Novel Methods to Reduce Muscle Fatigue Using Multi- Electrode Functional Electrical Stimulation in Isokinetic Knee Extension Contractions

Transcription

1 Novel Methods to Reduce Muscle Fatigue Using Multi- Electrode Functional Electrical Stimulation in Isokinetic Knee Extension Contractions by Vishvek Babbar A thesis submitted in conformity with the requirements for the degree of Master of Health Science Institute of Biomaterials and Biomedical Engineering University of Toronto Copyright by Vishvek Babbar 2015

2 Novel Methods to Reduce Muscle Fatigue Using Multi-Electrode Functional Electrical Stimulation in Isokinetic Knee Extension Contractions Abstract Vishvek Babbar Master of Health Science Institute of Biomaterials and Biomedical Engineering University of Toronto 2015 Muscle fatigue, a major limiting factor in the use of functional electrical stimulation, can be countered through multi-electrode sequential stimulation. To apply this method clinically, our lab developed a generic adapter. The purpose of this thesis project was (1) to test the adapter s performance and (2) to investigate whether two sequential techniques, namely Spatially Distributed Sequential Stimulation (SDSS) and Sequential Multiple Muscle-Head Stimulation (SMHS), are capable of inducing less fatigue than Single Electrode Stimulation (SES) in isokinetic knee extension contractions. Thirteen able-bodied individuals participated in a threeday study. Performance of the adapter was found to be accurate within acceptable error. Both SDSS and SMHS resulted in significantly less fatigue than SES as measured by fatigue index and torque peak mean. The less low frequency fatigue observed during SDSS and SMHS may explain the lower fatigue. SDSS and SMHS delivered via adapter pose an attractive alternative to SES during clinical applications. ii

3 Acknowledgments I thank my supervisor, Dr. Kei Masani, for his endless support and patience in guiding me through this project. I thank my committee members, Dr. Kei Masani, Dr. Milos R. Popovic, Dr. Paul Yoo, and Dr. Tom Chau, for their supervision. I thank Dr. Austin J. Bergquist and Ms. Saima Ali, the co-contributors of this project. I thank Mr. Azim Rashidi, Ms. Esther Oostdyk, Dr. Vera Zivanovic, and Mr. Carlos Buzelli for providing administrative and technical support at the Rehabilitation Engineering Laboratory. I thank Ms. Rhonda Marley and Mr. Jeffrey Little for providing administrative support through the Clinical Engineering program. I thank the students and staff at Rehabilitation Engineering Laboratory for making my time well spent. I thank those unnamed individuals who volunteered to participate in my experiment. Finally, I thank my family members, my mom, dad, and sister, and close friends for their endless support and motivation. iii

4 Table of Contents Table of Contents Acknowledgments... iii Table of Contents... iv List of Tables... viii List of Figures... ix List of Abbreviations... xiv Chapter Introduction Research Problem Motivations Research Method in Brief Roadmap of Thesis... 3 Chapter Background Functional Electrical Stimulation Nature of FES Systems FES Applications Fatigue in FES Central and Peripheral Fatigue Sources of Fatigue in FES Prevention of Fatigue in FES Random Modulation of Stimulation Parameters Progressively Altering Stimulation Parameters iv

5 2.3.3 Variable Frequency Trains Stimulation Over Nerve Trunk versus Muscle Belly Sequential Stimulation using Multiple Electrodes Considerations for Type of Contraction Requirement of Novel Tool for Multi-Electrode FES SDSS Adapter Clinical Application Chapter Purposes of Research Research Objective Research Purposes Purpose Purpose Chapter Methodology Apparatus Compex Stimulator SDSS Adapter Stimulation Electrodes Data Acquisition System Joint Dynamometer Study 1: Testing of SDSS Adapter Data Collection Statistical Analysis Study 2: Fatigue Reduction using Multi-Electrode FES Participants v

6 4.3.2 Target Muscle Group Electrode Placement Dynamometer Setting Experimental Protocol Data Analysis Chapter Results Study 1: Testing of SDSS Adapter Characterization of Pulses Similarity between the Adapter Outputs and the Source Stimulator Outputs Difference between the Adapter Outputs and the Source Stimulator Outputs Study 2: Fatigue Reduction using Multi-Electrode FES Participant Exclusion Initial Torque Values Change in Torque Fatigue Measures Low Frequency Fatigue Chapter Discussions Validity of SDSS Adapter as a Tool for Multi-Electrode FES Effectiveness of Sequential Multi-Electrode FES Initial torque Change in Torque Mechanism of Fatigue Reduction in Sequential Stimulation Limitations Future Recommendations vi

7 Chapter Conclusions References Appendices A. Consent Form B. Data Collection Sheet C. Normalized Change in Torque vii

8 List of Tables Table 1: Range of stimulation parameters used in FES applications Table 2: Range of specific parameters constraining the SDSS adapter Table 3: Demographical information on the 13 participants Table 4: Design of the fatigue study Table 5: Visits, rest between visits, and the time required Table 6: Order of collection of measurements and time taken to collect Table 7: Stimulation parameters during LFF and Fatigue Protocol viii

9 List of Figures Figure 1: Activating quadriceps and hamstring muscle via a) natural means in a healthy individual, and b) using FES in individual with damaged spinal cord [20] Figure 2: Typical biphasic asymmetric pulse where charge entering the tissue (product of A and C) is equal to charge leaving the tissue (product of B and D). fstim represents frequency of stimulation [20]... 7 Figure 3: Temporally asynchronous recruitment of muscle fibers to generate a tetanic contraction [20]... 9 Figure 4: [Ca 2+ ]-tension curve showing that Ca 2+ release at high frequencies is on horizontal part of curve, while release at low frequencies is at steep part. At low frequency, even small falls in [Ca 2+ ] would produce large drop in tension, resulting in LFF [35] Figure 5: Comparison of electrode placements of SMHS and SES. SMHS utilizes four cathodes placed far apart over distinct motor points. Anode is the same in both the cases [11] Figure 6: Comparison of electrode placements of SDSS and SES. SDSS utilizes four cathodes placed in a 4x4 matrix at the same location as SES. Anode in both cases is the same [7] [8] Figure 7: Stimulation pulses delivered during SES (top) and SDSS (bottom); 90 degree phase shift in SDSS results in each electrode in the 2x2 matrix to have one-fourth the frequency of the single SES electrode Figure 8: Difference in mechanism between SES and SDSS. Larger number of muscle fibers recruited and lower stimulation frequency result in less fatigue in SDSS than SES Figure 9: Schematic of SDSS adapter tool for distributing electrical stimulation signal. Top: single channel to single electrode in standard SES; Middle: four channels to four electrodes in sequential stimulation without adapter; Bottom: single channel to four electrodes in sequential stimulation with adapter ix

10 Figure 10: Schematic diagram of the various components of the SDSS adaptor unit. Red arrows: path that the stimulation pulse travels; Black arrows: components under influence of controller; Green arrow: light signal to LED Figure 11: SDSS adapter with single input from stimulator and four outputs to electrodes attached to the forearm Figure 12: Compex Motion stimulator with accompanying programmable cards [57] Figure 13: Graphic of PowerLab /30 Series data acquisition system [59] Figure 14: Graphic of Biodex System 3 with dynamometer, chair, controller, and knee extension attachment [60] Figure 15: Anatomy of quadriceps femoris muscle. Rectus femoris (green), vastus lateralis (red), and vastus medialis (blue) [61] Figure 16: A) Schematic representation of FES electrode placement during SES, SMHS, and SDSS. B) Schematic representation of pulse timing during FES at 40 Hz. Note that during SDSS and SMHS, each electrode is stimulated at 10 Hz (100 ms inter-stimulus interval), resulting in FES at a compound 40 Hz when considering all 4 electrodes together. Pulse intensity (PI) and pulse duration (PD) are shown for illustrative purposes only and are not drawn to the scale Figure 17: Electrode positioning during a) SDSS and b) SMHS. Both protocols use four active electrodes and one reference electrode. Position of the four active electrodes is different while that of single reference electrode is the same Figure 18: Sample biphasic pulses delivered by a) stimulator and b) channel 1, c) channel 2, d) channel 3, and e) channel 4 of the adapter at reasonable level of parameters (ie, 40 Hz, 250 µs, and 50 ma). Pulses are delivered sequentially by the adapter at one quarter the frequency of the stimulator Figure 19: One sample pulse delivered by a) stimulator and b) channel 1 of adapter at lowest most level of parameters (ie, 10 Hz, 100 µs, and 25 ma). Pulse delivered by stimulator and adapter is characteristically similar x

11 Figure 20: One sample pulse delivered by a) stimulator and b) channel 1 of adapter at highest most level of parameters (ie, 80 Hz, 300 µs, and 125 ma). Pulse delivered by stimulator and adapter is characteristically similar Figure 21: Frequency of pulses recorded from SDSS adapter versus Compex stimulator with target frequencies of 10, 20, 30, 40, 50, 60, 70, and 80 Hz across a) channel 1, b) channel 2, c) channel 3, and d) channel 4 of the adapter Figure 22: Duration of pulses (PD) recorded from SDSS adapter versus Compex stimulator with target durations of 100, 150, 200, 250, and 300 µs across a) channel 1, b) channel 2, c) channel 3, and d) channel 4 of the adapter Figure 23: Amplitude of pulses recorded from SDSS adapter versus Compex stimulator with target amplitudes of 25, 50, 75, 100, and 125 ma across a) channel 1, b) channel 2, c) channel 3, and d) channel 4 of the adapter Figure 24: Mean RMSE as percentage of target between four channels (n=4) of adapter and stimulator across a) eight target frequencies (10-80 Hz), b) five target durations ( µs), and c) five target amplitudes ( ma). Error bars indicate standard deviation Figure 25: Initial torque (average of first 10 contractions; n=10) values for SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants Figure 26: Example of typical a) isometric torque time series and b) isokinetic torque time series with 120 contractions for (from top to bottom) SES, SDSS, and SMHS Figure 27: Binned torque by 10 contractions (n=10) reported as Newton-meters (Nm) for SES, SDSS, and SMHS averaged across eleven participants (N=11) under a) isometric and b) isokinetic conditions. 12 points represent 12 bins from contraction 1 to 120. Error bars indicate standard error of the mean across eleven participants Figure 28: FI for SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven xi

12 participants. Only significant p values (p < 0.05) for single factor ANOVA and paired two-tailed t-tests with Bonferroni correction are shown Figure 29: TPM for SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants. Only significant p values (p < 0.05) for single factor ANOVA and paired two-tailed t-tests with Bonferroni correction are shown Figure 30: Pre- and Post-fatigue LFF ratio for SES, SDSS, and SMHS averaged across 11 participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants Figure 31: Change in LFF ratio from pre- to post-fatigue in SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants. Only significant p values (p < 0.05) for single factor ANOVA are shown Figure 32: Consent form page Figure 33: Consent form page Figure 34: Consent form page Figure 35: Consent form page Figure 36: Sample of Data Collection Sheet (front) Figure 37: Sample of Data Collection Sheet (back) Figure 38: Binned torque by 10 contractions (n=10) reported as percentage of static MVC (% MVC) for SES, SDSS, and SMHS averaged across eleven participants (N=11) under a) isometric and b) isokinetic conditions. 12 points represent 12 bins from contraction 1 to 120. Error bars indicate standard error of the mean across 11 participants Figure 39: Binned torque by 10 contractions (n=10) normalized to initial value for SES, SDSS, and SMHS averaged across eleven participants (N=11) under a) isometric and b) isokinetic xii

13 conditions. 12 points represent 12 bins from contraction 1 to 120. Error bars indicate standard error of the mean across 11 participants xiii

14 List of Abbreviations FES Functional Electrical Stimulation SDSS Spatially Distributed Sequential Stimulation SMHS Sequential multiple Muscle-Head Stimulation SES Single Electrode Stimulation SCI Spinal Cord Injury CNS Central Nervous System LFF Low Frequency Fatigue EMG Electromyography VL Vastus Lateralis VM Vastus Medialis RF Rectus Femoris MVC Maximum Voluntary Contraction FI Fatigue Index TPM Torque Peak Mean RMSE Root Mean Square Error xiv

15 1 Chapter 1 1 Introduction 1.1 Research Problem Spinal Cord Injury (SCI) is a neurological disorder that is caused by partial or complete damage of the spinal cord due to physical trauma or diseases. In individuals with SCI, neuromuscular signals from the brain to muscles are completely or partially ceased at the spinal cord resulting in paralysis inducing motor disability. The disuse of muscle due to paralysis over time can result in muscle atrophy, and disruptions of autonomic nervous system functions, such as regulation of blood pressure, heart rate, body temperature, and digestive processes. Further, secondary complications include pressure sores, muscle spasms, cardiovascular disease, and osteoporosis [1]. In Canada, over 85,000 persons are currently living with spinal cord injury (SCI), and approximately 11,000 more such injuries occur each year [2]. The annual economic liability associated with traumatic SCI individuals surviving initial hospitalization is estimated to be $2.67 billion [3]. Social impact of SCI is significant as an estimated 20-30% of people with SCI show clinically significant signs of depression, which in turn has negative impact on improvements in functioning and overall health [4]. Thus, SCI has large impact on the independence and quality of life of individuals and the society. Therefore, treatment and rehabilitation options for SCI have been actively investigated. Functional Electrical Stimulation (FES) is a promising tool to address the symptoms of SCI [1]. FES is a technology used to artificially generate muscle contractions to induce body movements such as grasping and walking [5] [6]. FES has been investigated and its usability has been proven for many different applications. There are two major types of usages, namely, functional (orthotic) and therapeutic uses. In functional uses, FES has been used as an assistive technology to help carry out tasks in persons daily life. In therapeutic uses, FES has been used during physical therapy to train muscular and/or cardiovascular systems, or to facilitate motor relearning [5] [6]. One major limitation of FES is that it causes rapid muscle fatigue, which severely restricts the duration over which it may be applied. To overcome this limitation, sequential stimulation was originally proposed in animal studies. Sequential stimulation activates fibers

16 2 individually allowing them to rest in between pulses to revive force-generating capacity. Our lab developed a novel method of stimulation called Spatially Distributed Sequential Stimulation (SDSS), and successfully showed its feasibility in isometric condition. However, the setting of SDSS is complicated and could be improved when applied in clinical settings. Moreover, another method of sequential stimulation, which activates multiple muscle heads, has been proposed by another group. This method has yet to be explored and compared against SDSS technique to determine the best strategy in fatigue reduction. 1.2 Motivations To promote sequential stimulation into a clinically applicable method, our lab has developed a generic adapter, called SDSS adapter, to be used with any commercial electrical stimulators, which realizes sequential stimulation with ease. This thesis project intended to test the adapter s performance with various stimulation conditions. SDSS was developed and tested in our lab, at first with a complete SCI individual [7], and then with 15 able-bodied participants [8] for lower leg muscles (i.e., plantarflexors, and dorsiflexors). Then SDSS was tested with 11 able-bodied and 17 spinal cord injured participants [9] for lower limb muscles (i.e., knee extensors, knee flexors, plantarflexors, and dorsiflexors). However, in all cases, the ankle or knee movements were in isometric condition, which is not often used in clinical settings. In clinical settings, isokinetic or isotonic condition is used more often. Further, another group of researchers proposed another sequential method [10] [11] [12], which, in this thesis, is named Sequential multiple Muscle-Head Stimulation (SMHS). Their method is different in a sense that electrodes are located on multiple muscle heads of a group of synergistic muscles. The electrode location of SDSS is the same as that of a conventional single electrode stimulation (SES), which is much simpler compared to SMHS. Although both methods have been demonstrated in reducing muscle fatigue during FES, the performance of SMHS and SDSS has never been compared. Thus, this thesis project also intended to investigate, using the SDSS adapter, whether SDSS was capable of reducing muscle fatigue in isokinetic condition in comparison to SMHS.

17 3 1.3 Research Method in Brief SDSS adapter was developed under collaboration between our lab and Tecnalia R&I Spain ( to deliver sequential stimulation by multiplexing the output of a single channel stimulator into multiple electrodes. In the first study, the performance of the adapter was tested to investigate its accuracy with various stimulation conditions by changing pulse frequency, duration, and amplitude. In the second study, thirteen able-bodied individuals underwent three FES protocols: 1) SES, 2) SDSS, and 3) SMHS. The three FES protocols were tested on three separate days under both isometric and isokinetic conditions. Participants were asked to sit in an electromechanical isokinetic dynamometer and stimulation was applied using surface electrodes on the quadriceps femoris. Knee extension contractions were induced under a fatiguing protocol, and knee joint torque generated during SES, SDSS, and SMHS was analyzed. Several fatigue measures, including fatigue index and torque peak mean, were calculated to determine the degree of fatigue produced during each protocol. 1.4 Roadmap of Thesis The present thesis has been structured in the format of a scientific paper and includes seven chapters. The first chapter is the Introduction in which FES, its applications, and shortcomings, as well as the motivation and purposes of the current project are presented. The second chapter is the Background and presents the previous knowledge necessary to understand the current project. Causes of muscle fatigue during FES, and several methods of reducing fatigue that have been applied by experimenters are described, including those pursued in this study. The third chapter, Purposes of Research, highlights the purposes of the project and presents the hypothesis. Methodology is the fourth chapter and describes the two studies incorporated into the project. The apparatus, experimental protocol, and methods of data analysis are presented here. The results achieved from the project are given in the fifth chapter (Results). Interpretation of the results and their significance are discussed in the sixth chapter (Discussions). Finally, concluding remarks are presented in the seventh chapter (Conclusions). Additional sections include Acknowledgements, List of Abbreviations, References, and Appendix.

18 4 Chapter 2 2 Background 2.1 Functional Electrical Stimulation Although the idea of electrical muscle stimulation dates back to Luigi Galvani (1791), the actual application of electrical currents to activate nerves innervating extremities affected by paralysis was first described in 1962 [13]. FES technology has been used since then to treat symptoms of many neurological disorders, including SCI [14]. The aim of FES is to stimulate motor nerves that innervate muscles to activate those muscles and produce functional movements. It applies short bursts of electrical impulses to cause muscle contraction [15]. FES has been used to generate various functional movements in individuals with SCI including standing, walking, reaching and grasping objects, rowing, cycling, sitting and standing, and swallowing [15] [16] [17] [18] [19]. In healthy individuals, voluntary muscle contractions occur when electrical signals from the central nervous system (CNS) are directed towards muscles in the form of action potentials that run down the spinal cord and motor neurons (Figure 1 a)) [20]. In SCI, the damaged spinal cord is unable to transmit electrical signals to motor neurons. Electrical current delivered via a stimulator presents an alternative source of stimulation, as shown in Figure 1 b) [20]. In the example in Figure 1 b), FES is being used to generate movement at the knee joint by causing contraction in quadriceps and hamstring muscle. Thus, FES can be applied to regain motor functions in individuals with upper motor neuron disorders, such as SCI. It should be noted that FES requires the lower motor neurons that innervate muscles to be intact, so external electrical signal may be converted to action potential and propagated to muscles [21].

19 5 Figure 1: Activating quadriceps and hamstring muscle via a) natural means in a healthy individual, and b) using FES in individual with damaged spinal cord [20] Nature of FES Systems Two main components of FES systems are the stimulator and the electrodes. The electrical stimulator delivers stimulation in the form of short current pulses via the electrodes. FES systems fall into three broad categories depending on whether the electrodes are transcutaneous (surface), percutaneous, or implanted [22]. Transcutaneous (surface) electrodes are applied to the skin and may be used to stimulate the nerve leading to the muscle, or the muscle itself. In nerve stimulation, small electrodes are typically placed over the nerve trunk at a site where it runs close to the skin (i.e. femoral triangle for femoral nerve innervating the quadriceps). In muscle stimulation, large electrodes are typically placed in close proximity to muscle motor point, activating intramuscular nerve branches [23] [24]. Motor point is an area above the muscle where a muscle twitch can be evoked with the least injected current. Stimulation at the motor point exhibits the most robust contraction [25]. Two electrodes are required to induce a current flow between the stimulator and the skin; one of them is the active electrode placed directly over the motor point, and the other is the reference electrode placed opposite to the active electrode distal to the muscle to be stimulated. The effects of stimulating the nerve trunk versus the muscle belly and the resulting fatigue have been studied [25] [26] [27]. Stimulation over muscle belly resulted in more fatigue

20 6 during isometric plantarflexion contractions due to H-reflexes analyzed through electromyographic analysis. Some problems associated with transcutaneous electrodes are difficulties in positioning, poor selectivity, and skin irritations [15] [21]. Percutaneous electrodes penetrate the skin to deliver stimulation more precisely to allow greater muscle selectivity, such as during stimulation of the intrinsic muscles of the hand [15]. Implanted electrodes are designed for long-term use and require open surgical procedures. This includes implantation of the stimulator that would receive power and instructions through a radiofrequency telemetry link from an externally located control unit [15]. The tension produced during muscle contraction depends on pulse frequency, duration, amplitude, and shape of pulse [20]. Thus, the force or joint torque produced by the muscle can be controlled by changing the pulse frequency, amplitude, and duration, which are collectively known as the stimulation parameters. The stimulation parameters may be determined in the ranges given in Table 1 [15]. Alteration of stimulation parameters has been studied extensively and will be revisited in Section Typically biphasic, asymmetric square-wave pulses are used in FES, because the total charge that is transferred into the tissue equals the total charge transferred out, thus preventing tissue damage (Figure 2 [20]). The stimulation parameters can be controlled in either an open-loop or a closed-loop manner. In an open-loop system, the control is characterized by a pre-set pattern of stimulation, i.e., all parameters are set and fixed in advance. A closed-loop system allows for continuous real-time modification of stimulation parameters based on sensory feedback [22]. Open-loop system is considered in this study. Table 1: Range of stimulation parameters used in FES applications. Frequency Hz Duration μs Amplitude ma

21 7 Figure 2: Typical biphasic asymmetric pulse where charge entering the tissue (product of A and C) is equal to charge leaving the tissue (product of B and D). fstim represents frequency of stimulation [20] FES Applications The use FES is classified into two categories: functional (orthotic) and therapeutic [5] [6]. Functional use refers to the one to activate paralyzed muscles during daily activities, in precise sequence and magnitude, to directly accomplish functional tasks as a neuroprosthesis [5] [6]. A neuroprosthesis is a device that incorporates an FES system to aid the user in achieving a particular function that they are unable to perform alone [22]. These functions include lower limb movement for mobility, upper limb movement for self-care tasks, bladder function, and respiratory control [28]. In these applications, the FES system needs to be used every time the user wants to perform the function. These applications allow the user to more or less rely on the system on a permanent basis and, for this reason, are also referred to as orthotic systems [5] [6]. Many neuroprostheses are commercially available, such as drop foot stimulators that users such as stroke survivors can wear while walking in their daily life to overcome drop foot gait [6] [29]. In therapeutic uses, FES is used for therapeutic purposes, including reduction of hemiplegic shoulder pain, muscle strengthening, prevention of muscle atrophy, prevention of deep venous thrombosis, improve hemodynamic functioning and cardiopulmonary conditioning [28]. Additionally, FES can be used to facilitate a user s motor-relearning instead of providing a function in daily life. This method is termed FES Therapy [5] [6]. The goal of FES Therapy is to stimulate neuroplasticity in the CNS of the user to help them re-learn how to voluntarily execute impaired functions [6].

22 8 2.2 Fatigue in FES While FES has shown significant contributions to muscle training and neuroplasticity, one aspect that limits its application is rapid muscle fatigue. Muscle fatigue may be defined as a reduction in the force-generating capacity of muscle due to recent contraction [23] Central and Peripheral Fatigue Physiologically, there are various possible locations for the underlying fatigue, which range from the brain to the muscle sarcolemma to the eventual generators of force and power, the crossbridge proteins [23]. There are two known mechanisms that may result in muscle fatigue: central fatigue and peripheral fatigue. Central fatigue is the result of failure of neural signal transmission in locations found in the brain, spinal cord and up to the neuromuscular junction. It may result from poor action potential transmission along axonal branch points. Peripheral fatigue may result from failure of transmission of neural signals at the neuromuscular junction, or failure of the muscle to respond to neural excitation due to certain deficits, resulting in inadequate excitation of motor neurons. In the case of SCI individuals, the central component is minor or absent and fatigue mostly results from problems within the motor unit, such as depletion of substances, accumulation of catabolites, or problems in excitation-contraction coupling [5] Sources of Fatigue in FES Random Recruitment of Muscle Fibers Slow-twitch fibers, which are fatigue-resistant, are dominantly recruited during low force contractions, while fast-twitch fibers, which are fatigue-unresistant, are recruited during higher force requirement. This natural recruitment pattern of muscle fiber is called size principle [15] [30]. Depending on size principle, low force contractions can be maintained for longer period than high force contractions. The nerve fiber recruitment properties experienced during FES differ from those caused by normal physiologic mechanisms. During normal physiologic mechanism, recruitment of fibers occurs according to the size principle. However, this process randomizes when applying external stimulation wherein the slow-twitch and fast-twitch fibers

23 9 are recruited randomly. This early recruitment of more easily fatigable fibers results in greater fatigue [30] [31] Synchronous Stimulation In physiological recruitment, a single pulse delivered to a motor unit results in a twitch contraction of the innervated fiber. To maintain a constant contraction, also known as tetanic contraction, a train of impulses is delivered at a reasonable frequency (6-8 Hz) in a sequential manner. Before one motor unit relaxes, another motor unit adjacent to it is activated to induce a fused contraction. This is referred to as asynchronous recruitment and allows various motor units to share the work of maintaining a contraction [30]. A visual representation of this phenomenon can be seen in Figure 3 where three impulses are delivered in a temporally asynchronous fashion which combine to result in a fused, tetanic contraction [20]. Muscles gradually get fatigued, since each motor unit is active only for a part of the time. Figure 3: Temporally asynchronous recruitment of muscle fibers to generate a tetanic contraction [20]. The problem is that the temporally asynchronous nature of recruitment does not occur when using FES. Since the stimulation parameters remain fixed, the muscle fibers are recruited at the same time in a more concentrated manner, which do not allow motor units to share the work of maintaining a contraction, leading to faster fatiguing. Also, since all muscle fibers are activated synchronously, a natural activation frequency of 6-8 Hz would not cause a sufficient fused

24 10 contraction. Therefore, to generate enough force it is required that frequency be increased from the 6-8 Hz range to Hz range, which further leads to greater fatigue [30] Changes in Muscles post SCI There is a large degree of morphological and contractile changes of muscles after SCI. In 4 to 7 months after SCI, a fiber type transformation away from slow-twitch begins and reaches a new steady state with predominantly fast-twitch glycolytic fibers a few years after the injury. There is progressive drop in proportion of slow myosin heavy chain isoform fibers, and rise in fibers that express both fast and slow isoforms. The decrease in slow-twitch fibers and myosin heavy chain isoforms results in fatigue occurring more quickly [32]. Furthermore, accumulation of catabolites and problems in excitation-contraction coupling also contribute to muscles of SCI individuals showing greater fatigability than healthy muscles, thus limiting applicability in SCI individuals [33] Low Frequency Fatigue Low frequency fatigue (LFF) is one mechanism of fatigue that may play a significant role in decline of force-generating capability of skeletal muscle. It may be caused by various forms of exercise including stretching or repeated contractions. It is called low frequency fatigue because it exhibits a preferential decline in force when tested at low (10-30 Hz) versus high ( Hz) frequencies after fatigue. Recovery of LFF is slow, taking hours or days, and for this reason it is also referred to as long-lasting fatigue. It should be noted that LFF is not caused by stimulations at low frequency but is only observed when probing the muscle at low frequency after fatigue [34]. The drop in force at low frequencies, i.e. LFF, can be explained by reduced release of Ca 2+ from sarcoplasmic reticulum. There exists a sigmoidal relationship between force (tension) and Ca 2+ (Figure 4). Since [Ca 2+ ] at high frequencies is on the horizontal part of the curve, moderate falls in [Ca 2+ ] have no effect on muscle tension. At low frequencies, where little [Ca 2+ ] is released, the steep portion of the curve means that even small falls in [Ca 2+ ] would produce large drop in tension [35].

25 11 Figure 4: [Ca 2+ ]-tension curve showing that Ca 2+ release at high frequencies is on horizontal part of curve, while release at low frequencies is at steep part. At low frequency, even small falls in [Ca 2+ ] would produce large drop in tension, resulting in LFF [35]. LFF is evaluated by recording torque at high frequency and low frequency after a fatiguing protocol, and comparing those with similar measures taken before fatiguing protocol. Pulses to potentiate the muscle are typically sent prior to high and low frequency. The ratio of the torque produced at low pulse frequency to that produced at high pulse frequency is evaluated. This ratio calculated before fatiguing protocol is compared to that calculated after. A decrease in the LFF ratio indicates the presence of LFF. The larger the decrease in ratio, the greater the presence of LFF [35]. LFF Ratio = torque at low freq torque at high freq LFF Ratio = LFF Ratio after LFF Ratio before LFF is present if: LFF Ratio after < LFF Ratio before or, LFF Ratio < 0

26 12 Presence of LFF indicates that at least some portion of fatigue observed is due to a low Ca 2+ release [36]. 2.3 Prevention of Fatigue in FES The reduction or prevention of muscle fatigue during FES has been approached using several techniques, including randomly modulating and progressively altering stimulation parameters, using variable frequency trains, and employing sequential stimulation using multiple electrodes Random Modulation of Stimulation Parameters Various combinations of the three prominent stimulation parameters namely, pulse frequency, duration, and amplitude, have been tested when applying FES. In a couple of studies [37] [38], random modulation of pulse frequency (mean 40 Hz), amplitude, and duration (mean 250 μs) in a range of +/- 15% was done to find out how random changes in one of the three parameters, while keeping the other two constant, would affect muscle fatigue. The study was done on 7 paraplegic subjects with stimulation applied to quadriceps and tibialis anterior. After testing 4 stimulation protocols (1 with constant parameters, and 3 with one modulated parameter and two constants), no significant difference in the level of fatigue was reported as measured by fatiguetime and force-time integral, and random modulation seemed to have no effect on fatigue Progressively Altering Stimulation Parameters Another study examined whether stimulation pulse frequency and duration should be kept low or high to minimize fatigue [39]. Three combinations of frequency and pulse duration were tested, where the first protocol involved low pulse frequency and long duration, the second involved medium pulse frequency as well as duration, and the third protocol involved high pulse frequency and short duration. The first protocol, using low frequency and long pulse duration, produced the least decline in peak force and the least muscle fatigue. The notion of higher pulse frequency resulting in higher fatigue was confirmed in another study where pulse frequency and duration were altered while keeping the total charge and torque output constant [40].

27 13 Pulse frequency and duration were again compared in a different study where three protocols of stepwise increase in pulse frequency, stepwise increase in pulse duration, and constant pulse frequency and duration were delivered to quadriceps femoris of 12 able-bodied individuals [41]. Isometric forces were measured and the peak force and force-time integrals indicated that the stepwise increase in pulse frequency showed better performance while producing a similar level of fatigue. The effect of pulse frequency on muscle fatigue was tested in a different study that measured changes in thenar muscle force and M-wave amplitude during three protocols of progressively increasing frequency (20-40 Hz), progressively decreasing frequency (40-20 Hz), and constant (20 Hz) frequency [42]. After testing on 23 able-bodied individuals there was no significant difference in force-time integral between the 3 protocols, suggesting that frequencies may vary significantly without changing overall force-time integral. Pulse amplitude alone was tested in a study that measured degree of fatigue in triceps surae muscle while keeping isometric torque-time integral constant in a protocol of voluntary contractions, and two FES protocols, one with constant pulse amplitude and other with gradually increasing pulse amplitude [24]. The degree of fatigue was similar in the two FES protocols as it was in the voluntary protocol, suggesting that torque-time integral may not be significantly affected by varying pulse amplitude. Another couple of studies tested the effect of progressively increasing pulse frequency or amplitude or both (in either order) in maintaining muscle force. The first study was on individuals with SCI [43] and the second one on able-bodied individuals [44]. The generated muscle force indicated that progressively increasing both frequency and amplitude was better than increasing only one of them. Also, increasing first the amplitude followed by frequency was better than increasing the frequency first and then the amplitude, and increasing the pulse frequency alone was better than increasing the amplitude alone. Another comprehensive study evaluated how pulse frequency, amplitude, and duration would affect fatigue and if the ratio of evoked torque to activated area could explain the degree of fatigue [45]. Seven able-bodied individuals underwent 4 protocols applied to knee extensors to measure isometric torque: 1) standard, 2) short pulse duration, 3) low pulse frequency, and 4) low pulse amplitude. It was found that decreasing the frequency in protocol 3 resulted in up to

28 14 39% decrease in fatigue, while decreasing amplitude and duration had no effect on fatigue. This finding furthers the previously developed conclusion that low frequency stimulation protocols are desired to minimize fatigue Variable Frequency Trains Variable frequency trains (VFT), where the interpulse intervals within each train are not kept constant, have also been tested and compared with constant frequency train (CFT) to determine the effect on force production and fatigue. VFTs have been more effective at reducing fatigue due to the catchlike property of muscles [46]. A recent study comparing mixed stimulation programs (MIX) that switch from CFT to VFT were tested against CFT alone in strength training-like conditions in 13 able-bodied individuals [47]. The mean torque was found to be 13% higher for MIX than CFT. The findings from various studies on altering stimulation parameters seem to indicate that keeping pulse frequency low and pulse duration high is ideal for minimizing fatigue while maintaining contraction force. Random modulation of parameters is not as reliable in reducing fatigue. Also, programs involving VFTs and those with mixture of VFT and CFT would produce higher torque than CFT alone Stimulation Over Nerve Trunk versus Muscle Belly Bergquist et al. demonstrated that neuromuscular electrical stimulation over a muscle belly (mnmes) resulted in more fatigue than stimulation over a nerve trunk (nnmes) [25]. mnmes generates contractions predominantly through M-waves and nnmes through H-reflexes [25]. Isometric plantarflexion torque and soleus electromyography (EMG) were recorded in 8 SCI individuals. mnmes cathode and anode were placed over triceps surae, nnmes cathode and anode were placed over tibial nerve trunk, and EMG electrodes were placed under the soleus. The fatigue index and peak twitch torque were significantly higher for nnmes protocol. Through the EMG analysis, H-reflexes were found to contribute to the drop in fatigue during nnmes.

29 Sequential Stimulation using Multiple Electrodes FES is conventionally applied through a single pair of electrodes over a muscle or a muscle group, which is named in this thesis as single electrode stimulation (SES), as defined above. To overcome the rapid muscle fatigue during SES, researchers have explored potential of sequential stimulation. This type of stimulation involves delivering pulses to many adjacent muscle heads/compartments within a synergistic group or within a muscle in a sequential manner so that they can share the work of maintaining a contraction. This is done through the use of multiple active electrodes to target multiple sites. The advantage of sequential stimulation in reducing muscle fatigue was first reported in animal studies. Sequential stimulation using an electrode array was demonstrated on the medial gastrocnemius muscle of a cat such that when the ventral roots corresponding to the muscle were divided into three parts, each caused almost the same strength of contraction [48] [49]. Another study applied sequential stimulation on multiple motor points of the long head of triceps I white rabbits [50]. In another study conducted on dogs, different parts of the gracilis muscles were stimulated in a sequential manner to compare effects on muscle fatigability and muscle blood perfusion with conventional whole muscle stimulation [51]. One of the first applications in human participants was by Pournezam et al. [52], who investigated periodic shifting of stimulation from one muscle to another, allowing each muscle to rest. The application was on the superficial knee extensors (vastus lateralis VL, vastus medialis VM, and rectus femoris RF) of SCI participant while the task was to sustain knee extension. Results showed that fatigue occurred at 9 minutes with sequential stimulation, while it occurred at about 1 minute with conventional stimulation. Reduced fatigue, prolonged muscle endurance, and improved muscle recovery time was reported under sequential stimulation Sequential Multiple Muscle-Head Stimulation, SMHS Malesevic et al. [11] demonstrated fatigue reduction in SCI participants using sequential stimulation of knee extensor muscles through four active surface electrodes distributed on the quadriceps. First protocol involved a large electrode delivering pulses at high pulse rate (HPR) of 30 Hz, and other involved four smaller electrodes at low pulse rate (LPR) of 16 Hz. Although HPR produced more force (due to higher frequency), LPR resulted in less fatigue, as the time

30 16 taken for torque reduction to 70% of maximum initial value in the sequential protocol was larger. Subsequently, another study carried out by the same group involved a similar experiment with similar protocols except HPR was at 40 Hz and LPR at 10 Hz [12]. Here, the finger flexion due to forearm muscle was analyzed. It was determined that the proposed surface-distributed lowfrequency asynchronous stimulation (sdlfas) doubled the time interval before onset of fatigue compared with conventional sequential stimulation. The conclusion was to combine multisurface electrodes with sdlfas to improve FES applications. Decker et al. [18] demonstrated that a stimulation protocol that alternates between muscle groups results in higher performance in FES induced cycling. The study involved 12 SCI individuals in which two types of protocols were tested to maximize the muscle performance. The alternating stimulation protocol which involved alternately stimulating the rectus femoris, vastus medialis, and vastus lateralis muscles resulted in longer ride times, longer distances traveled and also used lower stimulation intensity than the standard coactivation protocol. This was not an application of completely sequential stimulation because whole trains of pulses were first delivered to rectus femoris and then together to vastus lateralis and vastus medialis, instead of individual pulses being sequenced between the three muscle groups. However, it is still interesting to note the use of multiple electrodes to target multiple sites. The two groups mentioned above apply sequential stimulation using electrodes that are spaced far apart (Figure 5 [11]). Each of the four active electrodes (cathodes) targets one motor point on a muscle or muscle head of separate synergists rather than allowing different and potentially overlapping sets of motor units to be activated within a muscle or muscle head [9]. Thus, this method is applicable only for joint functions that can be realized by a large group of synergistic muscles, such as the knee extensors or flexors. In this thesis, this method of stimulation is termed as SMHS, i.e., sequential multiple muscle-head stimulation, as defined above, since it involves sequential stimulation by placing electrodes on individual muscle heads.

31 17 Figure 5: Comparison of electrode placements of SMHS and SES. SMHS utilizes four cathodes placed far apart over distinct motor points. Anode is the same in both the cases [11] Spatially Distributed Sequential Stimulation, SDSS Our lab proposed a novel method to reduce muscle fatigue by distributing the center of electrical field over a wider area within a single stimulation site using an array of electrodes (Figure 6) [7] [8] [9]. This method is unique in that it uses multiple surface electrodes arranged in an array that is placed at the exact same location and covers the same area as that used in conventional SES. Instead of targeting multiple motor points, only one or two motor points are targeted just like in the case with SES. This method of stimulation has been termed SDSS, i.e., spatially distributed sequential stimulation, as defined above. In a clinical setting, SDSS is advantageous to SMHS because there is no need to find individual motor points on which to place separate electrodes. Instead, only a single array electrode is placed at the conventional location on the muscle of interest. Moreover, SDSS is applicable in a greater variety of muscles than SMHS, where the latter is limited to large group of synergistic muscles.

32 18 Figure 6: Comparison of electrode placements of SDSS and SES. SDSS utilizes four cathodes placed in a 4x4 matrix at the same location as SES. Anode in both cases is the same [7] [8]. In our first study, FES was applied to the triceps surae muscle of one individual with complete SCI [7]. The study involved comparing fatigue index, fatigue-time, and torque-time integral, all as measures of fatigue, between SES and SDSS. The four active surface electrodes in SDSS were collocated at the same site and over the same area as during SES. Stimulation was applied to each of the multiple active electrodes with phase shift of 90 degrees. The 90 degree phase shift results in the individual 4 electrodes in the 2x2 matrix to have a frequency one-fourth of that of the single electrode used in SES. Figure 7 shows the difference between stimulation pulses delivered during SES and SDSS. In the study, sustained isometric contractions were carried out and all three measures of torque were greater for SDSS, indicating less fatigue and providing a first step in using multi-surface 2x2 electrodes to counter fatigue.

33 19 Figure 7: Stimulation pulses delivered during SES (top) and SDSS (bottom); 90 degree phase shift in SDSS results in each electrode in the 2x2 matrix to have one-fourth the frequency of the single SES electrode. In the second study, fatigue-reducing capability of SDSS was studied in more detail focusing on the muscle contractile properties. SDSS was delivered to plantarflexors of 15 able-bodied individuals to induce individual isometric contractions [8]. To capture muscle fatigue and chance of contractile properties, several measures, including peak torque, fatigue index, torque-time integral, torque rise time, rate of torque development, half-relaxation time, rate of torque relaxation, and power spectrum density were determined. The results demonstrated negligible torque decay during SDSS in contrast to SES. In the third study SDSS was assessed for its ability to reduce muscle fatigue compared to SES in both 11 able-bodied and 17 SCI individuals [9]. Both protocols were applied to major muscles of the lower limbs, including knee extensors and flexors, plantarflexors, and dorsiflexors while isometric ankle torque was measured. SDSS showed greater fatigue reducing ability as measured by fatigue parameters in all muscles except knee flexors of able-bodied individuals, and in all muscles of SCI individuals. Downey et al. recently demonstrated that low frequency asynchronous stimulation offers the most fatigue benefits as opposed to high frequency synchronous stimulation [53]. The term asynchronous is equivalent to sequential stimulation and synchronous is equivalent to SES. Four stimulation protocols were tested (asynchronous-8hz, asynchronous-16hz, synchronous-

34 20 32Hz S32, and synchronous-64 Hz) on 4 able-bodied and 4 SCI participants to generate isometric contractions of the quadriceps. The asynchronous electrode positioning matched that of SDSS in that four cathodes were placed in the same position as the single cathode in SES. Fatigue index and fatigue time for asynchronous-8hz were found to be significantly higher than the other three in both able-bodied and SCI participants. However, the four stimulation protocols were tested on the same day, which may result in a fatigue layover effect from one protocol to the next, despite randomization. In a subsequent study, Downey et al. studied the force ripple, which occurs at low frequency asynchronous stimulation [54]. After testing few different frequencies it was found that conventional 40 Hz and asynchronous 16 Hz induced contractions as smooth as volitional ones. Asynchronous 8, 10, and 12 Hz protocols resulted in significant ripple. The mechanism of SDSS remains unclear. In our second study [8], the mechanisms of SDSS were studied using array arranged EMG by placing electrodes in distinct locations of soleus muscle to show that during SDSS different compartments of muscle are activated alternatively by different electrodes. It was found that EMG amplitude was different depending on the location of active electrode within the array-arranged active electrode in SDSS, suggesting that different compartments within soleus are activated by different active electrode in SDSS. This result visually represents the hypothetical mechanism of SDSS with a comparison to SES. In SES, a smaller number of muscle fibers are activated continuously with a high frequency. In SDSS, a larger number of muscle fibers are activated alternatively with a low frequency (Figure 8).

35 21 Figure 8: Difference in mechanism between SES and SDSS. Larger number of muscle fibers recruited and lower stimulation frequency result in less fatigue in SDSS than SES. In summary, FES delivered using multi-surface electrodes has been pursued by different groups and has been applied in two different forms: 1. Sequential multiple muscle-head stimulation, SMHS 2. Spatially distributed sequential stimulation, SDSS As described above, both methods in their respective studies have been shown to perform better in reducing fatigue than SES. However, a single study that compares the two methods against each other has not been done. Also, all the studies applied sequential FES techniques to isometric contractions, which are not often used in clinical settings Considerations for Type of Contraction Two types of contractions carried out in this study are isometric and isokinetic. Isometric contractions are characterized by a constant muscle length with changing tension and expenditure of energy [55]. The velocity of contraction is zero. Isokinetic contractions are characterized by a constant expenditure of energy (velocity) with changing muscle length and tension [55]. Velocity of the contraction remains constant and they are typically done using an isokinetic dynamometer.

36 22 During electrical stimulation under isometric condition, the current is delivered to mostly the same muscle fibers throughout the contraction. These would be the fibers innervating the nerves at neuromuscular junctions located directly underneath the electrode. However, during isokinetic condition there is a shift of neuromuscular junctions through the course of the contraction due to muscle shortening. The neuromuscular junctions that started directly underneath the electrode in the beginning would have changed position and other neuromuscular junctions would occupy that position at the end. Thus, different muscle fibers would be stimulated through the contraction when there is movement involved in isokinetic condition. This would affect the degree of fatigue induced in isokinetic contractions. The movement of neuromuscular junctions beneath the electrodes would likely result in less fatigue because new fibers would be stimulated during the course of contraction. The moving fibers would have more time to recover from fatigue as compared to if they were in the same position in isometric condition. 2.4 Requirement of Novel Tool for Multi-Electrode FES One of the problems of multi-electrode sequential FES is associated with the hardware used to deliver stimulation. In previous studies, four channel stimulators have been used to deliver sequential stimulation to four electrodes. Two stimulators commonly encountered were the UNA-FET 4-channel stimulator [10] [11] [12] and Compex Motion stimulator [7] [8] [9], both of which were current-regulated. It is inconvenient to use a four-channel stimulator for sequential stimulation for a stimulation site due to the large amount of wiring involved. The stimulator would also require a complicated sequential FES program that would instruct it to deliver pulses to the four electrodes one at a time, thus increasing the chances of error. Many stimulators do not even have the capability to deliver sequential stimulation due to shortcomings in the software. A new FES system called Intelligent Functional Electrical Stimulation (INTFES) was developed to deliver asynchronous activation of synergistic muscles using multi-pad electrodes [56]. This system included two output active electrodes with 16 pads on each. It existed as a stimulator unit on its own that was restricted to two electrodes and could not be attached to pre-existing stimulators. A better solution would be a flexible adapter-like device that could be attached to any stimulator and would convert the single input pulses into sequentially distributed pulses.

37 SDSS Adapter A novel device was proposed to address the issue of flexibility. It is called the SDSS adapter and it is on-going in a collaboration with Drs. Keller and Mr. Bijelic at Tecnalia R&I Spain ( The design of the electric circuit was completed, and several prototypes were manufactured at Tecnalia. The first generation device was finalized in March 2014, while the second generation device was released in March The steps of testing the adapter were performed on the second generation device. The SDSS adapter was developed to accept one stimulation input from a single channel and send out four outputs to four different electrodes. The single input pulses would be sequentially distributed on the four electrodes. More importantly, it could be easily attached to and removed from the single channel of any stimulator, thus converting a conventional SES system to a sequential multi-electrode system. Figure 9 displays schematics of the electrode configuration used during conventional SES, sequential stimulation without SDSS adapter, and sequential stimulation with SDSS adapter. Figure 9: Schematic of SDSS adapter tool for distributing electrical stimulation signal. Top: single channel to single electrode in standard SES; Middle: four channels to four electrodes in sequential stimulation without adapter; Bottom: single channel to four electrodes in sequential stimulation with adapter.

38 24 The adapter consists of two modules: 1. Pulse Detection Module: detects the arrival of the new stimulation pulse and sends a signal to the Switching Circuit Module indicating that the pulse has been delivered. 2. Switching Circuit Module: receives information from first module and delivers a stimulation pulse to another electrode of the 2x2 matrix after a predetermined time period (500 μs). Table 2 shows the range of specific parameters that constrain the SDSS adapter. Table 2: Range of specific parameters constraining the SDSS adapter. Parameter Range Stimulation frequency 1 to 150 Hz Current amplitude 1 to 125 ma Stimulation voltage 1 to 250 V Pulse duration 50 to 1000 μs Pulse rise time 20 ns to 10 μs In addition, both symmetric and asymmetric bipolar pulses can be delivered with the adapter. The components comprising the adaptor and a brief description of each is given below: i. Pulse detection circuit transforms input current pulses into digital pulses sent to microcontroller. ii. Digital microcontroller performs all logical operations including control of switching circuit and LED indicator. iii. High voltage switching circuit routs stimulation pulses coming from stimulator to electrodes based on controller inputs. iv. Connectors single jack connected to input stimulator and four jacks connected to the four electrodes. v. LED indicator signals powering ON of the device. During stimulation pulses it flashes with f/8 of input frequency. vi. Battery replaceable 200 mah coin battery powers the device. The schematic diagram in Figure 10 displays various components and path of the stimulation pulse (red).

39 25 Figure 10: Schematic diagram of the various components of the SDSS adaptor unit. Red arrows: path that the stimulation pulse travels; Black arrows: components under influence of controller; Green arrow: light signal to LED. The adapter is connected to single stimulation channel via standard 2 mm jack, which is used in majority of stimulators. Exit pulses are routed to electrodes again via four 2 mm jacks. The adapter is powered by replaceable coin battery. Battery insertion and removal requires taking off the top cover of the device. Figure 11 gives visual representation of SDSS adapter. Figure 11: SDSS adapter with single input from stimulator and four outputs to electrodes attached to the forearm.

40 Clinical Application While significant advancement has been made in the field of FES therapy in the laboratory, efforts should be made to translate these techniques to the clinic. Development of the SDSS adapter is one step towards achieving this goal. The adapter makes the use of sequential FES significantly more convenient for both the care giver and receiver. The adapter introduces three advantages over the conventional four channel stimulator approach to deliver sequential FES: 1. It reduces the amount of wiring to arguably one quarter of that used with a four channel stimulator by using only one channel. 2. It simplifies programming of the stimulator thus making it more accessible to someone with a novice background. 3. It introduces a highly desired flexibility to convert any conventional FES system that delivers SES, to a multi-electrode system that can deliver SDSS, SMHS or any other type of sequential stimulation.

41 27 Chapter 3 3 Purposes of Research 3.1 Research Objective Researchers from our lab successfully showed the feasibility of SDSS in reducing muscle fatigue during FES, while other researchers showed similar effect for SMHS. The settings for applying SDSS and SMHS were complicated. To help with this, researchers from our lab developed the SDSS adapter. However, the device has not yet been tested. Both SDSS and SMHS were previously tested only under isometric condition. Isometric condition is rarely used in clinical settings, while dynamic joint movements including isokinetic condition are more often used. As mentioned above in Section 2.3.6, the contraction condition can affect the resultant fatigue, and the fatigue reduction by SDSS or SMHS in isokinetic condition may not be as good as one in isometric condition. Therefore, the effect of SDSS or SMHS should be tested in isokinetic condition to transfer this technique into clinical settings. Furthermore, SDSS and SHMS were proposed by different groups and have never been compared. Therefore, the objective of this thesis project was to take the required next steps to transfer these methods into clinical settings. The required next steps were (1) to complete the development of SDSS adapter, and (2) to test SDSS and SMHS in isokinetic condition and in the same environment. 3.2 Research Purposes Purpose 1 The first purpose of this thesis project was to test the adapter s performance. This included testing whether it performs within the specifications as outlined by the manufacturer, whether it indeed delivers pulses in a sequential manner, and whether characteristics of sequential pulses

42 28 delivered with the adapter are the same as those delivered using four channels of the source electrical stimulator alone Purpose 2 The second purpose was to investigate fatigue reducing capability of SDSS and SMHS when compared to SES under isokinetic condition. Isometric condition was also tested to ensure that previous findings were repeatable. Knee extension was selected as the target joint function, since it has been shown that SDSS has the largest effect of fatigue reduction in this joint function and SMHS has also been tested for this joint function Hypothesis It was hypothesized that both SDSS and SMHS would induce less muscle fatigue compared to SES. The sequential nature of the two types of stimulation will allow muscle fibers to rest in between and regain their force generating capacity. It was also hypothesized that SMHS would be more efficient than SDSS because it would activate more muscle fibers in quadriceps muscle groups than SDSS.

43 29 Chapter 4 4 Methodology Two separate studies were carried out through the course of this experiment. The first study, Study 1, involved testing the SDSS adapter for performance verification. The second study, Study 2, explored the fatigue reducing capability of multi-electrode FES, such as SDSS and SMHS, when compared to SES during knee extension contractions for both isometric and isokinetic condition. 4.1 Apparatus Compex Stimulator A programmable four channel FES stimulator (Compex Motion, Compex SA, Switzerland) was used to apply FES [57]. This stimulator was accompanied with chip cards that could be programmed through appropriate software on a PC, and inserted in the stimulator to deliver various types of stimulation protocols. The stimulator would be connected to PC via serial port. A push button was connected to the stimulator and used as a START/STOP button to start and stop the currently installed protocol. Four electrode wires protruded out of the four channels of the stimulator to be connected to externally supplied stimulation electrodes. The stimulator could apply stimulation with a pulse frequency between 0 and 100 Hz, duration between 0 and μs, and amplitude between 1 and 125 ma. Compex Motion was a current regulated stimulator. Compared to voltage-regulated stimulators, current-regulated ones generate more consistent and repeatable muscle contractions by delivering a constant charge, regardless of the variable impedance between the electrodes and motor neurons [28]. A graphic of the Compex Motion can be seen in Figure 12.

44 30 Figure 12: Compex Motion stimulator with accompanying programmable cards [57] SDSS Adapter Information on the SDSS adapter is given in Section Stimulation Electrodes Axelgaard ValuTrode Cloth self-adhesive surface electrodes with MultiStick Hydrogel were used to deliver electrical stimulation [58]. One set of electrodes was assigned to each participant and was used during their three visits. Each set consisted of two large electrodes (13 cm x 7.5 cm) and four small electrodes (6.5 cm x 3.75 cm). The small electrode was one quarter the area of large electrode so that, when combined, the four small electrodes covered the same area as one large electrode. These electrode sets were used to form active and reference electrode configurations for SES, SDSS, and SMHS. Active electrode was attached at the proximal end and the reference electrode at the distal end of the stimulation site. Reference electrode in all cases was one large electrode. Active electrode in SES was one large electrode and in SDSS and SMHS were four small electrodes Data Acquisition System A 16 channel data acquisition system (PowerLab /30 Series, ADInstruments, Colorado Springs) was used to collect all analog signals with appropriate software (LabChart, ADInstruments, Colorado Springs) in a laptop computer (Figure 13).

45 31 Figure 13: Graphic of PowerLab /30 Series data acquisition system [59] Joint Dynamometer An elecromechanical dynamometer (Biodex System 3, Biodex Medical Systems, USA) was used to measure the leg movement as well as the exerted torque [60]. The electromechanical dynamometer consisted of the rotating dynamometer and torque transducer, positioning chair, controller, and dynamometer attachments. Only the left and right knee extension attachment was used for this experiment. A graphic of the Biodex System 3 can be seen in Figure 14. Figure 14: Graphic of Biodex System 3 with dynamometer, chair, controller, and knee extension attachment [60]. 4.2 Study 1: Testing of SDSS Adapter Data Collection The input port of SDSS adapter was connected to the source electrical stimulator (i.e., Compex Motion II), and each output port of SDSS adapter was connected to the data acquisition system

46 32 via a custom made cable with a resistance, which converted the output current signal into the measured voltage signal. The measured signal was converted to the current output afterward using the used resistance value. In the same way, the output of source electrical stimulator was also measured. The signal was collected using a sampling rate of 100 khz. Performance of the SDSS adapter was assessed across the pulse frequency, duration, and amplitude. Output from all the four channels of SDSS adapter was compared against Compex stimulator for eight frequencies (10, 20, 30, 40, 50, 60, 70, and 80 Hz), five durations (100, 150, 200, 250, and 300 μs), and five amplitudes (25, 50, 75, 100, and 125 ma). To account for eight frequencies, five durations, and five amplitudes, 200 unique recordings were made (8 x 5 x 5 = 200). There were 25 recordings for each frequency, and 40 recordings for each duration and amplitude Statistical Analysis Two tailed paired t-tests were run between measurements from SDSS adapter and those from Compex for frequency, duration, and amplitude. P values with 95% confidence intervals were reported to signify whether the measurements between adapter and Compex were significantly different. 4.3 Study 2: Fatigue Reduction using Multi-Electrode FES Participants Thirteen healthy volunteers between the ages of 18 and 60 participated in this study, which included 10 males and 3 females. Several methods were employed to recruit the maximum number of participants. Recruitment posters were put up around the REL and Lyndhurst Centre with tearable contact information slips. s were sent out to students and staff of the Neural Engineering and Therapeutics team working at Lyndhurst Centre. Close friends and relatives were asked to volunteer for the experiment. Prior to starting the study an ethics application was cleared from University Health

47 33 Network s Research Ethics Board. All participants were asked to sign a Consent Form prior to participating. A copy of this form can be seen in Figure 32 through Figure 35 in Appendix A. Participants were asked to come to the lab on 3 separate occasions, where each visit would last 90 minutes. The visits were separated by a time period of at least 72 hours and at the most 7 days. During the time between visits and 72 hours prior to first visit, participants were asked to refrain from strenuous exercise of the lower body, especially strength training. Table 3 displays demographical information on the participants of this study, including age, gender, height, and weight. Age of the participants ranged from 18 to 53 years. Table 3: Demographical information on the 13 participants. Participant Age (years) Gender Height (cm) Weight (kg) 1 47 Male Male Female Male Male Male Male Female Female Male Male Male Male Target Muscle Group Knee extensors were chosen as the target muscle group. This was because it was shown that SDSS has the largest effect of fatigue reduction in this joint function and also SMHS has been tested for this joint function. Furthermore, from clinical perspectives, the reason of this selection was the large degree of FES based rehabilitation practices centered on cycling and rowing. This study aims to improve the outcomes of FES cycling and rowing by reducing fatigue in the quadriceps and prolonging rehabilitation. Knee extensors include vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) [55]. Anatomical drawing of quadriceps femoris is shown in Figure 15 [61].

48 34 Figure 15: Anatomy of quadriceps femoris muscle. Rectus femoris (green), vastus lateralis (red), and vastus medialis (blue) [61] Electrode Placement FES electrodes placement on the quadriceps under the three modes of stimulation are shown in Figure 16 a). Active electrodes are shown in black, and return electrodes in gray. The active electrodes in SES and SDSS cover the motor points of RF and proximal VL [7] [8] [9], while those in SMHS cover motor points of RF, VM, and proximal and distal VL [10] [11] [12]. Position of reference electrode is the same in all the three configurations. Figure 16 b) shows the pulse timing for the three modes of stimulation.

49 35 Figure 16: A) Schematic representation of FES electrode placement during SES, SMHS, and SDSS. B) Schematic representation of pulse timing during FES at 40 Hz. Note that during SDSS and SMHS, each electrode is stimulated at 10 Hz (100 ms inter-stimulus interval), resulting in FES at a compound 40 Hz when considering all 4 electrodes together. Pulse intensity (PI) and pulse duration (PD) are shown for illustrative purposes only and are not drawn to the scale Dynamometer Setting Participant was asked to sit comfortably and all the way back in the chair. Hip strap was attached to keep the participant s legs still. The shaft of the dynamometer was lined up square with the lateral portion of the knee cap by adjusting several settings of the chair (position, height, rotation, forward/back, seatback angle), and dynamometer (position, height, tilt, rotation). The knee attachment length was adjusted so the ankle rested firmly on the backrest with heel sticking out from bottom. Physical settings of the chair, dynamometer, and attachment length were adjusted during participant s first visit to achieve comfortable knee extensions, and were noted down on the Data Collection Sheet (sample given in Figure 36 and Figure 37 in the Appendix B) so they could be kept the same during future visits. Seatback angle was set at 120 degrees. During isometric mode, knee angle was set to 85 degrees (5 degrees knee extension) and rotation was locked. During isokinetic mode starting knee angle was set to 85 degrees and rotating speed to 180 degrees/second. This duty cycle was chosen because it is in accordance with the duty cycle typically used during FES cycling applications [18]. An 85 degree range of motion was

50 36 decided so that movement of knee was restricted between 85 degrees and 0 degrees (full extension). It should be noted that the leg did not move through the entire range of motion, as the stimulation ON time during each train was not high enough. The torque, velocity, and position transducer wires on the dynamometer were connected to the data acquisition system. The electrical dynamometer outputs including joint angle and joint torque were measured using the data acquisition system with a sampling frequency of 10 khz Experimental Protocol Study Design Each participant participated in 3 days of the experiment where, on each day, one out of the three protocols (SES, SDSS, SMHS) were tested. The order of testing was randomized. Table 4 shows the order of testing for each participant. Both legs of the participant were tested during each visit where one was isometric and other isokinetic, e.g., when left leg was isometric, right leg was isokinetic. The contraction mode assigned to the leg was kept the same for all the three visits. The order of a leg to be tested first was also randomized amongst the participants to avoid order effects, and was kept the same during all three visits. It was also ensured that during any given visit, the same configuration was not tested on both legs. Careful analysis of Table 4 reveals that at least 12 participants would be required to achieve a complete randomization across all parameters. This was achieved successfully as 13 individuals participated in the study. During recruitment participants were assigned a number from 1 to 12 using a random number generator. This was continued until all numbers from 1 to 12 were used. The last participant was assigned the number 13. Both modes were tested on each participant by assigning one leg as isometric and the other as isokinetic. This was randomized between left and right legs among participants. In between the three visits, a minimum rest period of 72 hours was required to ensure all fatigue occurring from the previous visit had subsided. It has been determined that at least 72 hours of rest is necessary in order to subside fatigue resulting from FES [53]. At the same time, the rest between visits would not go over 7 days to ensure no major metabolic changes occur in the quadriceps. Table 5 displays summary of the visits, rest periods, and time taken to complete.

51 37 Table 4: Design of the fatigue study. Person Mode Leg Day 1 Day 2 Day 3 Left Right tested first Left Right Left Right Left Right 1 Isometric Isokinetic Left SES SDSS SMHS SES SDSS SMHS 2 Isokinetic Isometric Left SDSS SMHS SES SDSS SMHS SES 3 Isometric Isokinetic Right SMHS SES SDSS SMHS SES SDSS 4 Isokinetic Isometric Right SES SDSS SMHS SES SDSS SMHS 5 Isometric Isokinetic Left SDSS SMHS SES SDSS SMHS SES 6 Isokinetic Isometric Left SMHS SES SDSS SMHS SES SDSS 7 Isometric Isokinetic Right SES SDSS SMHS SES SDSS SMHS 8 Isokinetic Isometric Right SDSS SMHS SES SDSS SMHS SES 9 Isometric Isokinetic Left SMHS SES SDSS SMHS SES SDSS 10 Isokinetic Isometric Left SES SDSS SMHS SES SDSS SMHS 11 Isometric Isokinetic Right SDSS SMHS SES SDSS SMHS SES 12 Isokinetic Isometric Right SMHS SES SDSS SMHS SES SDSS 13 Isometric Isokinetic Left SES SDSS SMHS SES SDSS SMHS Table 5: Visits, rest between visits, and the time required. Day 1 90 min Rest At least 72 hours; at most 7 days Day 2 90 min Rest At least 72 hours; at most 7 days Day 3 90 min Total (during visits) 270 min or 4.5 hours Protocol The entire protocol consisted of five steps: 1) setup, 2) electrode preparation, 3) warm-up and familiarization, 4) measurements, and 5) clean-up Electrode Preparation The participant s thigh was cleaned with isopropyl alcohol wipes and allowed to dry. Electrodes were placed on the appropriate positions on the thigh depending on the stimulation protocol being tested (Figure 17). Reference electrode was always placed at lower portion of the thigh above the knee cap. For SES and SDSS the active electrodes were placed in the estimated standard location where the motor points of RF and VL-proximal meet. For SMHS, a testing

52 38 electrode was used to find location of the four motor points (RF, VL-proximal, VM, VL-distal) by delivering pulses at a low frequency (10 Hz) and moving the electrode over the approximate location to check for muscle twitches. Locations with the highest degree of twitch (i.e., motor points) were marked down and active electrodes were placed over them. Electrode gel was applied to all electrodes before applying on the skin for better conductivity. Excess gel collected at electrode edges was cleaned with paper towel. Boundaries of the electrodes were marked with permanent marker to ensure position of the electrodes would remain the same during subsequent visits. Location of reference electrodes was kept the same, and location of SES and SDSS active electrodes was also kept the same. At the end of the visit participants were given their own marker to take home and asked to darken the lines periodically. a) SDSS electrode positioning b) SMHS electrode positioning Figure 17: Electrode positioning during a) SDSS and b) SMHS. Both protocols use four active electrodes and one reference electrode. Position of the four active electrodes is different while that of single reference electrode is the same Warm-up and Familiarization Biodex was set up and participant was seated with ankle placed in the knee attachment. After selecting appropriate contraction condition (isometric/isokinetic), participant was asked to carry out voluntary knee extension contractions to get familiarized with the force production and movement. Signal recorded in LabChart software in laptop was confirmed to be accurate. Participants were allowed to feel the electrical stimulation by sending test pulses. Participants were informed about the stimulator push button and comfort stop which they can press anytime to stop all stimulation and movement, respectively.

53 Measurements Three main measurements were collected during the trial: 1) Maximum voluntary contraction (MVC), 2) Low frequency fatigue (LFF), and 3) Torque readings from fatigue protocol. Table 6 displays steps taken to collect the measurements and the time taken during each step. Table 7 shows pulse frequency, duration, and amplitude delivered during LFF and Fatigue Protocol. Table 6: Order of collection of measurements and time taken to collect. MVC dynamic 1 ~ 10 sec Rest 1 min MVC static 1 ~ 10 sec Rest 1 min MVC dynamic 2 ~ 10 sec Rest 1 min MVC static 2 ~ 10 sec Rest 1 min Set LFF intensity ~ 2 min Rest 1 min Pre LFF ~ 10 sec Rest 1 min Set fatigue intensity ~ 2 min Rest 1 min Fatigue protocol 2 min Rest 1 min Post LFF 1 ~ 10 sec Rest 4 min Post LFF 2 ~ 10 sec Rest 5 min Post LFF 3 ~ 10 sec Total ~ 25 min Table 7: Stimulation parameters during LFF and Fatigue Protocol. Pulse Frequency Pulse Duration Pulse Amplitude LFF 20, 40, and 100 Hz 250 μs Variable Fatigue Protocol 40 Hz 250 μs Variable Maximum Voluntary Contraction Two types of MVCs were collected during each trial: dynamic and static. During dynamic MVC Biodex arm was set to isokinetic mode and participant was asked to extend the knee as fast as

54 40 possible. During static MVC dynamometer arm was set to isometric mode and participant was asked to sustain a contraction of maximum force for 3 seconds. A rest of 1 minute was given between MVCs to allow muscle to come to resting state. Note that the dynamic MVC was not used in the subsequent analysis and only static MVC was used to normalize torque as mentioned in Section Low Frequency Fatigue Assessment LFF assessment involved sending six trains of biphasic pulses at the following frequencies in the order mentioned: 1) 40 Hz; 2) 40 Hz; 3) 100 Hz; 4) 20 Hz; 5) 20 Hz; 6) 100 Hz. It generated six contractions of varying strength. The 100 Hz and 20 Hz readings are used for the high and low measurement, respectively, to calculate the ratio. They are delivered in a high-low-low-high manner to avoid order effects. The two 40 Hz trains allow for muscle potentiation prior to LFF assessment. Each train was delivered for 0.3 seconds ON and 0.7 seconds OFF one after another. The amplitude of the pulses was determined by carrying out a Set LFF Intensity protocol prior to the LFF assessment. This involved sending single train at 40 Hz starting at 5 ma to cause a contraction and increasing amplitude of subsequent trains (first by 10 ma, then by 5 ma) to find out the maximum tolerable intensity. 80% of the maximum tolerable intensity value was determined to be the LFF amplitude. Another train was sent at this amplitude to evaluate the torque generated. This torque was noted down as it would be used to match the torque that is to be reached for LFF assessments during visits 2 and 3 and for Fatigue protocol. All LFF assessments were delivered without using the adapter and instead using only a splitter. LFF assessment was carried out once before fatigue protocol and three times after. Fatigue Protocol Fatigue protocol involved sending 120 trains at 40 Hz with a 0.3 seconds ON and 0.7 seconds OFF pattern to induce 120 knee extension contractions. Because each train lasted 1 second, fatigue protocol lasted for 120 seconds. Pulse amplitude of trains during fatigue protocol was determined by carrying out a Set Fatigue Intensity protocol prior to the Fatigue protocol. Similar to Set LFF Intensity, single trains were sent one at a time with increasing amplitude until the torque matched the value noted down during Set LFF Intensity. The amplitude that

55 41 produced matched torque was set as the amplitude for Fatigue protocol. Fatigue protocol and Set Fatigue Intensity were delivered using the adapter during sequential stimulation. During fatigue protocol the experimenter stayed close to the participant in case they experienced severe discomfort and would need to stop the stimulation. In such a case the amplitude was lowered until it could be more tolerable Clean-up After collecting all measurements, the apparatus was turned off and electrodes removed from participant s legs. Paper towels were used to wipe clean any remaining gel Data Analysis All analyses were performed in a commercial numerical computing environment (MATLAB, The MathWorks, Inc., USA) Data Conversion Torque readings from the torque transducer were recorded in the LabChart software as units of magnitude or volts (V). Torque data were converted to Newton-meters (Nm) using a previously determined empirical relationship of the electrical dynamometer between magnitude (V) and torque (Nm). The factor of 144 Nm/V, determined empirically, allowed this conversion. Torque = 144 Nm V Magnitude Joint Torque Normalization The two dynamic MVC readings were deemed inconsistent during each visit as well as between visits of the same participant, and therefore were not used for subsequent analysis. Static MVC readings were used to convert torque into a secondary unit of percent-mvc. Each torque value was divided by the larger of the two static MVCs. In this way the torque data were normalized to the MVC value. Normalizing torque to MVC allowed us to examine how forceful the contractions were for each participant. Joint torque data was also normalized using the initial

56 42 torque. Note that these normalizations were used only for visualizing torque-time profile in Section 5.2.3, and did not affect Fatigue Measures as they are indexes Low Frequency Fatigue Average of the two high measurements and average of the two low measurements were used to calculate the LFF ratio. LFF ratio was calculated pre-stimulation and post-stimulation. A decrease in the LFF ratio from pre- to post-fatigue indicates the presence of LFF. The larger the decrease, the greater the presence of LFF [35]. To quantify the decrease, two measures of change in the LFF ratio, LFFdiff1 and LFFdiff10, were calculated by LFFdiff1 = Post 1 min Pre 1 min LFFdiff10 = Post 10 min Pre 1 min. These measures were compared between SES, SDSS, and SMHS Fatigue Measures Two fatigue measures were calculated to evaluate the degree of fatigue between the three types of stimulation. These measures are similar to those employed by Sayenko et al. [9]. 1. Fatigue Index (FI) was calculated as the ratio between the mean peak torque values of last 10 stimulus trains and those of initial 10 trains. FI = average of last 10 torques average of first 10 torques 2. Torque Peak Mean (TPM) was calculated as the mean of peak torques throughout the whole bout of fatiguing stimulation, normalized to mean peak torque values of the initial 10 stimulus trains. TPM = average of 120 torques average of first 10 torques

57 Statistical Methods The normality of each measure was tested using Kolmogorov-Smirnov tests, which confirmed normal distribution in each sample group. One way repeated measures ANOVAs and post-hoc Bonferroni tests were performed to compare SES, SDSS, and SMHS on initial torques, fatigue index, torque peak mean, and change of LFF ratio between SES, SDSS, and SMHS. P-value of <0.05 was used to show the statistical significance.

58 44 Chapter 5 5 Results 5.1 Study 1: Testing of SDSS Adapter Characterization of Pulses Sample biphasic pulses delivered by the stimulator (Figure 18 a)) and four channels of the adapter (Figure 18 b)-e)) are shown. These pulses were delivered by setting frequency at 40 Hz, duration at 250 µs, and amplitude at 50 ma, which is the same level of the parameters at which the fatigue protocol was delivered. It can be seen that pulses delivered by the adapter are indeed delivered in a sequential manner to the four channels, where the frequency at each channel is one quarter of that at the stimulator (in this case it would be 10 Hz). Although pulses at only one level of parameters are shown, the sequential nature of the adapter was found to be valid at all parameter levels. Shape of the pulses can be better visualized in Figure 19 and Figure 20. Figure 19 a) and b) display one sample pulse delivered by stimulator and first channel of adapter, respectively, at lowest most level of parameters (ie, 10 Hz, 100 µs, 25 ma). Figure 20 a) and b) display the same pulse delivered by stimulator and first channel of adapter, respectively, at highest most level of parameters (ie, 80 Hz, 300 µs, 125 ma). In case of both extremes of parameter level, it can be seen that the pulse delivered by stimulator and adapter is characteristically similar.

59 45 a) Stimulator (40 Hz, 250 μs, 50 ma) 10 ma 10 ms b) Adapter channel 1 c) Adapter channel 2 d) Adapter channel 3 e) Adapter channel 4 Figure 18: Sample biphasic pulses delivered by a) stimulator and b) channel 1, c) channel 2, d) channel 3, and e) channel 4 of the adapter at reasonable level of parameters (ie, 40 Hz, 250 µs, and 50 ma). Pulses are delivered sequentially by the adapter at one quarter the frequency of the stimulator.

60 46 a) Stimulator (10 Hz, 100 μs, 25 ma) a) Stimulator (80 Hz, 300 μs, 125 ma) 0.1 ms 5 ma 0.3 ms 25 ma b) Adapter channel 1 b) Adapter channel 1 Figure 19: One sample pulse delivered by a) stimulator and b) channel 1 of adapter at lowest most level of parameters (ie, 10 Hz, 100 µs, and 25 ma). Pulse delivered by stimulator and adapter is characteristically similar. Figure 20: One sample pulse delivered by a) stimulator and b) channel 1 of adapter at highest most level of parameters (ie, 80 Hz, 300 µs, and 125 ma). Pulse delivered by stimulator and adapter is characteristically similar Similarity between the Adapter Outputs and the Source Stimulator Outputs Figure 21 displays pulse frequency recorded from the adapter plotted as a function of frequency recorded from the source stimulator across eight target frequencies (10 to 80 Hz, increments of 10 Hz). The four plots in Figure 21 a)-d) represent channels 1-4 of the adapter. The plots contain eight clusters where each cluster contains 25 points that were recorded at the same pulse frequency but for various durations and amplitudes. Each point is the average of 10 pulses with the same stimulation conditions.

61 Adapter Ch 1 Freq (Hz) 47 a) Channel 1 b) Channel y = x R² = Stimulator Freq (Hz) c) Channel 3 d) Channel y = x R² = y = x R² = y = x R² = Figure 21: Frequency of pulses recorded from SDSS adapter versus Compex stimulator with target frequencies of 10, 20, 30, 40, 50, 60, 70, and 80 Hz across a) channel 1, b) channel 2, c) channel 3, and d) channel 4 of the adapter. Linear regression was performed on the points to produce a line of best fit. Equation of the line and R 2 value are shown on the plots. In the case when frequency delivered by adapter and stimulator is exactly equal, this regression line should have a slope of 0.25 and intercept of 0 as the frequency at each of the four channels of adapter is one quarter that delivered by the source stimulator (i.e., the pulse frequency of the source stimulator was 40 Hz while the one from a channel of the adapter was 10 Hz). Regression showed that slope was very close to 0.25, the intercept was close to 0, and R 2 was almost 1 for all channels. Figure 22 a)-d) displays similar types of plots for pulse duration (100 to 300 µs, increments of 50 µs) for channels 1-4 of adapter. Each cluster contains 40 points recorded at the same pulse duration and various frequencies and amplitudes. In the case when duration delivered by adapter and stimulator is exactly equal, the line of best fit should have a slope of 1 and intercept of 0. The plots showed that the slope was very close to 1, the intercept was close to 0, and R 2 was almost 1 for all channels.

62 Adapter Ch 1 PD (μs) 48 a) Channel 1 b) Channel y = x R² = Stimulator PD (μs) y = x R² = c) Channel 3 d) Channel y = 0.995x R² = y = x R² = Figure 22: Duration of pulses (PD) recorded from SDSS adapter versus Compex stimulator with target durations of 100, 150, 200, 250, and 300 µs across a) channel 1, b) channel 2, c) channel 3, and d) channel 4 of the adapter. Figure 23 a)-d) displays similar types of plots for pulse amplitudes (25 to 125 ma, increments of 25 ma) for channels 1-4 of adapter. Similar to pulse duration, the slope of line of best fit was very close to 1, the intercept was close to 0, and R 2 was 1 for all channels.

63 Adapter Ch 1 Amp (ma) 49 a) Channel 1 b) Channel y = x R² = y = x R² = 1 c) Channel 3 d) Channel Stimulator Amp (ma) y = x R² = y = x + 4E-05 R² = Figure 23: Amplitude of pulses recorded from SDSS adapter versus Compex stimulator with target amplitudes of 25, 50, 75, 100, and 125 ma across a) channel 1, b) channel 2, c) channel 3, and d) channel 4 of the adapter Difference between the Adapter Outputs and the Source Stimulator Outputs Mean frequency during 25 recordings across eight target pulse frequencies from 10 to 80 Hz was compared between adapter and stimulator. Frequency was found to be similar for target of 50 to 80 Hz but significantly different for target of 10 to 40 Hz (p 0.05). The root mean square error (RMSE) between four channels of adapter and stimulator across eight target frequencies ranged from to Hz (or 0.30 to 0.85 % of target frequency when calculated as relative error). Figure 24 a) displays relative error of mean RMSE as percentage of target between four channels of adapter and stimulator for the eight target frequencies. Mean duration during 40 recordings across five target pulse durations from 100 to 300 µs was compared between adapter and stimulator. Duration was found to be significantly different for all targets (p 0.05). RMSE ranged from to µs (or 0.75 to 0.47 % of target duration when calculated as relative error; Figure 24 b)).

64 RMSE (%) RMSE (%) RMSE (%) 50 Mean amplitude during 40 recordings across five target pulse amplitudes from 25 to 125 ma was compared between adapter and stimulator. Amplitude was also found to be significantly different for all targets (p 0.05). RMSE ranged from to ma (or 1.36 to 0.27 % of target amplitude when calculated as relative error; Figure 24 c)). a) Frequency b) Duration c) Amplitude 1.0% 1.0% 2.0% 0.8% 0.6% 0.4% 0.2% 0.0% Target Frequency (Hz) 0.8% 0.6% 0.4% 0.2% 0.0% Target Pulse Duration (μs) 1.5% 1.0% 0.5% 0.0% Target Amplitude (ma) Figure 24: Mean RMSE as percentage of target between four channels (n=4) of adapter and stimulator across a) eight target frequencies (10-80 Hz), b) five target durations ( µs), and c) five target amplitudes ( ma). Error bars indicate standard deviation. 5.2 Study 2: Fatigue Reduction using Multi-Electrode FES Prickly pain sensation was felt by all participants while stimulation was delivered to the muscle. This was true for all three stimulation protocols. In three cases, it was found in the middle of experiments that active electrodes were not placed directly over motor points of the muscle. In such cases motor points were found once again using the twitch method and fatigue protocol was resumed after some rest. In one case, there was a need to terminate the fatigue protocol because the participant could not tolerate the sensation. The participant was able to repeat that session on another day Participant Exclusion Results from certain participants were not used in the calculation of group fatigue measures based on two main exclusion criteria: 1. Any instance of greater than 70% decrease in torque between subsequent contractions after the maximum torque has been reached.

65 Torque (Nm) Torque (Nm) Inability to return to resting torque between subsequent contractions for more than 50% of contractions. Both of these criteria indicate inability of the participant to relax the knee extensor to a sufficient degree. The volitional contribution to induced torque would warrant these results to be irrelevant. As a result of applying these criteria, data from two participants was excluded. Thus, the results in this section refer to the data obtained from eleven out of thirteen participants Initial Torque Values During the experiment it was ascertained as best as possible that the initial torque between SES, SDSS, and SMHS was matched so that any fatigue would depend solely on the stimulation protocol and not on a high or low value of initial torque. Initial torque was compared among the stimulation protocols. Figure 25 a) and b) show the initial torque, i.e., the group average of the initial torque (calculated as the average of the peak torques of the first 10 trains) for SES, SDSS, and SMHS across eleven participants (N=11) for isometric (Figure 25 a)) and isokinetic (Figure 25 b)) conditions, respectively. There was no significant difference in the initial torque among SES, SDSS, and SMHS for isometric (ANOVA p = ) and isokinetic (ANOVA p = ) conditions. a) Isometric Initial Torque b) Isokinetic Initial Torque SES SDSS SMHS 0 SES SDSS SMHS Figure 25: Initial torque (average of first 10 contractions; n=10) values for SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants.

66 Change in Torque Torque time series is a raw plot of torque recorded by the dynamometer throughout the 120 contractions. Examples of torque profiles for SES, SDSS, and SMHS are shown in Figure 26 for a) isometric and b) isokinetic conditions, respectively. It should be noted that torque at the end of fatigue protocol is lower than in the beginning, due to fatigue. For both isometric and isokinetic conditions, SES seems to show greater decline in torque, and thus greater fatigue, than SDSS and SMHS. a) Isometric condition b) Isokinetic condition Figure 26: Example of typical a) isometric torque time series and b) isokinetic torque time series with 120 contractions for (from top to bottom) SES, SDSS, and SMHS. Figure 27 shows the group average time profile of torque reduction for a) isometric and b) isokinetic conditions. Each point indicates the group average (N=11) of 10 peaks of the exerted torque with standard deviation as the error bar, for SES, SDSS, and SMHS stimulation protocols. It can be seen that the SES torque was lower that the torque for SDSS and SMHS in both isometric and isokinetic conditions. In addition to the absolute values of peak torque, the normalized torque by MVC and by initial torque was plotted and was confirmed to be similar to Figure 27. These plots can be seen in Figure 38 and Figure 39 in Appendix C.

67 Torque (Nm) Torque (Nm) 53 a) Isometric Binned Torque reported as Nm b) Isokinetic Binned Torque reported as Nm SES SDSS SMHS SES SDSS SMHS Bin Number Bin Number Figure 27: Binned torque by 10 contractions (n=10) reported as Newton-meters (Nm) for SES, SDSS, and SMHS averaged across eleven participants (N=11) under a) isometric and b) isokinetic conditions. 12 points represent 12 bins from contraction 1 to 120. Error bars indicate standard error of the mean across eleven participants Fatigue Measures Figure 28 a) and b) show the average FI for SES, SDSS, and SMHS across eleven participants (N=11) for isometric and isokinetic conditions, respectively. ANOVAs revealed that FI was significantly different across SES, SDSS, and SMHS for isometric (ANOVA p = ) and isokinetic (ANOVA p = ) conditions. The post-hoc tests revealed that FIs of SDSS and SMHS were significantly larger than that of SES, while there was no significant difference between SDSS and SMHS for both isometric and isokinetic conditions (p-values in Figure 28).

68 Fatigue Index Fatigue Index 54 a) Isometric Fatigue Index b) Isokinetic Fatigue Index ANOVA: p= p= p= SES SDSS SMHS ANOVA: p= p= p= SES SDSS SMHS Figure 28: FI for SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants. Only significant p values (p < 0.05) for single factor ANOVA and paired twotailed t-tests with Bonferroni correction are shown. Figure 29 a) and b) show the average TPM for SES, SDSS, and SMHS across eleven participants (N=11) for isometric and isokinetic conditions, respectively. ANOVAs revealed that TPM was significantly different across SES, SDSS, and SMHS for isometric (ANOVA p = ) and isokinetic (ANOVA p = ) conditions. The post-hoc tests revealed that TPM of SDSS and SMHS were significantly larger than that of SES, while there was no significant difference between SDSS and SMHS for isometric condition (p-values in Figure 29 a)). For isokinetic condition, TPM of SDSS and SMHS were significantly larger than that of SES, and TPM of SMHS was significantly larger than that of SDSS (p-values in Figure 29 b)).

69 LFF Ratio LFF Ratio Torque Peak Mean Torque Peak Mean 55 a) Isometric Torque Peak Mean b) Isokinetic Torque Peak Mean 1.4 ANOVA: p= p= ANOVA: p= p= p= p= p= SES SDSS SMHS -0.1 SES SDSS SMHS Figure 29: TPM for SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants. Only significant p values (p < 0.05) for single factor ANOVA and paired two-tailed t-tests with Bonferroni correction are shown Low Frequency Fatigue LFF ratio was calculated one minute before start of fatiguing protocol (Pre 1 min), one minute after end of fatiguing protocol (Post 1 min), and ten minutes after end of fatiguing protocol (Post 10 min). Figure 30 a) and b) show average LFF ratios across eleven participants (N=11) for isometric and isokinetic conditions, respectively. A decrease in the LFF ratio from pre- to postfatigue indicates the presence of LFF. The larger the decrease the greater the presence of LFF [35]. In all cases LFF ratio decreased from pre- to post-fatigue. a) Isometric LFF Ratio b) Isokinetic LFF Ratio Pre 1 min Post 1 min Post 10 min Pre 1 min Post 1 min Post 10 min SES SDSS SMHS SES SDSS SMHS Figure 30: Pre- and Post-fatigue LFF ratio for SES, SDSS, and SMHS averaged across 11 participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants.

70 Change in LFF Ratio Change in LFF Ratio 56 Figure 31 a) and b) show the change in LFF ratio from Pre 1 min to Post 1 min (LFFdiff1) and Pre 1 min to Post 10 min (LFFdiff10) across eleven participants (N=11) under isometric and isokinetic conditions, respectively. ANOVA revealed that there was no difference in LFFdiff1 among SES, SDSS, and SMHS for both isometric and isokinetic conditions (p= and p= for isometric and isokinetic conditions, respectively) and no difference in LFFdiff10 for isokinetic condition (p=0.2800), while there was significant differences in LFFdiff10 for isometric (p=0.0323) condition. a) Isometric ΔLFF Ratio b) Isokinetic ΔLFF Ratio LFFdiff1 LFFdiff10 ANOVA: p= LFFdiff1 LFFdiff10 SES SDSS SMHS SES SDSS SMHS Figure 31: Change in LFF ratio from pre- to post-fatigue in SES, SDSS, and SMHS averaged across eleven participants (N=11) for a) isometric and b) isokinetic conditions. Error bars indicate standard deviation across eleven participants. Only significant p values (p < 0.05) for single factor ANOVA are shown.

71 57 Chapter 6 6 Discussions 6.1 Validity of SDSS Adapter as a Tool for Multi-Electrode FES Comparing the pulses delivered by Compex stimulator and SDSS adapter, it was determined that the adapter does indeed deliver pulses in a sequential manner at one quarter the frequency of the stimulator (Figure 18). The shape of the pulse produced by stimulator and adapter at lowest (Figure 19) and highest (Figure 20) most level of parameters is very similar. Thus, pulses delivered by stimulator and adapter are characteristically similar indicating that similar charge was being delivered by adapter and stimulator. Pulse frequency, duration, and amplitude recordings between SDSS adapter and Compex stimulator were almost equivalent. Lines fitted to scatter plot of the adapter pulse frequency versus the source stimulator pulse frequency for the four channels (Figure 21 a)-d)) resulted in the slope very close to 0.25, the intercept very close to 0, and R 2 very close to 1, as expected. Similar expected results were observed for pulse duration (Figure 22 a)-d)) and amplitude (Figure 23 a)-d)) where the slope was very close to 1, the intercept close 0, and R 2 close to 1. These findings support that SDSS adapter did not modify pulse frequency, duration and amplitude of the source stimulator much. In the case of pulse frequency, it is interesting to note that the clusters get more spread out at higher frequencies (Figure 21 a)-d)), indicating greater variability between adapter and stimulator with increases in frequency. This variability may be due to decreased accuracy in either adapter or stimulator at large frequencies. SDSS adapter is designed to deliver in the range of Hz and Compex stimulator in range of Hz. Although the highest frequency delivered (80 Hz) is well within the range of both, the effect of external errors could be magnified at higher frequencies. The increased variability was not observed to a large extent for pulse duration and amplitude. The difference of pulse frequency, duration and amplitude was compared between the SDSS adapter and the source stimulator. SDSS adapter was shown to be consistent with Compex stimulator for pulse frequencies 50 to 80 Hz but not for 10 to 40 Hz. It was not consistent for all

72 58 pulse durations (50 to 250 µs) and amplitudes (25 to 125 ma) tested. This implies that the adapter introduces a systematic bias as sequential stimulation delivered using the adapter would have had slightly different parameters than SES. However, as the differences of stimulation parameters between the adapter output and that of the source stimulator were very small, the difference would not be physiologically meaningful. For example, RMSE between adapter and stimulator across all frequencies ranged within Hz (Figure 24 a)). Although frequency is a major determinant of fatigue, a difference of the greatest RMSE of Hz would not be large enough to warrant an effect. Several studies that investigated the effect of frequency on fatigue reveal a frequency difference of at least 10 Hz is necessary to significantly affect fatigue [39] [40] [41] [42] [43] [44] [45]. For duration, a difference of the greatest RMSE of µs (Figure 24 b)) is also not large enough to warrant an effect on fatigue. It has been found that a difference of at least 100 µs is necessary to significantly affect fatigue [39] [40] [45]. Similarly for amplitude, a difference of the greatest RMSE of ma (Figure 24 c)) is not large enough to warrant an effect on fatigue. It has been found that a difference of at least 10 ma (when stimulating at the maximum delivered amplitude during fatigue protocol of 100 ma) is necessary to significantly affect fatigue [24] [43] [44] [45]. Thus, although the stimulation delivered with SDSS adapter attached was significantly different, the difference is not large enough to warrant an effect on induced fatigue during sequential stimulation. Thus, overall, the performance of SDSS adapter was verified according to purpose 1, i.e., the outputs of the adapter were very close to those of the source stimulator, and they were almost equivalent with ignorable differences. The SDSS adapter is a critical tool to transfer sequential stimulation methods such as SDSS and SMHS into clinical settings. The results of this study confirmed that the SDSS adapter can be effectively used in clinical settings. 6.2 Effectiveness of Sequential Multi-Electrode FES Initial torque The initial torque was controlled to be matched to ensure that muscle started with the same degree of fatigue (or lack thereof) in SES, SDSS, and SMHS, and any changes in fatigue were only due to the difference in stimulation protocol rather than initial fatigue level. For example, a

73 59 large initial torque would fatigue muscle quickly, resulting in less than expected torque at the end. Conversely, small initial torque would fatigue muscle slowly, resulting in more than expected torque at the end. As the results indicate, the initial torque was sufficiently matched between SES, SDSS, and SMHS (Figure 25) Change in Torque For isometric condition, the change in torque values in all three protocols resembles a sigmoidal shape (Figure 27 a)); torque changed slowly in the beginning, rapidly during the middle, and slowly at the end of the fatiguing protocol. This relates to Ca 2+ depletion within muscle [23] [62] [63]. Inorganic phosphate increases during fatigue and binds to Ca 2+, preventing its release from sarcoplasmic reticulum. During repetitive forceful contractions, concentration of Ca 2+ decreases slowly at first, taking some time for the effect to show on force production. Continuous force contractions rapidly depletes Ca 2+ and results in a strong reduction in force, explaining the steep portion of the sigmoid. The depletion of Ca 2+ eventually slows down, causing the decrease in force production to slow down at the end. However, under isokinetic condition, the sigmoidal shape is seen to a lesser degree (Figure 27 b)). This can be explained by the nature of isokinetic contraction in which there is a shifting of neuromuscular junctions through the course of contraction. During movement different muscle fibers are stimulated allowing previous fibers to recover from fatigue and replenish Ca 2+, and affecting the pattern of force production. Despite the same initial torque, the torque for SES decreased more rapidly than SDSS and SMHS for both isometric (Figure 27 a)) and isokinetic (Figure 27 b)) conditions, i.e., SES torque decreases from the start while SDSS and SMHS torque remains somewhat constant or increases during first 20 contractions. For both isometric and isokinetic conditions, FI was found to be significantly different between SES, SDSS, and SMHS, and FI for SES was found to be significantly lower than SDSS and SMHS (Figure 28). Further, TPM was found to be significantly different between SES, SDSS, and SMHS under both conditions, while TPM for SES was found to be significantly lower than SDSS and SMHS (Figure 29). It was demonstrated that SDSS and SMHS maintain the peak torques indicated by FI or the total exerted torque indicated by TPM more effectively than SES, suggesting that SDSS and SMHS are more fatigue resistant than SES. This clearly supports the hypothesis. It was previously shown that sequential stimulation such as SDSS ( [7] [8] [9] ) and SMHS ( [10] [11] [12] ) can effectively reduce muscle fatigue during FES in

74 60 isometric condition. The results of Study 2 confirmed the same evidence. Further, the results of Study 2 also confirmed that the fatigue reduction of SDSS and SMHS is effective in isokinetic condition. As dynamic conditions, such as isokinetic condition, are more often used in clinical settings, to prove the feasibility of SDSS and SMHS in a dynamic condition is an important step in transferring these sequential stimulation methods into clinical settings. SDSS ( [7] [8] [9] ) and SMHS ( [10] [11] [12] ) were separately proposed. In this study, the effects of SDSS and SMHS were directly compared. In most of the cases, both methods showed similar effects, while TPM for isokinetic condition showed that SMHS was able to reduce muscle fatigue more than SDSS (Figure 29 b)). The greater fatigue in SDSS than SMHS may be due to the greater motor unit recruitment overlap in SDSS than SMHS, which fatigues the muscle groups faster. This finding is in support of the hypothesis. Interestingly, this finding was only observed for TPM of isokinetic condition which may be due to the more sensitive nature of TPM than FI. The results suggest that SMHS is able to maintain the total exerted torque more effectively than SDSS. This can be an advantage of SMHS compared to SDSS. However, there are other advantages in SDSS compared to SMHS. These are, (1) SDSS requires simpler settings than SMHS, i.e., one multi-surface active electrode, instead of identification of motor points in multiple muscles and multiple active electrodes, and (2) SDSS has a potential to be applicable to joint movements which do not involve multiple muscle synergists. 6.3 Mechanism of Fatigue Reduction in Sequential Stimulation LFF was used to prove the underlining mechanism of fatigue reduction in SDSS and SMHS. At first, in all cases, the change in LFF ratio was negative at both Post 1 min (i.e., LFFdiff1) and Post 10 min (i.e, LFFdiff10) (Figure 31). This result indicates that the muscle fatigue during any stimulation mode was at least partially LFF, i.e., long lasting fatigue due to deficiencies in Ca 2+ release. The statistical results indicated that there was statistical difference in change of LFF ratio only in isometric condition comparing baseline LFF ratio and one at post-10 min (LFFdiff10) (Figure 31 a)). The average change of LFF ratio tended to be larger in SES compared to SDSS and SMHS. This may indicate that there was difference in the contribution of LFF to muscle fatigue in each

75 61 protocol, suggesting that fatigue related to deficiencies in Ca 2+ release was different among stimulation protocols. However, the results did not strongly support this explanation. The mechanism of SDSS ( [8] [9] ) and SMHS ( [11] [12] ) has been discussed previously, although it was not conclusive. In general, the sequential stimulation can activate different muscle fiber groups alternatively and can allow muscle fibers to rest in between pulses to replenish their force generating capacity (Figure 8). It was previously demonstrated in our lab that SDSS activates different sets of muscle fibers by each of the four electrodes [8]. That is, EMG readings taken from four electrodes placed under 4x4 cathode matrix revealed that during stimulation of the medial portion of muscle, the amplitude was larger under medial EMG electrodes and smaller under lateral EMG electrodes. Similarly, stimulation of the lateral portion of muscle resulted in a larger amplitude under lateral EMG electrodes. A similar parallel could be drawn towards SMHS where the electrodes deliver sequential stimulation to muscle fiber motor points. The result of LFF was expected to provide an additional aspect regarding the mechanism of sequential stimulation, but it was not clearly shown. Further testing with twitch EMG or mechanomyography would be required to provide for a similar mechanism for SDSS and SMHS. 6.4 Limitations Although both isometric and isokinetic contractions were carried out, a direct comparison between them was not made. This is because initial torques between the two types of contractions were not matched, even though they were matched within each type (i.e. between SES, SDSS, and SMHS). Matching of torque was not feasible because the torque necessary to induce fatigue between the two contraction types is very different. Isometric contractions require larger torques before fatiguing, and the same level of torque would not be possible under isokinetic condition due to severe pain caused by high stimulation intensity. The possibility of adjusting the duty cycle (i.e. resistance) was considered, but was not pursued and a duty cycle corresponding to the well-defined rehabilitation activity of FES cycling was used (i.e. 180 degrees/second) [18]. The advantage of doing this is to show that using sequential multielectrode FES is advantageous to conventional FES for direct FES applications, such as FES cycling.

76 Future Recommendations Since torques could not be matched between contraction types, it is recommended to do so in a different study to find out what role the type of contraction may play in inducing fatigue. The mechanism of fatigue reduction in SDSS has been studied using EMG and attributed to activation of individual muscle fibers. A similar recording of twitch EMG should be performed on SMHS to see if similar observation is evident. However, this could prove challenging since electrodes in SMHS are further apart and in distinct locations. Perhaps the usage of mechanomyography with accelerometers could prove useful in detecting selective activation of fibers. Two different multi-electrode configurations were studied in this experiment (i.e. SDSS and SMHS), both of which are useful in their own manner. Further studies on the number, shape, and location of electrodes could be carried out to recommend the best combination of electrode configuration for different muscles and types of contractions. The accuracy of proposed SDSS adapter has been tested against one type of stimulator. To label it as a generic adapter that can be attached to any stimulator, its accuracy must be tested against other commonly used stimulators as well. Future prototypes of the SDSS adapter should also be made more accurate by limiting outside artifact that may affect delivery of stimulation pulses.

77 63 Chapter 7 7 Conclusions The present study has demonstrated that multi-electrode sequential FES techniques are superior to conventional stimulation in reducing muscle fatigue for both isometric and isokinetic contractions. These techniques result in less muscle fatigue, which is a major limitation of FES. Although not significant, SMHS was found to induce less fatigue than SDSS, which can show the advantage of SMHS compared to SDSS. However, SDSS is more convenient to apply in a clinical setting. The results of LFF were not clear but show a tendency that the contribution of LFF in the induced muscle fatigue may differ between SES and sequential stimulation, suggesting that depletion of Ca 2+ may differ between these. Additionally, a novel device for convenient application of sequential FES in a clinical setting was tested. Although the device proved to be inconsistent with Compex stimulator, the difference in parameters was not large enough to significantly affect fatigue. Using the attachable novel adapter, the novel sequential stimulation methods can be conveniently applied in a clinical setting. The findings of this study could prove valuable in the application of FES for rehabilitation, therapy, and muscular exercise in individuals with upper motor neuron disorders, such as SCI. SDSS and SMHS would prolong the usage of FES before muscle fatigues, thus allowing the user to achieve a better result. Improved motor function would result in higher levels of independence for the user in daily activities. SDSS could be easily applied in clinical setting using the newly tested device with 2x2 patch electrodes to deliver fatigue resistant FES.

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86 72 Appendices A. Consent Form Figure 32: Consent form page 1.

87 Figure 33: Consent form page 2. 73

88 Figure 34: Consent form page 3. 74

89 Figure 35: Consent form page 4. 75

90 76 B. Data Collection Sheet Figure 36: Sample of Data Collection Sheet (front).