6th Annual International IEEE EMBS Conference on Neural Engineering San Diego, California, 6 - 8 November, 2013
Electrical Muscle Stimulation Increases Early Reinnervation Following Nerve Injury and Immediate Repair Michael P. Willand,Member, IEEE, Jennifer J. Zhang, Cameron D. Chiang, Gregory H. Borschel, and Tessa Gordon Abstract— Treating partially or completely denervated muscle following nerve injury using electrical muscle stimulation has been met with much controversy. Previous studies have shown that chronic electrical muscle stimulation or neuromuscular activity leads to impaired reinnervation of muscle end plates. In this study we investigated the use of a moderate stimulation paradigm delivered daily over a 2 week period and the effects on functional recovery and reinnervation. Rat gastrocnemius muscle was denervated by complete tibial nerve transection and immediately repaired using epineurial sutures. Electrical muscle stimulation was carried out 5 days per week in 1 hour sessions. Our results show that numbers of motoneurons reinnervating muscle and reinnervated endplates were significantly higher in animals that received daily muscle stimulation compared to those without stimulation. Other functional measurements such as muscle force, weight, and contractile properties were no different between groups. Our results provide evidence that the improved reinnervation may be due to antidromic depolarization of axons proximal to the repair site.
Negative results were also reported when extensively denervated muscles underwent daily chronic treadmill exercise [12]. These negative reports have led clinicians and therapists to limit or abandon the use of electrical muscle stimulation in favor of passive movement to treat denervated muscle following peripheral nerve injury. Recently, a short duration stimulation paradigm (1 hour per day, 5 days per week) was shown to increase muscle weight and force following one month of denervation [13]. A second study looked at one month of muscle stimulation following immediate nerve repair showing no differences in motor unit numbers but significant increases in contractile force [14]. However, muscles were assessed 2 months following the cessation of electrical stimulation which could potentially mask any early improvements in reinnervation. In this study, we set out to investigate the impact that electrical muscle stimulation has on early muscle reinnervation.
I. INTRODUCTION
II. METHODS
Peripheral nerve injuries can have long term debilitating consequences if left untreated [1]. Nerve injuries typically arise due to acute trauma such as sporting injuries, lacerations from glass fragments, or motor vehicle accidents. In these acute scenarios the best case for recovery is when microsurgical treatments are performed immediately or shortly after injury (days to weeks) [2]. While peripheral nerves do regenerate (approximately 1-3 mm per day), complete functional recovery is typically poor [3] as a majority of these injuries occur in proximal portions of the limb. The more proximal the injury, the longer it takes for new regenerating axons to make functional connections with their distal motor and sensory end organs, leaving a patient with compromised function for a considerable amount of time. Factors that contribute to the poor recovery include chronic Schwann cell and muscle denervation [4]. Long term muscle denervation results in progressive atrophy of muscle fibers resulting in diminished force output and reduced fiber diameter [5], [6]. One treatment method that has been used historically to reduce atrophy related changes and improve muscle excitability is electrical muscle stimulation [7]–[9]. However, there have been numerous reports that chronic electrical stimulation of partially denervated muscle fibers reduces terminal nerve sprouting and leads to compromised reinnervation [10], [11]. *This work was supported by MED-EL GmbH, Innsbruck, Austria M.P. Willand (
[email protected]), J.J. Zhang, C.D. Chiang, T. Gordon, and G.H. Borschel are with the Hospital for Sick Children, Toronto, ON M5G 1X8, CANADA.
978-1-4673-1969-0/13/$31.00 ©2013 IEEE
A. Animals and Surgical Procedures All experiments were performed on male thy1-GFP transgenic rats. These animals express green fluorescent protein (GFP) throughout their neural tissues when exposed to a 488 nm light source [15]. Surgical procedures, analgesia, and assessments were approved by The Hospital for Sick Children Laboratory Animal Services (LAS) and performed according to the Canadian Council on Animal Care Guidelines. Eights rats were randomly divided into two groups. The rats were anesthetized with 2% isofluorane and, using aseptic technique, the right tibial nerve was transected approximately 12-15 mm from the entry point to the gastrocnemius muscle. The transected nerve was then immediately repaired using two epineurial sutures. All rats had two intramuscular stimulating electrodes (Cooner Wire, AS 631) implanted in the gastrocnemius muscle with the cathode placed in the belly of the muscle and the anode more distally. Both electrodes were fixated in place using an electrode-suture complex [13]. For animals receiving stimulation, electrodes were then threaded subcutaneously and exposed at the neck to allow for connection to an external stimulator. B. Electrical Muscle Stimulation Animals receiving stimulation underwent a daily regimen that involved one hour of electrical stimulation. The paradigm involved 40 pulses at 100 Hz delivered every 6 seconds. Each stimulus pulse was biphasic with a 200 µs pulse width. Amplitudes were adjusted in order to maintain
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D. Immunohistochemistry Cryoprotected muscles were embedded in tissue freezing medium and frozen on dry ice. Serial sections (80 µm thickness) were cut with a cryostat at -20◦ C and mounted onto glass slides. Sections were stained with rhodamineα-bungarotoxin (α-BTX) conjugated to Alexa Fluor-594 (1:400, Invitrogen) and incubated overnight at 4◦ C to label the acetylcholine receptors in the post-synaptic membrane. Slides were imaged using a fluorescent microscope (Leica
To determine significant differences between the stimulated and unstimulated muscles an unpaired t-test was used. Where comparisons with the contralateral control limb were made a one way ANOVA was used with a post-hoc Tukey test used to observe differences between groups. In all cases significance was defined as p < 0.05. All values plotted in graphs are represented as the mean ± standard error of the mean (SEM). III. RESULTS Results in two groups of rats were compared: rats in which the gastrocnemius muscle was subjected to daily electrical stimulation 2 days after immediate repair of the transected tibial nerve (IR+ES) and rats which did not receive stimulation (IR). One animal in the stimulated group had complications with the electrode connection in the neck and was subsequently removed from all analyses. The estimated number of motor units (MUs) in the IR+ES group was significantly greater (2-fold) than the number of units in the IR group (Fig. 1A). Typical MU unit recordings are shown for one rat from each group in Fig. 1B and C. The responses show a dramatic increase in the number of MUs in the stimulated group of muscles.
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