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1.  Electrical Stimulation of Embryonic Neurons for 1 Hour Improves Axon Regeneration and the Number of Reinnervated Muscles that Function 
Motoneuron death following spinal cord injury or disease results in muscle denervation, atrophy, and paralysis. We have previously transplanted embryonic ventral spinal cord cells into peripheral nerve to reinnervate denervated muscles and to reduce muscle atrophy, but reinnervation was incomplete. Here, our aim was to determine whether brief electrical stimulation of embryonic neurons in peripheral nerve changes motoneuron survival, axon regeneration, and muscle reinnervation and function because neural depolarization is crucial for embryonic neuron survival and may promote activity-dependent axon growth. At 1 week after denervation by sciatic nerve section, embryonic day 14-15 cells were purified for motoneurons, injected into the tibial nerve of adult Fischer rats, and stimulated immediately for up to 1 hour. More myelinated axons were present in tibial nerves when transplants had been stimulated at 1 Hz for 1 hour at 10 weeks following transplantation. More muscles were reinnervated if the stimulation treatment lasted for 1 hour. Reinnervation reduced muscle atrophy, with or without the stimulation treatment. These data suggest that brief stimulation of embryonic neurons promotes axon growth, which has a long-term impact on muscle reinnervation and function. Muscle reinnervation is important because it may enable the use of functional electrical stimulation to restore limb movements.
doi:10.1097/NEN.0b013e318299d376
PMCID: PMC3760016  PMID: 23771218
Axon growth cone initiation; Cell transplantation; Motoneuron survival; Muscle denervation
2.  Acute Stimulation of Transplanted Neurons Improves Motoneuron Survival, Axon Growth, and Muscle Reinnervation 
Journal of Neurotrauma  2013;30(12):1062-1069.
Abstract
Few options exist for treatment of pervasive motoneuron death after spinal cord injury or in neurodegenerative diseases such as amyotrophic lateral sclerosis. Local transplantation of embryonic motoneurons into an axotomized peripheral nerve is a promising approach to arrest the atrophy of denervated muscles; however, muscle reinnervation is limited by poor motoneuron survival. The aim of the present study was to test whether acute electrical stimulation of transplanted embryonic neurons promotes motoneuron survival, axon growth, and muscle reinnervation. The sciatic nerve of adult Fischer rats was transected to mimic the widespread denervation seen after disease or injury. Acutely dissociated rat embryonic ventral spinal cord cells were transplanted into the distal tibial nerve stump as a neuron source for muscle reinnervation. Immediately post-transplantation, the cells were stimulated at 20 Hz for 1 h. Other groups were used to control for the cell transplantation and stimulation. When neurons were stimulated acutely, there were significantly more neurons, including cholinergic neurons, 10 weeks after transplantation. This led to enhanced numbers of myelinated axons, reinnervation of more muscle fibers, and more medial and lateral gastrocnemius muscles were functionally connected to the transplant. Reinnervation reduced muscle atrophy significantly. These data support the concept that electrical stimulation rescues transplanted motoneurons and facilitates muscle reinnervation.
doi:10.1089/neu.2012.2797
PMCID: PMC3689928  PMID: 23544978
axon regeneration; electrical stimulation; motoneuron transplantation; muscle denervation
3.  Age at spinal cord injury determines muscle strength 
As individuals with spinal cord injury (SCI) age they report noticeable deficits in muscle strength, endurance and functional capacity when performing everyday tasks. These changes begin at ~45 years. Here we present a cross-sectional analysis of paralyzed thenar muscle and motor unit contractile properties in two datasets obtained from different subjects who sustained a cervical SCI at different ages (≤46 years) in relation to data from uninjured age-matched individuals. First, completely paralyzed thenar muscles were weaker when C6 SCI occurred at an older age. Muscles were also significantly weaker if the injury was closer to the thenar motor pools (C6 vs. C4). More muscles were strong (>50% uninjured) in those injured at a younger (≤25 years) vs. young age (>25 years), irrespective of SCI level. There was a reduction in motor unit numbers in all muscles tested. In each C6 SCI, only ~30 units survived vs. 144 units in uninjured subjects. Since intact axons only sprout 4–6 fold, the limits for muscle reinnervation have largely been met in these young individuals. Thus, any further reduction in motor unit numbers with time after these injuries will likely result in chronic denervation, and may explain the late-onset muscle weakness routinely described by people with SCI. In a second dataset, paralyzed thenar motor units were more fatigable than uninjured units. This gap widened with age and will reduce functional reserve. Force declines were not due to electromyographic decrements in either group so the site of failure was beyond excitation of the muscle membrane. Together, these results suggest that age at SCI is an important determinant of long-term muscle strength, and fatigability, both of which influence functional capacity.
doi:10.3389/fnint.2014.00002
PMCID: PMC3899581  PMID: 24478643
motoneuron death; muscle reinnervation; motor axon sprouting; muscle use; muscle fatigue; muscle strength
4.  Motor unit firing rates during spasms in thenar muscles of spinal cord injured subjects 
Involuntary contractions of paralyzed muscles (spasms) commonly disrupt daily activities and rehabilitation after human spinal cord injury (SCI). Our aim was to examine the recruitment, firing rate modulation, and derecruitment of motor units that underlie spasms of thenar muscles after cervical SCI. Intramuscular electromyographic activity (EMG), surface EMG, and force were recorded during thenar muscle spasms that occurred spontaneously or that were triggered by movement of a shoulder or leg. Most spasms were submaximal (mean: 39%, SD: 33 of the force evoked by median nerve stimulation at 50 Hz) with strong relationships between EMG and force (R2 > 0.69). Unit recruitment occurred over a wide force range (0.2–103% of 50 Hz force). Significant unit rate modulation occurred during spasms (frequency at 25% maximal force: 8.8 Hz, 3.3 SD; at maximal force: 16.1 Hz, 4.1 SD). Mean recruitment frequency (7.1 Hz, 3.2 SD) was significantly higher than derecruitment frequency (5.4 Hz, 2.4 SD). Coactive unit pairs that fired for more than 4 s showed high (R2 > 0.7, n = 4) or low (R2:0.3–0.7, n = 12) rate-rate correlations, and derecruitment reversals (21 pairs, 29%). Later recruited units had higher or lower maximal firing rates than lower threshold units. These discrepant data show that coactive motoneurons are drive both by common inputs and by synaptic inputs from different sources during muscle spasms. Further, thenar motoneurons can still fire at high rates in response to various peripheral inputs after SCI, supporting the idea that low maximal voluntary firing rates and forces in thenar muscles result from reduced descending drive.
doi:10.3389/fnhum.2014.00922
PMCID: PMC4231945  PMID: 25452723
motor unit recruitment; motor unit derecruitment; motor unit firing rate modulation; afferent input; motoneuron; persistent inward current
5.  Motoneuron Replacement for Reinnervation of Skeletal Muscle in Adult Rats 
Reinnervation is needed to rescue muscle when motoneurons die in disease or injury. Embryonic ventral spinal cord cells transplanted into peripheral nerve reinnervate muscle and reduce atrophy but low motoneuron survival may limit motor unit formation. We tested whether transplantation of a purified population of embryonic motoneurons into peripheral nerve (mean ± SE: 146,458 ± 4011 motoneurons) resulted in more motor units and reinnervation than transplantation of a mixed population of ventral spinal cord cells (72,075 ± 12,329 motoneurons). Ten weeks after either kind of transplant, similar numbers of neurons expressed choline acetyl transferase and/or Islet-1. Motoneuron numbers always exceeded the reinnervated motor unit count. Most motor end plates were simple plaques. Reinnervation significantly reduced muscle fiber atrophy. These data show that the number of transplanted motoneurons or motoneuron survival do not limit muscle reinnervation. Incomplete differentiation of motoneurons in nerve and lack of muscle activity may result in immature neuromuscular junctions that limit reinnervation and function.
doi:10.1097/NEN.0b013e31826cf69a
PMCID: PMC3760019  PMID: 22964786
Axon regeneration; Motoneuron transplantation; Motor unit; Muscle denervation; Muscle reinnervation; Neuromuscular junction
6.  Characteristics of Lower Extremity Clonus after Human Cervical Spinal Cord Injury 
Journal of Neurotrauma  2012;29(5):915-924.
Abstract
Clonus can interfere with self-care and rehabilitation of people with spinal cord injury. Our aim was to characterize clonus and to evaluate factors that influence clonus duration in muscles paralyzed chronically by spinal cord injury. Electromyographic activity was recorded from soleus and 7 other limb muscles (5 ipsilateral, 2 contralateral) during clonus. In 14 subjects, clonus frequency in soleus averaged 5.4±0.9 Hz and was slower when the reflex path was longer. Contraction frequency slowed at the beginning and end of clonus (sometimes by 2 Hz). The magnitude of one cycle changed the timing and magnitude of the next cycle. These data suggest that afferent input influences the frequency and maintenance of clonus. Recording from many muscles revealed that clonus was prolonged (>40 sec) when only ipsilateral triceps surae or triceps surae and tibialis anterior were involved. Therefore, localized inputs to spinal circuits were important to sustain clonus. Clonus was intermediate (median: 21 sec) with activation of three or four ipsilateral muscles and these contractions were associated with greater activation of ipsilateral flexors. Clonus was short (<5 sec) when ipsilateral and contralateral muscles were activated (five or six muscles). Activation of extraneous afferent input, particularly contralateral muscles, may provide a way to shorten clonus after spinal cord injury.
doi:10.1089/neu.2010.1549
PMCID: PMC3303097  PMID: 21910643
muscle spasms; spasticity; stretch reflex
7.  Automatic analysis of EMG during clonus 
Journal of neuroscience methods  2011;204(1):35-43.
Clonus can disrupt daily activities after spinal cord injury. Here an algorithm was developed to automatically detect contractions during clonus in 24-hour electromyographic (EMG) records. Filters were created by non-linearly scaling a Mother (Morlet) wavelet to envelope the EMG using different frequency bands. The envelope for the intermediate band followed the EMG best (74.8–193.9 Hz). Threshold and time constraints were used to reduce the envelope peaks to one per contraction. Energy in the EMG was measured 50 ms either side of each envelope (contraction) peak. Energy values at 5 % and 95 % maximal defined EMG start and end time, respectively. The algorithm was as good as a person at identifying contractions during clonus (p = 0.946, n=31 spasms, 7 subjects with cervical spinal cord injury), and marking start and end times to determine clonus frequency (intra class correlation coefficient, α: 0.949), contraction intensity using root mean square EMG (α: 0.997) and EMG duration (α: 0.852). On average the algorithm was 574 times faster than manual analysis performed independently by two people (p≤ 0.001). This algorithm is an important tool for characterization of clonus in long-term EMG records.
doi:10.1016/j.jneumeth.2011.10.017
PMCID: PMC3249492  PMID: 22057220
spinal cord injury; muscle spasm; clonus; wavelet analysis; surface EMG
8.  Effects of baclofen on motor units paralysed by chronic cervical spinal cord injury 
Brain  2009;133(1):117-125.
Baclofen, a gamma-aminobutyric acid receptorB agonist, is used to reduce symptoms of spasticity (hyperreflexia, increases in muscle tone, involuntary muscle activity), but the long-term effects of sustained baclofen use on skeletal muscle properties are unclear. The aim of our study was to evaluate whether baclofen use and paralysis due to cervical spinal cord injury change the contractile properties of human thenar motor units more than paralysis alone. Evoked electromyographic activity and force were recorded in response to intraneural stimulation of single motor axons to thenar motor units. Data from three groups of motor units were compared: 23 paralysed units from spinal cord injured subjects who take baclofen and have done so for a median of 7 years, 25 paralysed units from spinal cord injured subjects who do not take baclofen (median: 10 years) and 45 units from uninjured control subjects. Paralysed motor unit properties were independent of injury duration and level. With paralysis and baclofen, the median motor unit tetanic forces were significantly weaker, twitch half-relaxation times longer and half maximal forces reached at lower frequencies than for units from uninjured subjects. The median values for these same parameters after paralysis alone were comparable to control data. Axon conduction velocities differed across groups and were slowest for paralysed units from subjects who were not taking baclofen and fastest for units from the uninjured. Greater motor unit weakness with long-term baclofen use and paralysis will make the whole muscle weaker and more fatigable. Significantly more paralysed motor units need to be excited during patterned electrical stimulation to produce any given force over time. The short-term benefits of baclofen on spasticity (e.g. management of muscle spasms that may otherwise hinder movement or social interactions) therefore have to be considered in relation to its possible long-term effects on muscle rehabilitation. Restoring the strength and speed of paralysed muscles to pre-injury levels may require more extensive therapy when baclofen is used chronically.
doi:10.1093/brain/awp285
PMCID: PMC2857957  PMID: 19903733
baclofen; spinal cord injury; muscle paralysis; muscle weakness; axon conduction velocity; intraneural motor axon stimulation
9.  Neurotrophic Factors Improve Motoneuron Survival and Function of Muscle Reinnervated by Embryonic Neurons 
Motoneuron death can occur over several spinal levels following muscle denervation due to disease or trauma. We tested whether co-transplantation of embryonic neurons with one or more neurotrophic factors into peripheral nerve improved axon regeneration, muscle fiber area, reinnervation and function to a greater degree than cell transplantation alone. Sciatic nerves of adult Fischer rats were cut to denervate muscles; 1 week later, embryonic day 14–15 ventral spinal cord cells were transplanted into the tibial nerve stump as the only source of neurons for muscle reinnervation. Factors that promote motoneuron survival (i.e. cardiotrophin-1; fibroblast growth factor-2; glial cell line-derived neurotrophic factor [GDNF]; insulin like growth factor-1 [IGF-1]; leukemia inhibitory factor; and hepatocyte growth factor [HGF]) were added to the transplant individually or in combinations. Inclusion of a single factor with the cells resulted in comparable myelinated axon counts, muscle fiber areas, and evoked electromyographic activity (EMG) to cells alone 10 weeks after transplantation. Only cell transplantation with GDNF, HGF and IGF-1 significantly increased motoneuron survival, myelinated axon counts, muscle reinnervation and evoked EMG compared to cells alone. Thus, immediate application of a specific combination of factors to dissociated embryonic neurons improves survival of motoneurons and the long-term function of reinnervated muscle.
doi:10.1097/NEN.0b013e3181a9360f
PMCID: PMC2727878  PMID: 19535998
Axon regeneration; Electromyographic activity; Motoneuron death; Motoneuron replacement; Muscle denervation; Muscle reinnervation; Neurotrophic factors
10.  Automatic classification of motor unit potentials in surface EMG recorded from thenar muscles paralyzed by spinal cord injury 
Journal of Neuroscience Methods  2009;185(1):165-177.
Involuntary electromyographic (EMG) activity has only been analyzed in the paralyzed thenar muscles of spinal cord injured (SCI) subjects for several minutes. It is unknown if this motor unit activity is ongoing. Longer duration EMG recordings can investigate the biological significance of this activity. Since no software is currently capable of classifying 24 hours of EMG data at a single motor unit level, the goal of this research was to devise an algorithm that would automatically classify motor unit potentials by tracking the firing behavior of motor units over 24-hours. Two-channels of thenar muscle surface EMG were recorded over 24-hours from 7 SCI subjects with a chronic cervical level injury using a custom data logging device with custom software. The automatic motor unit classification algorithm developed here employed multiple passes through these 24-hour EMG recordings to segment, cluster, form global templates and classify motor unit potentials, including superimposed potentials. The classification algorithm was able to track an average of 19 global classes in 7 24-hour recordings with a mean (± SE) accuracy of 89.9 % (± 0.98%) and classify potentials from these individual motor units with a mean accuracy of 90.3% (± 0.97%). The algorithm could analyze 24 hours of data in 2–3 weeks with minimal input from a person, while a human operator was estimated to take more than 2 years. This automatic method could be applied clinically to investigate the fasciculation potentials often found in motoneuron disorders such as amyotrophic lateral sclerosis.
doi:10.1016/j.jneumeth.2009.09.012
PMCID: PMC2904617  PMID: 19761794
EMG; long term recording; motor unit classification; spinal cord injury
11.  Mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency 
Human molecular genetics  2005;14(18):2737-2748.
We have created a mouse model with an isolated cytochrome c oxidase (COX) deficiency by disrupting the COX10 gene in skeletal muscle. Missense mutations in COX10 have been previously associated with mitochondrial disorders. Cox10p is a protoheme:heme-O-farnesyl transferase required for the synthesis of heme a, the prosthetic group of the catalytic center of COX. COX10 conditional knockout mice were generated by crossing a LoxP-tagged COX10 mouse with a transgenic mouse expressing cre recombinase under the myosin light chain 1f promoter. The COX10 knockout mice were healthy until approximately 3 months of age when they started developing a slowly progressive myopathy. Surprisingly, even though COX activity in COX10 KO muscles was <5% of control muscle at 2.5 months, these muscles were still able to contract at 80–100% of control maximal forces and showed only a 10% increase in fatigability, and no signs of oxidative damage or apoptosis were detected. However, the myopathy worsened with time, particularly in female animals. This COX10 KO mouse allowed us to correlate the muscle function with residual COX activity, an estimate that can help predict the progression pattern of human mitochondrial myopathies.
doi:10.1093/hmg/ddi307
PMCID: PMC2778476  PMID: 16103131

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