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Thoracic spinal cord injury affects long propriospinal neurons that interconnect the cervical and lumbar enlargements. These neurons are crucial for coordinating forelimb and hindlimb movements in a speed-dependent manner. At the same time, recovery from spinal cord injury is usually studied over a very limited range of speeds. To overcome this limitation, we investigated overground locomotion in rats over the full range of speeds pre-injury and after recovery from thoracic hemisection or contusion injuries. Intact rats expressed a speed-dependent continuum of alternating (walk and trot) and non-alternating (canter, gallop, half-bound gallop, and bound) gaits. After a lateral hemisection injury, rats recovered the ability to locomote over a wide range of speeds but lost the ability to move using the highest-speed gaits (half-bound gallop and bound) and predominantly used the limb contralateral to injury as lead during canter and gallop. A moderate contusion injury resulted in a greater reduction in maximal speed, loss of non-alternating gaits, and emergence of novel alternating gaits. Analysis of the gait structure in phase space and the variability of limb coupling suggests substantial reorganization of intraspinal pathways mediating interlimb coordination following recovery of locomotion after lateral hemisection or midline contusion injuries. This reorganization appears as an increased reliance on local enlargement circuitry and suggests that speed-dependent gait changes in the forelimbs rely on ascending information from the lumbar circuitry that is impaired post-injury. These observations highlight how investigating the full repertoire of locomotor speeds can reveal otherwise hidden aspects of spinal circuitry and post-injury reorganization.

Spinal cord injury (SCI) is a debilitating disease resulting in an estimated 18,000 new cases in the United States on an annual basis. Significant behavioral research on animal models has led to a large amount of data, some of which has been catalogued in the Open Data Commons for Spinal Cord Injury (ODC-SCI). More recently, high throughput sequencing experiments have been utilized to understand molecular mechanisms associated with SCI, with nearly 6,000 samples from over 90 studies available in the Sequence Read Archive. However, to date, no resource is available for efficiently mining high throughput sequencing data from SCI experiments. Therefore, we have developed a protocol for processing RNA-Seq samples from high-throughput sequencing experiments related to SCI resulting in both raw and normalized data that can be efficiently mined for comparisons across studies as well as homologous discovery across species. We have processed 1,196 publicly available RNA-seq samples from 50 bulk RNA-Seq studies across nine different species, resulting in an SQLite database that can be used by the SCI research community for further discovery. We provide both the database as well as a web-based front-end that can be used to query the database for genes of interest, differential gene expression, genes with high variance, and gene set enrichments.

Preclinical pilot study. To test the hypothesis that spinal opioidergic circuitry contributes to muscle stretch-induced locomotor deficits. Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA. A pilot study with eight female Sprague-Dawley rats that received 25 g-cm T10 contusion injuries and recovered for 5 weeks. Rats were divided into two groups with one group receiving subcutaneous injections of naltrexone dissolved in saline (15 mg/kg) or an equal volume of saline. Each group received a daily 24-minute stretching protocol during weeks 6, 8, and 11 post-injury. Locomotor function was assessed throughout using the BBB Open Field Locomotor Scale. Consistent with previous findings, stretching reduced locomotor function in both naltrexone and saline groups. However, the loss of locomotor function appeared earlier in the naltrexone group. Animals in both groups had a similar rate of recovery following the termination of stretching. Interestingly, the administration of naltrexone did not influence acute thermal cutaneous nociceptive responses as measured by a tail-flick assay but caused a significant increase in spasticity following stretch. The results of this study suggest that the endogenous opioid system plays a role in modulating the negative impact of muscle stretch on spinal cord motor circuitry that is vulnerable due to loss of descending input. The observed actions of the broad-spectrum opioid antagonist naltrexone imply that pharmaceuticals targeting the endogenous opioid system post-SCI may have unintended consequences.

Spinal interneurons are important facilitators and modulators of motor, sensory, and autonomic functions in the intact CNS. This heterogeneous population of neurons is now widely appreciated to be a key component of plasticity and recovery. This review highlights our current understanding of spinal interneuron heterogeneity, their contribution to control and modulation of motor and sensory functions, and how this role might change after traumatic spinal cord injury. We also offer a perspective for how treatments can optimize the contribution of interneurons to functional improvement.

Yucatan miniature pigs (YMPs) are similar to humans in spinal cord size as well as physiological and neuroanatomical features, making them a useful model for human spinal cord injury. However, little is known regarding pig gait kinematics, especially on a treadmill. In this study, 12 healthy YMPs were assessed during bipedal and/or quadrupedal stepping on a treadmill at six speeds (1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 km/h). Kinematic parameters, including limb coordination and proximal and distal limb angles, were measured. Findings indicate that YMPs use a lateral sequence footfall pattern across all speeds. Stride and stance durations decreased with increasing speed whereas swing duration showed no significant change. Across all speeds assessed, no significant differences were noted between hindlimb stepping parameters for bipedal or quadrupedal gait with the exception of distal limb angular kinematics. Specifically, significant differences were observed between locomotor tasks during maximum flexion (quadrupedal > bipedal), total excursion (bipedal > quadrupedal), and the phase relationship between the timing of maximum extension between the right and left hindlimbs (bipedal > quadrupedal). Speed also impacted maximum flexion and right-left phase relationships given that significant differences were found between the fastest speed (3.5 km/h) relative to each of the other speeds. This study establishes a methodology for bipedal and quadrupedal treadmill-based kinematic testing in healthy YMPs. The treadmill approach used was effective in recruiting primarily the spinal circuitry responsible for the basic stepping patterns as has been shown in cats. We recommend 2.5 km/h (0.7 m/sec) as a target walking gait for pre-clinical studies using YMPs, which is similar to that used in cats.

Neurons within the spinal cord are sensitive to environmental relations and can bring about a behavioral modification without input from the brain. For example, rats that have undergone a thoracic (T2) transection can learn to maintain a hind leg in a flexed position to minimize exposure to a noxious electrical stimulation (shock). Inactivating neurons within the spinal cord with lidocaine, or cutting communication between the spinal cord and the periphery (sciatic transection), eliminates the capacity to learn, which implies that it depends on spinal neurons. Here we show that these manipulations have no effect on the maintenance of the learned response, which implicates a peripheral process. EMG showed that learning augments the muscular response evoked by motoneuron output and that this effect survives a sciatic transection. Quantitative fluorescent imaging revealed that training brings about an increase in the area and intensity of ACh receptor labeling at the neuromuscular junction (NMJ). It is hypothesized that efferent motoneuron output, in conjunction with electrical stimulation of the tibialis anterior muscle, strengthens the connection at the NMJ in a Hebbian manner. Supporting this, paired stimulation of the efferent nerve and tibialis anterior generated an increase in flexion duration and augmented the evoked electrical response without input from the spinal cord. Evidence is presented that glutamatergic signaling contributes to plasticity at the NMJ. Labeling for vesicular glutamate transporter is evident at the motor endplate. Intramuscular application of an NMDAR antagonist blocked the acquisition/maintenance of the learned response and the strengthening of the evoked electrical response.SIGNIFICANCE STATEMENTThe neuromuscular junction (NMJ) is designed to faithfully elicit a muscular contraction in response to neural input. From this perspective, encoding environmental relations (learning) and the maintenance of a behavioral modification over time (memory) are assumed to reflect only modifications upstream from the NMJ, within the CNS. The current results challenge this view. Rats were trained to maintain a hind leg in a flexed position to avoid noxious stimulation. As expected, treatments that inhibit activity within the CNS, or disrupt peripheral communication, prevented learning. These manipulations did not affect the maintenance of the acquired response. The results imply that a peripheral modification at the NMJ contributes to the maintenance of the learned response.

Over the last 5 years, multiple stakeholders in the field of spinal cord injury (SCI) research have initiated efforts to promote publications standards and enable sharing of experimental data. In 2016, the National Institutes of Health/National Institute of Neurological Disorders and Stroke hosted representatives from the SCI community to streamline these efforts and discuss the future of data sharing in the field according to the FAIR (Findable, Accessible, Interoperable and Reusable) data stewardship principles. As a next step, a multi-stakeholder group hosted a 2017 symposium in Washington, DC entitled "FAIR SCI Ahead: the Evolution of the Open Data Commons for Spinal Cord Injury research." The goal of this meeting was to receive feedback from the community regarding infrastructure, policies, and organization of a community-governed Open Data Commons (ODC) for pre-clinical SCI research. Here, we summarize the policy outcomes of this meeting and report on progress implementing these policies in the form of a digital ecosystem: the Open Data Commons for Spinal Cord Injury (ODC-SCI.org). ODC-SCI enables data management, harmonization, and controlled sharing of data in a manner consistent with the well-established norms of scholarly publication. Specifically, ODC-SCI is organized around virtual "laboratories" with the ability to share data within each of three distinct data-sharing spaces: within the laboratory, across verified laboratories, or publicly under a creative commons license (CC-BY 4.0) with a digital object identifier that enables data citation. The ODC-SCI implements FAIR data sharing and enables pooled data-driven discovery while crediting the generators of valuable SCI data.

Long ascending propriospinal neurons (LAPNs) are a subpopulation of spinal cord interneurons that directly connect the lumbar and cervical enlargements. In uninjured animals, conditionally silencing LAPNs resulted in disrupted left-right coordination of the hindlimbs and forelimbs in a context-dependent manner, demonstrating that LAPNs secure alternation of the fore- and hindlimb pairs during overground stepping in the adult rat. Given their ventrolateral location in the spinal cord white matter, many LAPN axons likely remain intact following thoracic spinal cord injury (SCI), suggesting a potential role in the recovery of stepping. Thus, we hypothesized that silencing LAPNs after SCI would result in diminished hindlimb locomotor function. We found instead that silencing of spared LAPNs post-SCI restored the left-right hindlimb coordination associated with alternating gaits that was lost as a result of SCI. Several other fundamental characteristics of hindlimb stepping were also improved and the number of abnormal steps were reduced. However, hindlimb-forelimb coordination was not restored. These data suggest that the temporal information carried between the enlargements by the LAPNs after SCI may be detrimental to hindlimb locomotor function. These observations have implications for our understanding of the relationship between injury severity and functional outcome, for the efforts to develop neuro- and axo-protective therapeutic strategies, and also for the clinical study/implementation of spinal stimulation and neuromodulation.

Hemodynamic instability and cardiovascular (CV) dysfunction are hallmarks of patients living with cervical and high thoracic spinal cord injuries (SCI). Individuals experience bouts of autonomic dysreflexia (AD) and persistent hypotension which hamper the activities of daily living. Despite the widespread use of exercise training to improve health and CV function after SCI, little is known about how different training modalities impact hemodynamic stability and severity of AD in a model of incomplete SCI. In this study, we used implantable telemetry devices to assess animals with T2 contusions following 3.5 weeks of exercise training initiated 8 days post‐injury: passive hindlimb cycling (T2‐CYC, n = 5) or active forelimb swimming (T2‐SW, n = 6). Uninjured and non‐exercised SCI control groups were also included (CON, n = 6; T2‐CON, n = 7; T10‐CON, n = 6). Five weeks post‐injury, both T2‐CON and T2‐CYC presented with resting hypotension compared to uninjured CON and T10‐CON groups; no differences were noted in resting blood pressure in T2‐SW versus CON and T10‐CON. Furthermore, pressor responses to colorectal distention (AD) were larger in all T2‐injured groups compared to T10‐CON, and were not attenuated by either form of exercise training. Interestingly, when T2‐injured animals were re‐stratified based on terminal BBB scores (regardless of training group), animals with limited hindlimb recovery (T2‐LOW, n = 7) had more severe AD responses. Our results suggest that the spontaneous recovery of locomotor and autonomic function after severe but incomplete T2 SCI also influences the severity of AD, and that short periods (3.5 weeks) of passive hindlimb cycling or active forelimb swimming are ineffective in this model., Using a high‐thoracic, severe but incomplete model of spinal cord injury we found that passive cycling and swimming, initiated early post‐injury, do not significantly alter the severity of induced autonomic dysreflexia. We discovered that animals with the greatest recovery of hindlimb locomotor function had the smallest pressor responses during induced autonomic dysreflexia.

Multi-organ dysfunction is a major complication after spinal cord injury (SCI). In addition to local injury within the spinal cord, SCI causes major disruption to the peripheral organ innervation and regulation. The liver contains sympathetic, parasympathetic, and small sensory axons. The bi-directional signaling of sensory dorsal root ganglion (DRG) neurons that provide both efferent and afferent information is of key importance as it allows sensory neurons and peripheral organs to affect each other. SCI-induced liver inflammation precedes and may exacerbate intraspinal inflammation and pathology after SCI, which may be modulated by activity and exercise. In this study, we collected comprehensive gene expression data through RNA sequencing of liver tissue from rats with chronic SCI to determine the effects of activity and exercise on those expression patterns. The sequenced data are of high quality and show a high alignment rate to the Rn6 genome. Gene expression is demonstrated for genes associated with known liver pathologies. UCSC Genome Browser expression tracks are provided with the data to facilitate exploration of the samples., Design Type(s)gene expression analysis objective • stimulus or stress design • transcription profiling designMeasurement Type(s)gene expressionTechnology Type(s)RNA sequencingFactor Type(s)Spinal Cord Injury • Exercise • EnvironmentalVariable • biological replicateSample Characteristic(s)Rattus norvegicus • liver Machine-accessible metadata file describing the reported data (ISA-Tab format)

Within the cervical and lumbar spinal enlargements, central pattern generator (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that inter-connect the lumbar and cervical CPGs disrupts left-right limb coupling of each limb pair in the adult rat during overground locomotion on a high-friction surface. These perturbations occurred independent of the locomotor rhythm, intralimb coordination, and speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. These data reveal surprising flexibility and context-dependence in the control of interlimb coordination during locomotion.

Individuals with spinal cord injury (SCI) have been shown to exhibit systolic, and to a lesser extent, diastolic cardiac dysfunction. However, previous reports of cardiac dysfunction in this population are confounded by the changing loading conditions after SCI and as such, whether cardiac dysfunction per se is present is still unknown. Therefore, our aim was to establish if load-independent cardiac dysfunction is present after SCI, to understand the functional cardiac response to SCI, and to explore the changes within the cellular milieu of the myocardium. Here, we applied in vivo echocardiography and left-ventricular (LV) pressure-volume catheterization with dobutamine infusions to our Wistar rodent model of cardiac dysfunction 5 weeks following high (T2) thoracic contusion SCI, while also examining the morphological and transcriptional alterations of cardiomyocytes. We found that SCI significantly impairs systolic function independent of loading conditions (end-systolic elastance in control: 1.35 ± 0.15; SCI: 0.65 ± 0.19 mm Hg/μL). The reduction in contractile indices is accompanied by a reduction in width and length of cardiomyocytes as well as alterations in the LV extracellular matrix. Importantly, we demonstrate that the reduction in the rate (dP/dt(max)) of LV pressure rise can be offset with beta-adrenergic stimulation, thereby experimentally implicating the loss of descending sympatho-excitatory control of the heart as a principle cause of LV dysfunction in SCI. Our data provide evidence that SCI induces systolic cardiac dysfunction independent of loading conditions and concomitant cardiomyocyte atrophy that may be underpinned by changes in the genes regulating the cardiac extracellular matrix.

IntroductionActivity-based therapy (ABT) is an important aspect of rehabilitation following traumatic spinal cord injury (SCI). Unfortunately, it has never been adapted to acute care despite compelling preclinical evidence showing that it is safe and effective for promoting neurological recovery when started within days after SCI. This article provides the protocol for a study that will determine the feasibility and explore potential benefits of early ABT in the form of in-bed leg cycling initiated within 48 hours after the end of spinal surgery for SCI.Methods and analysisPROMPT-SCI (protocol for rapid onset of mobilisation in patients with traumatic SCI) is a single-site single-arm proof-of-concept trial. Forty-five patients aged 18 years or older with a severe traumatic SCI (American Spinal Injury Association Impairment Scale grade A, B or C) from C0 to L2 undergoing spinal surgery within 48 hours of the injury will be included. Participants will receive daily 30 min continuous sessions of in-bed leg cycling for 14 consecutive days, initiated within 48 hours of the end of spinal surgery. The feasibility outcomes are: (1) absence of serious adverse events associated with cycling, (2) completion of 1 full session within 48 hours of spinal surgery for 90% of participants and (3) completion of 11 sessions for 80% of participants. Patient outcomes 6 weeks and 6 months after the injury will be measured using neurofunctional assessments, quality of life questionnaires and inpatient length of stay. Feasibility and patient outcomes will be analysed with descriptive statistics. Patient outcomes will also be compared with a matched historical cohort that has not undergone in-bed cycling using McNemar and Student’s t-tests for binary and continuous outcomes, respectively.Ethics and disseminationPROMPT-SCI is approved by the Research Ethics Board of the CIUSSS NIM. Recruitment began in April 2021. Dissemination strategies include publications in scientific journals and presentations at conferences.Trial registration numberNCT04699474.

Injury to descending autonomic (sympathetic) pathways is common after high-level spinal cord injury (SCI) and associated with abnormal blood pressure and heart rate regulation. In individuals with high-level SCI, abnormal sympathovagal balance (such as during autonomic dysreflexia; paroxysmal hypertension provoked by sensory stimuli below the injury) is proarrhythmogenic. Exercise training is a key component of SCI rehabilitation and management of cardiovascular disease risk, but it is unclear whether exercise training influences susceptibility to cardiac arrhythmia. We aimed to evaluate: (i) whether susceptibility to arrhythmia increases in a rodent-model of SCI; (ii) the impact of the sympathomimetic drug dobutamine (DOB) on arrhythmia risk; (iii) whether exercise training ameliorates arrhythmia risk. Twenty-one Wistar rats were divided into 3 subgroups: T2-contusive SCI (T2, n = 7), T2-contusive SCI completing passive hindlimb cycling training (PHLC, n = 7), and T10-contusive SCI (T10, n = 7). Known electrocardiographic arrhythmia markers and heart rate variability parameters were evaluated before (PRE), 1-week (POST) and 5-weeks post-SCI (TERM) at baseline and during DOB infusion (30 μg/kg/min). Baseline markers of arrhythmia risk were increased in both T2 and T10 animals. DOB decreased R-R interval (p < 0.001), and increased markers of risk for ventricular arrhythmia, particularly in high-level (T2) animals (p < 0.05). Exercise training blunted the exacerbation of markers of arrhythmia risk in the presence of DOB. Markers of risk for cardiac arrhythmia are increased in experimental SCI, and DOB further increases arrhythmia risk in high-level SCI. Exercise training did not improve markers of arrhythmia risk at rest, but did ameliorate markers of arrhythmia risk during sympathetic stimulation.

After spinal cord injury (SCI) muscle contractures develop in the plegic limbs of many patients. Physical therapists commonly use stretching as an approach to avoid contractures and to maintain the extensibility of soft tissues. We found previously that a daily stretching protocol has a negative effect on locomotor recovery in rats with mild thoracic SCI. The purpose of the current study was to determine the effects of stretching on locomotor function at acute and chronic time points after moderately severe contusive SCI. Female Sprague-Dawley rats with 25 g-cm T10 contusion injuries received our standard 24-min stretching protocol starting 4 days (acutely) or 10 weeks (chronically) post-injury (5 days/week for 5 or 4 weeks, respectively). Locomotor function was assessed using the BBB (Basso, Beattie, and Bresnahan) Open Field Locomotor Scale, video-based kinematics, and gait analysis. Locomotor deficits were evident in the acute animals after only 5 days of stretching and increasing the perceived intensity of stretching at week 4 resulted in greater impairment. Stretching initiated chronically resulted in dramatic decrements in locomotor function because most animals had BBB scores of 0-3 for weeks 2, 3, and 4 of stretching. Locomotor function recovered to control levels for both groups within 2 weeks once daily stretching ceased. Histological analysis revealed no apparent signs of overt and persistent damage to muscles undergoing stretching. The current study extends our observations of the stretching phenomenon to a more clinically relevant moderately severe SCI animal model. The results are in agreement with our previous findings and further demonstrate that spinal cord locomotor circuitry is especially vulnerable to the negative effects of stretching at chronic time points. While the clinical relevance of this phenomenon remains unknown, we speculate that stretching may contribute to the lack of locomotor recovery in some patients.

Reduced muscle mass and increased fatiguability are major complications after spinal cord injury (SCI), and often hinder the rehabilitation efforts of patients. Such detriments to the musculoskeletal system, and the concomitant reduction in level of activity, contribute to secondary complications such as cardiovascular disease, diabetes, bladder dysfunction and liver damage. As a result of decreased weight-bearing capacity after SCI, muscles undergo morphological, metabolic, and contractile changes. Recent studies have shown that exercise after SCI decreases muscle wasting and reduces the burden of secondary complications. Here, we describe RNA sequencing data for detecting chronic transcriptomic changes in the rat soleus after SCI at two levels of injury severity, under conditions of restricted in-cage activity and two methods of applied exercise, swimming or shallow water walking. We demonstrate that the sequenced data are of good quality and show a high alignment rate to the Rattus norvegicus reference assembly (Rn6). The raw data, along with UCSC Genome Browser tracks created to facilitate exploration of gene expression, are available in the NCBI Gene Expression Omnibus (GEO; GSE129694).

Spinal cord injury (SCI) is a devastating clinical condition resulting in significant disabilities. Apart from local injury within the spinal cord, SCI patients develop a myriad of complications including multi-organ dysfunction. Some of the dysfunctions may be directly or indirectly related to the sensory neurons of the dorsal root ganglia (DRG), which signal to both the spinal cord and the peripheral organs. After SCI, some classes of DRG neurons exhibit sensitization and undergo axonal sprouting both peripherally and centrally. Such physiological and anatomical re-organization after SCI contributes to both adaptive and maladaptive plasticity processes, which may be modulated by activity and exercise. In this study, we collected comprehensive gene expression data in whole DRG below the levels of the injury to compare the effects of SCI with and without two different forms of exercise in rats., Design Type(s)gene expression analysis objective • stimulus or stress design • transcription profiling designMeasurement Type(s)gene expressionTechnology Type(s)RNA sequencingFactor Type(s)Spinal Cord Injury • Exercise • biological replicateSample Characteristic(s)Rattus norvegicus • dorsal root ganglion Machine-accessible metadata file describing the reported data (ISA-Tab format)

Within the cervical and lumbar spinal enlargements, central pattern generating (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that interconnect the lumbar and cervical CPGs disrupts left-right limb coupling at each girdle. These perturbations to interlimb coordination occurred independent of the locomotor rhythm, did not affect intralimb coordination, and did not disrupt the speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. Together, these data show that LAPNs provide temporal information important for interlimb coordination in a flexible, context-driven manner.

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.

Spinal cord injury (SCI) is a devastating condition that results in whole‐body dysfunction, notably cardiovascular (CV) disruption and disease. Injury‐induced destruction of autonomic pathways in conjunction with a progressive decline in physical fitness contribute to the poor CV status of SCI individuals. Despite the wide use of exercise training as a therapeutic option to reduce CV dysfunction, little is known about the acute hemodynamic responses to the exercise itself. We investigated CV responses to an exercise challenge (swimming) following both high and low thoracic contusion to determine if the CV system is able to respond appropriately to the challenge of swimming. Blood pressure (BP) telemetry and echocardiography were used to track the progression of dysfunction in rodents with T3 and T10 SCI (n = 8 each) for 10 weeks postcontusion. At 1 week postinjury, all animals displayed a drastic decline in heart rate (HR) during the exercise challenge, likely a consequence of neurogenic shock. Furthermore, over time, all groups developed a progressive inability to maintain BP within a narrow range during the exercise challenge despite displaying normal hemodynamic parameters at rest. Echocardiography of T10 animals revealed no persistent signs of cardiac dysfunction; T3 animals exhibited a transient decline in systolic function that returned to preinjury levels by 10 weeks postinjury. Novel evidence provided here illustrates that incomplete injuries produce hemodynamic instability that only becomes apparent during an exercise challenge. Further, this dysfunction lasts into the chronic phase of disease progression despite significant recovery of hindlimb locomotion and cardiac function.

A rarely explored subject in animal research is the effect of pre-injury variables on behavioural outcome post-SCI. Low reporting of such variables may underlie some discrepancies in findings between laboratories. Particularly, intensive task-specific training before a SCI might be important, considering that sports injuries are one of the leading causes of SCI. Thus, individuals with SCI often underwent rigorous training before their injuries. In the present study, we asked whether training before SCI on a grasping task or a swimming task would influence motor recovery in rats. Swim pre-training impaired recovery of swimming 2 and 4 weeks post-injury. This result fits with the idea of motor learning interference, which posits that learning something new may disrupt learning of a new task; in this case, learning strategies to compensate for functional loss after SCI. In contrast to swimming, grasp pre-training did not influence grasping ability after SCI at any time point. However, grasp pre-trained rats attempted to grasp more times than untrained rats in the first 4 weeks post-injury. Also, lesion volume of grasp pretrained rats was greater than that of untrained rats, a finding which may be related to stress or activity. The increased participation in rehabilitative training of the pre-trained rats in the early weeks post-injury may have potentiated spontaneous plasticity in the spinal cord and counteracted the deleterious effect of interference and bigger lesions. Thus, our findings suggest that pre-training plays a significant role in recovery after CNS damage and needs to be carefully controlled for.

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.

Neural circuitry in the lumbar spinal cord governs two principal features of locomotion, rhythm and pattern, which reflect intra- and interlimb movement. These features are functionally organized into a hierarchy that precisely controls stepping in a stereotypic, speed-dependent fashion. Here, we show that a specific component of the locomotor pattern can be independently manipulated. Silencing spinal L2 interneurons that project to L5 selectively disrupts hindlimb alternation allowing a continuum of walking to hopping to emerge from the otherwise intact network. This perturbation, which is independent of speed and occurs spontaneously with each step, does not disrupt multi-joint movements or forelimb alternation, nor does it translate to a non-weight-bearing locomotor activity. Both the underlying rhythm and the usual relationship between speed and spatiotemporal characteristics of stepping persist. These data illustrate that hindlimb alternation can be manipulated independently from other core features of stepping, revealing a striking freedom in an otherwise precisely controlled system., Intra- and interlimb coordination during locomotion is governed by hierarchically organized lumbar spinal networks. Here, the authors show that reversible silencing of spinal L2–L5 interneurons specifically disrupts hindlimb alternation leading to a continuum of walking to hopping.

Study Design Experimental Study Objectives To characterize the specific hindlimb electromyographic (EMG) patterns in response to muscle stretch and to measure the applied forces during stretching in the rat model of moderate SCI. Setting Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA Methods Female Sprague Dawley rats (n=4) were instrumented for telemetry-based EMG recording (right Rectus Femoris and Biceps Femoris) and received a moderate T10 spinal cord injury (SCI). The major hindlimb muscle groups were stretched using our clinically modeled protocol. The EMG responses were recorded biweekly for 8 weeks. The forces applied during stretching were measured using a custom-designed glove. Locomotor function was assessed using the BBB Open Field Locomotor Scale, 3D kinematics and gait analysis. Results Three main EMG patterns in response to stretch were identified: clonic-like, air-stepping and spasms. Torques applied during stretching ranged from 0.8–6 N*cm, and did not change significantly over the weeks of stretching. Two stretching sessions a week did not result in a significant disruption to locomotor function. Conclusions Stretching evokes EMG patterns in rats similar to those reported in humans including clonus and spasms. The torques used during stretching are comparable, based on the ratio of torque to body weight, to the few previously published studies that measured the forces and/or torques applied by physical therapists when stretching patients. Future studies are warranted to fully explore the impact of muscle stretch on spinal cord function after injury. Sponsorship DoD, KSCHIRT, NIH

Joint contractures and spasticity are two common secondary complications of a severe spinal cord injury (SCI), which can significantly reduce quality of life, and stretching is one of the top strategies for rehabilitation of these complications. We have previously shown that a daily static stretching protocol administered to rats at either acute or chronic time points after a moderate or moderate-severe T10 SCI significantly disrupts their hindlimb locomotor function. The objective of the current study was to examine the effects of dynamic range of motion (ROM) stretching on the locomotor function of rats with SCI as an alternative to static stretching. Starting at 6 weeks post-injury (T10 moderate contusion) eight adult Sprague–Dawley rats were subjected to hindlimb stretching for 4 weeks. Our standard stretching protocol (six maneuvers to stretch the major hindlimb muscle groups) was modified from 1 min static stretch-and-hold at the end ROM of each stretch position to a dynamic 2 sec hold, 1 sec release rhythm repeated for a duration of 1 min. Four weeks of daily (5 days/week) dynamic stretching led to significant disruption of locomotor function as assessed by the Basso, Beattie, Bresnahan (BBB) Open Field Locomotor Scale and three-dimensional (3D) kinematic and gait analyses. In addition, we identified and analyzed an apparently novel hindlimb response to dynamic stretch that resembles human clonus. The results of the current study extend the observation of the stretching phenomenon to a new modality of stretching that is also commonly used in SCI rehabilitation. Although mechanisms and clinical relevance still need to be established, our findings continue to raise concerns that stretching as a therapy can potentially hinder aspects of locomotor recovery.

Background. Stretching is a widely accepted standard-of-care therapy following spinal cord injury (SCI) that has not been systematically studied in animal models. Objective. To investigate the influence of a daily stretch-based physical therapy program on locomotor recovery in adult rats with moderate T9 contusive SCI. Methods. A randomized treatment and control study of stretching in an animal model of acute SCI. Moderate SCIs were delivered with the NYU Impactor. Daily stretching (30 min/day, 5 days/wk for 8 weeks) was provided by a team of animal handlers. Hindlimb function was assessed using the BBB Open Field Locomotor Scale and kinematically. Passive range-of-motion for each joint was determined weekly using a goniometer. Results. Declines in hindlimb function during overground stepping were observed for the first 4 weeks for stretched animals. BBB scores improved weeks 5 to 10 but remained below the control group. Stretched animals had significant deficits in knee passive range of motion starting at week 4 and for the duration of the study. Kinematic assessment showed decreased joint excursion during stepping that partially recovered beginning at week 5. Conclusion. Stretch-based therapy significantly impaired functional recovery in adult rats with a moderate contusive SCI at T10. The negative impact on function was greatest acutely but persisted even after the stretching ceased at 8 weeks postinjury.

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.

Active upper-limb and passive lower-limb exercise are two interventions used in the spinal cord injury (SCI) population. Although the global cardiac responses have been previously studied, it is unclear how either exercise influences contractile cardiac function. Here, the cardiac contractile and volumetric responses to upper-limb (swim) and passive lower-limb exercise were investigated in rodents with a severe high-thoracic SCI. Animals were divided into control (CON), SCI no exercise (NO-EX), SCI passive hindlimb cycling (PHLC), or SCI swim (SWIM) groups. Severe contusion SCI was administered at the T2 level. PHLC and SWIM interventions began on day 8 postinjury and lasted 25 days. Echocardiography and dobutamine stress echocardiography were performed before and after injury. Cardiac contractile indexes were assessed in vivo at study termination via a left ventricular pressure-volume conductance catheter. Stroke volume was reduced after SCI (91 µl in the NO-EX group vs. 188 µl in the CON group, P < 0.05) and was reversed at study termination in the PHLC (167 µl) but not SWIM (90 µl) group. Rates of contraction were reduced in NO-EX versus CON groups (6,079 vs. 9,225 mmHg, respectively, P < 0.05) and were unchanged by PHLC and SWIM training. Similarly, end-systolic elastance was reduced in the NO-EX versus CON groups (0.67 vs. 1.37 mmHg/µl, respectively, P < 0.05) and was unchanged by PHLC or SWIM training. Dobutamine infusion normalized all pressure indexes in each SCI group (all P < 0.05). In conclusion, PHLC improves flow-derived cardiac indexes, whereas SWIM training displayed no cardiobeneficial effect. Pressure-derived deficits were corrected only with dobutamine, suggesting that reduced β-adrenergic stimulation is principally responsible for the impaired cardiac contractile function after SCI. NEW & NOTEWORTHY This is the first direct comparison between the cardiac changes elicited by active upper-limb or passive lower-limb exercise after spinal cord injury. Here, we demonstrate that lower-limb exercise positively influences flow-derived cardiac indexes, whereas upper-limb exercise does not. Furthermore, neither intervention corrects the cardiac contractile dysfunction associated with spinal cord injury.

Propriospinal pathways, consisting of axons from interneurons that project to other spinal segments, have been implicated as likely candidates to mediate interlimb coordination in developing and adult mammals during quadrupedal locomotion. The superficial thoracic ventrolateral funiculus (VLF) contains both ascending and descending axons, and when stimulated can induce alternating rhythmic locomotor-like activity in the lumbar ventral roots of the isolated neonatal rat spinal cord. The goal of this work was to characterize the synaptic inputs onto cervical neurons from ascending axons in the VLF. Sprague-Dawley rats (P4-P7) were deeply anesthesized with halothane and their spinal cords were isolated, removed, and maintained in vitro. Intracellular recordings were made from 68 cervical (C5-C7) neurons having 71 latency classifications in response to thoracic VLF stimulation. Antidromic (n=35), monosynaptic (n=2), di-or tri-synaptic (n=18) and long-latency polysynaptic (n=16) responses were recorded. Recordings from reduced preparations (mid-sagittal section at C5-C7) suggest that much of the delay in the long-latency polysynaptic responses require a bilaterally intact cervical spinal cord. Fifty-three percent (17/32) of the VLF responsive cervical interneurons tested also exhibited long latency excitatory responses to lumbar dorsal root stimulation suggesting that many of the cervical VLF responsive interneurons receive indirect input from lumbar primary afferents. We hypothesize that the VLF contains a population of ascending axons originating from lumbar propriospinal interneurons that can influence cervical inter- and motoneurons. These ascending VLF axons may participate in interlimb coordination by providing moment-by-moment feedback to the cervical enlargement of lumbar central pattern generator and/or hindlimb proprioceptive activity.

There is general consensus that spinal cord injuries (SCI) above T6 result in altered sympathetic control of the heart, which negatively influences cardiac structure and function. To by-pass disrupted circuitry and investigate cardiac responses under enhanced sympathetic activity we utilized dobutamine (DOB) stress echocardiography. Animals were divided into a T2, 25 g-cm contusive SCI (SCI) or an uninjured control (CON) group. Echocardiography was performed pre-SCI and at 1, 2 and 6 weeks post-SCI. Increasing doses of DOB (5, 10 & 20 µg/min/kg) were infused intravenously pre-SCI and at 1 and 6 weeks post-SCI. Parasternal-short axis images were used to compare group differences in systolic function and track changes in response to SCI and DOB over time. One week post-SCI, stroke volume (SV), end diastolic volume (EDV), cardiac output (CO) and ejection fraction (EF) were all reduced compared to CON and these deficits persisted to 6weeks. We also found an increase in collagen deposition at 6 weeks post SCI. Pre-SCI, DOB elicited a decrease in EDV and increases in CO, EF and HR but not SV. At 6 weeks following SCI, in addition to increases in CO, EF and HR, DOB also induced increases in SV. This is the first report, to our knowledge, of DOB responses in a contusive SCI model with persistent cardiac impairments. The return of CO to pre-SCI levels and the substantial increase in SV at low DOB dosages shows that impaired descending control of the heart is directly contributing to reduced resting SV after SCI.

We have recently documented that treatment with the alternative biofuel, acetyl-l-carnitine (ALC, 300 mg/kg), as late as 1 hr after T10 contusion spinal cord injury (SCI), significantly maintained mitochondrial function 24 hrs after injury. Here we report that following more severe contusion SCI centered on the L1/L2 segments that are postulated to contain lamina X neurons critical for locomotion (the “central pattern generator”), ALC treatment resulted in significant improvements in acute mitochondrial bioenergetics and long-term hindlimb function. While control-injured rats were only able to achieve slight movements of hindlimb joints, ALC-treated animals produced consistent weight-supported plantar steps one month after injury. Such landmark behavioral improvements were significantly correlated with increased tissue sparing of both gray and white matter proximal to the injury, as well as preservation of choline acetyltransferase (ChAT)-positive neurons in lamina X rostral to the injury site. These findings signify that functional improvements with ALC treatment are mediated, in part, by preserved locomotor circuitry rostral to upper lumbar contusion SCI. Based on beneficial effects of ALC on mitochondrial bioenergetics after injury, our collective evidence demonstrate that preventing mitochondrial dysfunction acutely “promotes” neuroprotection that may be associated with the milestone recovery of plantar, weight-supported stepping.

The ventrolateral funiculus in the spinal cord has been identified as containing important ascending and descending pathways related to locomotion and interlimb coordination. The purpose of this descriptive study was to investigate the patterns of axon termination of long ascending and descending ventrolateral pathways within the cervical and lumbar enlargements of the adult rat spinal cord. To accomplish this, we made discrete unilateral injections of the tracer biotinylated dextran-amine (BDA) into the ventrolateral white matter at T9. Although some BDA-labeled axons with varicosities were found bilaterally at all cervical levels, particularly dense BDA labeling was observed in laminae VIII and IX ipsilaterally at the C6 and C8 levels. In the same animals, dense terminal labeling was found in the lumbar enlargement in medial lamina VII and ventromedial laminae VIII and IX contralaterally. This labeling was most apparent in the more rostral lumbar segments. These observations continue the characterization of inter-enlargement (long propriospinal) pathways, illustrating a substantial and largely reciprocal inter-enlargement network with large numbers of both ascending and descending ventrolateral commissural neurons. These pathways are anatomically well-suited to the task of interlimb coordination and to participate in the remarkable recovery of locomotor function seen in the rat following thoracic spinal cord injuries that spare as little as 20% of the total white matter cross sectional area.

To study the pathophysiology of spinal cord injury (SCI), we used the LISA-Vibraknife to generate a precise and reproducible dorsal laceration SCI in the mouse. The surgical procedure involved a T9 laminectomy, dural resection, and a spinal cord laceration to a precisely controlled depth. Four dorsal hemisection injuries with lesion depths of 0.5, 0.8, 1.1, and 1.4 mm, as well as normal, sham (laminectomy and dural removal only), and transection controls were examined. Assessments including the Basso Mouse Scale (BMS), footprint analysis, beam walk, toe spread reflex, Hargreaves' test, and transcranial magnetic motor-evoked potential (tcMMEP) analysis were performed to assess motor, sensorimotor, and sensory function. These outcome measures demonstrated significant increases in functional deficits as the depth of the lesion increased, and significant behavioral recovery was observed in the groups over time. Quantitative histological examination showed significant differences between the injury groups and insignificant lesion depth variance within each of the groups. Statistically significant differences were additionally found in the amount of ventral spared tissue at the lesion site between the injury groups. This novel, graded, reproducible laceration SCI model can be used in future studies to look more closely at underlying mechanisms that lead to functional deficits following SCI, as well as to determine the efficacy of therapeutic intervention strategies in the injury and recovery processes following SCI.

Rolipram, an inhibitor of phosphodiesterase 4 (PDE4) proteins that hydrolyze cAMP, increases axonal regeneration following spinal cord injury (SCI). Recent evidence indicates that rolipram also protects against a multitude of apoptotic signals, many of which are implicated in secondary cell death post-SCI. In the present study, we used immunohistochemistry and morphometry to determine potential spinal cord targets of rolipram and to test its protective potential in rats undergoing a cervical spinal cord contusive injury. We found that 3 PDE4 subtypes (PDE4A, B, D) were expressed by spinal cord oligodendrocytes. OX-42 immunopositive microglia only expressed the PDE4B subtype. Oligodendrocyte somata were quantified within the cervical ventrolateral funiculus, a white matter region critical for locomotion, at varying time points after SCI in rats receiving rolipram or vehicle treatments. We show that rolipram significantly attenuated oligodendrocyte death at 24 hours post-SCI continuing through 72 hours, the longest time point examined. These results demonstrate for the first time that spinal cord glial cells express PDE4 subtypes and that the PDE4 inhibitor rolipram protects oligodendrocytes from secondary cell death following a contusive SCI. They also indicate that further investigations into neuroprotection and axonal regeneration with rolipram are warranted for treating SCI.

In the mammalian spinal cord, the ventrolateral funiculus (VLF) has been identified as critical to postural control and locomotor function, in part due to the reticulospinal pathways it contains. The primary purpose of this descriptive study was to investigate the distribution of neurons in the medulla labeled retrogradely from the VLF and the intermediate gray matter of specific lumbar and cervical spinal cord segments in the adult rat. We made discrete injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5/6, 7/8 or lumbar (L) 2 segmental levels followed by a single injection of Fluoro-Gold (FG) into the right VLF at T9. Double-labeled medullary neurons were found primarily in the gigantocellular group of nuclei (Gi), distributed both ipsilaterally and contralaterally following cervical or lumbar FR injections. In addition, a substantial population of neurons contained within the vestibular group of nuclei was double labeled both ipsilaterally and contralaterally. We also identified a substantial population of Gi-related neurons located ipsilateral to the VLF injections that were double labeled following left unilateral FR injections at C5/6, C7/8 or L2. These results describe a substantial population of ipsilateral and commissural medullary neurons that project to both cervical and thoracolumbar segments. Two different populations of commissural neurons are described, one with axons that cross the midline rostral to T9, and one with axons that cross the midline caudal to T9. These observations provide strong additional evidence for a pattern of reticulo- and vestibulospinal projections that include substantial numbers of commissural neurons and project to multiple cervical and thoracolumbar levels.

Assessment of locomotor function of rodents may be supplemented using electrophysiological tests which monitor the integrity of ascending and descending tracts as well as the focal circuitry of the spinal cord in non-sedated rodents. Magnetically induced SSEPs (M-SSEPs) were elicited in rats by activating the hindpaw using magnetic stimulation (MS). M-SSEP response latencies were slightly longer than those elicited by electrical stimulation. M-SSEPs were eliminated following selective dorsal column lacerations of the spinal cord, indicating that they were transmitted via this tract. Magnetically induced motor evoked potentials (M-MEPs) were elicited in mice following transcranial MS and recorded from the gastrocnemius muscles. M-MEPs performed on myelin deficient mice demonstrated longer onset latencies and smaller amplitudes than in wild-type mice. Magnetically induced H-reflexes (MH-reflexes) which assess local circuitry in the lumbosacral area of the spinal cord were performed in rats. This response disappeared following an L3 contusion spinal cord injury, however, kainic acid (KA) injection at L3, known to selectively destroy interneurons, caused a shorter latency and an increase in the amplitude of the MH-reflex. M-SSEPs and MH-reflexes in rats and M-MEPs in mice compliment locomotor evaluation in assessing the functional integrity of the spinal cord under normal and pathological conditions in the non-sedated animal.

The ventrolateral funiculus (VLF) in the spinal cord contains important ascending and descending pathways related to locomotion and interlimb coordination. The primary purpose of this descriptive study was to investigate the distribution of inter-enlargement pathways in the adult rat spinal cord with an emphasis on the VLF. We made discrete unilateral injections of Fluoro-Gold (FG) into the right VLF at thoracic segment (T) 9, and either unilateral or bilateral injections of Fluoro-Ruby (FR) into the intermediate gray matter at the cervical (C) 5-6, C7-8, or lumbar (L) 2 segmental levels. Inter-enlargement neurons with ascending axons in the right VLF were found bilaterally in laminae VII and VIII throughout the rostral lumbar spinal cord (L1-L3) and predominantly contralaterally in the caudal lumbosacral (L4-S1) spinal cord. Following left unilateral FR injections at C5-6 or C7-8 and right unilateral VLF injections of FG at T9, very few double-labeled neurons could be found anywhere in the lumbar spinal cord. Similar injections of FR at L2 revealed an almost symmetrical bilateral distribution of double-labeled neurons throughout the cervical spinal cord (C1-8). These results describe ascending and descending pathways within the spinal cord that interconnect the two enlargements and involve both commissural and ipsilateral interneurons. The majority of inter-enlargement neurons had axons within the VLF at T9. These observations support the hypothesis that the VLF contains long ascending and descending axons with propriospinal inter-enlargement, commissural and ipsilateral connections that are anatomically well-suited to mediate interlimb coordination.

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.

Functional impairment after spinal cord injury (SCI) is partially attributed to neuronal cell death, with further degeneration caused by the accompanying apoptosis of myelin-forming oligodendrocytes. The Eph receptor protein tyrosine kinase family and its cognate ligands, the ephrins, have been identified to be involved in axonal outgrowth, synapse formation, and target recognition, mainly mediated by repulsive activity. Recent reports suggest that ephrin/Eph signaling might also play a role as a physiological trigger for apoptosis during embryonic development. Here, we investigated the expression profile of EphA7, after SCI, by using a combination of quantitative real-time PCR (QRT-PCR) and immunohistochemical techniques. QRT-PCR analysis showed an increase in the expression of full-length EphA7 at 7 days postinjury (DPI). Receptor immunoreactivity was shown mostly in astrocytes of the white matter at the injury epicenter. In control animals, EphA7 expression was observed predominantly in motor neurons of the ventral gray matter, although some immunoreactivity was seen in white matter. Furthermore, blocking the expression of EphA7 after SCI using antisense oligonucleotides resulted in significant acceleration of hindlimb locomotor recovery at 1 week. This was a transient effect; by 2 weeks postinjury, treated animals were not different from controls. Antisense treatment also produced a return of nerve conduction, with shorter latencies than in control treated animals after transcranial magnetic stimulation. We identified EphA7 receptors as putative regulators of apoptosis in the acute phase after SCI. These results suggest a functional role for EphA7 receptors in the early stages of SCI pathophysiology.