Neuroprotective Pentapeptide, CN-105, Improves Outcomes in Translational Models of Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a devastating form of cerebrovascular disease for which there are no approved pharmacological interventions that improve outcomes. Apolipoprotein E (apoE) has emerged as a promising therapeutic target given its isoform-specific neuroprotective properties and ability to modify neuroinflammatory responses. We developed a 5-amino acid peptide, CN-105, that mimics the polar face of the apoE helical domain involved in receptor interactions, readily crosses the blood-brain barrier, and improves outcomes in well-established preclinical ICH models. In the current study, we investigated the therapeutic potential of CN-105 in translational ICH models that account for hypertensive comorbidity, sex, species, and age. METHODS: In three separate experiments, we delivered three intravenous doses of CN-105 (up to 0.20 mg/kg) or vehicle to hypertensive male BPH/2 J mice, spontaneously hypertensive female rats, or 11-month-old male mice within 24-h of ICH. Neuropathological and neurobehavioral outcomes were determined over 3, 7, and 9 days, respectively. RESULTS: In spontaneously hypertensive male mice, there was a significant dose-dependent effect of CN-105 on vestibulomotor function at 0.05 and 0.20 mg/kg doses (p < 0.05; 95% CI: 0.91-153.70 and p < 0.001; 95% CI: 49.54-205.62), while 0.20 mg/kg also improved neuroseverity scores (p < 0.05; 95% CI: 0.27-11.00) and reduced ipsilateral brain edema (p < 0.05; 95% CI: - 0.037 to - 0.001). In spontaneously hypertensive female rats, CN-105 (0.05 mg/kg) had a significant effect on vestibulomotor function (p < 0.01; η2 = 0.093) and neuroseverity scores (p < 0.05; η2 = 0.083), and reduced contralateral edema expansion (p < 0.01; 95% CI: - 1.41 to - 0.39). In 11-month-old male mice, CN-105 had a significant effect on vestibulomotor function (p < 0.001; η2 = 0.111) but not neuroseverity scores (p > 0.05; η2 = 0.034). CONCLUSIONS: Acute treatment with CN-105 improves outcomes in translational ICH models independent of sex, species, age, or hypertensive comorbidity.

Microglia Promote Increased Pain Behavior through Enhanced Inflammation in the Spinal Cord during Repeated Social Defeat Stress.

Clinical studies indicate that psychosocial stress contributes to adverse chronic pain outcomes in patients, but it is unclear how this is initiated or amplified by stress. Repeated social defeat (RSD) is a mouse model of psychosocial stress that activates microglia, increases neuroinflammatory signaling, and augments pain and anxiety-like behaviors. We hypothesized that activated microglia within the spinal cord facilitate increased pain sensitivity following RSD. Here we show that mechanical allodynia in male mice was increased with exposure to RSD. This stress-induced behavior corresponded with increased mRNA expression of several inflammatory genes, including IL-1ß, TNF-α, CCL2, and TLR4 in the lumbar spinal cord. While there were several adhesion and chemokine-related genes increased in the lumbar spinal cord after RSD, there was no accumulation of monocytes or neutrophils. Notably, there was evidence of microglial activation selectively within the nociceptive neurocircuitry of the dorsal horn of the lumbar cord. Elimination of microglia using the colony stimulating factor 1 receptor antagonist PLX5622 from the brain and spinal cord prevented the development of mechanical allodynia in RSD-exposed mice. Microglial elimination also attenuated RSD-induced IL-1ß, CCR2, and TLR4 mRNA expression in the lumbar spinal cord. Together, RSD-induced allodynia was associated with microglia-mediated inflammation within the dorsal horn of the lumbar spinal cord.SIGNIFICANCE STATEMENT Mounting evidence indicates that psychological stress contributes to the onset and progression of adverse nociceptive conditions. We show here that repeated social defeat stress causes increased pain sensitivity due to inflammatory signaling within the nociceptive circuits of the spinal cord. Studies here mechanistically tested the role of microglia in the development of pain by stress. Pharmacological ablation of microglia prevented stress-induced pain sensitivity. These findings demonstrate that microglia are critical mediators in the induction of pain conditions by stress. Moreover, these studies provide a proof of principle that microglia can be targeted as a therapeutic strategy to mitigate adverse pain conditions.

Optimization of a translational murine model of closed-head traumatic brain injury.

Traumatic brain injury (TBI) from closed-head trauma is a leading cause of disability, with limited effective interventions. Many TBI models impact brain parenchyma directly, and are limited by the fact that these forces do not recapitulate clinically relevant closed head injury. However, applying clinically relevant injury mechanics to the intact skull may lead to variability and as a result, preclinical modeling TBI remains a challenge. Current models often do not explore sex differences in TBI, which is critically important for translation to clinical practice. We systematically investigated sources of variability in a murine model of closed-head TBI and developed a framework to reduce variability across severity and sex. We manipulated pressure, dwell time, and displacement to determine effects on motor coordination, spatial learning, and neuronal damage in 10-week-old male and female mice. Increasing pressure beyond 70 psi had a ceiling effect on cellular and behavioral outcomes, while manipulating dwell time only affected behavioral performance. Increasing displacement precisely graded injury severity in both sexes across all outcomes. Physical signs of trauma occurred more frequently at higher displacements. Stratifying severity based on day-1 rotarod performance retained histological relationships and separated both sexes into injury severity cohorts with distinct patterns of behavioral recovery. Utilizing this stratification strategy, within-group rotarod variability over 6 days post-injury was reduced by 50%. These results have important implications for translational research in TBI and provide a framework for using this clinically relevant translational injury model in both male and female mice.

Spinal cord repair is modulated by the neurogenic factor Hb-egf under direction of a regeneration-associated enhancer.

Unlike adult mammals, zebrafish regenerate spinal cord tissue and recover locomotor ability after a paralyzing injury. Here, we find that ependymal cells in zebrafish spinal cords produce the neurogenic factor Hb-egfa upon transection injury. Animals with hb-egfa mutations display defective swim capacity, axon crossing, and tissue bridging after spinal cord transection, associated with disrupted indicators of neuron production. Local recombinant human HB-EGF delivery alters ependymal cell cycling and tissue bridging, enhancing functional regeneration. Epigenetic profiling reveals a tissue regeneration enhancer element (TREE) linked to hb-egfa that directs gene expression in spinal cord injuries. Systemically delivered recombinant AAVs containing this zebrafish TREE target gene expression to crush injuries of neonatal, but not adult, murine spinal cords. Moreover, enhancer-based HB-EGF delivery by AAV administration improves axon densities after crush injury in neonatal cords. Our results identify Hb-egf as a neurogenic factor necessary for innate spinal cord regeneration and suggest strategies to improve spinal cord repair in mammals.

Effect of Acute Physical Interventions on Pathophysiology and Recovery After Spinal Cord Injury: A Comprehensive Review of the Literature.

Physical rehabilitation is essential for enhancing recovery in individuals with spinal cord injury (SCI); however, aside from early surgical intervention and hemodynamic management, there are no proven interventions for promoting recovery in the acute phase. In general, early rehabilitation is considered beneficial, but optimal parameters and potential contraindications for implementing rehabilitation at very early time points are unclear. Moreover, clinical trials to date are limited to studies initiating rehabilitation 2 weeks after injury and later. To address these gaps, this article reviews the preclinical literature on physical interventions initiated within the first 8 days postinjury. Effects of early rehabilitation on molecular and structural nervous system changes, behavioral function, and body systems are considered. Most studies utilized treadmill or cycle training as the primary intervention. Treadmill training initiated within the first 3 days and terminated by 1 week after injury worsened autonomic function, inflammation, and locomotor outcomes, while swim training during this period increased microvascular dysfunction. In contrast, lower-intensity rehabilitation such as reach training, ladder training, or voluntary wheel or ball training showed benefits when implemented during the first 3 days. Rehabilitation initiated at 4 days postinjury was also associated with enhanced motor recovery. Cycling appears to have the greatest risk-benefit ratio; however, the effects of cycle training in the first 3 days were not investigated. Overall, research suggests that lower intensity or voluntary rehabilitation during the hyperacute phase is more appropriate until at least 4 days postinjury, at which point higher-intensity activity becomes safer and more beneficial for recovery.

Surgical Considerations to Improve Recovery in Acute Spinal Cord Injury.

Acute traumatic spinal cord injury (SCI) can be a devastating and costly event for individuals, their families, and the health system as a whole. Prognosis is heavily dependent on the physical extent of the injury and the severity of neurological dysfunction. If not treated urgently, individuals can suffer exacerbated secondary injury cascades that may increase tissue injury and limit recovery. Initial recognition and rapid treatment of acute SCI are vital to limiting secondary injury, reducing morbidity, and providing the best chance of functional recovery. This article aims to review the pathophysiology of SCI and the most up-to-date management of the acute traumatic SCI, specifically examining the modern approaches to surgical treatments along with the ethical limitations of research in this field.

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.

Sparing of Descending Axons Rescues Interneuron Plasticity in the Lumbar Cord to Allow Adaptive Learning After Thoracic Spinal Cord Injury.

This study evaluated the role of spared axons on structural and behavioral neuroplasticity in the lumbar enlargement after a thoracic spinal cord injury (SCI). Previous work has demonstrated that recovery in the presence of spared axons after an incomplete lesion increases behavioral output after a subsequent complete spinal cord transection (TX). This suggests that spared axons direct adaptive changes in below-level neuronal networks of the lumbar cord. In response to spared fibers, we postulate that lumbar neuron networks support behavioral gains by preventing aberrant plasticity. As such, the present study measured histological and functional changes in the isolated lumbar cord after complete TX or incomplete contusion (SCI). To measure functional plasticity in the lumbar cord, we used an established instrumental learning paradigm (ILP). In this paradigm, neural circuits within isolated lumbar segments demonstrate learning by an increase in flexion duration that reduces exposure to a noxious leg shock. We employed this model using a proof-of-principle design to evaluate the role of sparing on lumbar learning and plasticity early (7 days) or late (42 days) after midthoracic SCI in a rodent model. Early after SCI or TX at 7 days, spinal learning was unattainable regardless of whether the animal recovered with or without axonal substrate. Failed learning occurred alongside measures of cell soma atrophy and aberrant dendritic spine expression within interneuron populations responsible for sensorimotor integration and learning. Alternatively, exposure of the lumbar cord to a small amount of spared axons for 6 weeks produced near-normal learning late after SCI. This coincided with greater cell soma volume and fewer aberrant dendritic spines on interneurons. Thus, an opportunity to influence activity-based learning in locomotor networks depends on spared axons limiting maladaptive plasticity. Together, this work identifies a time dependent interaction between spared axonal systems and adaptive plasticity in locomotor networks and highlights a critical window for activity-based rehabilitation.

Lumbar Myeloid Cell Trafficking into Locomotor Networks after Thoracic Spinal Cord Injury.

Spinal cord injury (SCI) promotes inflammation along the neuroaxis that jeopardizes plasticity, intrinsic repair and recovery. While inflammation at the injury site is well-established, less is known within remote spinal networks. The presence of bone marrow-derived immune (myeloid) cells in these areas may further impede functional recovery. Previously, high levels of the gelatinase, matrix metalloproteinase-9 (MMP-9) occurred within the lumbar enlargement after thoracic SCI and impeded activity-dependent recovery. Since SCI-induced MMP-9 potentially increases vascular permeability, myeloid cell infiltration may drive inflammatory toxicity in locomotor networks. Therefore, we examined neurovascular reactivity and myeloid cell infiltration in the lumbar cord after thoracic SCI. We show evidence of region-specific recruitment of myeloid cells into the lumbar but not cervical region. Myeloid infiltration occurred with concomitant increases in chemoattractants (CCL2) and cell adhesion molecules (ICAM-1) around lumbar vasculature 24h and 7days post injury. Bone marrow GFP chimeric mice established robust infiltration of bone marrow-derived myeloid cells into the lumbar gray matter 24h after SCI. This cell infiltration occurred when the blood-spinal cord barrier was intact, suggesting active recruitment across the endothelium. Myeloid cells persisted as ramified macrophages at 7days post injury in parallel with increased inhibitory GAD67 labeling. Importantly, macrophage infiltration required MMP-9.

Method for assessing brain changes associated with gluteus maximus activation.

STUDY DESIGN: Reliability study. OBJECTIVES: To determine the feasibility and reliability of using transcranial magnetic stimulation (TMS) to assess corticomotor excitability (CE) of the gluteus maximus. BACKGROUND: Sport-specific skill training targeting greater utilization of the gluteus maximus has been proposed as a method to reduce the incidence of noncontact knee injuries. The use of TMS to assess changes in CE may help to determine training-induced central mechanisms associated with gluteus maximus activation. METHODS: Within- and between-day reliability was measured in 10 healthy adults. The CE was measured by stimulating the gluteus maximus ìhotspotî at 120% and 150% of motor threshold, while subjects performed a double-leg bridge. An intraclass correlation coefficient (model 2,1), standard error of measurement, and minimal detectable change were calculated to determine the within- and between-day reliability for the following TMS variables: peak-to-peak motor-evoked potential (MEP) amplitudes, cortical silent period, and MEP latency. RESULTS: It is feasible to measure the CE of the gluteus maximus with TMS. The intraclass correlation coefficients for all TMS outcome measures ranged from 0.73 to 0.97. The ranges of minimal detectable change, with respect to mean values for each TMS variable, were larger for MEP amplitude (304.7-585.4 µV) compared to those for cortical silent period duration (25.3-40.8 milliseconds) and MEP latency (1.1-2.1 milliseconds). CONCLUSION: The present study demonstrated a feasible method for using TMS to measure CE of the gluteus maximus. Small minimal detectable change values for the cortical silent period and MEP latency provide a reference for future studies.