Motoneuron Death after Human Spinal Cord Injury.

The severe muscle weakness and atrophy measured after human spinal cord injury (SCI) may relate to chronic muscle denervation due to motoneuron death and/or altered muscle use. The aim of this study was to estimate motoneuron death after traumatic human SCI. The diameter and number of myelinated axons were measured in ventral roots post-mortem because ventral roots contain large diameter (> 7 µm) myelinated axons that typically arise from motoneurons and innervate skeletal muscle. In four cases (SCI levels C7, C8, T4, and L1) involving contusion (n = 3) or laceration (n = 1), there was a significant reduction in the number of large diameter myelinated axons at the lesion epicenter (mean ± standard error [SE]: 45 ± 11% Uninjured), one level above (51 ± 14%), and one (27 ± 12%), two (45 ± 40%), and three (54 ± 23%) levels below the epicenter. Reductions in motoneuron numbers varied by side and case. These deficits result from motoneuron death because the gray matter was destroyed at and near the lesion epicenter. Muscle denervation must ensue. In seven cases, ventral roots at or below the epicenter had large diameter myelinated axons with unusually thin myelin, a sign of incomplete remyelination. The mean ± SE g ratio (axon diameter/fiber diameter) was 0.60 ± 0.01 for axons of all diameters in five above-lesion ventral roots, but increased significantly for large diameter fibers (≥ 12 µm) in three roots at the lesion epicenter. Motoneuron death after human SCI will coarsen muscle force gradation and control, while extensive muscle denervation will stifle activity-based treatments.

3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates

The histological assessment of spinal cord tissue in three dimensions has previously been very time consuming and prone to errors of interpretation. Advances in tissue clearing have significantly improved visualization of fluorescently labelled axons. While recent proof-of-concept studies have been performed with transgenic mice in which axons were prelabeled with GFP, investigating axonal regeneration requires stringent axonal tracing methods as well as the use of animal models in which transgenic axonal labeling is not available. Using rodent models of spinal cord injury, we labeled axon tracts of interest using both adeno-associated virus and chemical tracers and performed tetrahydrofuran-based tissue clearing to image multiple axon types in spinal cords using light sheet and confocal microscopy. Using this approach, we investigated the relationships between axons and scar-forming cells at the injury site as well as connections between sensory axons and motor pools in the spinal cord. In addition, we used these methods to trace axons in nonhuman primates. This reproducible and adaptable virus-based approach can be combined with transgenic mice or with chemical-based tract-tracing methods, providing scientists with flexibility in obtaining axonal trajectory information from transparent tissue.

Electrical stimulation of transplanted motoneurons improves motor unit formation.

Motoneurons die following spinal cord trauma and with neurological disease. Intact axons reinnervate nearby muscle fibers to compensate for the death of motoneurons, but when an entire motoneuron pool dies, there is complete denervation. To reduce denervation atrophy, we have reinnervated muscles in Fisher rats from local transplants of embryonic motoneurons in peripheral nerve. Since growth of axons from embryonic neurons is activity dependent, our aim was to test whether brief electrical stimulation of the neurons immediately after transplantation altered motor unit numbers and muscle properties 10 wk later. All surgical procedures and recordings were done in anesthetized animals. The muscle consequences of motoneuron death were mimicked by unilateral sciatic nerve section. One week later, 200,000 embryonic day 14 and 15 ventral spinal cord cells, purified for motoneurons, were injected into the tibial nerve 10-15 mm from the gastrocnemii muscles as the only neuron source for muscle reinnervation. The cells were stimulated immediately after transplantation for up to 1 h using protocols designed to examine differential effects due to pulse number, stimulation frequency, pattern, and duration. Electrical stimulation that included short rests and lasted for 1 h resulted in higher motor unit counts. Muscles with higher motor unit counts had more reinnervated fibers and were stronger. Denervated muscles had to be stimulated directly to evoke contractions. These results show that brief electrical stimulation of embryonic neurons, in vivo, has long-term effects on motor unit formation and muscle force. This muscle reinnervation provides the opportunity to use patterned electrical stimulation to produce functional movements.

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.

Electrical stimulation of embryonic neurons for 1 hour improves axon regeneration and the number of reinnervated muscles that function.

Motoneuron death after spinal cord injury or disease results in muscle denervation, atrophy, and paralysis. We have previously transplanted embryonic ventral spinal cord cells into the 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 the peripheralnerve 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 to 15 cells were purified for motoneurons, injected into the tibial nerve of adult Fischer rats, and stimulated immediatelyfor up to 1 hour. More myelinated axons were present in tibial nerves 10 weeks after transplantation when transplants had been stimulated acutely at 1 Hz for 1 hour. 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.

Acute stimulation of transplanted neurons improves motoneuron survival, axon growth, and muscle reinnervation.

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.

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 ± 4,011 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 plate were simple plaques. Reinnervation significantly reduced muscle fiber atrophy. These data show that the number of transplanted motoneurons and 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.

Neurotrophic factors improve muscle reinnervation from embryonic neurons.

Motoneurons die in diseases like amyotrophic lateral sclerosis and after spinal cord trauma, inducing muscle denervation. We tested whether transplantation of embryonic cells with neurotrophic factors into peripheral nerve of adult rats improves muscle reinnervation and motor unit function more than cells alone. One week after sciatic nerve section, embryonic ventral spinal cord cells were transplanted into the tibial nerve with or without glial cell line-derived neurotrophic factor, hepatocyte growth factor, and insulin-like growth factor-1. These cells represented the only neuron source for muscle reinnervation. Ten weeks after transplantation, all medial gastrocnemius muscles contracted in response to electrical stimulation of cell transplants with factors. Only 80% of muscles responded with cells alone. Factors and cells resulted in survival of more motoneurons and reinnervation of more muscle fibers for a given axon (motor unit) number. Greater reinnervation from embryonic cells may enhance muscle excitation by patterned electrical stimulation.

Neurotrophic factors improve motoneuron survival and function of muscle reinnervated by embryonic neurons.

Motoneuron death can occur over several spinal levels with disease or trauma, resulting in muscle denervation. We tested whether cotransplantation of embryonic neurons with 1 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 ventral spinal cord cells (days 14-15) were transplanted into the tibial nerve stump as the only source of neurons for muscle reinnervation. Factors that promote motoneuron survival (cardiotrophin 1; fibroblast growth factor 2; glial cell line-derived neurotrophic factor; insulin-like growth factor 1; leukemia inhibitory factor; and hepatocyte growth factor) 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 to cells alone 10 weeks after transplantation. Only cell transplantation with glial cell line-derived neurotrophic factor, hepatocyte growth factor, and insulin-like growth factor 1 significantly increased motoneuron survival, myelinated axon counts, muscle reinnervation, and evoked electromyographic activity compared with 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.

Embryonic neurons transplanted into the tibial nerve reinnervate muscle and reduce atrophy but NCAM expression persists.

OBJECTIVE: The aim of this study was to use the glycogen depletion technique to determine whether reinnervated muscle fibers could be distinguished from denervated muscle fibers by their size or by neural cell adhesion molecule (NCAM) expression. METHODS: Medial gastrocnemius muscles of five adult Fischer rats were reinnervated from embryonic neurons transplanted into the distal stump of the tibial nerve. Ten weeks later, the transplants were stimulated repeatedly to deplete reinnervated muscle fibers of glycogen. Areas of reinnervated (glycogen-depleted) muscle fibers were measured and assessed for NCAM expression. The areas of muscle fibers from reinnervated, denervated (n=5) and unoperated control muscles (n=5) were compared. RESULTS: Mean reinnervated muscle fiber area was significantly larger than the mean for denervated fibers (mean +/- SE: 40 +/- 6 and 10 +/- 1% of unoperated control fibers, respectively). NCAM was expressed in 55 +/- 7% of reinnervated fibers (mean +/- SE; range: 42-77%). The mean areas of reinnervated fibers that did or did not express NCAM were similar. NCAM was only expressed in some fibers in completely denervated muscles. DISCUSSION: Our data show that NCAM expression does not differentiate muscle denervation or reinnervation. Quantifying the area of large fibers did distinguish reinnervated muscle fibers from denervated fibers and showed that reinnervation of muscle from neurons placed in peripheral nerve is a strategy to rescue muscle from atrophy.

Long-term delivery of FGF-6 changes the fiber type and fatigability of muscle reinnervated from embryonic neurons transplanted into adult rat peripheral nerve.

Motoneuron death leads to muscle denervation and atrophy. Transplantation of embryonic neurons into peripheral nerves results in reinnervation and provides a strategy to rescue muscles from atrophy independent of neuron replacement in a damaged or diseased spinal cord. But the count of regenerating axons always exceeds the number of motor units in this model, so target-derived trophic factor levels may limit reinnervation. Our aim was to examine whether long-term infusion of fibroblast growth factor-6 (FGF-6) into denervated medial gastrocnemius muscles improved the function of muscles reinnervated from neurons transplanted into nerve of adult Fischer rats. Factor delivery (10 microg, 4 weeks) began after sciatic nerve transection. After a week of nerve degeneration, 1 million embryonic day 14-15 ventral spinal cord cells were transplanted into the distal tibial stump as a neuron source. Ten weeks later, neurons that expressed motoneuron markers survived in the nerves. More myelinated axons were in nerves to saline-treated muscles than in FGF-6-treated muscles. However, each group showed comparable reductions in muscle fiber atrophy because of reinnervation. Mean reinnervated fiber area was 43%-51% of non-denervated fibers. Denervated fiber area averaged 11%. FGF-6-treated muscles were more fatigable than other reinnervated muscles but had stronger motor units and fewer type I fibers than did saline-treated muscles. FGF-6 thus influenced function by changing the type of fiber reinnervated by transplanted neurons. Deficits in FGF-6 may also contribute to the increase in type I fibers in muscles reinnervated from peripheral axons, suggesting that the effects of FGF-6 on fiber type are independent of the neuron source used for reinnervation.

Embryonic cerebral cortex cells retain CNS phenotypes after transplantation into peripheral nerve.

Differentiation of stem cells depends on environmental cues. In this study, acutely dissociated or expanded cells derived from embryonic day 14 (E14) rat cerebral cortex were transplanted into the distal tibial nerve stump of adult Fischer rats to determine whether a peripheral nervous system (PNS) environment would influence cell differentiation. Acutely dissociated cells, which included neural precursors and post-mitotic neurons, were transplanted immediately after harvest. Expanded cortical cells were transplanted after 8 days of culture with fibroblast growth factor-2 (FGF-2), a process that yields a population of neural stem cells and/or neural precursors. After 2 or 10 weeks in peripheral nerve, the majority of the transplanted cells was astrocytes, as judged from glial fibrillary acid protein (GFAP) expression. Only acutely dissociated transplants had cells that exhibited neuronal phenotypes. Those neurons present in transplants at 10 weeks stained positive for glutamate decarboxylase and did not reinnervate muscle. Maintenance of this cortical phenotype in peripheral nerve suggests that it is necessary to transplant cells with neural phenotypes appropriate for muscle to restore its function.

Properties of medial gastrocnemius motor units and muscle fibers reinnervated by embryonic ventral spinal cord cells.

Severe muscle atrophy occurs after complete denervation. Here, Embryonic Day 14-15 ventral spinal cord cells were transplanted into the distal tibial nerve stump of adult female Fischer rats to provide a source of neurons for muscle reinnervation. Our aim was to characterize the properties of the reinnervated motor units and muscle fibers. Some reinnervated motor units contracted spontaneously. Electrical stimulation of the transplants at increasing intensity produced an average (+/- SE) of 7 +/- 1 electromyographic and force steps. Each signal increment represented the excitation of another motor unit. These reinnervated units exerted an average force of 12.0 +/- 1.5 mN, strength similar to that of control fatigue-resistant units. Repeated transplant stimulation depleted 17% of the muscle fibers of glycogen, an indication of some functional reinnervation. Reinnervated (glycogen-depleted), denervated (no cells transplanted), and control fibers were of histochemical type I, IIA, or IIB. Fibers of the same type were grouped after reinnervation. The proportion of fiber types also changed. Reinnervated fibers were primarily type IIA, whereas most fibers in denervated and control muscles were type IIB. Reinnervated fibers of each type had significantly larger cross-sectional areas than the corresponding fiber types in denervated muscles. These data suggest that neurons with different properties can reside in the unusual environment of the adult rat peripheral nerve, make functional connections with muscle, specify muscle fiber type, and reduce the amount that each type atrophies.

Muscle reinnervation with delayed or immediate transplant of embryonic ventral spinal cord cells into adult rat peripheral nerve.

Muscle denervation is common in various neuromuscular diseases and after trauma. It induces skeletal muscle atrophy. Only muscle reinnervation leads to functional recovery. In previous studies, denervated adult rat muscles were rescued by transplantation of embryonic day 14-15 (E14-15) ventral spinal cord cells into a nearby peripheral nerve. In the present study, changes were made in the environment into which the cells were placed to test whether reinnervation was improved by: 1) prior nerve degeneration, induced by sciatic nerve transection 1 week before cell transplantation; 2) transplantation of 1 million versus 5 million cells; 3) addition of nerve growth factor (NGF) to the transplant. Ten weeks after cell transplantation, axons had grown from all of the transplants. The numbers of myelinated axons that regenerated into the tibial, medial (MG), and lateral gastrocnemius-soleus (LGS) nerves were similar across treatments. The mean diameters of large LGS axons (>6 microm) were significantly larger with nerve degeneration before transplantation. The mean diameters of MG and LGS axons were significantly larger with transplantation of 1 million versus 5 million cells. Silver-stained experimental and control lateral gastronemius (LG) muscles showed axons that terminated at motor end plates. Nodal and terminal sprouts were more common in reinnervated muscles (45-63% of all end plates) than in control muscles (10%). Electrical stimulation of the transplants induced weak contractions in 39 of 47 MG muscles (83%) and 33 of 46 LG muscles (72%) but at higher voltages than needed to excite control muscles. The threshold for MG contraction was lower with transplantation of 1 million cells, while LG thresholds were lower without NGF. The cross-sectional area of whole LG muscles was significantly larger with cell transplantation (immediate or delayed) than with media alone, but all of these muscle areas were reduced significantly compared with control muscle areas. These data suggest that delayed transplantation of fewer cells without NGF assists regeneration of larger diameter axons and prevents some muscle atrophy.