Mouse Model of Spinal Cord Hypoperfusion with Immediate Paralysis Caused by Endovascular Repair of Thoracic Aortic Aneurysm.

BACKGROUND: A clinically relevant mouse model of thoracic endovascular aortic repair-induced ischemic spinal cord injury has been lacking since the procedure was first employed in 1991. The hypothesis was that ligation of mouse intercostal arteries would simulate thoracic endovascular aortic repair-induced ischemic spinal cord injury and behavioral deficit. The aim was to create a mouse model of thoracic endovascular aortic repair-induced spinal cord hypoperfusion by ligating five pairs of mouse intercostal vessels. METHODS: Mice were divided into sham (n = 53) and ligation (n = 60) groups. The procedures called for double ligation of three pairs and single ligation of two pairs of thoracic intercostal arteries in adult C57BL/6 mice. A laser Doppler probe was used in vivo on the spinal cords and intercostal arteries to document the extent of arterial ligation and spinal cord hypoperfusion. The Basso Mouse Scale for Locomotion, histological studies, and electron microscopy demonstrated postligation locomotive and histopathological changes. RESULTS: Ligation induced a significant and instantaneous drop in blood flow in the intercostal arteries (% change; mean = -63.81; 95% CI, -72.28 to -55.34) and the thoracic spinal cord (% change; mean = -68.55; 95% CI, -80.23 to -56.87). Paralysis onset was immediate and of varying degree, with behavioral deficit stratified into three groups: 9.4% exhibited severe paralysis, 37.5% moderate paralysis, and 53.1% mild paralysis at day 1 (n = 32; P < 0.001). Mild and moderate paralysis was transient, gradually improving over time. Severe paralysis showed no improvement and exhibited a higher mortality rate (83%; n = 15 of 18) compared to moderately (33%; n = 6 of 18) and mildly (24%; n = 6 of 25) paralyzed mice (P < 0.001). The overall ligation group survival rate (84%; n = 46 of 55) was significantly lower than the sham group (100%; n = 48 of 48) with P = 0.003. CONCLUSIONS: The mouse model generates reproducible spinal cord hypoperfusion and accompanying histopathological ischemic spinal cord damage. The resulting anatomical changes and variable behavioral deficits mimic the variability in radiological and clinical findings in human patients.

Eccentric rehabilitation induces white matter plasticity and sensorimotor recovery in chronic spinal cord injury.

Experience-dependent white matter plasticity offers new potential for rehabilitation-induced recovery after neurotrauma. This first-in-human translational experiment combined myelin water imaging in humans and genetic fate-mapping of oligodendrocyte lineage cells in mice to investigate whether downhill locomotor rehabilitation that emphasizes eccentric muscle actions promotes white matter plasticity and recovery in chronic, incomplete spinal cord injury (SCI). In humans, of 20 individuals with SCI that enrolled, four passed the imaging screen and had myelin water imaging before and after a 12-week (3 times/week) downhill locomotor treadmill training program (SCI + DH). One individual was excluded for imaging artifacts. Uninjured control participants (n = 7) had two myelin water imaging sessions within the same day. Changes in myelin water fraction (MWF), a histopathologically-validated myelin biomarker, were analyzed in a priori motor learning and non-motor learning brain regions and the cervical spinal cord using statistical approaches appropriate for small sample sizes. PDGFRα-CreERT2:mT/mG mice, that express green fluorescent protein on oligodendrocyte precursor cells and subsequent newly-differentiated oligodendrocytes upon tamoxifen-induced recombination, were either naive (n = 6) or received a moderate (75 kilodyne), contusive SCI at T9 and were randomized to downhill training (n = 6) or unexercised groups (n = 6). We initiated recombination 29 days post-injury, seven days prior to downhill training. Mice underwent two weeks of daily downhill training on the same 10% decline grade used in humans. Between-group comparison of functional (motor and sensory) and histological (oligodendrogenesis, oligodendroglial/axon interaction, paranodal structure) outcomes occurred post-training. In humans with SCI, downhill training increased MWF in brain motor learning regions (postcentral, precuneus) and mixed motor and sensory tracts of the ventral cervical spinal cord compared to control participants (P < 0.05). In mice with thoracic SCI, downhill training induced oligodendrogenesis in cervical dorsal and lateral white matter, increased axon-oligodendroglial interactions, and normalized paranodal structure in dorsal column sensory tracts (P < 0.05). Downhill training improved sensorimotor recovery in mice by normalizing hip and knee motor control and reducing hyperalgesia, both of which were associated with new oligodendrocytes in the cervical dorsal columns (P < 0.05). Our findings indicate that eccentric-focused, downhill rehabilitation promotes white matter plasticity and improved function in chronic SCI, likely via oligodendrogenesis in nervous system regions activated by the training paradigm. Together, these data reveal an exciting role for eccentric training in white matter plasticity and sensorimotor recovery after SCI.

Bone Marrow-Derived Monocytes Drive the Inflammatory Microenvironment in Local and Remote Regions after Thoracic Spinal Cord Injury.

Spinal cord injury (SCI) produces a toxic inflammatory microenvironment that negatively affects plasticity and recovery. Recently, we showed glial activation and peripheral myeloid cell infiltration extending beyond the epicenter through the remote lumbar cord after thoracic SCI. The presence and role of infiltrating monocytes is important, especially in the lumbar cord where locomotor central pattern generators are housed. Therefore, we compared the inflammatory profile of resident microglia and peripheral myeloid cells after SCI. Bone marrow chimeras received midthoracic contusive SCI, and trafficking was determined 1-7 days later. Fluorescence-activated cell (FAC) sorting showed similar infiltration timing of both neutrophils and macrophages in epicenter and lumbar regions. While neutrophil numbers were attenuated by day 3, macrophages remained unchanged at day 7, suggesting that macrophages have important long-term influence on the microenvironment. Nanostring gene array identified a strong proinflammatory profile of infiltrating macrophages relative to microglia at both epicenter and lumbar sites. Macrophages had elevated expression of inflammatory cytokines (IL-1ß, IFNγ), chemokines (CCL2, CXCL2), mediators (COX-1, MMP-9), and receptors (CCR2, Ly6C), and decreased expression of growth promoting genes (GDNF, BDNF). Importantly, lumbar macrophages had elevated expression of active trafficking genes (CCR2, l-selectin, MMP-9) compared with epicenter macrophages. Further, acute rehabilitation exacerbated the inflammatory profile of infiltrated macrophages in the lumbar cord. Such high inflammatory potential and negative response to rehabilitation of infiltrating macrophages within lumbar locomotor central pattern generators likely impedes activity-dependent recovery. Therefore, limiting active trafficking of macrophages into the lumbar cord identifies a novel target for SCI therapies to improve locomotion.

Unique Sensory and Motor Behavior in Thy1-GFP-M Mice before and after Spinal Cord Injury.

Sensorimotor recovery after spinal cord injury (SCI) is of utmost importance to injured individuals and will rely on improved understanding of SCI pathology and recovery. Novel transgenic mouse lines facilitate discovery, but must be understood to be effective. The purpose of this study was to characterize the sensory and motor behavior of a common transgenic mouse line (Thy1-GFP-M) before and after SCI. Thy1-GFP-M positive (TG+) mice and their transgene negative littermates (TG-) were acquired from two sources (in-house colony, n = 32, Jackson Laboratories, n = 4). C57BL/6J wild-type (WT) mice (Jackson Laboratories, n = 10) were strain controls. Moderate-severe T9 contusion (SCI) or transection (TX) occurred in TG+ (SCI, n = 25, TX, n = 5), TG- (SCI, n = 5), and WT (SCI, n = 10) mice. To determine responsiveness to rehabilitation, a cohort of TG+ mice with SCI (n = 4) had flat treadmill (TM) training 42-49 days post-injury (dpi). To characterize recovery, we performed Basso Mouse Scale, Grid Walk, von Frey Hair, and Plantar Heat Testing before and out to day 42 post-SCI. Open field locomotion was significantly better in the Thy1 SCI groups (TG+ and TG-) compared with WT by 7 dpi (p < 0.01) and was maintained through 42 dpi (p < 0.01). These unexpected locomotor gains were not apparent during grid walking, indicating severe impairment of precise motor control. Thy1 derived mice were hypersensitive to mechanical stimuli at baseline (p < 0.05). After SCI, mechanical hyposensitivity emerged in Thy1 derived groups (p < 0.001), while thermal hyperalgesia occurred in all groups (p < 0.001). Importantly, consistent findings across TG+ and TG- groups suggest that the effects are mediated by the genetic background rather than transgene manipulation itself. Surprisingly, TM training restored mechanical and thermal sensation to baseline levels in TG+ mice with SCI. This behavioral profile and responsiveness to chronic training will be important to consider when choosing models to study the mechanisms underlying sensorimotor recovery after SCI.

Adaptation of the Basso-Beattie-Bresnahan locomotor rating scale for use in a clinical model of spinal cord injury in dogs.

BACKGROUND: Naturally occurring acute spinal cord injury (SCI) in pet dogs provides an important clinical animal model through which to confirm and extend findings from rodent studies; however, validated quantitative outcome measures for dogs are limited. NEW METHOD: We adapted the Basso Beattie Bresnahan (BBB) scale for use in a clinical dog model of acute thoracolumbar SCI. Based on observation of normal dogs, modifications were made to account for species differences in locomotion. Assessments of paw and tail position, and trunk stability were modified to produce a 19 point scale suitable for use in dogs, termed the canine BBB scale (cBBB). Pet dogs with naturally occurring acute SCI were assigned cBBB scores at 3, 10 and 30days after laminectomy. RESULTS: Scores assigned via the cBBB were stable across testing sessions in normal dogs but increased significantly between days 3 and 30 in SCI-affected dogs (p=0.0003). The scale was highly responsive to changes in locomotor recovery over a 30day period, with a standardized response mean of 1.34. COMPARISON WITH EXISTING METHODS: Concurrent validity was good, with strong correlations observed between the cBBB and two other locomotor scales, the OSCIS (r=0.94; p<0.001) and the MFS (r=0.85; p<0.0001). cBBB scores inversely correlated with other assessments of recovery including mechanical sensory threshold (r=-0.68; p<0.0001) and coefficient of variation of stride length (r=-0.49; p<0.0001). CONCLUSIONS: These results support the use of the cBBB to assess locomotor recovery in canine clinical translational models of SCI.

Quantitative evaluation of 3D mouse behaviors and motor function in the open-field after spinal cord injury using markerless motion tracking.

Thousands of scientists strive to identify cellular mechanisms that could lead to breakthroughs in developing ameliorative treatments for debilitating neural and muscular conditions such as spinal cord injury (SCI). Most studies use rodent models to test hypotheses, and these are all limited by the methods available to evaluate animal motor function. This study's goal was to develop a behavioral and locomotor assessment system in a murine model of SCI that enables quantitative kinematic measurements to be made automatically in the open-field by applying markerless motion tracking approaches. Three-dimensional movements of eight naïve, five mild, five moderate, and four severe SCI mice were recorded using 10 cameras (100 Hz). Background subtraction was used in each video frame to identify the animal's silhouette, and the 3D shape at each time was reconstructed using shape-from-silhouette. The reconstructed volume was divided into front and back halves using k-means clustering. The animal's front Center of Volume (CoV) height and whole-body CoV speed were calculated and used to automatically classify animal behaviors including directed locomotion, exploratory locomotion, meandering, standing, and rearing. More detailed analyses of CoV height, speed, and lateral deviation during directed locomotion revealed behavioral differences and functional impairments in animals with mild, moderate, and severe SCI when compared with naïve animals. Naïve animals displayed the widest variety of behaviors including rearing and crossing the center of the open-field, the fastest speeds, and tallest rear CoV heights. SCI reduced the range of behaviors, and decreased speed (r = .70 p<.005) and rear CoV height (r = .65 p<.01) were significantly correlated with greater lesion size. This markerless tracking approach is a first step toward fundamentally changing how rodent movement studies are conducted. By providing scientists with sensitive, quantitative measurement methods, subjectivity and human error is reduced, potentially providing insights leading to breakthroughs in treating human disease.

Elevated MMP-9 in the lumbar cord early after thoracic spinal cord injury impedes motor relearning in mice.

Spinal cord injury results in distant pathology around putative locomotor networks that may jeopardize the recovery of locomotion. We previously showed that activated microglia and increased cytokine expression extend at least 10 segments below the injury to influence sensory function. Matrix metalloproteinase-9 (MMP-9) is a potent regulator of acute neuroinflammation. Whether MMP-9 is produced remote to the injury or influences locomotor plasticity remains unexamined. Therefore, we characterized the lumbar enlargement after a T9 spinal cord injury in C57BL/6 (wild-type [WT]) and MMP-9-null (knock-out [KO]) mice. Within 24 h, resident microglia displayed an activated phenotype alongside increased expression of progelatinase MMP-3 in WT mice. By 7 d, increases in active MMP-9 around lumbar vasculature and production of proinflammatory TNF-α were evident. Deletion of MMP-9 attenuated remote microglial activation and restored TNF-α expression to homeostatic levels. To determine whether MMP-9 impedes locomotor plasticity, we delivered lumbar-focused treadmill training in WT and KO mice during early (2-9 d) or late (35-42 d) phases of recovery. Robust behavioral improvements were observed by 7 d, when only trained KO mice stepped in the open field. Locomotor improvements were retained for 4 weeks as identified using state of the art mouse kinematics. Neither training nor MMP-9 depletion alone promoted recovery. The same intervention delivered late was ineffective, suggesting that lesion site sparing is insufficient to facilitate activity-based training and recovery. Our work suggests that by attenuating remote mechanisms of inflammation, acute treadmill training can harness endogenous spinal plasticity to promote robust recovery.

Characterization of recovered walking patterns and motor control after contusive spinal cord injury in rats.

Currently, complete recovery is unattainable for most individuals with spinal cord injury (SCI). Instead, recovery is typically accompanied by persistent sensory and motor deficits. Restoration of preinjury function will likely depend on improving plasticity and integration of these impaired systems. Eccentric muscle actions require precise integration of sensorimotor signals and are predominant during the yield (E2) phase of locomotion. Motor neuron activation and control during eccentric contractions is impaired across a number of central nervous system (CNS) disorders, but remains unexamined after SCI. Therefore, we characterized locomotor recovery after contusive SCI using hindlimb (HL) kinematics and electromyographic (EMG) recordings with specific consideration of eccentric phases of treadmill (TM) walking. Deficits in E2 and a caudal shift of locomotor subphases persisted throughout the 3-week recovery period. EMG records showed notable deficits in the semitendinosus (ST) during yield. Unlike other HL muscles, recruitment of ST changed with recovery. At 7 days, the typical dual-burst pattern of ST was lost and the second burst (ST2) was indistinct. By 21 days, the dual-burst pattern returned, but latencies remained impaired. We show that ST2 burst duration is highly predictive of open field Basso, Beattie, Bresnahan (BBB) scores. Moreover, we found that simple changes in locomotor specificity which enhance eccentric actions result in new motor patterns after SCI. Our findings identify a caudal shift in stepping kinematics, irregularities in E2, and aberrant ST2 bursting as markers of incomplete recovery. These residual impairments may provide opportunities for targeted rehabilitation.

Acute and chronic tactile sensory testing after spinal cord injury in rats.

Spinal cord injury (SCI) impairs sensory systems causing allodynia. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia(3). Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

Validity of acute and chronic tactile sensory testing after spinal cord injury in rats.

Spinal cord injury (SCI) impairs sensory systems causing allodynia. Measuring the development of allodynia in rodent models of SCI is challenging due to spinal shock and marked motor impairments. Assessment of SCI-induced allodynia is not standardized across labs, making interpretation of results difficult. Therefore, we validated sensory threshold assessment after SCI and developed a novel assessment of allodynia prior to motor recovery in a rat SCI model. One hundred fifty-six Sprague-Dawley rats received T8 laminectomy or mild to moderate SCI using the OSU SCI device (0.3 mm to 1.3 mm cord displacement). To determine tactile thresholds, von Frey hairs (VFH) were applied in Up-Down or ascending order to the dorsal or plantar hindpaw. The most efficient and valid procedures that maintain high sensitivity and specificity were identified. Ten Up-Down VFH applications yielded stable thresholds; reducing the risk of threshold decay and unnecessary exposure to painful stimuli. Importantly, distraction of SCI-rats with food revealed differential decay of thresholds than when distraction is not provided. The new test uses dorsal VFH stimulation and is independent of trunk or hindlimb control. Acute dorsal VFH thresholds collected before recovery of hindlimb weight support accurately predicted plantar VFH thresholds measured at late timepoints (chi(2)=8.479; p<0.05). Thus, standardized testing early after SCI using the dorsal VFH test or later using 10 stimuli in the Up-Down test produces valid measures of tactile sensation across many SCI severities. Early detection of allodynia in experimental SCI will allow identification of mechanisms responsible for pain development and determine targets for therapeutic interventions.

Remote activation of microglia and pro-inflammatory cytokines predict the onset and severity of below-level neuropathic pain after spinal cord injury in rats.

Spinal cord injury (SCI) impairs sensory systems causing chronic allodynia. Mechanisms underlying neuropathic pain have been more extensively studied following peripheral nerve injury (PNI) than after central trauma. Microglial activation, pro-inflammatory cytokine production and activation of p38 MAP kinase pathways may induce at-level allodynia following PNI. We investigated whether midthoracic SCI elicits similar behavioral and cellular responses below the level of injury (lumbar spinal cord; L5). Importantly, we show that anatomical connections between L5 and supraspinal centers remain intact after moderate SCI allowing direct comparison to a well-established model of peripheral nerve injury. We found that SCI elicits below-level allodynia of similar magnitude to at-level pain caused by a peripheral nerve injury. Moreover, the presence of robust microglial activation in L5 cord predicted allodynia in 86% of rats. Also increased phosphorylation of p38 MAP kinase occurred in the L5 dorsal horn of allodynic rats. For below-level allodynia after SCI, TNF-alpha and IL-1beta increased in the L5 dorsal horn by 7 dpo and returned to baseline by 35 dpo. Interestingly, IL-6 remains at normal levels early after SCI and increases at chronic time points. Increased levels of pro-inflammatory cytokines also occurred in the thalamus after SCI-induced allodynia. These data suggest that remote microglial activation is pivotal in the development and maintenance of below-level allodynia after SCI. Fractalkine, a known activator of microglia, and astrocytes were not primary modulators of below-level pain. Although the mechanisms of remote microglial activation are unknown, this response may be a viable target for limiting or preventing neuropathic pain after SCI in humans.

Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.

Genetically engineered mice are used extensively to examine molecular responses to spinal cord injury (SCI). Inherent strain differences may confound behavioral outcomes; therefore, behavioral characterization of several strains after SCI is warranted. The Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) for rats has been widely used for SCI mice, but may not accurately reflect their unique recovery pattern. This study's purpose was to develop a valid locomotor rating scale for mice and to identify strain differences in locomotor recovery after SCI. We examined C57BL/6, C57BL/10, B10.PL, BALB/c, and C57BL/6x129S6 F1 strains for 42 days after mild, moderate, and severe contusive SCI or transection of the mid thoracic spinal cord. Contusions were created using the Ohio State University electromagnetic SCI device which is a displacement-driven model, and the Infinite Horizon device, which is a force-driven model. Attributes and rankings for the Basso Mouse Scale for Locomotion (BMS) were determined from frequency analyses of seven locomotor categories. Mouse recovery differed from rats for coordination, paw position and trunk instability. Disagreement occurred across six expert raters using BBB (p < 0.05) but not BMS to assess the same mice. BMS detected significant differences in locomotor outcomes between severe contusion and transection (p < 0.05) and SCI severity gradations resulting from displacement variations of only 0.1 mm (p < 0.05). BMS demonstrated significant face, predictive and concurrent validity. Novice BMS raters with training scored within 0.5 points of experts and demonstrated high reliability (0.92-0.99). The BMS is a sensitive, valid and reliable locomotor measure in SCI mice. BMS revealed significantly higher recovery in C57BL/10, B10.PL and F1 than the C57BL/6 and BALB/c strains after moderate SCI (p < 0.05). The differing behavioral response to SCI suggests inherent genetic factors significantly impact locomotor recovery and must be considered in studies with inbred or genetically engineered mouse strains.

Stepwise motor and all-or-none sensory recovery is associated with nonlinear sparing after incremental spinal cord injury in rats.

Spinal cord injury (SCI) causes motor and sensory deficits that impair functional performance. While more functional recovery occurs with greater white matter sparing (WMS), it is unclear which locomotor features are more vulnerable to SCI than others, if recovery of certain features depends on specific amounts of WMS, and whether motor recovery patterns differ from sensory recovery. Locomotor and sensory recovery after graded contusive SCI with cord displacements of 0.3, 0.5, 0.7, 0.9, 1.1, 1.25, and 1.3 mm was examined for 6 weeks in 80 female Sprague-Dawley rats. Seven SCI gradations resulted in three locomotor performance levels measured with BBB (P < 0.01): High: laminectomy (LAM) controls and 0.3 (19.87 +/- 0.35 SEM); Intermediate: 0.5-0.9 (13.71 +/- 0.32); and Low: 1.1-1.3 (9.23 +/- 0.36). Normal paw position was most susceptible to SCI requiring 90% WMS, while consistent plantar stepping was least susceptible depending on 10% WMS. A threshold at the 0.9 severity for coordination, toe clearance, and nearly normal trunk stability and tail usage required 25% WMS. Analysis of interlimb coordination using new phase dispersion (PD) techniques delineated three recovery patterns: synchronous (0.3), modified concordance (0.5, 0.7), and disengaged (0.9, 1.1). Lesion severity correlated to WMS (r(2) = 0.96) and to BBB (r(2) = 0.87) by nonlinear polynomial regressions. Mechanical allodynia developed only after injuries resulting in < or =10% WMS. Nonlinear motor and sensory recovery patterns suggest that small reparative changes may substantially improve function in individuals with SCI. A hierarchical locomotor recovery based on simple segmental versus complex supraspinal motor control is proposed.

Passive or active immunization with myelin basic protein impairs neurological function and exacerbates neuropathology after spinal cord injury in rats.

Myelin-reactive T-cells are activated by traumatic spinal cord injury (SCI) in rodents and humans. Despite the historical association of these cells with experimental and clinical neuropathology, recent data suggest a neuroprotective role for myelin-reactive T-cells. Because of the biological and therapeutic implications of these findings, we attempted to reproduce the original neuroprotective vaccine protocols in a model of rat SCI. Specifically, MBP-reactive T-cell function was enhanced in SCI rats via passive or active immunization. Locomotor function was assessed using a standardized locomotor rating scale (Basso-Beattie-Bresnahan scale) and was correlated with myelin and axon sparing. The functional and anatomical integrity of the rubrospinal pathway also was analyzed using the inclined plane test and anatomical tract tracing. MBP-immunized rats exhibited varying degrees of functional impairment, exacerbated lesion pathology, greater rubrospinal neuron loss, increased intraspinal T-cell accumulation, and enhanced macrophage activation relative to SCI control groups. These data are consistent with the conventional view of myelin-reactive T-cells as pathological effector cells.