Gordon, T., N. Tyreman, V. F. Rafuse, and J. B. Munson. Fast-to-slow conversion following chronic low-frequency activation of medial gastrocnemius muscle in cats. I. Muscle and motor unit properties. J. Neurophysiol. 77: 2585–2604, 1997. This study of cat medial gastrocnemius (MG) muscle and motor unit (MU) properties tests the hypothesis that the normal ranges of MU contractile force, endurance, and speed are directly associated with the amount of neuromuscular activity normally experienced by each MU. We synchronously activated all MUs in the MG muscle with the same activity (20 Hz in a 50% duty cycle) and asked whether conversion of whole muscle contractile properties is associated with loss of the normal heterogeneity in MU properties. Chronically implanted cuff electrodes on the nerve to MG muscle were used for 24-h/day stimulation and for monitoring progressive changes in contractile force, endurance, and speed by periodic recording of maximal isometric twitch and tetanic contractions under halothane anesthesia. Chronic low-frequency stimulation slowed muscle contractions and made them weaker, and increased muscle endurance. The most rapid and least variable response to stimulation was a decline in force output of the muscle and constituent MUs. Fatigue resistance increased more slowly, whereas the increase in time to peak force varied most widely between animals and occurred with a longer time course than either force or endurance. Changes in contractile force, endurance, and speed of the whole MG muscle accurately reflected changes in the properties of the constituent MUs both in extent and time course. Normally there is a 100-fold range in tetanic force and a 10-fold range in fatigue indexes and twitch time to peak force. After chronic stimulation, the range in these properties was significantly reduced and, even in MU samples from single animals, the range was shown to correspond with the slow (type S) MUs of the normal MG. In no case was the range reduced to less than the type S range. The same results were obtained when the same chronic stimulation pattern of 20 Hz/50% duty cycle was imposed on paralyzed muscles after hemisection and unilateral deafferentation. The findings that the properties of MUs still varied within the normal range of type S MUs and were still heterogeneous despite a decline in the variance in any one property indicate that the neuromuscular activity can account only in part for the wide range of muscle properties. It is concluded that the normal range of properties within MU types reflects an intrinsic regulation of properties in the multinucleated muscle fibers.
Progressive atrophy of Schwann cells in denervated nerve stumps is a major reason for progressive failure of functional recovery after peripheral nerve injury and surgical repair.To examine whether side-to-side nerve bridges between an intact donor nerve and a recipient denervated distal nerve stump promote nerve growth and in turn, protect distal nerve stumps to improve axon regeneration after delayed surgical repair.In Sprague-Dawley rats, 1 or 3 side-to-side common peroneal (CP) nerve bridges were used to bridge between the donor intact tibial (TIB) nerve and a recipient denervated CP distal nerve stump in the contralateral hind limb. No bridges were placed in control animals. After 4 months, either a fluorescent retrograde dye was applied to back-label TIB motoneurons with axons that had grown into the CP nerve stump or the proximal and distal CP nerve stumps were resutured in experimental and control animals to encourage CP nerve regeneration for 5 months. Retrograde dyes were again applied to count CP motoneurons that regenerated their axons through protected and unprotected nerve stumps.Significantly more donor TIB motoneurons regenerated axons into the recipient denervated CP nerve stump through 3 side-to-side CP nerve bridges compared with 1 bridge. This TIB nerve protection significantly increased the number of CP motoneurons regenerating axons through the denervated CP nerve stumps, the number of regenerated axons, and the weight of the reinnervated muscles.Multiple side-to-side nerve bridges protect chronically denervated nerve stumps to improve axon regeneration and target reinnervation after delayed nerve repair.
Abstract The overall goal of this work was to create a high-resolution MRI atlas of the lumbosacral enlargement of the spinal cord of the rat (Sprague-Dawley), cat, domestic pig, rhesus monkey, and human. These species were chosen because they are commonly used in basic and translational research in spinal cord injuries and diseases. Six spinal cord specimens from each of the studied species (total of 30 specimens) were fixed, extracted, and imaged. Sizes of the spinal cord segments, cross-sectional dimensions, and locations of the spinal cord gray and white matter were quantified and compared across species. The obtained atlas establishes a reference for the neuroanatomy of the intact lumbosacral spinal cord in these species. It can also be used to guide the planning of surgical procedures of the spinal cord, technology design and development of spinal cord neuroprostheses, and the precise delivery of cells/drugs into target regions within the spinal cord parenchyma.
OBJECTIVE The goal of this study was to assess the safety of mapping spinal cord locomotor networks using penetrating stimulation microelectrodes in Yucatan minipigs (YMPs) as a clinically translational animal model. METHODS Eleven YMPs were trained to walk up and down a straight line. Motion capture was performed, and electromyographic (EMG) activity of hindlimb muscles was recorded during overground walking. The YMPs underwent a laminectomy and durotomy to expose the lumbar spinal cord. Using an ultrasound-guided stereotaxic frame, microelectrodes were inserted into the spinal cord in 8 animals. Pial cuts were made to prevent tissue dimpling before microelectrode insertion. Different locations within the lumbar enlargement were electrically stimulated to map the locomotor networks. The remaining 3 YMPs served as sham controls, receiving the laminectomy, durotomy, and pial cuts but not microelectrode insertion. The Porcine Thoracic Injury Behavioral Scale (PTIBS) and hindlimb reflex assessment results were recorded for 4 weeks postoperatively. Overground gait kinematics and hindlimb EMG activity were recorded again at weeks 3 and 4 postoperatively and compared with preoperative measures. The animals were euthanized at the end of week 4, and the lumbar spinal cords were extracted and preserved for immunohistochemical analysis. RESULTS All YMPs showed transient deficits in hindlimb function postoperatively. Except for 1 YMP in the experimental group, all animals regained normal ambulation and balance (PTIBS score 10) at the end of weeks 3 and 4. One animal in the experimental group showed gait and balance deficits by week 4 (PTIBS score 4). This animal was excluded from the kinematics and EMG analyses. Overground gait kinematic measures and EMG activity showed no significant (p > 0.05) differences between preoperative and postoperative values, and between the experimental and sham groups. Less than 5% of electrode tracks were visible in the tissue analysis of the animals in the experimental group. There was no statistically significant difference in damage caused by pial cuts between the experimental and sham groups. Tissue damage due to the pial cuts was more frequently observed in immunohistochemical analyses than microelectrode tracks. CONCLUSIONS These findings suggest that mapping spinal locomotor networks in porcine models can be performed safely, without lasting damage to the spinal cord.
Background Spinal cord injury (SCI) affects locomotion and quality of life. Two spinal cord stimulation approaches are currently under investigation for restoring standing and walking following SCI: epidural spinal cord stimulation (ESCS) and intraspinal microstimulation (ISMS). In ESCS, electrodes are placed on the dura mater and in ISMS, ultrafine wires are inserted into the cord. These modalities likely activate the locomotor regions in the ventral horn through different pathways. Objective The goal of this study is to examine the difference in the distribution of neuronal activation and the type of neurons activated by ESCS and ISMS. Methods The first step was to establish the needed immunohistochemical (IHC) staining protocols. Domestic pigs were divided into naïve (n=2) and positive control (n=1) groups. The naïve control animals were anesthetized for 5 hrs. The positive control animal was anesthetized for 2 hrs and injected with hypertonic saline in hindlimb muscles. The animals were then euthanized, and the spinal cord removed for IHC analysis. Antibodies against cFos, a maker of neuronal activation, and NeuN, a neuronal marker were used. Results Preliminary results indicate that ESCS activates neurons in the dorsal horn with scattered activation in the intermediate and ventral regions. ISMS primarily activates neurons in the intermediate and ventral regions where locomotor-related networks reside. Significance To the best of our knowledge, this is the first time the type and sites of activation of ESCS and ISMS are investigated. This will provide a foundational understanding of the mechanism of action of these stimulation modalities.
Nerve sprouting to reinnervate partially denervated muscles is important in several disease and injury states. To examine the effectiveness of sprouting of active and inactive motor units (MUs) and the basis for a limit to sprouting, one of three rat lumbar spinal roots was cut under normal conditions and when the spinal cord was hemisected at T12. Muscle and MU isometric contractile forces were recorded and muscle fibres in glycogen-depleted single muscle units enumerated 23 to 380 days after surgery. Enlargement of intact MUs by sprouting was effective in compensating for up to 80% loss of innervation. For injuries that removed >70-80% of the intact MUs, muscle contractile force and weight dropped sharply. For partial denervation of <70%, all MUs increased contractile force by the same factor in both normally active muscles and muscles whose activity was reduced by T12 hemisection. Direct measurements of MU size by counting glycogen-depleted muscle fibres in physiologically and histochemically defined muscle units, provided direct evidence for a limit in MU size, whether or not the activity of the muscles was reduced by spinal cord hemisection. Analysis of spatial distribution of muscle fibres within the outer boundaries of the muscle unit demonstrated a progressive increase in fibres within the territory to the limit of sprouting when most of the muscle unit fibres were adjacent to each other. We conclude that the upper limit of MU enlargement may be explained by the reinnervation of denervated muscle fibres by axon sprouts within the spatial territory of the muscle unit, formerly distributed in a mosaic pattern.
The cell adhesion molecule N-CAM is localized to the adult neuromuscular junction but is also expressed in the extrajunctional membrane of denervated muscles concurrent with extrajunctional acetylcholine receptors. Here we used N-CAM immunohistochemistry to determine whether we could detect early denervation in hindlimb muscles of the G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS). In denervated wild type mouse muscles, N-CAM immunoreactivity on the sarcolemma of all fiber types and within the sarcoplasm of only type IIA fibers was detected at day 2: ∼30% of the muscle fibers in cross-section were fully circumscribed by N-CAM immunoreactivity and ∼25% of fibers were incompletely circumscribed. The proportion of the latter fibers remained constant over the next 8 days as the proportions of the former fibers increased exponentially. Thereafter, fully circumscribed muscle fibers increased to a maximum by 30 days with a concomitant fall in the incompletely circumscribed fibers. Hence, early muscle denervation was detected by the incomplete circumscription of fiber membranes by N-CAM immunoreactivity with full circumscription and intracellular localization indicating more long-term denervation. In the G93A transgenic mouse, rapid denervation of fast-twitch muscles was readily detected by a corresponding proportion of muscle fibers in cross-section with positive N-CAM immunoreactivity. The proportions of incompletely and completely circumscribed muscle fibers corresponded well with the rate of decline in intact motor units and reduced muscle contractile forces. Progressively more fully circumscribed muscle fibers became evident with age. We conclude that the N-CAM immunoreactivity on muscle fiber membranes in muscle cross-sections provides a sensitive means of detecting early muscle fiber denervation.
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