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In the course of investigating how common clinical treatments and adaptive technologies affect recovery after spinal cord injury (SCI), we discovered that a clinically-modeled hindlimb stretching protocol dramatically, but transiently, reduces locomotor function. Nociceptive sensory input is capable of altering motor output at the spinal level, and nociceptive neurons are sensitized after SCI. Here we tested the hypotheses that stretch-induced locomotor deficits are dependent on nociceptive afferents by depleting TRPV1+ sensory afferents using capsaicin injections in neonatal rats. Following maturation, animals received 25g-cm contusive SCI at T10. After plateau of locomotor recovery at 6 weeks, daily stretching was performed for 3 weeks, followed by 2 weeks without stretch, and again for two additional weeks. Animals were sacrificed 2 h after the last stretching session for histological assessments. Consistent with previous findings, stretch-induced drops in locomotor function were observed in nociceptor-intact animals but were nearly absent in nociceptor-depleted animals. These functional changes were accompanied by corresponding increases in the number of c-Fos+ nuclei throughout the lumbar enlargement. As expected, nociceptor-depleted animals had very little CGRP+ axonal innervation of the dorsal horn. Nociceptor-intact stretched animals had significantly higher levels of CGRP+ as compared to non-stretched SCI rats, suggesting that stretching promoted intraspinal CGRP+ sprouting. These results indicate that stretch-induced locomotor dysfunction in animals with incomplete SCI involves C-fibers, adding a negative post-SCI role to their adaptive roles (e.g., bladder control), and suggesting that the clinical use of muscle stretching to combat contractures and spasticity may be unintentionally detrimental to locomotor function.

Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.

Mitochondrial dysfunction is becoming a pivotal target for neuroprotective strategies following contusion spinal cord injury (SCI) and the pharmacological compounds that maintain mitochondrial function confer neuroprotection and improve long-term hindlimb function after injury. In the current study we evaluated the efficacy of cell-permeating thiol, N-acetylcysteineamide (NACA), a precursor of endogenous antioxidant glutathione (GSH), on mitochondrial function acutely, and long-term tissue sparing and hindlimb locomotor recovery following upper lumbar contusion SCI. Some designated injured adult female Sprague-Dawley rats (n=120) received either Vehicle or NACA (75, 150, 300 or 600 mg/kg) at 15min and 6hrs post-injury. After 24hr the total, synaptic, and non-synaptic mitochondrial populations were isolated from a single 1.5cm spinal cord segment (centered at injury site) and assessed for mitochondrial bioenergetics. Results showed compromised total mitochondrial bioenergetics following acute SCI that was significantly improved with NACA treatment in a dose-dependent manner, with maximum effects at 300 mg/kg (n=4/group). For synaptic and non-synaptic mitochondria, only 300 mg/kg NACA dosage showed efficacy. Similar dosage (300mg/kg) also maintained mitochondrial GSH near normal levels. Other designated injured rats (n=21) received continuous NACA (150 or 300mg/kg/day) treatment starting at 15min post-injury for one week to assess long-term functional recovery over 6 weeks post-injury. Locomotor testing and novel gait analyses showed significantly improved hindlimb function with NACA that were associated with increased tissue sparing at the injury site. Overall, NACA treatment significantly maintained acute mitochondrial bioenergetics and normalized GSH levels following SCI, and prolonged delivery resulted in significant tissue sparing and improved recovery of hindlimb function.

Ethidium bromide (EB) has been extensively used in the rat as a model of spinal cord demyelination. However, this lesion has not been addressed in the adult mouse, a model with unlimited genetic potential. Here we characterize behavioral function, inflammation, myelin status and axonal viability following bilateral injection of 0.20 mg/mL ethidium bromide or saline into the ventral white matter (VWM) of female C57Bl/6 mice. EB-induced VWM demyelination significantly reduced spared VWM and Basso Mouse Scale (BMS) scores persisting out to 2 months. Chronic hindlimb dysfunction was accompanied by a persistent inflammatory response (demonstrated by CD45(+) immunofluorescence) and axonal loss (demonstrated by NF-M immunofluorescence and electron microscopy; EM). These cellular responses differ from the rat where inflammation resolves by 3-4 weeks and axon loss is minimal following EB demyelination. As these data suggest that EB-injection in the mouse spinal cord is a non-remyelinating lesion, we sought to ask whether wheel running could promote recovery by enhancing plasticity of local lumbar circuitry independent of remyelination. This did not occur as BMS and Treadscan assessment revealed no significant effect of wheel running on recovery. However, this study defines the importance of descending ventral motor pathways to locomotor function in the mouse as VWM loss results in a chronic hindlimb deficit.

The aim of the present study was to characterize a novel electrophysiological assessment, the magnetically evoked interenlargement response (MIER), by defining the anatomical location of the fast conducting (large myelinated) ascending axons that mediate the response and the relationship between the response and locomotor function following experimental spinal cord injury. Electromyographic (EMG) responses were recorded from the triceps muscles following magnetic stimulation of one hip. Short-latency (approximately 6 ms) EMGs were recorded from triceps muscles in normal controls and following different laceration injuries (dorsal, lateral or dorsal and lateral hemisections) or a 150-kilodyne (kd) contusion injury at the T9 level. The amplitude of the triceps MIER was significantly correlated to the area of spared white matter in the lateral funiculus and to hindlimb function during open field locomotion (r2 = 0.55). Following a complete lateral hemisection, MIERs were present in the triceps bilaterally following stimulation of either hip. Responses could also be recorded from the masseter muscles indicating that the influence of this pathway extends beyond the spinal cord. Anatomical evidence of a bilaterally distributed propriospinal pathway was found when biotinylated dextran amine (BDA) was injected into the lateral white matter on one side of the spinal cord at T9. BDA-labeled axons with varicosities were found bilaterally in the intermediate and ventral gray matter of the caudal region of the cervical enlargement. These observations suggest that MIERs may be useful to quantitatively assess neurotransmission and functional recovery over time after experimental spinal cord injury.

Growth factor-expanded neural precursor cells isolated from the mammalian central nervous system can differentiate into neurons and glia. Although the morphological and neurochemical development of these neural precursor cells has been investigated, little attention has been paid to their electrophysiology. This study examined the electrophysiological properties of neurons and glia derived from neural precursor cells isolated from the adult rat spinal cord (SC) and subventricular zone (SVZ). Cells were cultured in medium containing epidermal growth factor and/or fibroblast growth factor-2. After at least two passages, spheres of neural precursor cells were plated on coated coverslips and maintained in culture for up to 6 weeks. Whole-cell patch recordings were made using standard current clamp techniques. Immature action potentials were observed within hours of plating for both SC and SVZ cells. Input resistance and time constants decreased over the first week after plating and no further changes were found at later times. At similar times following plating, however, SVZ cells had a lower input resistance and shorter time constant compared to SC cells. SVZ cells also had higher resting membrane potentials and smaller after hyperpolarizations than those of SC cells, despite no significant difference in the amplitude of action potentials. Neither the SC nor the SVZ cells were capable of eliciting more than a single action potential in response to injected current. While all SC cells tested were depolarized by glutamate, the response of SVZ cells to glutamate varied considerably. This study revealed that neural precursor cells from SC and SVZ differ in both active and passive membrane properties. It appears also that the electrophysiological development of SC and SVZ precursor-derived neurons is incomplete under the conditions used. These observations suggest that the neural precursor cells from different anatomical locations may be physiologically diverse and may exhibit some differences in commitment toward neuronal or glial phenotypes.

The majority of human spinal cord injuries involve gray matter loss from the cervical or lumbar enlargements. However, the deficits that arise from gray matter damage are largely masked by the severe deficits due to associated white matter damage. We have developed a model to examine gray matter-specific deficits and therapeutic strategies that uses intraspinal injections of the excitotoxin kainic acid into the T9 and L2 regions of the spinal cord. The resulting deficits have been compared to those from standard contusion injuries at the same levels. Injuries were assessed histologically and functional deficits were determined using the Basso, Beattie, and Bresnahan (BBB) 21-point open field locomotor scale and transcranial magnetic motor evoked potentials (tcMMEPs). Kainic acid injections into T9 resulted in substantial gray matter damage; however, BBB scores and tcMMEP response latencies were not different from those of controls. In contrast, kainic acid injections into L2 resulted in paraplegia with BBB scores similar to those following contusion injuries at either T9 or L2, without affecting tcMMEP response latencies. These observations demonstrate that gray matter loss can result in significant functional deficits, including paraplegia, in the absence of a disruption of major descending pathways.

Repairing upper extremity function would significantly enhance the quality of life for persons with cervical spinal cord injury (SCI). Repair strategy development requires investigations of the deficits and the spontaneous recovery that occurs when cervical spinal cord axonal pathways are damaged. The present study revealed that both anatomically and electrophysiologically complete myelotomies of the C4 spinal cord dorsal columns significantly increased the adult rat's averaged times to first attend to adhesive stickers placed on the palms of their forepaws at 1 week. Complete bilateral myelotomies of the dorsal funiculi and dorsal hemisection, but not bilateral dorsolateral funiculi injuries, also similarly increased these times at 1 week. These data extend a previous finding by showing that a forepaw tactile sensory deficit that occurred in the adult rat after bilateral C4 spinal cord dorsal funiculi injury is due to damage of the dorsal columns. Averaged times to first attend to the stickers also decreased to those of sham-operated rats at 3 and 4 weeks post-dorsal hemisection with weekly testing. In contrast, a separate group of rats with dorsal hemisections had significantly increased times when tested only at 4 weeks. These data indicate that frequent assessment of this particular behavior in rats with dorsal hemisections extinguishes it and/or engenders a learned response in the absence of sensory axons in the dorsal columns and dorsolateral funiculi. This finding contrasted with weekly testing of grid walking where increased forelimb footfall numbers persisted for 4 weeks post-dorsal hemisection.