Open Field Locomotor Scale Research Articles

Combining clinically common drugs with hindlimb stretching in spinal cord injured rodents.

Preclinical pilot study. To explore peripheral and central nociceptive mechanisms that contribute to muscle stretch-induced locomotor deficits following spinal cord injury. Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA. Ten female Sprague-Dawley rats received moderate, 25 g/cm T10 contusion injuries and recovered for 4 weeks. Rats were divided into three groups: Morphine/Ibuprofen-treated, Acetaminophen/Baclofen-treated, and saline control. Each group received daily hindlimb muscle stretching during weeks 4, 5, 9, and 10 post-injury and drugs were administered with stretching during weeks 4 and 9 only. Locomotor function was assessed throughout the experiment using the BBB Open Field Locomotor Scale. Hindlimb responses including spasticity, writhing, and clonic-like vibrations during muscle stretching were classified and scored. Consistent with our previous studies, hindlimb muscle stretching caused significant deficits in locomotor recovery following spinal cord injury. Baclofen and Ibuprofen partially mitigated the stretching effect, but none of the drugs significantly prevented the drop in locomotor function during stretching. Interestingly, treatment with Baclofen or Ibuprofen significantly reduced hindlimb responses such as spasticity and writhing during stretching, while Morphine exacerbated clonic-like vibrations in response to stretching maneuvers. These findings suggest that stretching may inhibit locomotor recovery through combined mechanisms of peripheral inflammation and sensitization of nociceptive afferents. When combined with central sprouting and loss of descending controls after SCI, this results in exaggerated nociceptive input during stretching. The inability of the applied clinical drugs to mitigate the detrimental effects of stretching highlights the complexity of the stretching phenomenon and emphasizes the need for further investigation.

Upregulation of TRPC6 inhibits astrocyte activation and proliferation after spinal cord injury in rats by suppressing AQP4 expression

AimsThis work investigates the effects and mechanisms of inhibiting TRPC6 (a non-selective cation channel) downregulation on rat astrocyte activation and proliferation following spinal cord injury (SCI) by suppressing AQP4 expression. We used HYP9 (TRPC6-specific agonist) and TGN-020 (AQP4-specific inhibitor) to explore the relationship between TRPC6 and AQP4 and their probable protective effects on SCI. Methods: In a rat SCI model, we randomly assigned female Sprague-Dawley rats into the following four groups: Sham, SCI, SCI+HYP9, and SCI+TGN-020. Western blotting and immunofluorescence staining were used to determine protein expression among groups following SCI. TUNEL and immunofluorescence staining were used to identify changes in the rate of apoptosis and the fraction of surviving neurons after SCI. The Basso-Beattie-Bresnahan open-field locomotor scale was used to identify changes in motor function after SCI. In vitro astrocyte scratch model, we first used the CCK8 assay to test the effects of varying doses of HYP9 or TGN-020 on astrocytes and then split the astrocytes into four groups: Con, Scratch, Scratch+HYP9, and Scratch+TGN-020. Western blotting and immunofluorescence were used to identify changes in the expression of target proteins. Results: In vivo and in vitro models, SCI dramatically decreased TRPC6 while considerably upregulating AQP4, glial fibrillary acidic protein (GFAP), and proliferating cell nuclear antigen (PCNA) expression. However, HYP9 or TGN-020 significantly suppressed activation of astrocytes, promoted neurons survival in the anterior horn of the spinal cords, and benefited the recovery of motor function in the hind limbs of rats following SCI. Interestingly, TRPC6 agonists dramatically suppressed AQP4 overexpression, indicating that the probable mechanism of HYP9 benefiting alleviation of SCI may be connected to AQP4 inhibition and astrocyte activation and proliferation reduction. Conclusion: we discovered for the first time that HYP9 inhibits astrocyte activation and proliferation by inhibiting AQP4 in SCI rats in vivo and in vitro models and that it preserves neuronal survival and functional recovery after SCI.

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

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.

TGN-020 alleviates edema and inhibits astrocyte activation and glial scar formation after spinal cord compression injury in rats

AimsIdentifying drugs that inhibit edema and glial scar formation and increase neuronal survival is crucial to improving outcomes after spinal cord injury (SCI). Here, we used 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), a potent selective inhibitor of aquaporin 4 (AQP4), to investigate the effects of TGN-020 on SCI in Sprague-Dawley rats. Main methodsWe compressed the spinal cord at T10 using a sterile impounder (35 g, 5 min), to induce moderate injury. TGN-020 (100 mg/kg) or an equal volume of 10% dimethyl sulfoxide was then administered via intraperitoneal injection. Neurological function was evaluated using the Basso-Beattie-Bresnahan open-field locomotor scale 1, 3, 7, 14, 21, and 28 days after SCI. The degree of edema was assessed via determination of the precise spinal cord water content 3 days after SCI. Expression levels of AQP4, glial fibrillary acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), and growth-associated protein-43 (GAP-43) were determined via western blotting and immunofluorescence staining 3 days after SCI and 4 weeks after SCI. Numbers of surviving neurons and glial scar sizes were determined using Nissl and hematoxylin-eosin staining, respectively. Key findingsOur results showed that TGN-020 promoted functional recovery at days 3, 7, 14, 21, and 28, as well as reduced the degree of edema and inhibited the expression of AQP4, GFAP, PCNA at days 3 after SCI. Furthermore, observations 4 weeks after SCI revealed that TGN-020 inhibited the glial scar formation and upregulated GAP-43 expression. SignificanceTGN-020 can alleviate spinal cord edema, inhibit glial scar formation, and promote axonal regeneration, conferring beneficial effects on recovery in rats.

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

Study DesignExperimental StudyObjectivesTo 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.SettingKentucky Spinal Cord Injury Research Center, Louisville, KY, USAMethodsFemale 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.ResultsThree 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.ConclusionsStretching 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.SponsorshipDoD, KSCHIRT, NIH

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.

Metabolomic Changes in Rat Model of Cauda Equina Injury.

To show the differences of metabolomic changes in a rat model of cauda equina injury (CEI) and find potent metabolic biomarkers of CEI. A total of 28 Sprague-Dawley rats were used in this study. After the rats were given anesthesia and fixed in a prone position, a piece of silicone block was placed into the epidural space below the lamina. Behavior tests including the Basso, Beattie, and Bresnahan open field locomotor scale and an inclined plane test were conducted 1 day and 2 days after surgery. The cauda equina tissue was collected 12 hours, 1 day, and 2 days after surgery. Ultraperformance liquid chromatography coupled with quadruple time-of-flight mass spectrometry was used for a quantitative analysis of cauda equine metabolic changes in rats from different groups. The differences between the metabolic profiles of the rats in 4 groups were analyzed using partial least squares discriminant analysis. In behavior tests and histologic analyses given 2 days after surgery, the animals showed remarkable organ dysfunction and pathologic damage. Metabolic profiles showed remarkable differences between the control and model groups. Thirty-four potential CEI metabolite biomarkers were identified between the control group and different time-point model groups. These potential biomarkers appeared in 15 metabolic pathways. Our results may improve the cause of CEI and provide a basis for clinical diagnosis and locating biomarkers in the early stages of the pathologic process ofCEI.

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.

Xenon-delayed postconditioning attenuates spinal cord ischemia/reperfusion injury through activation AKT and ERK signaling pathways in rats

Previous studies have shown that xenon-delayed postconditioning for up to 2h after reperfusion provides protection against spinal cord ischemia/reperfusion (I/R) injury in rats. This study was designed to determine the roles of phosphatidylinositol 3-kinase (PI3K)–Akt and extracellular signal-regulated kinase (ERK) in this neuroprotection. The rats were randomly assigned to the following nine groups (n=16∗9): 1) I/R+N2 group, 2) I/R+Xe group, 3) I/R+PD98059+N2 group (ERK blocking agent), 4) I/R+wortmannin+N2 group (PI3K-Akt blocking agent), 5) I/R+PD98059+Xe group, 6) I/R+wortmannin+Xe group, 7) I/R+DMSO+Xe group (dimethyl sulfoxide, vehicle control), 8) I/R+DMSO+N2 group, and 9) sham group (no spinal cord ischemia and no xenon). Spinal cord ischemia was induced for 25min in male Sprague-Dawley rats. Neurological function was assessed using the Basso, Beattie, and Bresnahan (BBB) open-field locomotor scale at 6, 12, 24 and 48h after reperfusion. Histological examination of the lumbar spinal cord was performed using Nissl staining and TUNEL staining at 4 (n=8) and 48 (n=8)h after reperfusion. Western blotting was performed to evaluate p-Akt and p-ERK expression in the spinal cord at 4 (n=8) and 48 (n=8) h after reperfusion. Compared with the sham group, all rats in the I/R groups had lower BBB scores, fewer normal motor neurons, more apoptotic neurons and lower p-Akt and p-ERK levels at each time point (P<0.05). Compared with the I/R group, rats in the I/R+Xe group had higher neurological scores, more normal motor neurons, fewer apoptotic neurons and significantly higher levels of p-Akt and p-ERK at each time point (P<0.05). Compared with the I/R+Xe group, the I/R+PD98059+Xe and I/R+wortmannin+Xe groups showed worse neurological outcomes and less p-Akt and p-ERK at each time point (P<0.05). These results suggest that xenon-delayed postconditioning improves neurological outcomes to spinal cord I/R injury in rats through the activation of the AKT and ERK signaling pathways.

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.

Transplantation of schwann cells differentiated from adipose stem cells improves functional recovery in rat spinal cord injury.

When the spinal cord is damaged, medical procedures are vital to prevent of improvement of the lesion. Because of poor regeneration ability of central nervous tissue, the most injuries are irreversible. One of encouraging interventions for treatment of spinal cord injury is Schwann cell transplantation. However, isolation of Schwann cell for clinical interventions is complicated approach with low cells yield and purity. Thus, easily accessed sources like Adipose mesenchymal stem cells have been taken notice. Therefore, this study was planned to assess the effect of adipose stromal cell-derived Schwann cell transplantation in functional recovery after lateral hemisection in adult rats. After isolation, adipose stem cells were differentiated to Schwann cells. The differentiation was verified by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Then, we loaded the cells into collagen scaffolds with parallel aligned canals and transplanted into rats with 3 mm lesions at T9 - T10 level. Motor and sensory improvement were evaluated by open field locomotor scale, narrow beam, and tail flick tests for 60 days. Subsequently, conventional histology and immunohistochemistry were performed. In vitro results revealed that mesenchymal stem cells after differentiation gained Schwann cells morphology and markers. Schwann cell-grafted group had significantly higher locomotor and sensory scores in comparison with the control and scaffold without cell groups. Histological observations showed differentiated cells have the ability to improve axonal regeneration and remyelination. Our study proved that adipose tissue- derived Schwann cells can change the rough environment of damaged spinal cord and support axon regeneration and enhance functional recovery, and possibly be helpful for people suffering from spinal cord injuries.

Promotion of spinal cord axon regeneration by 3D nanofibrous core–sheath scaffolds

Since spinal cord injury is a complicated problem, neural tissue repair, and regeneration strategies have received a great deal of attention. In this study, a three-dimensional (3D) nanofibrous core-sheath scaffold with nanorough sheath and aligned core were fabricated by a combined electrospinning method with water vortex and two-nozzle system. In vitro and in vivo biological tests were carried out on the poly(lactic-co-glycolic acid) (PLGA) scaffolds. The cell morphology and proliferation evaluation of nerve cells on 3D PLGA scaffolds were studied. Cells were properly orientated along the aligned fiber direction of the scaffold. In animal studies, adult rats received a complete lateral hemisection at the T9-T10 level. Scaffolds were engrafted to bridge 3 mm defects of 10 adult rat spinal cords; 10 rats were used as controls. For 8 weeks, motor and sensory recovery by open field locomotor scale, narrow beam and tail flick tests were assessed. Locomotor and sensory scores of grafted animals were significantly better than the control group. Histological findings demonstrated that the scaffold supports the axonal regeneration of injured spinal cords and regenerating axons were seen to enter the graft and extend along its length.

Mesenchymal stem cells as an alternative for Schwann cells in rat spinal cord injury.

Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Mesenchymal stem cells were cultured from adult rats' bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment.

α4β1 integrin blockade after spinal cord injury decreases damage and improves neurological function

The extent of disability caused by spinal cord injury (SCI) relates to secondary tissue destruction arising partly from an intraspinal influx of neutrophils and monocyte/macrophages after the initial injury. The integrin α4β1, expressed by these leukocytes, is a key to their activation and migration into/within tissue. Therefore, blocking this integrin's functions may afford significant neuroprotection. Rats were treated intravenously with a blocking monoclonal antibody (mAb) to the α4 subunit of α4β1 at 2 and 24 h after thoracic clip-compression SCI. Anti-α4β1 treatment significantly decreased neutrophil and monocyte/macrophage influx at 3 d by 47% and 53%, respectively, and decreased neutrophil influx by 61% at 7 d after SCI. Anti-α4β1 treatment also significantly reduced oxidative activity in injured cord homogenates at 3 d. For example, myeloperoxidase activity decreased by 38%, inducible nitric oxide by 44%, dichlorofluorescein (marking free radicals) by 33% and lipid peroxidation (malondialdehyde) by 42%. At 2–8 weeks after SCI, motor function improved by up to 2 points on an open-field locomotor scale. Treated rats supported weight with their hind paws instead of sweeping. At 2–4 weeks after SCI, anti-α4β1 treatment decreased blood pressure responses during autonomic dysreflexia by as much as 43% and, at 2–8 weeks, decreased mechanical allodynia elicited from the trunk and hind paw by up to 54% and 40%, respectively. This improved functional recovery correlated with spared myelin-containing white matter and > 10-fold more bulbospinal serotonergic axons below the injury than were in controls. The significant neurological improvement offered by this neuroprotective strategy underscores the potential for an anti-integrin treatment for SCI.

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.

Homologous Transplantation of Neural Stem Cells to the Injured Spinal Cord of Mice

Murine neural stem cells (NSCs) were homografted onto the injured spinal cord (SC) to assess their potential to improve motor behavior, to differentiate as neurons, and to establish synapse-like contacts with the descending axonal paths of the host. In addition, we investigated whether transduced NSCs over-expressing vascular endothelial growth factor might exert any angiogenetic effect in the injured SC. NSCs derived from mouse embryos were transduced to express either green fluorescent protein or vascular endothelial growth factor. The cells were engrafted in mice where an extended dorsal funiculotomy had been performed at the T8-T9 level. At intervals from 4 to 12 weeks after grafting, motor behavior was assessed using an open field locomotor scale and footprint analysis. At the same time points, the SC was studied by conventional histology, immunohistochemistry, and fluorescence microscopy. The interactions between the grafted NSCs and descending axonal paths were investigated using anterogradely transported fluorescent axonal tracers. By the 12-week time point, mice engrafted with NSCs significantly improved both their locomotor score on open field test and their base of support on footprint analysis. Histological studies showed that green fluorescent protein-positive NSCs survived as long as 12 weeks after grafting, migrated from the grafting site with a tropism toward the lesion, and either remained undifferentiated or differentiated into the astrocytic phenotype without neuronal or oligodendrocytic differentiation. Interestingly, the NSC-derived astrocytes expressed vimentin, suggesting that these cells differentiated as immature astrocytes. The tips of severed descending axonal paths came adjacent to grafted NSCs without forming synapse-like structures. When genetically engineered to over-express vascular endothelial growth factor, the grafted NSCs significantly increased vessel density in the injured area. In the traumatically injured mice SC, NSC grafting improves motor recovery. Although differentiation of engrafted NSCs is restricted exclusively toward the astrocytic phenotype, the NSC-derived astrocytes show features that are typical of the early phase after SC injury when the glial scar is still permissive to regenerating axons. The immature phenotype of the NSC-derived astrocytes suggests that these cells might support neurite outgrowth by the host neurons. Thus, modifying the glial scar with NSCs might enhance axonal regeneration in the injured area. The use of genetically engineered NSCs that express trophic factors appears to be an attractive tool in SC transplantation research.

A Simple Post Hoc Transformation that Improves the Metric Properties of the BBB Scale for Rats with Moderate to Severe Spinal Cord Injury

The Basso, Beattie, Bresnahan (BBB) open field locomotor scale is a popular measure of functional recovery following spinal cord injury (SCI). To examine the metric properties of the scale, we performed detailed analyses of BBB scores from 643 rats with moderate and severe SCI (12.5, 25, or 50 mm MASCIS) from two different laboratories. The analyses revealed that the BBB scale is ordinal in the most frequently used portion of the scale. Higher scores (14 and greater) were not frequently assigned in the dataset as animals with mild injuries were not sampled, making the ordinal nature of the upper end of the scale difficult to assess. The rare scores assigned in this range disproportionately increased variance. Under these conditions collapsing scores above 14 into one category increased effect size. Analysis of the lower region of the scale revealed that some scores (2 and 3) were rarely assigned, implying a discontinuity in the scale. The discontinuous nature of the lower portion of the scale presents a problem for both parametric and nonparametric statistical analyses. Pooling scores 2/3/4 eliminated the gap, enhancing the metric properties of the scale. Under the injury conditions evaluated, the transformation helped assure that the data were continuous and ordered. Further, interval durations were comparable across the entire range of the transformed scale, allowing application of parametric statistical techniques. The transformation should be applied in a post hoc fashion to reduce variability and increase power in cases where few scores fall in upper portion of the scale.

A Simple Post Hoc Transformation that Improves the Metric Properties of the BBB Scale for Rats with Moderate to Severe Spinal Cord Injury

The Basso, Beattie, Bresnahan (BBB) open field locomotor scale is a popular measure of functional recovery following spinal cord injury (SCI). To examine the metric properties of the scale, we performed detailed analyses of BBB scores from 643 rats with moderate and severe SCI (12.5, 25, or 50 mm MASCIS) from two different laboratories. The analyses revealed that the BBB scale is ordinal in the most frequently used portion of the scale. Higher scores (14 and greater) were not frequently assigned in the dataset as animals with mild injuries were not sampled, making the ordinal nature of the upper end of the scale difficult to assess. The rare scores assigned in this range disproportionately increased variance. Under these conditions collapsing scores above 14 into one category increased effect size. Analysis of the lower region of the scale revealed that some scores (2 and 3) were rarely assigned, implying a discontinuity in the scale. The discontinuous nature of the lower portion of the scale presents a problem for both parametric and nonparametric statistical analyses. Pooling scores 2/3/4 eliminated the gap, enhancing the metric properties of the scale. Under the injury conditions evaluated, the transformation helped assure that the data were continuous and ordered. Further, interval durations were comparable across the entire range of the transformed scale, allowing application of parametric statistical techniques. The transformation should be applied in a post hoc fashion to reduce variability and increase power in cases where few scores fall in upper portion of the scale.

Spinal cord repair in neonatal rats: a correlation between axonal regeneration and functional recovery.

The present study aimed to analyse how anatomical regeneration contributes to functional recovery after experimental spinal cord repair. Thoracic spinal cord of neonatal rats was completely transected to make a gap and repaired by grafting a section of embryonic spinal cord. Six weeks after surgery, outcome of locomotor performance was assessed using an open field locomotor scale (BBB scale). Axonal regeneration across the repaired site was quantitatively assessed in the raphe, vestibular, and red nuclei and the sensorimotor cortex by a retrograde tracing method. The rats that had no labelled neurons in any of the supraspinal nuclei showed no hind-forelimb coordination. The rats that had labelled neurons in the brainstem nuclei but not in the sensorimotor cortex showed hind-forelimb coordination of varying grades depending on the amount of regeneration. The rats that had labelled neurons in all of the examined nuclei showed almost normal locomotion. In addition to a relationship between distribution of the labelled neurons and functional recovery, a positive correlation was observed between number of the labelled neurons in each of the supraspinal nuclei and locomotor performance of the rat. Thus the grade of restored function appeared to be regulated by distribution and number of fibres regenerated across the repaired site and into the target region. These results suggest that accurate reconstruction of neural connections is essential for significant functional recovery after spinal cord repair.