BMAL1 loss in oligodendroglia contributes to abnormal myelination and sleep.

Myelination depends on the maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that OPC-specific proliferation, morphology, and BMAL1 are time-of-day dependent. Knockout of Bmal1 in mouse OPCs during development disrupts the expression of genes associated with circadian rhythms, proliferation, density, morphology, and migration, leading to changes in OPC dynamics in a spatiotemporal manner. Furthermore, these deficits translate into thinner myelin, dysregulated cognitive and motor functions, and sleep fragmentation. OPC-specific Bmal1 loss in adulthood does not alter OPC density at baseline but impairs the remyelination of a demyelinated lesion driven by changes in OPC morphology and migration. Lastly, we show that sleep fragmentation is associated with increased prevalence of the demyelinating disorder multiple sclerosis (MS), suggesting a link between MS and sleep that requires further investigation. These findings have broad mechanistic and therapeutic implications for brain disorders that include both myelin and sleep phenotypes.

Human Spinal Oligodendrogenic Neural Progenitor Cells Enhance Pathophysiological Outcomes and Functional Recovery in a Clinically Relevant Cervical Spinal Cord Injury Rat Model.

Traumatic spinal cord injury (SCI) results in the loss of neurons, oligodendrocytes, and astrocytes. Present interventions for SCI include decompressive surgery, anti-inflammatory therapies, and rehabilitation programs. Nonetheless, these approaches do not offer regenerative solutions to replace the lost cells, fiber tracts, and circuits. Neural stem/progenitor cell (NPC) transplantation is a promising strategy that aims to encourage regeneration. However, NPC differentiation remains inconsistent, thus, contributing to suboptimal functional recovery. As such, we have previously engineered oligodendrogenically biased NPCs (oNPCs) and demonstrated their efficacy in a thoracic model of SCI. Since the majority of patients with SCI experience cervical injuries, our objective in the current study was to generate human induced pluripotent stem cell-derived oNPCs (hiPSC-oNPCs) and to characterize these cells in vitro and in vivo, utilizing a clinically relevant rodent model of cervical SCI. Following transplantation, the oNPCs engrafted, migrated to the rostral and caudal regions of the lesion, and demonstrated preferential differentiation toward oligodendrocytes. Histopathological evaluations revealed that oNPC transplantation facilitated tissue preservation while diminishing astrogliosis. Moreover, oNPC transplantation fostered remyelination of the spared tissue. Functional analyses indicated improved forelimb grip strength, gait, and locomotor function in the oNPC-transplanted rats. Importantly, oNPC transplantation did not exacerbate neuropathic pain or induce tumor formation. In conclusion, these findings underscore the therapeutic potential of oNPCs in promoting functional recovery and histopathological improvements in cervical SCI. This evidence warrants further investigation to optimize and advance this promising cell-based therapeutic approach.

Circadian Control of Glial Cell Homeodynamics.

The molecular mechanisms that maintain circadian rhythms in mammalian as well as non-mammalian systems are well documented in neuronal populations but comparatively understudied in glia. Glia are highly dynamic in form and function, and the circadian clock provides broad dynamic ranges for the maintenance of this homeostasis, thus glia are key to understanding the role of circadian biology in brain function. Here, we highlight the implications of the molecular circadian clock on the homeodynamic nature of glia, underscoring the current gap in understanding the role of the circadian system in oligodendroglia lineage cells and subsequent myelination. Through this perspective, we will focus on the intersection of circadian and glial biology and how it interfaces with global circadian rhythm maintenance associated with normative and aberrant brain function.

The endogenous progenitor response following traumatic brain injury: a target for cell therapy paradigms.

Although there is ample evidence that central nervous system progenitor pools respond to traumatic brain injury, the reported effects are variable and likely contribute to both recovery as well as pathophysiology. Through a better understanding of the diverse progenitor populations in the adult brain and their niche-specific reactions to traumatic insult, treatments can be tailored to enhance the benefits and dampen the deleterious effects of this response. This review provides an overview of endogenous precursors, the associated effects on cognitive recovery, and the potential of exogenous cell therapeutics to modulate these endogenous repair mechanisms. Beyond the hippocampal dentate gyrus and subventricular zone of the lateral ventricles, more recently identified sites of adult neurogenesis, the meninges, as well as circumventricular organs, are also discussed as targets for endogenous repair. Importantly, this review highlights that progenitor proliferation alone is no longer a meaningful outcome and studies must strive to better characterize precursor spatial localization, transcriptional profile, morphology, and functional synaptic integration. With improved insight and a more targeted approach, the stimulation of endogenous neurogenesis remains a promising strategy for recovery following traumatic brain injury.

The Protein Kinase Inhibitor Midostaurin Improves Functional Neurological Recovery and Attenuates Inflammatory Changes Following Traumatic Cervical Spinal Cord Injury.

Traumatic spinal cord injury (SCI) impairs neuronal function and introduces a complex cascade of secondary pathologies that limit recovery. Despite decades of preclinical and clinical research, there is a shortage of efficacious treatment options to modulate the secondary response to injury. Protein kinases are crucial signaling molecules that mediate the secondary SCI-induced cellular response and present promising therapeutic targets. The objective of this study was to examine the safety and efficacy of midostaurin-a clinically-approved multi-target protein kinase inhibitor-on cervical SCI pathogenesis. High-throughput analyses demonstrated that intraperitoneal midostaurin injection (25 mg/kg) in C6/7 injured Wistar rats altered the local inflammasome and downregulated adhesive and migratory genes at 24 h post-injury. Treated animals also exhibited enhanced recovery and restored coordination between forelimbs and hindlimbs after injury, indicating the synergistic impact of midostaurin and its dimethyl sulfoxide vehicle to improve functional recovery. Furthermore, histological analyses suggested improved tissue preservation and functionality in the treated animals during the chronic phase of injury. This study serves as a proof-of-concept experiment and demonstrates that systemic midostaurin administration is an effective strategy for mitigating cervical secondary SCI damage.

Freshly Thawed Cryobanked Human Neural Stem Cells Engraft within Endogenous Neurogenic Niches and Restore Cognitive Function after Chronic Traumatic Brain Injury.

Human neural stem cells (hNSCs) have potential as a cell therapy after traumatic brain injury (TBI). While various studies have demonstrated the efficacy of NSCs from ongoing culture, there is a significant gap in our understanding of freshly thawed cells from cryobanked stocks-a more clinically relevant source. To address these shortfalls, the therapeutic potential of our previously validated Shef-6.0 human embryonic stem cell (hESC)-derived hNSC line was tested after long-term cryostorage and thawing before transplant. Immunodeficient athymic nude rats received a moderate unilateral controlled cortical impact (CCI) injury. At four weeks post-injury, 6 × 105 freshly thawed hNSCs were transplanted into six injection sites (two ipsi- and four contra-lateral) with 53.4% of cells surviving three months post-transplant. Interestingly, most hNSCs were engrafted in the meninges and the lining of lateral ventricles, associated with high CXCR4 expression and a chemotactic response to SDF1alpha (CXCL12). While some expressed markers of neuron, astrocyte, and oligodendrocyte lineages, the majority remained progenitors, identified through doublecortin expression (78.1%). Importantly, transplantation resulted in improved spatial learning and memory in Morris water maze navigation and reduced risk taking in an elevated plus maze. Investigating potential mechanisms of action, we identified an increase in ipsilateral host hippocampus cornu ammonis (CA) neuron survival, contralateral dentate gyrus (DG) volume, and DG neural progenitor morphology as well as a reduction in neuroinflammation. Together, these findings validate the potential of hNSCs to improve function after TBI and demonstrate that long-term biobanking of cells and thawing aliquots before use may be suitable for clinical deployment.

Harnessing the Secretome of Mesenchymal Stromal Cells for Traumatic Spinal Cord Injury: Multicell Comparison and Assessment of In Vivo Efficacy.

Cell therapy offers significant promise for traumatic spinal cord injury (SCI), which despite many medical advances, has limited treatment strategies. Able to address the multifactorial and dynamic pathophysiology of SCI, cells present various advantages over standard pharmacological approaches. However, the use of live cells is also severely hampered by logistical and practical considerations. These include specialized equipment and expertise, standardization of cell stocks, sustained cell viability post-thawing, and cryopreservation-induced delayed-onset cell death. For this reason, we suggest a novel and clinically translatable alternative to live-cell systemic infusion, which retains the efficacy of the latter while overcoming many of its limitations. This strategy involves the administration of concentrated cell secretome and exploits the trophic mechanism by which stromal cells function. In this study, we compare the efficacy of intravenously delivered concentrated conditioned media (CM) from human umbilical cord matrix cells (HUCMCs), bone marrow mesenchymal stromal cells, as well as newborn and adult fibroblasts in a rat model of moderately severe cervical clip compression/contusion injury (C7--T1, 35 g). This is further paired with a thorough profile of the CM cytokines, chemokines, and angiogenic factors. The HUCMC-derived CM was most effective at limiting acute (48 h post-SCI) vascular pathology, specifically lesion volume, and functional vascularity. Principle component analysis (PCA), hierarchical clustering, and interaction analysis of proteins highly expressed in the HUCMC secretome suggest involvement of the MAPK/ERK, JAK/STAT, and immune cell migratory pathways. This "secretotherapeutic" strategy represents a novel and minimally invasive method to target multiple organ systems and several pathologies shortly after traumatic SCI.

The effects of mouse strain and age on a model of unilateral cervical contusion spinal cord injury.

There are approximately 1.2 million people currently living with spinal cord injury (SCI), with a majority of cases at the cervical level and half involving incomplete injuries. Yet, as most preclinical research has been focused on bilateral thoracic models, there remains a disconnect between bench and bedside that limits translational success. Here, we profile a clinically relevant model of unilateral cervical contusion injury in the mouse (30kD with 0, 2, 5, or 10 second dwell time). We demonstrate sustained behavioral deficits in performance on grip strength, cylinder reaching, horizontal ladderbeam and CatWalk automated gait analysis tasks. Beyond highlighting reliable parameters for injury assessment, we also explored the effect of mouse strain and age on injury outcome, including evaluation of constitutively immunodeficient mice relevant for neurotransplantation and cellular therapy testing. Comparison of C57Bl/6 and immunodeficient Rag2gamma(c)-/- as well as Agouti SCIDxRag2Gamma(c)-/- hybrid mouse strains revealed fine differences in post-injury ipsilateral grip strength as well as total number of rearings on the cylinder task. Differences in post-SCI contralateral forepaw duty cycle and regularity index as measured by CatWalk gait analysis between the two immunodeficient strains were also observed. Further, assessment of young (3-4 months old) and aging (16-17 months old) Rag2gamma(c)-/- mice identified age-related pre-injury differences in strength and rearing that were largely masked following cervical contusion injury; observations that may help interpret previous results in aged rodents as well as human clinical trials. Collectively, the work provides useful insight for experimental design and analysis of future pre-clinical studies in a translational unilateral cervical contusion injury model.

GDNF rescues the fate of neural progenitor grafts by attenuating Notch signals in the injured spinal cord in rodents.

Neural progenitor cell (NPC) transplantation is a promising strategy for the treatment of spinal cord injury (SCI). In this study, we show that injury-induced Notch activation in the spinal cord microenvironment biases the fate of transplanted NPCs toward astrocytes in rodents. In a screen for potential clinically relevant factors to modulate Notch signaling, we identified glial cell-derived neurotrophic factor (GDNF). GDNF attenuates Notch signaling by mediating delta-like 1 homolog (DLK1) expression, which is independent of GDNF's effect on cell survival. When transplanted into a rodent model of cervical SCI, GDNF-expressing human-induced pluripotent stem cell-derived NPCs (hiPSC-NPCs) demonstrated higher differentiation toward a neuronal fate compared to control cells. In addition, expression of GDNF promoted endogenous tissue sparing and enhanced electrical integration of transplanted cells, which collectively resulted in improved neurobehavioral recovery. CRISPR-induced knockouts of the DLK1 gene in GDNF-expressing hiPSC-NPCs attenuated the effect on functional recovery, demonstrating that this effect is partially mediated through DLK1 expression. These results represent a mechanistically driven optimization of hiPSC-NPC therapy to redirect transplanted cells toward a neuronal fate and enhance their integration.

The effects of human immunoglobulin G on enhancing tissue protection and neurobehavioral recovery after traumatic cervical spinal cord injury are mediated through the neurovascular unit.

BACKGROUND: Spinal cord injury (SCI) is a condition with few effective treatment options. The blood-spinal cord barrier consists of pericytes, astrocytes, and endothelial cells, which are collectively termed the neurovascular unit. These cells support spinal cord homeostasis by expressing tight junction proteins. Physical trauma to the spinal cord disrupts the barrier, which leads to neuroinflammation by facilitating immune cell migration to the damaged site in a process involving immune cell adhesion. Immunosuppressive strategies, including methylprednisolone (MPSS), have been investigated to treat SCI. However, despite some success, MPSS has the potential to increase a patient's susceptibility to wound infection and impaired wound healing. Hence, immunomodulation may be a more attractive approach than immunosuppression. Approved for modulating neuroinflammation in certain disorders, including Guillain-Barre syndrome, intravenous administration of human immunoglobulin G (hIgG) has shown promise in the setting of experimental SCI, though the optimal dose and mechanism of action remain undetermined. METHODS: Female adult Wistar rats were subjected to moderate-severe clip compression injury (35 g) at the C7-T1 level and randomized to receive a single intravenous (IV) bolus of hIgG (0.02, 0.2, 0.4, 1, 2 g/kg), MPSS (0.03 g/kg), or control buffer at 15 min post-SCI. At 24 h and 6 weeks post-SCI, molecular, histological, and neurobehavioral effects of hIgG were analyzed. RESULTS: At 24 h post-injury, human immunoglobulin G co-localized with spinal cord pericytes, astrocytes, and vessels. hIgG (2 g/kg) protected the spinal cord neurovasculature after SCI by increasing tight junction protein expression and reducing inflammatory enzyme expression. Improvements in vascular integrity were associated with changes in spinal cord inflammation. Interestingly, hIgG (2 g/kg) increased serum expression of inflammatory cytokines and co-localized (without decreasing protein expression) with spinal cord vascular cell adhesion molecule-1, a protein used by immune cells to enter into inflamed tissue. Acute molecular benefits of hIgG (2 g/kg) led to greater tissue preservation, functional blood flow, and neurobehavioral recovery at 6 weeks post-SCI. Importantly, the effects of hIgG (2 g/kg) were superior to control buffer and hIgG (0.4 g/kg), and comparable with MPSS (0.03 g/kg). CONCLUSIONS: hIgG (2 g/kg) is a promising therapeutic approach to mitigate secondary pathology in SCI through antagonizing immune cell infiltration at the level of the neurovascular unit.

Endogenous Interleukin-10 Deficiency Exacerbates Vascular Pathology in Traumatic Cervical Spinal Cord Injury.

Although the majority of traumatic spinal cord injuries (SCIs) take place at the cervical level, pre-clinical studies have been disproportionally focused on thoracic insults. With differences in anatomy, physiology, and immune response between spinal cord levels, there is evidence that injury pathophysiology may vary, requiring tailored treatment paradigms. Further, as only a few therapies have been successfully translated to the clinic, cervical models are increasingly recognized as essential for the characterization of trauma and therapy. Using a novel and clinically relevant cervical contusion-compression mouse model of bilateral incomplete injury, this study aimed to assess the role of interleukin10 (IL-10), a potent cytokine with broad anti-inflammatory effects, in SCI vascular pathology. While the effects of IL-10 loss have been previously evaluated, the vascular changes are poorly characterized. Here, using in vivo high-resolution ultrasound imaging, we demonstrate that IL-10 deficiency is associated with increased acute vascular damage. Importantly, the loss of endogenous IL-10 led to significant differences in the acute systemic response to SCI, specifically the circulating levels of IL-12 (p70), LIX (CXCL5), IL-1ß, tumor necrosis factor (TNF)-α, and IL-6 relative to genotype sham controls. These effects also fostered modest impairments in long-term functional recovery, assessed by the Basso Mouse Scale, as well as histological outcomes.

Early Intravenous Infusion of Mesenchymal Stromal Cells Exerts a Tissue Source Age-Dependent Beneficial Effect on Neurovascular Integrity and Neurobehavioral Recovery After Traumatic Cervical Spinal Cord Injury.

Localized vascular disruption after traumatic spinal cord injury (SCI) triggers a cascade of secondary events, including inflammation, gliosis, and scarring, that can further impact recovery. In addition to immunomodulatory and neurotrophic properties, mesenchymal stromal cells (MSCs) possess pericytic characteristics. These features make MSCs an ideal candidate for acute cell therapy targeting vascular disruption, which could reduce the severity of secondary injury, enhance tissue preservation and repair, and ultimately promote functional recovery. A moderately severe cervical clip compression/contusion injury was induced at C7-T1 in adult female rats, followed by an intravenous tail vein infusion 1 hour post-SCI of (a) term-birth human umbilical cord perivascular cells (HUCPVCs); (b) first-trimester human umbilical cord perivascular cells (FTM HUCPVCs); (c) adult bone marrow mesenchymal stem cells; or (d) vehicle control. Weekly behavioral testing was performed. Rats were sacrificed at 24 hours or 10 weeks post-SCI and immunohistochemistry and ultrasound imaging were performed. Both term and FTM HUCPVC-infused rats displayed improved (p < .05) grip strength compared with vehicle controls. However, only FTM HUCPVC-infusion led to significant weight gain. All cell infusion treatments resulted in reduced glial scarring (p < .05). Cell infusion also led to increased axonal, myelin, and vascular densities (p < .05). Although post-traumatic cavity volume was reduced with cell infusion, this did not reach significance. Taken together, we demonstrate selective long-term functional recovery alongside histological improvements with HUCPVC infusion in a clinically relevant model of cervical SCI. Our findings highlight the potential of these cells for acute therapeutic intervention after SCI.

Methylprednisolone treatment enhances early recovery following surgical decompression for degenerative cervical myelopathy without compromise to the systemic immune system.

BACKGROUND: Degenerative cervical myelopathy (DCM) is caused by degenerative or congenital changes to the discs and soft tissues of the cervical spine, which leads to chronic compression of the spinal cord. The current treatment for moderate to severe DCM consists of surgical decompression, which, while effective in most cases, can result in neuroinflammation and spinal cord reperfusion injury, leading to perioperative neurological complications and suboptimal neurological recovery. The primary objective of this study was to assess, in a translationally relevant animal model of DCM, the efficacy of perioperative methylprednisolone (MP) in enhancing neurological recovery and to evaluate its effect on the inflammatory response following decompression. METHODS: DCM was induced in C57BL/6 mice. Briefly, an aromatic polyether material was implanted underneath the C5-C6 laminae to cause progressive compression of the cervical spinal cord due to focal ossification. Decompressive surgery was undertaken at 12 weeks post initial biomaterial implantation. Animals received one dose of MP (30 mg/kg) or vehicle 30 min before decompression and at 2 weeks after decompression. Acute analysis of secreted cytokines and spinal cord microvasculature was complemented with immunohistochemistry for glial and neuronal cell markers. Locomotor outcomes were measured using the CatWalk system. The composition of circulating white blood cells was analyzed by flow cytometry. RESULTS: A single dose of MP before decompression significantly sped locomotor recovery (*p < 0.05) and reduced the incidence of perioperative motor complications, without affecting the composition of circulating white blood cells. Histological assessment of the spinal cord showed significant neuronal preservation and a modest reduction in parenchymal inflammation. CONCLUSIONS: Our data suggest that MP reduces perioperative neurological complications following decompressive surgery for DCM by protecting neurons from inflammation, without compromising the composition of circulating immune cells. We propose that MP, which is commonly used for neurological disorders including spinal cord injury, be considered as a perioperative adjunct to decompressive surgery to attenuate neurological complications.

Splenic involvement in umbilical cord matrix-derived mesenchymal stromal cell-mediated effects following traumatic spinal cord injury.

BACKGROUND: The spleen plays an important role in erythrocyte turnover, adaptive immunity, antibody production, and the mobilization of monocytes/macrophages (Mφ) following tissue injury. In response to trauma, the spleen initiates production of inflammatory cytokines, which in turn recruit immune cells to the inflamed tissue, exacerbating damage. Our previous work has shown that intravenous mesenchymal stromal cell (MSC) infusion has potent immunomodulatory effects following spinal cord injury (SCI), associated with the transplanted cells homing to and persisting within the spleen. Therefore, this work aimed to characterize the relationship between the splenic inflammatory response and SCI pathophysiology, emphasizing splenic involvement in MSC-mediated effects. METHODS: Using a rodent model of cervical clip-compression SCI, secondary tissue damage and functional recovery were compared between splenectomised rodents and those with a sham procedure. Subsequently, 2.5 million MSCs from the term human umbilical cord matrix cells (HUCMCs) were infused via tail vein at 1-h post-SCI and the effects were assessed in the presence or absence of a spleen. RESULTS: Splenectomy alone had no effect on lesion volume, hemorrhage, or inflammation. There was also no significant difference between the groups in functional recovery and those in lesion morphometry. Yet, while the infusion of HUCMCs reduced spinal cord hemorrhage and increased systemic levels of IL-10 in the presence of a spleen, these effects were lost with splenectomy. Further, HUCMC infusion was shown to alter the expression levels of splenic cytokines, suggesting that the spleen is an important target and site of MSC effects. CONCLUSIONS: Our results provide a link between MSC function and splenic inflammation, a finding that can help tailor the cells/transplantation approach to enhance therapeutic efficacy.

Promising neuroprotective strategies for traumatic spinal cord injury with a focus on the differential effects among anatomical levels of injury.

Traumatic spinal cord injury (SCI) is a devastating condition of motor, sensory, and autonomic dysfunction. The significant cost associated with the management and lifetime care of patients with SCI also presents a major economic burden. For these reasons, there is a need to develop and translate strategies that can improve outcomes following SCI. Given the challenges in achieving regeneration of the injured spinal cord, neuroprotection has been at the forefront of clinical translation. Yet, despite many preclinical advances, there has been limited translation into the clinic apart from methylprednisolone (which remains controversial), hypertensive therapy to maintain spinal cord perfusion, and early decompressive surgery. While there are several factors related to the limited translational success, including the clinical and mechanistic heterogeneity of human SCI, the misalignment between animal models of SCI and clinical reality continues to be an important factor. Whereas most clinical cases are at the cervical level, only a small fraction of preclinical research is conducted in cervical models of SCI. Therefore, this review highlights the most promising neuroprotective and neural reparative therapeutic strategies undergoing clinical assessment, including riluzole, hypothermia, granulocyte colony-stimulating factor, glibenclamide, minocycline, Cethrin (VX-210), and anti-Nogo-A antibody, and emphasizes their efficacy in relation to the anatomical level of injury. Our hope is that more basic research will be conducted in clinically relevant cervical SCI models in order to expedite the transition of important laboratory discoveries into meaningful treatment options for patients with SCI.

Spinal cord injuries: how could cell therapy help?

INTRODUCTION: Spinal cord injury (SCI) is a devastating condition, where regenerative failure and cell loss lead to paralysis. The heterogeneous and time-sensitive pathophysiology has made it difficult to target tissue repair. Despite many medical advances, there are no effective regenerative therapies. As stem cells offer multi-targeted and environmentally responsive benefits, cell therapy is a promising treatment approach. Areas covered: This review highlights the cell therapies being investigated for SCI, including Schwann cells, olfactory ensheathing cells, mensenchymal stem/stromal cells, neural precursors, oligodendrocyte progenitors, embryonic stem cells, and induced pluripotent stem cells. Through mechanisms of cell replacement, scaffolding, trophic support and immune modulation, each approach targets unique features of SCI pathology. However, as the injury is multifaceted, it is increasingly recognized that a combinatorial approach will be necessary to treat SCI. Expert opinion: Most preclinical studies, and an increasing number of clinical trials, are finding that single cell therapies have only modest benefits after SCI. These considerations, alongside issues of therapy cost-effectiveness, need to be addressed at the bench. In addition to exploring combinatorial strategies, researchers should consider cell reproducibility and storage parameters when designing animal experiments. Equally important, clinical trials must follow strict regulatory guidelines that will enable transparency of results.

Early Intravenous Delivery of Human Brain Stromal Cells Modulates Systemic Inflammation and Leads to Vasoprotection in Traumatic Spinal Cord Injury.

UNLABELLED: : Spinal cord injury (SCI) is a life-threatening condition with multifaceted complications and limited treatment options. In SCI, the initial physical trauma is closely followed by a series of secondary events, including inflammation and blood spinal cord barrier (BSCB) disruption, which further exacerbate injury. This secondary pathology is partially mediated by the systemic immune response to trauma, in which cytokine production leads to the recruitment/activation of inflammatory cells. Because early intravenous delivery of mesenchymal stromal cells (MSCs) has been shown to mitigate inflammation in various models of neurologic disease, this study aimed to assess these effects in a rat model of SCI (C7-T1, 35-gram clip compression) using human brain-derived stromal cells. Quantitative polymerase chain reaction for a human-specific DNA sequence was used to assess cell biodistribution/clearance and confirmed that only a small proportion (approximately 0.001%-0.002%) of cells are delivered to the spinal cord, with the majority residing in the lung, liver, and spleen. Intriguingly, although cell populations drastically declined in all aforementioned organs, there remained a persistent population in the spleen at 7 days. Furthermore, the cell infusion significantly increased splenic and circulating levels of interleukin-10-a potent anti-inflammatory cytokine. Through this suppression of the systemic inflammatory response, the cells also reduced acute spinal cord BSCB permeability, hemorrhage, and lesion volume. These early effects further translated into enhanced functional recovery and tissue sparing 10 weeks after SCI. This work demonstrates an exciting therapeutic approach whereby a minimally invasive cell-transplantation procedure can effectively reduce secondary damage after SCI through systemic immunomodulation. SIGNIFICANCE: Central nervous system pericytes (perivascular stromal cells) have recently gained significant attention within the scientific community. In addition to being recognized as major players in neurotrauma, pericytes have been discovered to share a common origin and potentially function with traditionally defined mesenchymal stromal cells (MSCs). Although there have been several in vitro comparisons, the in vivo therapeutic application of human brain-derived stromal cells has not been previously evaluated. This study demonstrates that these cells not only display a MSC phenotype in vitro but also have similar in vivo immunomodulatory effects after spinal cord injury that are more potent than those of non-central nervous system tissue-derived cells. Therefore, these cells are of great interest for therapeutic use in spinal cord injury.

A New Acute Impact-Compression Lumbar Spinal Cord Injury Model in the Rodent.

Traumatic injury to the lumbar spinal cord results in complex central and peripheral nervous tissue damage causing significant neurobehavioral deficits and personal/social adversity. Although lumbar cord injuries are common in humans, there are few clinically relevant models of lumbar spinal cord injury (SCI). This article describes a novel lumbar SCI model in the rat. The effects of moderate (20 g), moderate-to-severe (26 g) and severe (35 g, and 56 g) clip impact-compression injuries at the lumbar spinal cord level L1-L2 (vertebral level T11-T12) were assessed using several neurobehavioral, neuroanatomical, and electrophysiological outcome measures. Lesions were generated after meticulous anatomical landmarking using microCT, followed by laminectomy and extradural inclusion of central and radicular elements to generate a traumatic SCI. Clinically relevant outcomes, such as MR and ultrasound imaging, were paired with robust morphometry. Analysis of the lesional tissue demonstrated that pronounced tissue loss and cavitation occur throughout the acute to chronic phases of injury. Behavioral testing revealed significant deficits in locomotion, with no evidence of hindlimb weight-bearing or hindlimb-forelimb coordination in any injured group. Evaluation of sensory outcomes revealed highly pathological alterations including mechanical allodynia and thermal hyperalgesia indicated by increasing avoidance responses and decreasing latency in the tail-flick test. Deficits in spinal tracts were confirmed by electrophysiology showing increased latency and decreased amplitude of both sensory and motor evoked potentials (SEP/MEP), and increased plantar H-reflex indicating an increase in motor neuron excitability. This is a comprehensive lumbar SCI model and should be useful for evaluation of translationally oriented pre-clinical therapies.

Contrast enhanced ultrasound imaging for assessment of spinal cord blood flow in experimental spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 µl) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described "ischemic penumbra zone". This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Very high resolution ultrasound imaging for real-time quantitative visualization of vascular disruption after spinal cord injury.

Spinal cord injury (SCI) is characterized by vascular disruption with intramedullary hemorrhage, alterations in blood-spinal cord barrier integrity, and perilesional ischemia. A safe and easily applied imaging technique to quantify evolving intraspinal vascular changes after SCI is lacking. We evaluated the utility of very high resolution ultrasound (VHRUS) imaging to assess SCI-induced vascular disruption in a clinically relevant rodent model. The spinal cords of Wistar rats were lesioned at the 11th thoracic vertebra (Th11) by a 35 g 1-minute clip compression. Three-dimensional quantification of intraspinal hemorrhage using VHRUS (at an acute 90-min and subacute 24-h time point post-SCI) was compared with lesional hemoglobin and extravasated Evans blue dye measured spectrophotometrically. The anatomy of hemorrhage was comparatively assessed using VHRUS and histology. Time-lapse videos demonstrated the evolution of parenchymal hemorrhage. VHRUS accurately depicted the structural (gray and white matter) and vascular anatomy of the spinal cord (after laminectomy) and was safely repeated in the same animal. After SCI, a hyperechoic signal extended from the lesion epicenter. Significant correlations were found between VHRUS signal and hemorrhage in the acute (r=0.88, p<0.0001) and subacute (r=0.85, p<0.0001) phases and extravasated Evans blue (a measure of vascular disruption) in the subacute phase (r=0.94, p<0.0001). Time-lapse videos demonstrated that the expanding parenchymal hemorrhage is preceded by new perilesional hemorrhagic foci. VHRUS enables real-time quantitative live anatomical imaging of acute and subacute vascular disruption after SCI in rats. This technique has important scientific and clinical translational applications.