Moderate Contusion Spinal Cord Injury Research Articles

NADPH oxidase isoform expression is temporally regulated and may contribute to microglial/macrophage polarization after spinal cord injury.

Spinal cord injury (SCI) results in both acute and chronic inflammation, as a result of activation of microglia, invasion of macrophages and activation of the NADPH oxidase (NOX) enzyme. The NOX enzyme is a primary source of reactive oxygen species (ROS) and is expressed by microglia and macrophages after SCI. These cells can assume either a pro- (M1) or anti-inflammatory (M2) polarization phenotype and contribute to tissue response to SCI. However, the contribution of NOX expression and ROS production to this polarization and vice versa is currently undefined. We therefore investigated the impact of SCI on NOX expression and microglial/macrophage polarization over time in a mouse model of contusion injury. Adult C57Bl/6 mice were exposed to a moderate T9 contusion SCI and tissue was assessed at acute, sub-acute and chronic time points for NOX isoform expression and co-expression with M1 and M2 microglia/macrophage polarization markers. Two NOX isoforms were increased after injury and were associated with both M1 and M2 markers, with an M1 preference for NOX2 acutely and NOX4 chronically. M2 cells were primarily found at acute time points only; the peak of NOX2 expression was associated with the decline in M2 polarization. In vitro, NOX2 inhibition shifted microglial polarization toward the M2 phenotype. These results now show that microglial/macrophage expression of NOX isoforms is independent of polarization state, but that NOX activity can influence subsequent polarization. These data can contribute to the therapeutic targeting of NOX as a therapy for SCI.

Temporal and Spatial Evolution of Raised Intraspinal Pressure after Traumatic Spinal Cord Injury.

Traumatic spinal cord injury (SCI) often leads to permanent neurological impairment. Currently, the only clinically effective intervention for patients with acute SCI is surgical decompression by removal of impinging bone fragments within 24 h after injury. Recent clinical studies suggest that elevated intraparenchymal spinal pressure (ISP) limits functional recovery following SCI. Here, we report on the temporal and spatial patterns of elevated ISP following a moderate rodent contusion SCI. Compared with physiological ISP in the intact cord (2.7 ± 0.5 mm Hg), pressures increase threefold 30 min following injury (8.9 ± 1.1 mm Hg, p < 0.001) and remain elevated for up to 7 days (4.3 ± 0.8 mm Hg). Measurements of rostrocaudal ISP distribution reveal peak pressures in the injury center and in segments rostral to the injury during the acute phase(≤ 24 h). During the subacute phase(≥ 72 h), peak ISP decreases while a 7.5 mm long segment of moderately elevated ISP remains, centered on the initial contusion site. Interestingly, the contribution of the dural and pial compartments toward increased ISP changes with time after injury: Dural and pial linings contribute almost equally to increased ISP during the acute phase, whereas the dural lining is primarily responsible for elevated ISP during the subacute phase (78.9%). Our findings suggest that a rat contusion SCI model in combination with novel micro-catheters allows for direct measurement of ISP after SCI. Similarly to traumatic brain injury, raised tissue pressure is likely to have detrimental effects on spontaneous recovery following SCI.

18F-FDG-PET imaging of rat spinal cord demonstrates altered glucose uptake acutely after contusion injury

Spinal cord injury (SCI) results in an acute reduction in neuronal and glial cell viability, disruption in axonal tract integrity, and prolonged increases in glial activity and inflammation, all of which can influence regional metabolism and glucose utilization. To date, the understanding of glucose uptake and utilization in the injured spinal cord is limited. Positron emission tomography (PET)-based measurements of glucose uptake may therefore serve as a novel biomarker for SCI. This study aimed to determine the acute and sub-acute glucose uptake pattern after SCI to determine its potential as a novel non-invasive tool for injury assessment and to begin to understand the glucose uptake pattern following acute SCI. Briefly, adult male Sprague-Dawley rats were subjected to moderate contusion SCI, confirmed by locomotor function and histology. PET imaging with [18F] Fluorodeoxyglucose (FDG) was performed prior to injury and at 6 and 24h and 15days post-injury (dpi). FDG-PET imaging revealed significantly depressed glucose uptake at 6h post-injury at the lesion epicenter that returned to sham/naïve levels at 24h and 15 dpi after moderate injury. FDG uptake at 15 dpi was likely influenced by a combination of elevated glial presence and reduced neuronal viability. These results show that moderate SCI results in acute depression in glucose uptake followed by an increase in glucose uptake that may be related to neuroinflammation. This acute and sub-acute uptake, which is dependent on cellular responses, may represent a therapeutic target.

Early-onset treadmill training reduces mechanical allodynia and modulates calcitonin gene-related peptide fiber density in lamina III/IV in a mouse model of spinal cord contusion injury.

Below-level central neuropathic pain (CNP) affects a large proportion of spinal cord injured individuals. To better define the dynamic changes of the spinal cord neural network contributing to the development of CNP after spinal cord injury (SCI), we characterized the morphological and behavioral correlates of CNP in female C57BL/6 mice after a moderate T11 contusion SCI (50 kdyn) and the influence of moderate physical activity. Compared with sham-operated animals, injured mice developed mechanical allodynia 2 weeks post injury when tested with small-diameter von Frey hair filaments (0.16 g and 0.4 g filament), but presented hyporesponsiveness to noxious mechanical stimuli (1.4 g filament). The mechano-sensory alterations lasted up to 35 days post injury, the longest time point examined. The response latency to heat stimuli already decreased significantly 10 days post injury reaching a plateau 2 weeks later. In contrast, injured mice developed remarkable hyposensitivity to cold stimuli. Animals that underwent moderate treadmill training (2 × 15 minutes; 5 d/wk) showed a significant reduction in the response rate to light mechanical stimuli as early as 6 days after training. Calcitonin gene-related peptide (CGRP) labeling in lamina III-IV of the dorsal horn revealed significant increases in CGRP-labeling density in injured animals compared with sham control animals. Importantly, treadmill training reduced CGRP-labeling density by about 50% (P < 0.01), partially reducing the injury-induced increases. Analysis of IB4-labeled nonpeptidergic sensory fibers revealed no differences between experimental groups. Abnormalities in temperature sensation were not influenced by physical activity. Thus, treadmill training partially resolves signs of below-level CNP after SCI and modulates the density of CGRP-labeled fibers.

Serial Diffusion Tensor Imaging In Vivo Predicts Long-Term Functional Recovery and Histopathology in Rats following Different Severities of Spinal Cord Injury.

The current study demonstrates the feasibility of using serial magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) in vivo to quantify temporally spinal cord injury (SCI) pathology in adult female Sprague-Dawley rats that were scanned prior to a moderate or severe upper lumbar contusion SCI. Injured rats were behaviorally tested for hind limb locomotion (Basso, Beattie, Bresnahan [BBB] scores) weekly for 4 weeks and scanned immediately after each session, ending with terminal gait analyses prior to euthanasia. As a measure of tissue integrity, fractional anisotropy (FA) values were significantly lower throughout the spinal cord in both injury cohorts at all time-points examined versus pre-injury. Moreover, FA values were significantly lower following severe versus moderate SCI at all time-points, and FA values at the injury epicenters at all time-points were significantly correlated with both spared white and gray matter volumes, as well as lesion volumes. Critically, quantified FA values at subacute (24 h) and all subsequent time-points were highly predictive of terminal behavior, reflected in significant correlations with both weekly BBB scores and terminal gait parameters. Critically, the finding that clinically relevant subacute (24 h) FA values accurately predict long-term functional recovery may obviate long-term studies to assess the efficacy of therapeutics tested experimentally or clinically. In summary, this study demonstrates a reproducible serial MRI procedure to predict the long-term impact of contusion SCI on both behavior and histopathology using subacute DTI metrics obtained in vivo to accurately predict multiple terminal outcome measures, which can be particularly valuable when comparing experimental interventions.

A controlled spinal cord contusion for the rhesus macaque monkey

Most in vivo spinal cord injury (SCI) experimental models use rodents. Due to the anatomical and functional differences between rodents and humans, reliable large animal models, such as non-human primates, of SCI are critically needed to facilitate translation of laboratory discoveries to clinical applications. Here we report the establishment of a controlled spinal contusion model that produces severity-dependent functional and histological deficits in non-human primates. Six adult male rhesus macaque monkeys underwent mild to moderate contusive SCI using 1.0 and 1.5mm tissue displacement injuries at T9 or sham laminectomy (n=2/group). Multiple assessments including motor-evoked potential (MEP), somatosensory-evoked potential (SSEP), MR imaging, and monkey hindlimb score (MHS) were performed. Monkeys were sacrificed at 6months post-injury, and the lesion area was examined for cavitation, axons, myelin, and astrocytic responses. The MHS demonstrated that both the 1.0 and 1.5mm displacement injuries created discriminative neurological deficits which were severity-dependent. The MEP response rate was depressed after a 1.0mm injury and was abolished after a 1.5mm injury. The SSEP response rate was slightly decreased following both the 1.0 and 1.5mm SCI. MRI imaging demonstrated an increase in T2 signal at the lesion site at 3 and 6months, and diffusion tensor imaging (DTI) tractography showed interrupted fiber tracts at the lesion site at 4h and at 6months post-SCI. Histologically, severity-dependent spinal cord atrophy, axonal degeneration, and myelin loss were found after both injury severities. Notably, strong astrocytic gliosis was not observed at the lesion penumbra in the monkey. In summary, we describe the development of a clinically-relevant contusive SCI model that produces severity-dependent anatomical and functional deficits in non-human primates. Such a model may advance the translation of novel SCI repair strategies to the clinic.

Training-Induced Functional Gains following SCI.

We previously demonstrated that daily, hour-long training sessions significantly improved both locomotor (limb kinematics, gait, and hindlimb flexor-extensor bursting patterns) and nonlocomotor (bladder function and at-level mechanical allodynia) functions following a moderate contusive spinal cord injury. The amount of training needed to achieve this recovery is unknown. Furthermore, whether this recovery is induced primarily by neuronal activity below the lesion or other aspects related to general exercise is unclear. Therefore, the current study objectives were to (1) test the efficacy of 30 minutes of step training for recovery following a clinically relevant contusion injury in male Wistar rats and (2) test the efficacy of training without hindlimb engagement. The results indicate that as little as 30 minutes of step training six days per week enhances overground locomotion in male rats with contusive spinal cord injury but does not alter allodynia or bladder function. Thirty minutes of forelimb-only exercise did not alter locomotion, allodynia, or bladder function, and neither training protocol altered the amount of in-cage activity. Taken together, locomotor improvements were facilitated by hindlimb step training for 30 minutes, but longer durations of training are required to affect nonlocomotor systems.

Astrocytic CCAAT/Enhancer-Binding Protein Delta Contributes to Glial Scar Formation and Impairs Functional Recovery After Spinal Cord Injury.

After spinal cord injury, inflammatory reaction induces the aggregation of astrocytes to form a glial scar that eventually blocks axonal regeneration. Transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) is a regulatory protein of genes responsive to inflammatory factors, but its role in glial scar formation after spinal cord injury remains unknown. By using a model of moderate spinal cord contusion injury at the mid-thoracic level, we found that C/EBPδ was expressed mostly in the reactive astrocytes bordering the lesion in wild-type mice from 7 days after the injury. C/EBPδ-deficient mice showed reduced glial scar formation, more residual white matter, and better motor function recovery compared with wild-type mice 28 days after the injury. Upon interleukin (IL)-1β stimulation in vitro, the increased expression of C/EBPδ in reactive astrocytes inhibited RhoA expression and, subsequently, the ability of astrocyte migration. However, these reactive astrocytes also produced an increased amount of matrix metalloproteinase-3, which promoted the migration of non-IL-1β-treated, inactive astrocytes. Although the involvement of other non-astroglial C/EBPδ cannot be entirely excluded, our studies suggest that astrocytic C/EBPδ is integral to the inflammatory cascades leading to glial scar formation after spinal cord injury.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-015-9486-6) contains supplementary material, which is available to authorized users.

Treadmill training induced lumbar motoneuron dendritic plasticity and behavior recovery in adult rats after a thoracic contusive spinal cord injury

Spinal cord injury (SCI) is devastating, causing sensorimotor impairments and paralysis. Persisting functional limitations on physical activity negatively affect overall health in individuals with SCI. Physical training may improve motor function by affecting cellular and molecular responses of motor pathways in the central nervous system (CNS) after SCI. Although motoneurons form the final common path for motor output from the CNS, little is known concerning the effect of exercise training on spared motoneurons below the level of injury. Here we examined the effect of treadmill training on morphological, trophic, and synaptic changes in the lumbar motoneuron pool and on behavior recovery after a moderate contusive SCI inflicted at the 9th thoracic vertebral level (T9) using an Infinite Horizon (IH, 200 kDyne) impactor. We found that treadmill training significantly improved locomotor function, assessed by Basso–Beattie–Bresnahan (BBB) locomotor rating scale, and reduced foot drops, assessed by grid walking performance, as compared with non-training. Additionally, treadmill training significantly increased the total neurite length per lumbar motoneuron innervating the soleus and tibialis anterior muscles of the hindlimbs as compared to non-training. Moreover, treadmill training significantly increased the expression of a neurotrophin brain-derived neurotrophic factor (BDNF) in the lumbar motoneurons as compared to non-training. Finally, treadmill training significantly increased synaptic density, identified by synaptophysin immunoreactivity, in the lumbar motoneuron pool as compared to non-training. However, the density of serotonergic terminals in the same regions did not show a significant difference between treadmill training and non-training. Thus, our study provides a biological basis for exercise training as an effective medical practice to improve recovery after SCI. Such an effect may be mediated by synaptic plasticity, and neurotrophic modification in the spared lumbar motoneuron pool caudal to a thoracic contusive SCI.

Acute administration of ucf-101 ameliorates the locomotor impairments induced by a traumatic spinal cord injury

Secondary death of neural cells plays a key role in the physiopathology and the functional consequences of traumatic spinal cord injury (SCI). Pharmacological manipulation of cell death pathways leading to the preservation of neural cells is acknowledged as a main therapeutic goal in SCI. In the present work, we hypothesize that administration of the neuroprotective cell-permeable compound ucf-101 will reduce neural cell death during the secondary damage of SCI, increasing tissue preservation and reducing the functional deficits. To test this hypothesis, we treated mice with ucf-101 during the first week after a moderate contusive SCI. Our results reveal that ucf-101 administration protects neural cells from the deleterious secondary mechanisms triggered by the trauma, reducing the extension of tissue damage and improving motor function recovery. Our studies also suggest that the effects of ucf-101 may be mediated through the inhibition of HtrA2/OMI and the concomitant increase of inhibitor of apoptosis protein XIAP, as well as the induction of ERK1/2 activation and/or expression. In vitro assays confirm the effects of ucf-101 on both pathways as well as on the reduction of caspase cascade activation and apoptotic cell death in a neuroblastoma cell line. These results suggest that ucf-101 can be a promising therapeutic tool for SCI that deserves more detailed analyses.

A novel device for studying weight supported, quadrupedal overground locomotion in spinal cord injured rats

BackgroundProviding weight support facilitates locomotion in spinal cord injured animals. To control weight support, robotic systems have been developed for treadmill stepping and more recently for overground walking. New methodWe developed a novel device, the body weight supported ambulatory rodent trainer (i.e. BART). It has a small pneumatic cylinder that moves along a linear track above the rat. When air is supplied to the cylinder, the rats are lifted as they perform overground walking. We tested the BART device in rats that received a moderate spinal cord contusion injury and in normal rats. Locomotor training with the BART device was not performed. ResultsAll of the rats learned to walk in the BART device. In the contused rats, significantly greater paw dragging and dorsal stepping occurred in the hindlimbs compared to normal. Providing weight support significantly raised hip position and significantly reduced locomotor deficits. Hindlimb stepping was tightly coupled to forelimb stepping but only when the contused rats stepped without weight support. Three weeks after the contused rats received a complete spinal cord transection, significantly fewer hindlimb steps were performed. Comparison with existing methodsRelative to rodent robotic systems, the BART device is a simpler system for studying overground locomotion. The BART device lacks sophisticated control and sensing capability, but it can be assembled relatively easily and cheaply. ConclusionsThese findings suggest that the BART device is a useful tool for assessing quadrupedal, overground locomotion which is a more natural form of locomotion relative to treadmill locomotion.

Early Intervention for Spinal Cord Injury with Human Induced Pluripotent Stem Cells Oligodendrocyte Progenitors

Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.

Characterization of recovery, repair, and inflammatory processes following contusion spinal cord injury in old female rats: is age a limitation?

BackgroundAlthough the incidence of spinal cord injury (SCI) is steadily rising in the elderly human population, few studies have investigated the effect of age in rodent models. Here, we investigated the effect of age in female rats on spontaneous recovery and repair after SCI. Young (3 months) and aged (18 months) female rats received a moderate contusion SCI at T9. Behavioral recovery was assessed, and immunohistocemical and stereological analyses performed.ResultsAged rats demonstrated greater locomotor deficits compared to young, beginning at 7 days post-injury (dpi) and lasting through at least 28 dpi. Unbiased stereological analyses revealed a selective increase in percent lesion area and early (2 dpi) apoptotic cell death caudal to the injury epicenter in aged versus young rats. One potential mechanism for these differences in lesion pathogenesis is the inflammatory response; we therefore assessed humoral and cellular innate immune responses. No differences in either acute or chronic serum complement activity, or acute neutrophil infiltration, were observed between age groups. However, the number of microglia/macrophages present at the injury epicenter was increased by 50% in aged animals versus young.ConclusionsThese data suggest that age affects recovery of locomotor function, lesion pathology, and microglia/macrophage response following SCI.

Peripheral noxious stimulation reduces withdrawal threshold to mechanical stimuli after spinal cord injury: Role of tumor necrosis factor alpha and apoptosis

We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered 1day after SCI on mechanical withdrawal responses to von Frey stimuli from 1 to 28days after treatment. In addition, because the proinflammatory cytokine tumor necrosis factor alpha (TNFα) is implicated in numerous injury-induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNFα, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation after SCI induced mechanical sensitivity by 24h. These behavioral changes were accompanied by increased expression of TNFα. Cellular assessments of downstream targets of TNFα revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12kDa) of caspase 3, indicative of active apoptosis at a time point consistent with the onset of mechanical allodynia. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24h after stimulation. Interestingly, expression of the inflammatory mediator NFκB was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNFα signaling.

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.

Characterization of the expression and inflammatory activity of NADPH oxidase after spinal cord injury

Reactive oxygen species (ROS) and the NADPH oxidase (NOX) enzyme are both up-regulated after spinal cord injury (SCI) and play significant roles in promoting post-injury inflammation. However, the cellular and temporal expression profile of NOX isotypes, including NOX2, 3, and 4, after SCI is currently unclear. The purpose of this study was to resolve this expression profile and examine the effect of inhibition of NOX on inflammation after SCI. Briefly, adult male rats were subjected to moderate contusion SCI. Double immunofluorescence for NOX isotypes and CNS cellular types was performed at 24 h, 7 days, and 28 days post-injury. NOX isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on injury status. NOX2 and 4 were found in all cell types assessed, while NOX3 was positively identified in neurons only. NOX2 was the most responsive to injury, increasing in both microglia and astrocytes. The biggest increases in expression were observed at 7 days post-injury and increased expression was maintained through 28 days. NOX2 inhibition by systemic administration of gp91ds-tat at 15 min, 6 h or 7 days after injury reduced both pro-inflammatory cytokine expression and evidence of oxidative stress in the injured spinal cord. This study therefore illustrates the regional and temporal influence on NOX isotype expression and the importance of NOX activation in SCI. This information will be useful in future studies of understanding ROS production after injury and therapeutic potentials.

Long-term survival, axonal growth-promotion, and myelination of Schwann cells grafted into contused spinal cord in adult rats

Schwann cells (SCs) have been considered to be one of the most promising cell types for transplantation to treat spinal cord injury (SCI) due to their unique growth-promoting properties. Despite the extensive use as donor cells for transplantation in SCI models, the fate of SCs is controversial due in part to the lack of a reliable marker for tracing the grafted SCs. To precisely assess the fate and temporal profile of transplanted SCs, we isolated purified SCs from sciatic nerves of adult transgenic rats overexpressing GFP (SCs-GFP). SCs-GFP were directly injected into the epicenter of a moderate contusive SCI at the mid-thoracic level at 1week post-injury. The number of SCs-GFP or SCs-GFP labeled with Bromodeoxyuridine (BrdU) was quantified at 5min, 1day, and 1, 2, 4, 12 and 24weeks after cell injection. Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, footfall error, thermal withdrawal latency, and footprint analysis were performed before and after the SCs-GFP transplantation. After transplantation, SCs-GFP quickly filled the lesion cavity. A remarkable survival of grafted SCs-GFP up to 24weeks post-grafting was observed with clearly identified SC individuals. SCs-GFP proliferated after injection, peaked at 2weeks (26% of total SCs-GFP), decreased thereafter, and ceased at 12weeks post-grafting. Although grafted SCs-GFP were mainly confined within the border of surrounding host tissue, they migrated along the central canal for up to 5.0mm at 4weeks post-grafting. Within the lesion site, grafted SCs-GFP myelinated regenerated axons and expressed protein zero (P0) and myelin basic protein (MBP). Within the SCs-GFP grafts, new blood vessels were formed. Except for a significant decrease of angle of rotation in the footprint analysis, we did not observe significant behavioral improvements in BBB locomotor rating scale, thermal withdrawal latency, or footfall errors, compared to the control animals that received no SCs-GFP. We conclude that SCs-GFP can survive remarkably well, proliferate, migrate along the central canal, and myelinate regenerated axons when being grafted into a clinically-relevant contusive SCI in adult rats. Combinatorial strategies, however, are essential to achieve a more meaningful functional regeneration of which SCs may play a significant role.

In vivo 31P NMR spectroscopy assessment of skeletal muscle bioenergetics after spinal cord contusion in rats

Muscle paralysis after spinal cord injury leads to muscle atrophy, enhanced muscle fatigue, and increased energy demands for functional activities. Phosphorus magnetic resonance spectroscopy ((31)P-MRS) offers a unique non-invasive alternative of measuring energy metabolism in skeletal muscle and is especially suitable for longitudinal investigations. We determined the impact of spinal cord contusion on in vivo muscle bioenergetics of the rat hind limb muscle using (31)P-MRS. A moderate spinal cord contusion injury (cSCI) was induced at the T8-T10 thoracic spinal segments. (31)P-MRS measurements were performed weekly in the rat hind limb muscles for 3 weeks. Spectra were acquired in a Bruker 11 T/470 MHz spectrometer using a 31P surface coil. The sciatic nerve was electrically stimulated by subcutaneous needle electrodes. Spectra were acquired at rest (5 min), during stimulation (6 min), and recovery (20 min). Phosphocreatine (PCr) depletion rates and the pseudo first-order rate constant for PCr recovery (k PCr) were determined. The maximal rate of PCr resynthesis, the in vivo maximum oxidative capacity (V max) and oxidative adenosine triphosphate (ATP) synthesis rate (Q max) were subsequently calculated. One week after cSCI, there was a decline in the resting total creatine of the paralyzed muscle. There was a significant reduction (~24 %) in k PCr measures of the paralyzed muscle, maximum in vivo mitochondrial capacity (V max) and the maximum oxidative ATP synthesis rate (Q max) at 1 week post-cSCI. During exercise, the PCr depletion rates in the paralyzed muscle one week after injury were rapid and to a greater extent than in a healthy muscle. Using in vivo MRS assessments, we reveal an acute oxidative metabolic defect in the paralyzed hind limb muscle. These altered muscle bioenergetics might contribute to the host of motor dysfunctions seen after cSCI.

Fenbendazole improves pathological and functional recovery following traumatic spinal cord injury

During a study of spinal cord injury (SCI), mice in our colony were treated with the anthelmintic fenbendazole to treat pinworms detected in other mice not involved in the study. As this was not part of the original experimental design, we subsequently compared pathological and functional outcomes of SCI in female C57BL/6 mice who received fenbendazole (150ppm, 8mg/kg body weight/day) for 4weeks prior to moderate contusive SCI (50kdyn force) as compared to mice on the same diet without added fenbendazole. The fenbendazole-treated mice exhibited improved locomotor function, determined using the Basso mouse scale, as well as improved tissue sparing following contusive SCI. Fenbendazole may exert protective effects through multiple possible mechanisms, one of which is inhibition of the proliferation of B lymphocytes, thereby reducing antibody responses. Autoantibodies produced following SCI contribute to the axon damage and locomotor deficits. Fenbendazole pretreatment reduced the injury-induced CD45R-positive B cell signal intensity and IgG immunoreactivity at the lesion epicenter 6weeks after contusive SCI in mice, consistent with a possible effect on the immune response to the injury. Fenbendazole and related benzimadole antihelmintics are FDA approved, exhibit minimal toxicity, and represent a novel group of potential therapeutics targeting secondary mechanisms following SCI.

Tissue sparing, behavioral recovery, supraspinal axonal sparing/regeneration following sub-acute glial transplantation in a model of spinal cord contusion

BackgroundIt has been shown that olfactory ensheathing glia (OEG) and Schwann cell (SCs) transplantation are beneficial as cellular treatments for spinal cord injury (SCI), especially acute and sub-acute time points. In this study, we transplanted DsRED transduced adult OEG and SCs sub-acutely (14 days) following a T10 moderate spinal cord contusion injury in the rat. Behaviour was measured by open field (BBB) and horizontal ladder walking tests to ascertain improvements in locomotor function. Fluorogold staining was injected into the distal spinal cord to determine the extent of supraspinal and propriospinal axonal sparing/regeneration at 4 months post injection time point. The purpose of this study was to investigate if OEG and SCs cells injected sub acutely (14 days after injury) could: (i) improve behavioral outcomes, (ii) induce sparing/regeneration of propriospinal and supraspinal projections, and (iii) reduce tissue loss.ResultsOEG and SCs transplanted rats showed significant increased locomotion when compared to control injury only in the open field tests (BBB). However, the ladder walk test did not show statistically significant differences between treatment and control groups. Fluorogold retrograde tracing showed a statistically significant increase in the number of supraspinal nuclei projecting into the distal spinal cord in both OEG and SCs transplanted rats. These included the raphe, reticular and vestibular systems. Further pairwise multiple comparison tests also showed a statistically significant increase in raphe projecting neurons in OEG transplanted rats when compared to SCs transplanted animals. Immunohistochemistry of spinal cord sections short term (2 weeks) and long term (4 months) showed differences in host glial activity, migration and proteoglycan deposits between the two cell types. Histochemical staining revealed that the volume of tissue remaining at the lesion site had increased in all OEG and SCs treated groups. Significant tissue sparing was observed at both time points following glial SCs transplantation. In addition, OEG transplants showed significantly decreased chondroitin proteoglycan synthesis in the lesion site, suggesting a more CNS tolerant graft.ConclusionsThese results show that transplantation of OEG and SCs in a sub-acute phase can improve anatomical outcomes after a contusion injury to the spinal cord, by increasing the number of spared/regenerated supraspinal fibers, reducing cavitation and enhancing tissue integrity. This provides important information on the time window of glial transplantation for the repair of the spinal cord.