There is increasing motivation to develop clinically relevant experimental models for cervical SCI in rodents and techniques to assess deficits in forelimb function. Here we describe a bilateral cervical contusion model in rats. Female Sprague-Dawley rats received mild or moderate cervical contusion injuries (using the Infinite Horizons device) at C5, C6, or C7/8. Forelimb motor function was assessed using a grip strength meter (GSM); sensory function was assessed by the von Frey hair test; the integrity of the corticospinal tract (CST) was assessed by biotinylated dextran amine (BDA) tract tracing. Mild contusions caused primarily dorsal column (DC) and gray matter (GM) damage while moderate contusions produced additional damage to lateral and ventral tissue. Forelimb and hindlimb function was severely impaired immediately post-injury, but all rats regained the ability to use their hindlimbs for locomotion. Gripping ability was abolished immediately after injury but recovered partially, depending upon the spinal level and severity of the injury. Rats exhibited a loss of sensation in both fore- and hindlimbs that partially recovered, and did not exhibit allodynia. Tract tracing revealed that the main contingent of CST axons in the DC was completely interrupted in all but one animal whereas the dorsolateral CST (dlCST) was partially spared, and dlCST axons gave rise to axons that arborized in the GM caudal to the injury. Our data demonstrate that rats can survive significant bilateral cervical contusion injuries at or below C5 and that forepaw gripping function recovers after mild injuries even when the main component of CST axons in the dorsal column is completely interrupted.

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.

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.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors increases phrenic motor output. Ampakines are a class of drugs that are positive allosteric modulators of AMPA receptors. We hypothesized that 1) ampakines can stimulate phrenic activity after incomplete cervical spinal cord injury (SCI), and 2) pairing ampakines with brief hypoxia could enable sustained facilitation of phrenic bursting. Phrenic activity was recorded ipsilateral (IL) and contralateral (CL) to C2 spinal cord hemisection (C2Hx) in anesthetized adult rats. Two weeks after C2Hx, ampakine CX717 (15 mg/kg, i.v.) increased IL (61 ± 46% baseline, BL) and CL burst amplitude (47 ± 26%BL) in 8 of 8 rats. After 90 min, IL and CL bursting remained above baseline (BL) in 7 of 8 rats. Pairing ampakine with a single bout of acute hypoxia (5-min, arterial partial pressure of O2 ~ 50 mmHg) had a variable impact on phrenic bursting, with some rats showing a large facilitation that exceeded the response of the ampakine alone group. At 8 weeks post-C2Hx, 7 of 8 rats increased IL (115 ± 117%BL) and CL burst amplitude (45 ± 27%BL) after ampakine. The IL burst amplitude remained above BL for 90-min in 7 of 8 rats; CL bursting remained elevated in 6 of 8 rats. The sustained impact of ampakine at 8 weeks was not enhanced by hypoxia exposure. Intravenous vehicle (10% 2-Hydroxypropyl-β-cyclodextrin) did not increase phrenic bursting at either time point. We conclude that ampakines effectively stimulate neural drive to the diaphragm after cervical SCI. Pairing ampakines with a single hypoxic exposure did not consistently enhance phrenic motor facilitation.

The importance of mitochondria in spinal cord injury has mainly been attributed to their participation in apoptosis at the site of injury. But another aspect of mitochondrial function is the generation of more than 90% of cellular energy in the form of ATP, mediated by the oxidative phosphorylation (OxPhos) process. Cytochrome c oxidase (CcO) is a central OxPhos component and changes in its activity reflect changes in energy demand. A recent study suggests that respiratory muscle function in chronic obstructive pulmonary disease (COPD) patients is compromised via alterations in mitochondrial function. In an animal model of cervical spinal cord hemisection (C2HS) respiratory dysfunction, we have shown that theophylline improves respiratory function. In the present study, we tested the hypothesis that theophylline improves respiratory function at the cellular level via improved mitochondrial function in the C2HS model. We demonstrate that CcO activity was significantly (33%) increased in the spinal cord adjacent to the site of injury (C3-C5), and that administration of theophylline (20mg/kg 3x daily orally) after C2HS leads to an even more pronounced increase in CcO activity of 62% compared to sham-operated animals. These results are paralleled by a significant increase in cellular ATP levels (51% in the hemidiaphragm ipsilateral to the hemisection). We conclude that C2HS increases energy demand and activates mitochondrial respiration, and that theophylline treatment improves energy levels through activation of the mitochondrial OxPhos process to provide energy for tissue repair and functional recovery after paralysis in the C2HS model.

A C2 cervical spinal cord hemisection (SH) interrupts descending inspiratory-related drive to phrenic motoneurons located between C3 and C5 in rats, paralyzing the ipsilateral hemidiaphragm muscle. There is gradual recovery of rhythmic diaphragm muscle activity ipsilateral to cervical spinal cord injury over time, consistent with neuroplasticity and strengthening of spared, contralateral descending premotor input to phrenic motoneurons. Brain-derived neurotrophic factor (BDNF) signaling through the tropomyosin related kinase receptor subtype B (TrkB) plays an important role in neuroplasticity following spinal cord injury. We hypothesized that 1) increasing BDNF/TrkB signaling at the level of the phrenic motoneuron pool by intrathecal BDNF delivery enhances functional recovery of rhythmic diaphragm activity after SH, and 2) inhibiting BDNF/TrkB signaling by quenching endogenous neurotrophins with the soluble fusion protein TrkB-Fc or by knocking down TrkB receptor expression in phrenic motoneurons using intrapleurally-delivered siRNA impairs functional recovery after SH. Diaphragm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of SH and 3days post-SH. After SH surgery in adult rats, an intrathecal catheter was placed at C4 to chronically infuse BDNF or TrkB-Fc using an implanted mini-osmotic pump. At 14days post-SH, all intrathecal BDNF treated rats (n=9) displayed recovery of ipsilateral hemidiaphragm EMG activity, compared to 3 out of 8 untreated SH rats (p<0.01). During eupnea, BDNF treated rats exhibited 76±17% of pre-SH root mean squared EMG vs. only 5±3% in untreated SH rats (p<0.01). In contrast, quenching endogenous BDNF with intrathecal TrkB-Fc treatment completely prevented functional recovery up to 14days post-SH (n=7). Immunoreactivity of the transcription factor cAMP response element-binding protein (CREB), a downstream effector of TrkB signaling, increased in phrenic motoneurons following BDNF treatment (n=6) compared to artificial cerebrospinal fluid treatment (n=6; p<0.001). Intrapleural injections of non-sense or TrkB siRNA were administered after SH to specifically target phrenic motoneurons. At 14days post-SH, none out of 9 TrkB siRNA treated rats displayed functional recovery compared to 5 out of 9 non-sense siRNA treated rats. These results indicate that BDNF/TrkB signaling in phrenic motoneuron pool plays a critical role in functional recovery after cervical spinal cord injury.

Spinal cord injury is a severe condition, resulting in specific neurological symptoms depending on the level of damage. Approximately 60% of spinal cord injuries affect the cervical spinal cord, resulting in complete or incomplete tetraplegia and higher mortality rates than injuries of the thoracic or lumbar region. Although cervical spinal cord injuries frequently occur in humans, there are few clinically relevant models of cervical spinal cord injury. Animal models are critical for examining the cellular and molecular manifestations of human cervical spinal cord injury, which is not feasible in the clinical setting, and to develop therapeutic strategies. There is a limited number of studies using cervical, bilateral contusion SCI and providing a behavioral assessment of motor and sensory functions, which is partly due to the high mortality rate and severe impairment observed in severe cervical SCI models. The goal of this study was to develop a mouse model of cervical contusion injury with moderate severity, resulting in an apparent deficit in front and hindlimb function but still allowing for self-care of the animals. In particular, we aimed to characterize a mouse cervical injury model to be able to use genetic models and a wide range of viral techniques to carry out highly mechanistic studies into the cellular and molecular mechanisms of cervical spinal cord injury. After inducing a bilateral, cervical contusion injury at level C5, we followed the recovery of injured and sham-uninjured animals for eight weeks post-surgery. Hindlimb and forelimb motor functions were significantly impaired immediately after injury, and all mice demonstrated partial improvement over time that remained well below that of uninjured control mice. Mice also displayed a significant loss in their sensory function throughout the testing period. This loss of sensory and motor function manifested as a reduced ability to perform skilled motor tasks in all of the injured mice. Here, we describe a new mouse model of moderate bilateral cervical spinal cord injury that does not lead to mortality and provides a comprehensive assessment of histological and behavioral assessments. This model will be useful in enhancing our mechanistic understanding of cervical spinal cord injury and in the development of treatments targeted at promoting neuroprotection, neuroplasticity, and functional recovery after cervical SCI.

Cervical injuries are the most common form of SCI. In this study, we used a neuromodulatory approach to promote skilled movement recovery and repair of the corticospinal tract (CST) after a moderately severe C4 midline contusion in adult rats. We used bilateral epidural intermittent theta burst (iTBS) electrical stimulation of motor cortex to promote CST axonal sprouting and cathodal trans-spinal direct current stimulation (tsDCS) to enhance spinal cord activation to motor cortex stimulation after injury. We used Finite Element Method (FEM) modeling to direct tsDCS to the cervical enlargement. Combined iTBS-tsDCS was delivered for 30min daily for 10days. We compared the effect of stimulation on performance in the horizontal ladder and the Irvine Beattie and Bresnahan forepaw manipulation tasks and CST axonal sprouting in injury-only and injury+stimulation animals. The contusion eliminated the dorsal CST in all animals. tsDCS significantly enhanced motor cortex evoked responses after C4 injury. Using this combined spinal-M1 neuromodulatory approach, we found significant recovery of skilled locomotion and forepaw manipulation skills compared with injury-only controls. The spared CST axons caudal to the lesion in both animal groups derived mostly from lateral CST axons that populated the contralateral intermediate zone. Stimulation enhanced injury-dependent CST axonal outgrowth below and above the level of the injury. This dual neuromodulatory approach produced partial recovery of skilled motor behaviors that normally require integration of posture, upper limb sensory information, and intent for performance. We propose that the motor systems use these new CST projections to control movements better after injury.

To promote skilled forelimb function following a spinal cord injury, we have evaluated whether long-term voluntary sensorimotor rehabilitation can promote substantial reaching and grasping recovery. Long-Evans rats were trained to reach single pellets and then received a moderate 100 kdyn contusion to the C5 lateral funiculi. During the first eight months post-injury, a group of animals was enrolled in daily skilled reaching rehabilitation consisting of grabbing and manipulating seeds from the bottom of a grid. Single-pellet reaching and grasping recovery was tested biweekly throughout the functional follow-up and the recovery was compared to a second group of contused but non-rehabilitated animals. Following the injury, reaching and grasping success dropped to zero in both groups and remained absent for three months post-injury, followed by a slight recovery that remained constant until the end of the follow-up. No differences in reaching success were found between groups. Nevertheless, the type of gesture errors in the failed attempts were categorized and scored. The errors ranged from the animal's inability to lift the paw and initiate the movement to the final stage of the attempt, in which the pellet falls during grasping and retraction of the paw towards the mouth. Both groups of animals exhibited similar types of errors but the animals with rehabilitation showed less error variability and those that occurred at the latest stages of the attempt predominated compared to those performed by the non-trained animals. Histological examination of the injury showed that injury severity was similar between groups and that the damage was circumscribed to the site of impact, affecting mainly the dorsal and medial region of the lateral funiculi, with preservation of the dorsal component of the corticospinal tract and the interneurons and motoneurons of the spinal segments beyond the site of injury. The results indicate that activity-dependent plasticity driven by voluntary rehabilitation decreases task error variability and drives the recovery of the movement gestures. However, the plasticity achieved is insufficient to attain full functional recovery to successfully reach, grasp and release the pellets in the mouth, indicating the necessity for additional interventional therapies to promote repair.

Mid-cervical spinal cord contusion disrupts both the pathways and motoneurons vital to the activity of inspiratory muscles. The present study was designed to determine if a rat contusion model could result in a measurable deficit to both ventilatory and respiratory motor function under "normal" breathing conditions at acute to chronic stages post trauma. Through whole body plethysmography and electromyography we assessed respiratory output from three days to twelve weeks after a cervical level 3 (C3) contusion. Contused animals showed significant deficits in both tidal and minute volumes which were sustained from acute to chronic time points. We also examined the degree to which the contusion injury impacted ventilatory pattern variability through assessment of Mutual Information and Sample Entropy. Mid-cervical contusion significantly and robustly decreased the variability of ventilatory patterns. The enduring deficit to the respiratory motor system caused by contusion was further confirmed through electromyography recordings in multiple respiratory muscles. When isolated via a lesion, these contused pathways were insufficient to maintain respiratory activity at all time points post injury. Collectively these data illustrate that, counter to the prevailing literature, a profound and lasting ventilatory and respiratory motor deficit may be modelled and measured through multiple physiological assessments at all time points after cervical contusion injury.

Cervical spinal cord injury (SCI) impairs arm and hand function largely by interrupting descending tracts. Most SCI spare some axons at the lesion, including the corticospinal tract (CST), which is critical for voluntary movement. We targeted descending motor connections with paired electrical stimulation of motor cortex and cervical spinal cord in the rat. We sought to replicate the previously published effects of intermittent theta burst stimulation of forelimb motor cortex combined with trans-spinal direct current stimulation placed on the skin over the neck to target the cervical enlargement. We hypothesized that paired stimulation would improve performance in skilled walking and food manipulation (IBB) tasks. Rats received a moderate C4 spinal cord contusion injury (200 kDynes), which ablates the main CST. They were randomized to receive paired stimulation for 10 consecutive days starting 11 days after injury, or no stimulation. Behavior was assessed weekly from weeks 4-7 after injury, and then CST axons were traced. Rats with paired cortical and spinal stimulation achieved significantly better forelimb motor function recovery, as measured by fewer stepping errors on the horizontal ladder task (34 ± 9% in stimulation group vs. 51 ± 18% in control, p = .013) and higher scores on the food manipulation task (IBB, 0-9 score; 7.2 ± 0.8 in stimulated rats vs. 5.2 ± 2.6 in controls, p = .025). The effect size for both tasks was large (Cohen's d = 1.0 and 0.92, respectively). The CST axon length in the cervical spinal cord did not differ significantly between the groups, but there was denser and broader ipsilateral axons distribution distal to the spinal cord injury. The large behavioral effect and replication in an independent laboratory validate this approach, which will be trialed in cats before being tested in people using non-invasive methods.

Following cervical spinal cord injury at C(2) (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability.

Unilateral cervical spinal cord hemisection at C2 (C2SH) interrupts descending bulbospinal inputs to phrenic motoneurons, paralyzing the diaphragm muscle. Recovery after C2SH is enhanced by brain derived neurotrophic factor (BDNF) signaling via the tropomyosin-related kinase subtype B (TrkB) receptor in phrenic motoneurons. The role for gene therapy using adeno-associated virus (AAV)-mediated delivery of TrkB to phrenic motoneurons is not known. The present study determined the therapeutic efficacy of intrapleural delivery of AAV7 encoding for full-length TrkB (AAV-TrkB) to phrenic motoneurons 3 days post-C2SH. Diaphragm EMG was recorded chronically in male rats (n=26) up to 21 days post-C2SH. Absent ipsilateral diaphragm EMG activity was verified 3 days post-C2SH. A greater proportion of animals displayed recovery of ipsilateral diaphragm EMG activity during eupnea by 14 and 21 days post-SH after AAV-TrkB (10/15) compared to AAV-GFP treatment (2/11; p=0.031). Diaphragm EMG amplitude increased over time post-C2SH (p<0.001), and by 14 days post-C2SH, AAV-TrkB treated animals displaying recovery achieved 48% of the pre-injury values compared to 27% in AAV-GFP treated animals. Phrenic motoneuron mRNA expression of glutamatergic AMPA and NMDA receptors revealed a significant, positive correlation (r(2)=0.82), with increased motoneuron NMDA expression evident in animals treated with AAV-TrkB and that displayed recovery after C2SH. Overall, gene therapy using intrapleural delivery of AAV-TrkB to phrenic motoneurons is sufficient to promote recovery of diaphragm activity, adding a novel potential intervention that can be administered after upper cervical spinal cord injury to improve impaired respiratory function.

Schwann cells (SCs) hold promise for spinal cord injury (SCI) repair; however, there are limitations for its use as a lone treatment. We showed that acute inhibition of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) by bisperoxovanadium (bpV) was neuroprotective and enhanced function following cervical hemicontusion SCI. We hypothesized that combining acute bpV therapy and delayed SC engraftment would further improve neuroprotection and recovery after cervical SCI. Adult female Sprague-Dawley (SD) rats were randomly sorted into 5 groups: sham, vehicle, bpV, SC transplantation, and bpV+SC transplantation. SCs were isolated from adult green fluorescent protein (GFP)-expressing SD rats (GFP-SCs). 200 μg/kg bpV(pic) was administered intraperitoneally (IP) twice daily for 7 days post-SCI in bpV-treated groups. GFP-SCs (1×10(6) in 5 μl medium) were transplanted into the lesion epicenter at the 8th day post-SCI. Forelimb function was tested for 10 weeks and histology was assessed. bpV alone significantly reduced lesion (by 40%, p<0.05) and cavitation (by 65%, p<0.05) and improved functional recovery (p<0.05) compared to injury alone. The combination promoted similar neuroprotection (p<0.01 vs. injury); however, GFP-SCs alone did not. Both SC-transplanted groups exhibited remarkable long-term SC survival, SMI-31(+) axon ingrowth and RECA-1(+) vasculature presence in the SC graft; however, bpV+SCs promoted an 89% greater axon-to-lesion ratio than SCs only. We concluded that bpV likely contributed largely to the neuroprotective and functional benefits while SCs facilitated considerable host-tissue interaction and modification. The combination of the two shows promise as an attractive strategy to enhance recovery after SCI.

Neuroplasticity following spinal cord injury contributes to spontaneous recovery over time. Recent studies highlight the important role of brain-derived neurotrophic factor (BDNF) signaling via the high-affinity tropomyosin-related kinase (Trk) receptor subtype B (TrkB) in recovery of rhythmic diaphragm activity following unilateral spinal hemisection at C2 (C2SH). We hypothesized that TrkB kinase activity is necessary for spontaneous recovery of diaphragm activity post-C2SH. A chemical-genetic approach employing adult male TrkB(F616A) mice (n=49) was used to determine the impact of inhibiting TrkB kinase activity by the phosphoprotein phosphatase 1 inhibitor derivative 1NMPP1 on recovery of ipsilateral hemidiaphragm EMG activity. In mice, C2SH was localized primarily to white matter tracts comprising the lateral funiculus. The extent of damaged spinal cord (~27%) was similar regardless of the presence of functional recovery, consistent with spontaneous recovery reflecting neuroplasticity primarily of contralateral spared descending pathways to the phrenic motor pools. Ipsilateral hemidiaphragm EMG activity was verified as absent in all mice at 3days post-C2SH. By 2weeks after C2SH, ipsilateral hemidiaphragm EMG activity was present in 39% of vehicle-treated mice compared to 7% of 1NMPP1-treated mice (P=0.03). These data support the hypothesis that BDNF/TrkB signaling involving TrkB kinase activity plays a critical role in spontaneous recovery of diaphragm activity following cervical spinal cord injury.

Despite extensive gray matter loss following spinal cord injury (SCI), little attention has been given to neuronal replacement strategies and their effects on specific functional circuits in the injured spinal cord. In the present study, we assessed breathing behavior and phrenic nerve electrophysiological activity following transplantation of microdissected dorsal or ventral pieces of rat fetal spinal cord tissue (FSC(D) or FSC(V), respectively) into acute, cervical (C2) spinal hemisections. Transneuronal tracing demonstrated connectivity between donor neurons from both sources and the host phrenic circuitry. Phrenic nerve recordings revealed differential effects of dorsally vs. ventrally derived neural progenitors on ipsilateral phrenic nerve recovery and activity. These initial results suggest that local gray matter repair can influence motoneuron function in targeted circuits following spinal cord injury and that outcomes will be dependent on the properties and phenotypic fates of the donor cells employed.

Both glibenclamide and riluzole reduce necrosis and improve outcome in rat models of spinal cord injury (SCI). In SCI, gene suppression experiments show that newly upregulated sulfonylurea receptor 1 (Sur1)-regulated NC(Ca-ATP) channels in microvascular endothelial cells are responsible for "persistent sodium currents" that cause capillary fragmentation and "progressive hemorrhagic necrosis". Glibenclamide is a potent blocker of Sur1-regulated NC(Ca-ATP) channels (IC(50), 6-48 nM). Riluzole is a pleotropic drug that blocks "persistent sodium currents" in neurons, but in SCI, its molecular mechanism of action is uncertain. We hypothesized that riluzole might block the putative pore-forming subunits of Sur1-regulated NC(Ca-ATP) channels, Trpm4. In patch clamp experiments, riluzole blocked Sur1-regulated NC(Ca-ATP) channels in endothelial cells and heterologously expressed Trpm4 (IC(50), 31 μM). Using a rat model of cervical SCI associated with high mortality, we compared the effects of glibenclamide and riluzole administered beginning at 3h and continuing for 7 days after impact. During the acute phase, both drugs reduced capillary fragmentation and progressive hemorrhagic necrosis, and both prevented death. At 6 weeks, modified (unilateral) Basso, Beattie, Bresnahan locomotor scores were similar, but measures of complex function (grip strength, rearing, accelerating rotarod) and tissue sparing were significantly better with glibenclamide than with riluzole. We conclude that both drugs act similarly, glibenclamide on the regulatory subunit, and riluzole on the putative pore-forming subunit of the Sur1-regulated NC(Ca-ATP) channel. Differences in specificity, dose-limiting potency, or in spectrum of action may account for the apparent superiority of glibenclamide over riluzole in this model of severe SCI.

Spinal cord injury (SCI) causes an interruption of descending motor and autonomic nervous tracts. However, a partial injury, and particularly a unilateral section, is generally followed by spontaneous locomotor and respiratory recovery. Although locomotor functional recovery has been correlated to spontaneous anatomical plasticity of the corticospinal tract, the remodeling of the bulbospinal tract that sustains respiratory improvement is unknown and has therefore been investigated here after chronic lateral cervical injury in rats (90 days post-lesion by comparison to 7 days post-lesion). We show that chronic lateral C2 SCI leads both to a decreased thickness of the ipsilateral ventrolateral funiculus at sus and sub-lesional levels and to an opposite effect on the contralateral side. At C1 level, the number of ventrolateral bulbospinal fibers, stained with anterograde tracer was reduced within the ipsilateral ventrolateral funiculi while collateral arborization toward the gray matter and growth associated protein-43 levels was increased. At C2 lesional level, fibers rerouting toward the gray matter were also identified for 5% of the axotomized axon terminals. Despite these chronic sprouting processes respiratory bulbospinal projections to ipsilateral phrenic nucleus remained poor (less than 10% compared to non-injured conditions). Retrograde labeling of projections onto the phrenic nucleus revealed, after chronic injury, an increased recruitment of C1 propriospinal interneurons which moreover received more contacts from bulbospinal collaterals. This chronic remodeling was correlated with chronic diaphragm recovery under conditions of respiratory stress. Thus, despite extensive axonal loss and absence of direct phrenic reinnervation by bulbospinal respiratory neurons, sprouting processes toward cervical propriospinal neurons may contribute to the observed partial respiratory recovery.

Following spinal cord injury (SCI), intraspinal transplantation of neural progenitor cells (NPCs) harvested from the forebrain sub-ventricular zone (SVZ) can improve locomotor outcomes. Cervical SCI often results in respiratory-related impairments, and here we used an established model cervical SCI (C2 hemisection, C2Hx) to confirm the feasibility of mid-cervical transplantation of SVZ-derived NPCs and the hypothesis that that this procedure would improve spontaneous respiratory motor recovery. NPCs were isolated from the SVZ of enhanced green fluorescent protein (GFP) expressing neonatal rats, and then intraspinally delivered immediately caudal to an acute C2Hx lesion in adult non-GFP rats. Whole body plethysmography conducted at 4 and 8wks post-transplant demonstrated increased inspiratory tidal volume in SVZ vs. sham transplants during hypoxic (P=0.003) or hypercapnic respiratory challenge (P=0.019). Phrenic nerve output was assessed at 8wks post-transplant; burst amplitude recorded ipsilateral to C2Hx was greater in SVZ vs. sham rats across a wide range of conditions (e.g., quiet breathing through maximal chemoreceptor stimulation; P<0.001). Stereological analyses at 8wks post-injury indicated survival of ~50% of transplanted NPCs with ~90% of cells distributed in ipsilateral white matter at or near the injection site. Peak inspiratory phrenic bursting after NPC transplant was positively correlated with the total number of surviving cells (P<0.001). Immunohistochemistry confirmed an astrocytic phenotype in a subset of the transplanted cells with no evidence for neuronal differentiation. We conclude that intraspinal transplantation of SVZ-derived NPCs can improve respiratory recovery following high cervical SCI.

Task specific rehabilitation training is commonly used to treat motor dysfunction after neurological injures such as spinal cord injury (SCI), yet the use of task specific training in preclinical animal studies of SCI is not common. This is due in part to the difficulty in training animals to perform specific motor tasks, but also due to the lack of knowledge about optimal rehabilitation training parameters to maximize recovery. The single pellet reaching, grasping and retrieval (SPRGR) task (a.k.a. single pellet reaching task or Whishaw task) is a skilled forelimb motor task used to provide rehabilitation training and test motor recovery in rodents with cervical SCI. However, the relationships between the amount, duration, intensity, and timing of training remain poorly understood. In this study, using automated robots that allow rats with cervical SCI ad libitum access to self-directed SPRGR rehabilitation training, we show clear relationships between the total amount of rehabilitation training, the intensity of training (i.e., number of attempts/h), and performance in the task. Specifically, we found that rats naturally segregate into High and Low performance groups based on training strategy and performance in the task. Analysis of the different training strategies showed that more training (i.e., increased number of attempts in the SPRGR task throughout rehabilitation training) at higher intensities (i.e., number of attempts per hour) increased performance in the task, and that improved performance in the SPRGR task was linked to differences in corticospinal tract axon collateral densities in the injured spinal cords. Importantly, however, our data also indicate that rehabilitation training becomes progressively less efficient (i.e., less recovery for each attempt) as both the amount and intensity of rehabilitation training increases. Finally, we found that Low performing animals could increase their training intensity and transition to High performing animals in chronic SCI. These results highlight the rehabilitation training strategies that are most effective to regain skilled forelimb motor function after SCI, which will facilitate pre-clinical rehabilitation studies using animal models and could be beneficial in the development of more efficient clinical rehabilitation training strategies.