Construction of a Searchable Database for Gene Expression Changes in Spinal Cord Injury Experiments.

Spinal cord injury (SCI) is a debilitating condition with an estimated 18,000 new cases annually in the United States. The field has accepted and adopted standardized databases such as the Open Data Commons for Spinal Cord Injury (ODC-SCI) to aid in broader analyses, but these currently lack high-throughput data despite the availability of nearly 6000 samples from over 90 studies available in the Sequence Read Archive. This limits the potential for large datasets to enhance our understanding of SCI-related mechanisms at the molecular and cellular level. 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 1196 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.

Construction of a searchable database for gene expression changes in spinal cord injury experiments.

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.

Broad opioid antagonism amplifies disruption of locomotor function following therapy-like hindlimb stretching in spinal cord injured rats.

STUDY DESIGN: Preclinical pilot study. OBJECTIVES: To test the hypothesis that spinal opioidergic circuitry contributes to muscle stretch-induced locomotor deficits. SETTING: Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Nociceptor-dependent locomotor dysfunction after clinically-modeled hindlimb muscle stretching in adult rats with spinal cord injury.

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.

Temporal analysis of cardiovascular control and function following incomplete T3 and T10 spinal cord injury in rodents.

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.

Electromyographic patterns of the rat hindlimb in response to muscle stretch after spinal cord injury.

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 0.4-8 N•cm, and with the exception of the quadriceps, 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.

Challenging cardiac function post-spinal cord injury with dobutamine.

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, 25g-cm contusive SCI (SCI) or an uninjured control (CON) group. Echocardiography was performed pre-SCI and at 1, 2 and 6weeks post-SCI. Increasing doses of DOB (5, 10 & 20µg/min/kg) were infused intravenously pre-SCI and at 1 and 6weeks 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 6weeks post SCI. Pre-SCI, DOB elicited a decrease in EDV and increases in CO, EF and HR but not SV. At 6weeks 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.

Dynamic "Range of Motion" Hindlimb Stretching Disrupts Locomotor Function in Rats with Moderate Subacute Spinal Cord Injuries.

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.

Disruption of Locomotion in Response to Hindlimb Muscle Stretch at Acute and Chronic Time Points after a Spinal Cord Injury in Rats.

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.

Challenges of animal models in SCI research: Effects of pre-injury task-specific training in adult rats before lesion.

A rarely explored subject in animal research is the effect of pre-injury variables on behavioral 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 pre-trained 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.

Hindlimb stretching alters locomotor function after spinal cord injury in the adult rat.

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.

Hindlimb immobilization in a wheelchair alters functional recovery following contusive spinal cord injury in the adult rat.

BACKGROUND: Locomotor training of rats with thoracic contusion spinal cord injuries can induce task-specific changes in stepping but rarely results in improved overground locomotion, possibly due to a ceiling effect. Thus, the authors hypothesize that incompletely injured rats maximally retrain themselves while moving about in their cages over the first few weeks postinjury. OBJECTIVE: To test the hypothesis using hindlimb immobilization after mild thoracic contusion spinal cord injury in adult female rats. A passive stretch protocol was included as an independent treatment. METHODS: Wheelchairs were used to hold the hindlimbs stationary in an extended position leaving the forelimbs free. The wheelchairs were used for 15 to 18 hours per day, 5 days per week for 8 weeks, beginning at 4 days postinjury. A 20-minute passive hindlimb stretch therapy was applied to half of the animals. RESULTS: Hindlimb locomotor function of the wheelchair group was not different from controls at 1 week postinjury but declined significantly over the next 4 weeks. Passive stretch had no influence on wheelchair animals but limited functional recovery of normally housed animals, preventing them from regaining forelimb-hindlimb coordination. Following 8 weeks of wheelchair immobilization and stretch therapy, only the wheelchair group displayed an improvement in function when returned to normal housing but retained significant deficits in stepping and coordination out to 16 weeks. CONCLUSION: Hindlimb immobilization and passive stretch may hinder or conceal the normal course of functional recovery of spinal cord injured rats. These observations have implications for the management of acute clinical spinal cord injuries.

Anterograde labeling of ventrolateral funiculus pathways with spinal enlargement connections in the adult rat spinal cord.

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.

Swim training initiated acutely after spinal cord injury is ineffective and induces extravasation in and around the epicenter.

Activity-based rehabilitation is a promising strategy for improving functional recovery following spinal cord injury (SCI). While results from both clinical and animal studies have shown that a variety of approaches can be effective, debate still exists regarding the optimal post-injury period to apply rehabilitation. We recently demonstrated that rats with moderately severe thoracic contusive SCI can be re-trained to swim when training is initiated 2 weeks after injury and that swim training had no effect on the recovery of overground locomotion. We concluded that swim training is a task-specific model of post-SCI activity-based rehabilitation. In the present study, we ask if re-training initiated acutely is more or less effective than when initiated at 2 weeks post-injury. Using the Louisville Swim Scale, an 18-point swimming assessment, supplemented by kinematic assessment of hindlimb movement during swimming, we report that acute re-training is less effective than training initiated at 2 weeks. Using the bioluminescent protein luciferase as a blood-borne macromolecular marker, we also show a significant increase in extravasation in and around the site of SCI following only 8 min of swimming at 3 days post-injury. Taken together, these results suggest that acute re-training in a rat model of SCI may compromise rehabilitation efforts via mechanisms that may involve one or more secondary injury cascades, including acute spinal microvascular dysfunction.

Use of environmentally enriched housing for rats with spinal cord injury: the need for standardization.

A variety of rehabilitation methods that increase social interaction and locomotor activity are reported to yield positive benefits in humans and animals with spinal cord injury (SCI). Environmental enrichment often incorporates group housing, increased cage size, and objects to increase social interaction and stimulate locomotor activity of animals. Others have reported that adult rats housed in enriched environments immediately after moderate contusion thoracic SCI show improvements in locomotion, but not in neurotransmission through or anatomy at the SCI site. In the present study, in contrast to previous reports, environmental enrichment did not improve the locomotion of rats with contusion thoracic SCI. Furthermore, as in previous reports, improvements were not observed for either electrophysiologic measures of neurotransmission through (transcranial magnetic motor-evoked potentials) and caudal to (magnetic-evoked interlimb reflex) the injury site or the amount of spared white matter at the epicenter. Determining the effectiveness of environmental enrichment to improve locomotor recovery in the SCI model requires standardization of housing procedures, outcome measures, and analyses.

The Louisville Swim Scale: a novel assessment of hindlimb function following spinal cord injury in adult rats.

The majority of animal studies examining the recovery of function following spinal cord injury use the BBB Open-Field Locomotor Scale as a primary outcome measure. However, it is now well known that rehabilitation strategies can bring about significant improvements in hindlimb function in some animal models. Thus, improvements in walking following spinal cord injury in rats may be influenced by differences in activity levels and housing conditions during the first few weeks post-injury. Swimming is a natural form of locomotion that animals are not normally exposed to in the laboratory setting. We hypothesized that deficits in, and functional recovery of, swimming would accurately represent the locomotor capability of the nervous system in the absence of any retraining effects. To test this hypothesis, we have compared the recovery of walking and swimming in rats following a range of standardized spinal cord injuries and two different retraining strategies. In order to assess swimming, we developed a rating system we call the Louisville Swimming Scale (LSS) that evaluates three characteristics of swimming that are highly altered by spinal cord injury--namely, hindlimb movement, forelimb dependency, and body position. The data indicate that the LSS is a sensitive and reliable method of determining swimming ability and the improvement in hindlimb function after standardized contusion injury of the thoracic spinal cord. Furthermore, the data suggests that when used in conjunction with the BBB Open-field Locomotor Scale, the LSS assesses locomotor capabilities that are not influenced by a retraining effect.

Effects of swimming on functional recovery after incomplete spinal cord injury in rats.

One of the most promising rehabilitation strategies for spinal cord injury is weight-supported treadmill training. This strategy seeks to re-train the spinal cord below the level of injury to generate a meaningful pattern of movement. However, the number of step cycles that can be accomplished is limited by the poor weight-bearing capability of the neuromuscular system after injury. We have begun to study swimming as a rehabilitation strategy that allows for high numbers of steps and a high step-cycle frequency in a standard rat model of contusive spinal cord injury. The purpose of the present study was to evaluate the effect of swimming as a rehabilitation strategy in rats with contusion injuries at T9. We used a swimming strategy with or without cutaneous feedback based on original work in the chick by Muir and colleagues. Adult female rats (n=27) received moderately-severe contusion injuries at T9. Walking and swimming performance were evaluated using the Open-Field Locomotor Scale (BBB; Basso et al., 1995) and a novel swimming assessment, the Louisville Swimming Scale (LSS). Rats that underwent swim-training with or without cutaneous feedback showed a significant improvement in hindlimb function during swimming compared to untrained animals. Rats that underwent swim-training without cutaneous feedback showed less improvement than those trained with cutaneous feedback. Rats in the non-swimming group demonstrated little improvement over the course of the study. All three groups showed the expected improvement in over-ground walking and had similar terminal BBB scores. These findings suggest that animals re-acquire the ability to swim only if trained and that cutaneous feedback improves the re-training process. Further, these data suggest that the normal course of recovery of over-ground walking following moderately-severe contusion injuries at T9 is the result of a re-training process.