT and epidural 
stimulation will affect the levels of bladder neurotrophins in a manner that enhances functional recovery. We 
hypothesize that SCI will induce an increase in NGF and BDNF in urine and biopsied bladder tissue and this will 
be reversed with LT but not with non-weight bearing training or stand training. Note that a study in rats using 
different exercise paradigms (treadmill training, swim training, stand training) found significant improvements on 
central pain and normalization of lumbar spinal and soleus muscle BDNF levels for the LT group only 83. Bladder 
tissue from our preliminary study in rats was analyzed for NGF mRNA using real-time PCR (Figure 4). Multiple 
regression analysis of selected cystometry parameters with NGF revealed that two of the cystometry measures 
and bladder weight were good predictors of NGF with significant group differences. ICI and MAC were significant 
predictors of NGF (R2=.638, p<.05, N=14). ICI and bladder weight were significant predictors 
of NGF (R2=.689, p=.004, N=14). Therefore, training modified the relationship of NGF to ICI, MAC, and bladder 
weight. These relationships are demonstrated with 3D graphs in Figure 4b-d. 
Lower NGF levels significantly correlates with lower residual volume, indicating higher voiding efficiency and 
reduces the potential for recurring urinary tract infections. The relationship between NGF and residual volume is 
apparent only in association with LT, which may be due to altered circuitry that controls micturition after SCI. We 
chose to examine NGF first because previous reports have shown that it is up-regulated in the bladder following 
SCI and it is highly associated with bladder dysfunction. NGF delivery causes bladder afferents to become hyper-
excited and results in detrusor hyperreflexia 90, while NGF removal, via antibody treatment, relieves detrusor 
hyperreflexia and detrusor-sphincter dyssynergia 91, 92. Treatment that lowered bladder BDNF levels reduced 
over-activity in a cystitis rodent model 93 and thus is an additional neurotrophin that we will target. In our current 
animal studies, to broaden potential targets, we are conducting a focused neurotrophin qPCR array using bladder 
tissues from trained versus non-trained male rats. The results could be used to identify one or two additional 
targets to examine in the human tissues we obtain for the current study. Thus, our new rat data (Figure 4) 
indicates for the first time that the post-SCI bladder NGF levels and bladder function can be affected in tandem by 
LT. This finding would indicate the need to maintain some daily form of training, perhaps in combination with a 
drug therapy (e.g., anti-NGF). These experiments will provide new knowledge of the role of training on the 
neurotrophin regulation in humans after SCI. 
Similar results are anticipated for urine biomarkers for NGF and BDNF, an area of investigation that has 

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received a lot of recent attention for overactive bladder. Several studies have been published indicating elevated 
urinary levels of NGF and BDNF (but not GDNF) concentrations (measured by ELISA and normalized to 
creatinine) in patients with overactive bladder relative to healthy controls 89, 101-104. These levels have been shown 
to decrease back to normal levels with a variety of treatments, including antimuscarinic therapy 101, 105 and a 3 
month lifestyle intervention (bladder training - limited and select fluid intake) 101. As with overactive bladder, 
urinary biomarkers may be used for the evaluation of bladder dysfunction in SCI individuals. In addition, we may 
find a strong correlation with the neurotrophin levels in the biopsy samples (urinary biomarkers would make it 
much easier to monitor the effects of our therapeutic interventions). 
We also hypothesize that after SCI, bladder filling at rest (tested while doing cystometry) will result in co- 
activation of lower limb flexors and extensors which will be significantly reduced after LT but not for non-weight 
bearing training or stand training. We hypothesize that the locomotor activity generated during stepping 
influences the bladder circuitry and improvements in bladder function will correlate to improvements in EMG 
activity during stepping (measured as part of a study focused exclusively on locomotor circuitry) and reduce the 
aberrant activity during bladder filling. We may record EMG activity (see details in Methods below) of the lower 
leg muscles during the cystometric testing and during stepping on a treadmill (bladder is emptied prior to each 60 
minute session, so it will not be fully distended at any point during training). Specifically, we expect that pre-
training, increased tibialis anterior EMG activity (hyperactive flexion reflex) and co-contraction of extensor and 
flexors will occur in response to the bladder contractions. A fully distended bladder (filling during cystometry 
session) is expected to trigger spasticity or clonus. Such a finding would explain the observed need for the 
bladders of human SCI individuals to be emptied immediately prior to rehabilitative step training on a treadmill. A 
full bladder may inhibit training by increasing EMG activity within flexors, thereby causing clonus or spasticity. We 
propose that this functional reorganization occurs with the LT intervention corresponding to improvements in EMG 
activity during stepping. These experiments will further our knowledge about vesicosomatic reflexes and 
understanding about how visceral organs may affect other systems, challenging our view of the locomotor system 
and the sources of sensory inputs to the central pattern generator. An example showing the benefits of LT on 
EMG activity of the lower leg muscles is provided in Figure 5 from both our human and animal study. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
V 
Figure 5. Exemplary data demonstrating activation of flexors and extensors during assisted stepping from an 
individual with a clinically incomplete SCI (Panels A-D) and unassisted stepping from a trained and non-trained 
adult rat (E-H). EMG activity (μV) from the soleus (SOL), tibialis anterior (TA), semitendinosus (ST), and vastus 
lateralis (VL) from both left and right legs of a human pre- (A & C) and post-training (B & D) and from the left 
hindlimb of a trained and non-trained animal at Day 0 (E & G) and Day 81 (F & H). 

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b. Specific Aim 2: To determine the effects of weight-bearing task-specific training for locomotion (stepping on a 
treadmill) after traumatic incomplete and complete SCI in humans on erectile function (stand-only and non-weight 
bearing training once again will serve as controls). Subjects will be the same as for Aim 1. 
SCI research participants are expected to exhibit marked deficits in sexual function (erection; ejaculation) and 
thus low levels of sexual satisfaction upon entering the study 106, 107.  Many are likely to be using drugs or sexual 
aids to enhance function. It is hypothesized that LT but not non-weight bearing or stand training will result in a 
significant improvement in erectile function and sexual satisfaction as evidenced by a significant score increase in 
the International Index of Erectile Function (IIEF), a grade increase on the Erectile Hardness Grading Scale, and 
a significant reduction in the need to enhance erectile function with medications and/or aids. Note that the IIEF is 
the “gold standard” for evaluation of erectile function in clinical trials 108, 109 and is the preferred 
instrument for measurement in the SCI population 110. Also, the Erection Hardness Grading Scale is often used 
along with the IIEF 111, 112. Importantly, studies show that scores obtained with subjective grading of erectile 
hardness by patients is positively associated with psychosocial outcome measures (emotional well-being and 
sexual satisfaction) 112. Preliminary assessments were obtained for one study subject before and after 
undergoing epidural stimulation plus LT (in addition to the many anecdotal reports from subjects undergoing LT at 
several rehabilitation centers that are part of the Neural Recovery Network). These preliminary findings are 
provided under Specific Aim 3 (section c below). 
 
c. Specific Aim 3:  To assess the added effect of epidural stimulation with standing, CV, VM and LT on 
urodynamic parameters, bladder urinary neurotrophin levels and sexual function after severe complete SCI. 
It is possible that for individuals with motor complete SCI (AIS A, B) who generate very minimal levels of 
EMG activity during stepping, significant bladder recovery will not occur, as does not occur for standing and 
stepping, even after intensive LT. We can further test the hypothesis of activation of the spinal circuitry as a 
mechanism for improved bladder function by also studying a cohort of individuals who first undergo LT and then 
receive an epidural implant for the intent of generating standing and stepping (Hypotheses 3A – 3D).  Dr. 
Harkema is the investigator of a NBIB funded study that focuses on the evolution of standing and stepping in 
humans using epidural stimulation combined with LT as well as a Craig Neilsen Foundation funded study 
looking at the recovery of CV function in combination with epidural stimulation113. We can leverage these 
already funded and ongoing studies to further our knowledge of activating spinal circuitry in the recovery of 
physiological functions,including bladder and sexual function. We will complete the same assessments as in 
Aims 1 and 2 on subjects with motor complete SCI already enrolled in this study. These experiments will provide 
information on whether 
in the most severely injured individuals a combination of epidural stimulation and LT, CV, VM, and stand 
training will be sufficient to evoke bladder changes.   An example showing improved bladder function from the 
first study participant to have 
urodynamic assessments before and after epidural plus LT (recently completed) is presented in Figure 6. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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Figure 6.  Cystometry recordings from a 32 year old male with a C7 neuro level SCI, AIS Grade B (motor 
complete, sensory incomplete; full pre- recording shown in Figure 2). Note the reduced pressure during filling 
and some ability to voluntarily void post-epidural plus LT. Urgency threshold was at 160 cc’s pre-epidural+LT 
and 260 cc’s post-epidural + LT. Note that during the voiding phase of the post-epidural + LT assessment, the 
subject commented (i) that it was cold in the room, and (ii) there were lots of people around. Perhaps the void 
would have been more in a different environment. Asked if he tried to void on his own at home, the subject 
stated that he did not try as he did not want to risk getting a UTI. 
 
In terms of sexual function, the same outcome measures will be done as per Aim 2. An assessment was 
done on a male subject (AIS Grade A; neuro level T4) receiving epidural stimulation + LT. Prior to enrollment in 
the study, the subject reported being a Grade 2 (increase in size and slight increase in hardness, but insufficient 
for sexual intercourse) and used the maximum dosage of Viagra (100 mg) 100% of the time to achieve a Grade 
4 (fully hard [rigid] erection) on the Erection Hardness Grading Scale. After completion of the study and returning 
for a six month follow-up (had access to a trainer at home and did one hour sessions of stand training 5 days a 
week), the subject reported a Grade 4 score, and only needed to use a low dose of Viagra (25 mg) less than 
50% of the time. He reported through interview that Viagra was needed on long work days when he did not have 
time or energy to do his daily training. He also reported that he noticed the changes in erectile function after 
about 3 to 4 weeks of epidural plus LT training during the study. 
Note that all the preliminary data reported above comes from three of the four subjects to date. The first 
subject, whose locomotor outcome data was published in 2011 in Lancet 114, was trained prior to the start of 
collecting urodynamic data for bladder function. Also, Figure 3 (pre- and post-training example) is from the 
current study participant who just recently received an epidural implant. Although bladder function data is 
provided above for one subject (AIS B, Figures 2 and 6) and sexual function data for the other (AIS A described 
in previous paragraph), both participants had improvements in bladder and sexual function. The C7 
AIS B participant, for example, had the ability to ejaculate but experienced head thumping during it (i.e., 
autonomic dysreflexia). The subject reported that the head thumping went away about one month into training 
plus epidural stimulation. The first study subject, a T2 AIS B, went from a score of 12 (mild to moderate erectile 
dysfunction) on the Sexual Health Inventory for Men (SHIM; short form of the IIEF) to a score of 24 (normal 
erectile function), with satisfaction rating for attempted sexual intercourse increasing from “a few times” to “many 
times” before versus after training plus epidural stimulation. All four subjects report increased 
awareness of bladder fullness after just LT, including chills down the legs (per description in Figure 3 legend), a 
sense of pressure below the umbilicus, and a sense of the penis retracting. The C7 AIS B subject, for example, 
would get a tingling sensation in his face and the top of his head (both sides) when his bladder is full (prior to 
enrollment in the study). After training, in addition to these above lesion level sensations, he also reported a 
tingling sensation in his groin/penis as his bladder reached capacity during filling while undergoing cystometry, 
which he reported began occurring after about 3 weeks of LT. Both subjects who completed the study with all 
measurements also reported ability to void during their bowel program after undergoing LT plus epidural 
stimulation. For example, prior to enrollment in the study, the C7 AIS B subject would leak a few drops of urine 
while doing his morning bowel program. Since participating in the study, he is able to void 50 to 75% of the 
bladder volume while doing his bowel program (estimated by amount of residual volume he collects upon 
catheterization afterwards). This particular finding, in addition to information regarding Viagra usage changes 
described above for the T4 AIS A subject, emphasizes the need for an interview component in this study, 
which will also provide useful data for testing new hypotheses in animal models (bedside to bench, then 
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3. Specific Methods. 
 
a. Interventions (Those provided only as part of this study). 
 
 
Non-weight bearing exercise: 80 sessions of arm crank (RT300 cycle) will occur at the same intensity and 
duration (60 minutes) as the weight-bearing interventions. Individuals will be sitting in their wheelchair 
propelling an arm crank at similar velocities to the stepping (35-55 rpm) using motor driven assistance when 
needed (only for those with cervical injuries whose arm function has been impaired). Resistance will be 
continuously increased over the course of the training sessions as the individuals are able to propel the arm 
crank continuously for an hour.  
 
For non-weight bearing training, research participants may also include those receiving 80 one-hour sessions 
of UE-NMES which consists of electrical stimulation applied via the XCite system (Restorative Therapies, Inc 
(RTI), Baltimore, MD), a portable, digital, dual channel, interferential device. Bifurcated leads and self-adhesive 
reusable surface electrodes (PALS® Platinum Neurostimulation Electrodes (Restorative Therapies, Inc)) will 
be placed over the bilateral arm and upper trunk muscles during the training sessions, while the research 
participant is seated in his/her wheelchair or on a mat.  
 
Stand Training (weight bearing without stepping):  Approximately 80 sessions of standing uses body weight 
support for 60 minutes each session and suspension may be provided by a harness and lift at the minimum 
support at which limb buckling and trunk collapse can be avoided 115 or research participants may use the 
custom designed standing apparatus, comprised of horizontal bars anterior and lateral to the individual. These 
bars will be used for upper extremity support and balance assistance as needed. Bungee cords will be placed 
across the upper tibias and hips for dynamic support.  The research participant will begin the sitting-to-
standing transition by using the horizontal bars of the standing apparatus for assistance and support; trainers 
positioned at the trunk, pelvis and knees will manually assist as needed during this transition. In case the 
upper limbs and trunk control is insufficient for safely using the standing apparatus, research participants will 
be placed on the treadmill, and a body weight support system with a harness will be used to avoid trunk 
collapse and knee buckling. In this case, the level of body weight support will be continuously reduced over 
the course of the training sessions as the individuals increase their ability to bear weight on the lower limbs. A 
trainer positioned behind the research participant will aid in pelvis and trunk stabilization; the trainer will 
ensure that the trunk and pelvis are not flexed or hyper-extended during standing. Trainer(s) positioned at the 
lower limb will provide manual facilitation using a customized technique developed by this research team that 
facilitates knee extension during standing. Trainer(s) promote knee extension by applying gentle pressure at 
the tibial tuberosity and stimulation of the patellar tendon. Manual facilitation at the trunk-pelvis and at the legs 
will be used only when needed. Research participants will undergo 80 sessions of stand training (1 hour, 4-5 
sessions per week) and will be encouraged to stand for as long as possible throughout the training session, 
with the goal to stand for 60 minutes with the least amount of assistance. Seated resting periods will occur 
when requested by the individuals. 
 
b. Procedures. 
A voiding diary with frequency/volume of each void and/or catheterization will be generated for two 
consecutive 24 hour periods once per week by using standard voiding diaries. The number of voids, voided 
volume, and distribution between daytime and night-time will be obtained. In addition, urine samples will be 
tested once a week for the presence of blood, urobilinogen, glucose, ketones, bilirubin, protein, nitrites, 
leukocytes, pH, and specific gravity using DiaScreen reagent strips for urinalysis. Vasopressin levels also will be 
tested from the urine samples using the Arginine Vasopressin ELISA kit (Cayman Chemical). 

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Urodynamics: You may be asked to refrain from taking any bladder medications 24-hours prior to 
Urodynamics. Urodynamic study will include an initial measurement of the urinary flow (uroflow: voided volume, 
maximum flow rate, time to maximum flow rate and total voiding time). This will be followed by cystometry 
evaluation (measuring bladder pressure during filling, possible uninhibited bladder contraction and maximum 
cystometric capacity), with determination of the leak point pressure and postvoid residual volume when voiding is 
possible at the end of the study when a second uroflow can be obtained. A 12 French straight catheter will 
be used to empty the bladder completely and then a 7 French three way urodynamic catheter will be placed in 
the bladder to fill the bladder as well as to measure and record intra-vesical pressure. Another catheter with a 
balloon will be placed in the rectum to record the intra-abdominal pressure. Detrusor pressures will be calculated 
by subtracting the intra-abdominal pressure from the intra-vesical pressure. The bladder will be filled at a slow 
rate with body temperature water. Each subject will be asked to cough to verify intra- abdominal catheter 
position, and will be instructed to communicate when s/he first feels a full bladder (first sensation); when s/he first 
feels the desire to urinate (first urge to void); and when s/he can no longer wait to void (maximum capacity). The 
volume of water and bladder pressure will be recorded. Uninhibited bladder contractions will be identified. Next, 
the individual will be asked to empty their bladder and voiding bladder pressures recorded. The Urodynamic 
equipment, and with a rectal balloon catheter in place, may also record pressure measurements of the anal 
sphincter muscles, the sensation in the rectum, and activity of the reflex pathways that are needed for normal 
bowel movements. 
During the time when cystometry evaluation is being done, EMG surface patch electrodes will be placed on  
the skin at 3 and 9 o'clock on either side of the urethra in order to record the urethral sphincter activity. This will 
evaluate coordination of the urethra and anal sphincters during the voiding phase and during possible 
uninhibited contraction episodes. Detrusor-sphincter dyssynergia will be evaluated and classified according to 
the Blaivas classification into: type 
1 DSD characterized by a crescendo increase of the sphincter activity that reaches its maximum at the peak of 
the detrusor contraction (as the detrusor pressure begins to decline, sudden complete external sphincter 
relaxation occurs); type 2 DSD characterized by clonic contractions of the external urethral sphincter 
interspersed throughout the detrusor contraction (these patients usually void with an interrupted spurting 
stream); type 3 DSD, characterized by external urethral sphincter contraction persists throughout the entire 
detrusor contraction (these patients void with an obstructive stream or cannot void at all) 117, 118. EMG 
electrodes may also be placed during cystometry on the medial hamstrings, vastus lateralis, soleus and tibialis 
anterior muscles. EMG amplitudes, durations and co-activation will be calculated during filling and voiding 
phases of the pressure-flow study.  Detailed methods have been published.  
After urodynamic testing, the skin of the participant will be inspected and ensured that all areas exposed during 
the experiment are dry. Blood pressure (BP) parameters may be monitored throughout urodynamic testing using 
either the Dinamap Carescape V100 (GE Healthcare) or continuous arterial BP acquired from a finger cuff placed 
around the left middle finger, index finger, or thumb (Portapres-2;Finapres MedicalSystems). Beat-to-beat 
systolic and diastolic recordings will be calculated from acquired continuous BP measurements. Overall, 
urodynamic assessments should last approximately 1.5 to 2 hours.  
Urinary Biomarkers: Urine samples will be obtained using a sterile 12 French straight catheter that is inserted 
through the urethra into the bladder for cystometry. The bladder is always emptied prior to filling for cystometry 
recordings, so once the volume is measured, urine samples will be placed in sterile storage vials and stored at 
4C for less than 3 hours.  Procedures will follow similar methods established and used by multiple groups of 
investigators 101, 104, 119.  Briefly, samples will be centrifuged (3,000 rpm for 10 minutes) and 1 ml supernatant 
aliquoted into 1.5 ml tubes, with some used to determine creatine concentration. Aliquots will be frozen at -80C for 
later processing. Urinary NGF and BDNF concentrations will then be determined on the thawed samples 

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using the Emax ImmunoAssay System with specific ELISA kit per manufacturer instructions 101, 104, 119. The 
concentration of NGF and BDNF in each urine sample will be extracted from a standard curve and normalized 
to the concentration of urinary creatine (NGF/Cr, BDNF/Cr). All samples will be run in triplicate and the values 
averaged. 
 
Bladder biopsies: Bladder biopsies are an optional procedure. If conducted, Dr. Mohamed will perform bladder 
biopsies using standard procedures at the University of Louisville Urology clinic. A random bladder biopsy will 
be obtained during cystoscopy, in order to capture the epithelium, basement membrane, up to the superficial 
layers of the detrusor muscle. The sample will be obtained in standard surgical sterile technique under local 
anesthesia. All layers of the bladder wall with exception of the outer serosa should be represented in the biopsy 
specimen (Figure 7). During this procedure, a 16 French Olympus Flexible Cystoscope (a lighted instrument 
used to look at the bladder) is passed through the urethra into the bladder, which may cause a feeling of slight 
discomfort. The bladder will be filled with normal saline through the cystoscope, producing an uncomfortable 
sensation, similar to a strong urge to 
urinate. A biopsy forceps grasper is then inserted through the cystoscope working channel into the bladder and a 
very small amount of bladder tissue is excised. The forceps cup has a 2 mm outer diameter but the cup itself has 
a 5 mm diameter. Participants may feel a pinch during the biopsy. Depending on the amount of pressure applied 
with this size forceps cup, a single full 1 cm biopsy sample can be obtained (i.e., plenty of tissue needed for 
assessments). Next, the affected blood vessels are cauterized in order to seal them and stop bleeding within the 
bladder wall, through a Bugbee electrode placed through the cystoscope. This may induce 
a burning sensation. 
After the cystoscope is removed, the participant’s urethra may be sore and they may report a burning 
sensation during urination for 1-2 days following the procedure. Viscous Lidocane will be applied through the 
urethra in attempt to damper this sensation. Samples will be immediately frozen in liquid nitrogen for later 
analyses conducted by members of the research team. To assay bladder biopsies for NGF and BDNF, frozen 
bladder will be homogenized and protein extracted using a lysis buffer, and subsequently run in duplicates 
(triplicates if tissue samples adequate) in 96-well plate antibody-sandwich ELISA with internal standard per 
manufacturer (Emax® ImmunoAssay System, Promega, Madision WI, USA). This procedure should last 
approximately 1 hour. 
 
 
 
 
 
 
 
 
 
 
 
Figure 7. Layers of the bladder wall 
 
 
120. 

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EMG activity during stepping: All methods have been published previously 47, 51.  In brief, EMG and kinematics 
may be acquired during stepping at four speeds (0.27, 0.45, 0.89, 1.34 m/s) using the maximum body weight load 
at which knee-buckling and trunk collapse can be avoided. Manual facilitation will be provided only if needed.  
Another stepping bout will be implemented at the load and speed where the most independence from manual 
facilitation occurs at the trunk, hips and legs. EMG amplitudes, durations and co-activation will be calculated.  
Detailed methods have been published previously. This assessment should last approximately 2-3 hours. 
 
 
Measures of Erectile Function: The full International Index of Erectile Function (IIEF) questionnaire is readily 
available in many publications 108. It has been validated in 32 languages and can be used cross-culturally 121. 
Briefly, there are 15 questions that can be divided into 5 unique domains: erectile function, intercourse 
satisfaction, orgasmic function, sexual desire, and overall satisfaction 108. The Erection Hardness Grading Scale 
(Grades 1-4) is defined as follows: increase in size of penis but no hardness (rigidity)as Grade 1; increase in 
size and slight increase in hardness (rigidity), but insufficient for sexual intercourse as Grade 2; increase in 
hardness (rigidity) sufficient for sexual intercourse but not fully hard (rigid) as Grade 3; fully hard (rigid) as 
Grade 4 111, 112. In conjunction with measures of erectile function, the International Spinal Cord Injury Data Set 
questionnaires for bowel, bladder and sexual function (male and female) also will be incorporated to assess 
management and dysfunction of these systems. These questionnaires should take approximately 10-20 
minutes to complete. 
 
4. Statistical Analysis Plan. Many of the analyses involve paired comparisons of pre-training measurements 
(voiding frequency, bladder capacity, NGF levels, etc.) with mid- and/or post-training measurements (LT, stand, 
non-weight bearing training). We will evaluate the null hypotheses of no improvement due to training with paired 
t-tests. Standard 
F-tests will be conducted to detect variance heterogeneity, and Welch’s correction subsequently applied as 
needed. The Wilcoxon signed rank test will be used in place of the t-test should the normality assumption be 
violated.  Hypothesis 1C suggests an association between urodynamic parameters and EMG activity during 
stepping after LT. This hypothesis will be evaluated by a nonparametric correlation analysis (e.g. Spearman, 
Kendall correlations) comparing pre- to mid- and/or post-training changes in urodynamic parameters to EMG 
activity post- training. Power estimations using EMG co-activation data from previous human locomotor training 
studies indicate that a sample size of 10 per group would achieve a significant difference at the 0.05 level 
between the groups with a power of 80%. 
 
Hypotheses 3A-3D are specific to Group 2 (LT + epidural stimulation) and each involves comparing the 
change in outcome variables over 80 LT sessions to changes over 80 stand + epidural stimulation sessions 
then 80 LT + epidural stimulation sessions. We will use paired t-tests to compare the improvement over 80 
sessions of LT to the improvement over 80 sessions of stand + epidural stimulation, and like-wise for 80 
sessions of stand + epidural stimulation to the improvement over 80 sessions of LT + epidural stimulation. 
Comparisons of the changes in outcome variables over 80 sessions of training with cardiovascular plus 
epidural stimulation (CV, VM, and stand) may also be performed. Welch’s correction and the Wilcoxon signed 
rank test will be applied as described above. 
Descriptive statistics for each outcome will be calculated for each study group at each assessment. In 
particular, we will generate mean and standard error estimates for each outcome variable pre-training, post- 
training, and for the pre-post training difference, so that these quantities can be used in planning future research.  
All hypothesis tests will be conducted at the .05 level. It is possible that results derived from Specific Aim 3 (study 
of LT + epidural stimulation) may understate the effect of LT + epidural stimulation, as the effect 
of epidural stimulation added to LT may be partially masked by the gains experienced over the initial 80 
sessions of LT received. 
 

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5. Limitations and potential pitfalls. Time since injury will vary by years. It is difficult to confine time since injury 
to within a few months and also recruit a sufficient number of SCI research participants. It is possible that the time 
since injury would influence the outcomes. However, we have shown neural changes in individuals’ even 
decades after clinically complete SCI 113 with the most significant change in bladder function reported 
from an individual 8 years post injury.  A key element of risk is that we have relied on patient reports of bladder 
improvements coincident with the intense LT. Improved bladder function, even incremental, would have a 
dramatic impact on health and quality of life for those suffering the lifelong consequences of neurologic injury. 
Also, individuals will vary in terms of their continence and voiding as well as sexual function, including 
opportunities for sexual activity, types of stimulation that work, and positioning problems with paralysis. For these 
reasons, every participant has pre- and post- measurements for all aspects of the study, so that each subject can 
also serve as their own control (in addition to quantification of group trends). There may exist a high variability in 
the magnitude of outcomes. However, if a significant number of individuals recover even some bladder function it 
would have a tremendous impact on their quality of life and potentially reduce infections and longer term bladder 
complications. 
 
6. Data Safety and Monitoring Plan 
 
An independent Data Safety Monitoring Board (DSMB) has been chartered to monitor the safety of 
research participants as well as the data validity and integrity of studies conducted at the Kentucky Spinal Cord 
Injury Research Center, University of Louisville and the Frazier Rehab Institute. Members of the DSMB serve in 
an individual capacity and will convene yearly to provide their expertise including recommendations regarding the 
continuation, modification, or termination of any or all phases of this study. The DSMB will review cumulative 
study data to evaluate safety, study conduct, scientific validity and data integrity. DSMB members may review 
current versions of the protocol and Informed Consent Form, and any subsequent amendments to ensure an 
understanding of a study’s objectives and design. Day-to-day oversight of the study will be provided by the 
Principal Investigators. They will review all study data and any adverse events, and report all adverse events to 
the University of Louisville IRB, DSMB chairperson and sponsor as appropriate. Medical events that occur while 
the research participants are engaged in the research protocols also will be logged by a research nurse, physical 
therapists, physicians and/or staff. Any adverse events (AE) are collected on an Adverse Event Form and will be 
reported at the yearly DSMB meeting. An AE report will be generated for each event and will include a description 
of the event, when and how it was reported, as well as any official chart records or documentation to corroborate 
the event and a determination of attribution. Any AE that the PI determines to be definitely, probably, or possibly 
related to the research intervention, or serious in nature, and unexpected will be reported to the IRB and sponsor 
as appropriate, within 5 business days of the PI gaining knowledge of the event. Any unanticipated problems 
involving risks to research participants or others will include a corrective plan and measures to prevent 
reoccurrence. Such events will be reported to appropriate regulatory agencies as required within 5 business days 
of the PI gaining knowledge of the event.