Midthoracic Spinal Cord Injury Research Articles

Cypin Inhibition as a Therapeutic Approach to Treat Spinal Cord Injury-Induced Mechanical Pain.

Cypin (cytosolic postsynaptic density protein 95 interactor) is the primary guanine deaminase in the central nervous system (CNS), promoting the metabolism of guanine to xanthine, an important reaction in the purine salvage pathway. Activation of the purine salvage pathway leads to the production of uric acid (UA). UA has paradoxical effects, specifically in the context of CNS injury as it confers neuroprotection, but it also promotes pain. Since neuropathic pain is a comorbidity associated with spinal cord injury (SCI), we postulated that small molecule cypin inhibitor B9 treatment could attenuate SCI-induced neuropathic pain, potentially by interfering with UA production. However, we also considered that this treatment could hinder the neuroprotective effects of UA and, in doing so, exacerbate SCI outcomes. To address our hypothesis, we induced a moderate midthoracic contusion SCI in female mice and assessed whether transient intrathecal administration of B9, starting at 1 d postinjury (dpi) until 7 dpi, attenuates mechanical pain in hindlimbs at 3 weeks pi. We also evaluated the effects of B9 on the spontaneous recovery of locomotor function. We found that B9 alleviates mechanical pain but does not affect locomotor function. Importantly, B9 does not exacerbate lesion volume at the epicenter. In accordance with these findings, B9 does not aggravate glutamate-induced excitotoxic death of SC neurons in vitro. Moreover, SCI-induced increased astrocyte reactivity at the glial scar is not altered by B9 treatment. Our data suggest that B9 treatment reduces mechanical pain without exerting major detrimental effects following SCI.

PI3K signaling promotes formation of lipid-laden foamy macrophages at the spinal cord injury site

After spinal cord injury (SCI), infiltrating macrophages undergo excessive phagocytosis of myelin and cellular debris, forming lipid-laden foamy macrophages. To understand their role in the cellular pathology of SCI, investigation of the foamy macrophage phenotype in vitro revealed a pro-inflammatory profile, increased reactive oxygen species (ROS) production, and mitochondrial dysfunction. Bioinformatic analysis identified PI3K as a regulator of inflammation in foamy macrophages, and inhibition of this pathway decreased their lipid content, inflammatory cytokines, and ROS production. Macrophage-specific inhibition of PI3K using liposomes significantly decreased foamy macrophages at the injury site after a mid-thoracic contusive SCI in mice. RNA sequencing and in vitro analysis of foamy macrophages revealed increased autophagy and decreased phagocytosis after PI3K inhibition as potential mechanisms for reduced lipid accumulation. Together, our data suggest that the formation of pro-inflammatory foamy macrophages after SCI is due to the activation of PI3K signaling, which increases phagocytosis and decreases autophagy.

Using uniaxial tensile testing to evaluate the biomechanical properties of bladder tissue after spinal cord injury in rat model

To investigate the biomechanical properties of rat bladder tissue after spinal cord injury (SCI) using uniaxial tensile testing.Evidence suggests the bladder wall undergoes remodeling following SCI. There is limited data describing the biomechanical properties of bladder wall after SCI. This study describes the changes in elastic and viscoelastic mechanical properties of bladder tissue using a rat model after SCI.Seventeen adult rats received mid-thoracic SCI. Basso, Beattie, and Bresnahan (BBB) locomotor testing was performed on the rats 7–14 days after injury quantifying the degree of SCI. Bladder tissue samples were collected from controls and spinal injured rats at 2- and 9-weeks post-injury. Tissue samples underwent uniaxial stress relaxation to determine instantaneous and relaxation modulus as well as monotonic load-to failure to determine Young’s modulus, yield stress and strain, and ultimate stress.SCI resulted in abnormal BBB locomotor scores. Nine weeks post-injury, instantaneous modulus decreased by 71.0% (p = 0.03) compared to controls. Yield strain showed no difference at 2 weeks post-injury but increased 78% (p = 0.003) in SCI rats at 9 weeks post-injury. Compared to controls, ultimate stress decreased 46.5% (p = 0.05) at 2 weeks post-injury in SCI rats but demonstrated no difference at 9 weeks post-injury.The biomechanical properties of rat bladder wall 2 weeks after SCI showed minimal difference compared to controls. By week 9, SCI bladders had a reduction in instantaneous modulus and increased yield strain. The findings indicate biomechanical differences can be identified between control and experimental groups at 2- and 9-week intervals using uniaxial testing.

Intravenous transplantation of amnion-derived mesenchymal stem cells promotes functional recovery and alleviates intestinal dysfunction after spinal cord injury.

Spinal cord injury (SCI) is often accompanied by gastrointestinal dysfunction due to the disconnection of the spinal autonomic nervous system. Gastrointestinal dysfunction reportedly upregulates intestinal permeability, leading to bacterial translocation of the gut microbiome to the systemic circulation, which further activates systemic inflammation, exacerbating neuronal damage. Mesenchymal stem cells (MSC) reportedly ameliorate SCI. Here, we aimed to investigate their effect on the associated gastrointestinal dysfunction. Human amnion-derived MSC (AMSCs) were intravenously transplanted one day after a rat model of midthoracic SCI. Biodistribution of transplanted cells, behavioral assessment, and histological evaluations of the spinal cord and intestine were conducted to elucidate the therapeutic effect of AMSCs. Bacterial translocation of the gut microbiome was examined by in situ hybridization and bacterial culture of the liver. Systemic inflammations were examined by blood cytokines, infiltrating immune cells in the spinal cord, and the size of the peripheral immune tissue. AMSCs released various neurotrophic factors and were mainly distributed in the liver and lung after transplantation. AMSC-transplanted animals showed smaller spinal damage and better neurological recovery with preserved neuronal tract. AMSCs transplantation ameliorated intestinal dysfunction both morphologically and functionally, which prevented translocation of the gut microbiome to the systemic circulation. Systemic inflammations were decreased in animals receiving AMSCs in the chronic phase. Intravenous AMSC administration during the acute phase of SCI rescues both spinal damage and intestinal dysfunction. Reducing bacterial translocation may contribute to decreasing systemic inflammation.

Daily acute intermittent hypoxia therapy 1‐week after moderate contusion spinal cord injury improves lower urinary tract function in rats

There is an increasing interest in the use of non-invasive acute intermittent hypoxia (AIH) as a therapeutic intervention to improve respiratory and locomotor function in spinal cord injury (SCI). SCI, however, also significantly disrupts normal bladder function, leading to detrusor overactivity, decreased voiding efficiency, and increased residual volume. Ongoing studies in our laboratory investigating the effects of AIH exposure in rats as a strategy for improving lower urinary tract (LUT) function following mid-thoracic SCI indicate that a single bout of AIH decreases the incidence of non-voiding bladder contractions (NVCs) and increases voiding efficiency. In the current study, we begin to evaluate the potential for AIH to be used as a therapy for inducing long-lasting improvements in SCI-induced LUT dysfunction. To accomplish this, we examined the effects of daily AIH (dAIH) administered for 7 consecutive days on both spontaneous awake micturition patterns and reflex micturition event (rME) behaviors in adult female Sprague-Dawley rats with mid-thoracic moderate contusion (200 kdyn) SCI. For these experiments, dAIH composed of five alternating 3-min exposures of hypoxia (12% O2) and normoxia (21% O2) was initiated 1-week after SCI, with awake micturition patterns being evaluated by overnight metabolic chamber measurements weekly for 2-3-weeks before and 4 weeks after SCI and rME behaviors elicited by continuous infusion of saline (0.04-0.09 ml/min) into the bladder being evaluated by cystometry at 4-weeks after SCI; daily normoxia (dNx; 21% O2 episodes) served as a control. The impact of SCI on hindlimb motor and LUT function prior to initiating dAIH or dNx treatment was evaluated using BBB scores and manually expressed urine volumes during post-operative care, and these measurements revealed that deficits and initial stages of recovery were similar (as expected) in both treatment groups. Serial metabolic chamber measurements in both dAIH- and dNx-treated rats that exhibited functional voids at 1-week post-SCI demonstrated that most rats exhibited some degree of functional recovery of micturition pattern behaviors. Only dAIH-treated rats, however, exhibited a recovery of void frequency pattern (i.e., number of voids and inter-void interval)to pre-SCI levels albeit with slightly increased void volumes; dNx-treated rats also had increased void volumes. Intravesical bladder pressure (BP) measurements during terminal cystometry experiments in urethane-anesthetized (1.4 g/kg) rats revealed that dAIH-treated rats exhibited a decreased incidence of NVCs and that voiding contractions had increased delta BP (peak BP-minimum BP) and dynamic voiding efficiency (void volume/fill volume) compared to dNx-treated rats. Taken together, these findings suggest that dAIH treatment produces beneficial improvements in LUT function lasting at least two weeks that include more efficient voiding and decreased NVCs, and further support the use of dAIH therapy to treat SCI-induced LUT dysfunction.

Association of intravenous administration of human Muse cells with deficit amelioration in a rat model of spinal cord injury.

Multilineage-differentiating stress-enduring (Muse) cells are pluripotent stem cells, which can be harvested from the bone marrow. After transplantation, Muse cells can migrate to an injured site of the body and exert repair effects. However, it remains unknown whether Muse cell transplantation can be an effective treatment in spinal cord injury (SCI). The authors used a rat model of thoracic spinal cord contusion injury. For Muse cell transplantation, the clinical product CL2020 containing 300,000 Muse cells was administered intravenously 1 day after midthoracic SCI. Animals were divided into CL2020 (n = 11) and vehicle-treated (n = 15) groups. Behavioral and histological evaluations were conducted over a period of 8 weeks to see whether intravenous CL2020 administration provided therapeutic effects for SCI. The effects of human-selective diphtheria toxin on reversion of the therapeutic effects of CL2020 were also investigated. Hindlimb motor function significantly improved after CL2020 transplantations. Importantly, the effects were reverted by the human-selective diphtheria toxin. In immunohistochemical analyses, the cystic cavity formed after the injury was smaller in the CL2020 group. Furthermore, higher numbers of descending 5-hydroxytryptamine (5-HT) fibers were preserved distal to the injury site after CL2020 administration. Eight weeks after the injury, Muse cells in CL2020 were confirmed to differentiate most predominantly into neuronal cells in the injured spinal cord. Following SCI, Muse cells in CL2020 can reach the injured spinal cord after intravenous administration and differentiate into neuronal cells. Muse cells in CL2020 facilitated nerve fiber preservation and exerted therapeutic potential for severe SCI.

Epidural Electrical Stimulation of the Lumbosacral Spinal Cord Improves Trunk Stability During Seated Reaching in Two Humans With Severe Thoracic Spinal Cord Injury

Background: Quality of life measurements indicate that independent performance of activities of daily living, such as reaching to manipulate objects, is a high priority of individuals living with motor impairments due to spinal cord injury (SCI). In a small number of research participants with SCI, electrical stimulation applied to the dorsal epidural surface of the spinal cord, termed epidural spinal electrical stimulation (ES), has been shown to improve motor functions, such as standing and stepping. However, the impact of ES on seated reaching performance, as well as the approach to identifying stimulation parameters that improve reaching ability, have yet to be described.Objective: Herein, we characterize the effects of ES on seated reaching performance in two participants with chronic, complete loss of motor and sensory functions below thoracic-level SCI. Additionally, we report the effects of delivering stimulation to discrete cathode/anode locations on a 16-contact electrode array spanning the lumbosacral spinal segments on reach distance while participants were seated on a mat and/or in their wheelchair.Methods: Two males with mid-thoracic SCI due to trauma, each of which occurred more than 3 years prior to study participation, were enrolled in a clinical trial at Mayo Clinic, Rochester, MN, USA. Reaching performance was assessed, with and without ES, at several time points throughout the study using the modified functional reach test (mFRT). Altogether, participant 1 performed 1,164 reach tests over 26-time points. Participant 2 performed 480 reach tests over 17-time points.Results: Median reach distances during ES were higher for both participants compared to without ES. Forward reach distances were greater than lateral reach distances in all environments, mat or wheelchair, for both participants. Stimulation delivered in the caudal region of the array resulted in improved forward reach distance compared to stimulation in the rostral region. For both participants, when stimulation was turned off, no significant changes in reach distance were observed throughout the study.Conclusion: ES enhanced seated reaching-performance of individuals with chronic SCI. Additionally, electrode configurations delivering stimulation in caudal regions of the lumbosacral spinal segments may improve reaching ability compared to rostral regions.

A closed-loop self-righting controller for seated balance in the coronal and diagonal planes following spinal cord injury

Spinal cord injury (SCI) often results in loss of the ability to keep the trunk erect and stable while seated. Functional neuromuscular stimulation (FNS) can cause muscles paralyzed by SCI to contract and assist with trunk stability. We have extended the results of a previously reported threshold-based controller for restoring upright posture using FNS in the sagittal plane to more challenging displacements of the trunk in the coronal plane. The system was applied to five individuals with mid-thoracic or higher SCI, and in all cases the control system successfully restored upright sitting. The potential of the control system to maintain posture in forward-sideways (diagonal) directions was also tested in three of the subjects. In all cases, the controller successfully restored posture to erect. Clinically, these results imply that a simple, threshold based control scheme can restore upright sitting from forward, lateral or diagonal leaning without a chest strap; and that removal of barriers to upper extremity interaction with the surrounding environment could potentially allow objects to be more readily retrieved from around the wheelchair. Technical performance of the system was assessed in terms of three variables: response time, recovery time and percent maximum deviation from erect. Overall response and recovery times varied widely among subjects in the coronal plane (415±213 ms and 1381±883 ms, respectively) and in the diagonal planes (530±230 ms and 1800±820 ms, respectively). Average response time was significantly lower (p < 0.05) than the recovery time in all cases. The percent maximum deviation from erect was of the order of 40% or less for 9 out of 10 cases in the coronal plane and 5 out of 6 cases in diagonal directions.

Inspiratory motor activity and burst‐to‐burst variability during single and repeated bout hypoxic exposures in spontaneously breathing urethane‐anesthetized adult female rats with mid‐thoracic moderate contusion SCI

Spinal cord injury (SCI) at either the cervical or thoracic level can lead to inefficient ventilation by disrupting descending inputs to respiratory motoneurons resulting in altered mechanics of breathing and a reduced ability to adequately and appropriately respond to ventilatory complications. Under these conditions, individuals with SCI may experience individual or repeated bouts of acute hypoxia that vary in severity and/or duration. Recently, the use of acute intermittent hypoxia (AIH) has been proposed as a potential therapeutic intervention to induce spinal motor plasticity and improve respiratory motor function following incomplete cervical (C2) SCI in both rodents and humans; however, much less is known about the effects of either single or repeated hypoxic exposures on respiratory motor output following mid‐thoracic SCI, which spares phrenic motoneurons. To begin to address this issue, we examined diaphragm EMG activity in response to 15‐min of hypoxia (12% O2) delivered as either a single 15‐min bout (protocol 1) or as three 5‐min bouts separated by 5‐min normoxic intervals (protocol 2) in spontaneously breathing urethane‐anesthetized naïve (control; n=6 protocol 1; n=8 protocol 2) or mid‐thoracic SCI (n=6 protocol 1; n=7 protocol 2) rats. Diaphragm EMG burst frequency and amplitude were measured and quantified at 15‐min intervals under baseline (BL) normoxic conditions and at 5‐min intervals during and following the hypoxic exposures. In addition, burst‐to‐burst frequency and amplitude variability were determined using coefficient of variation (CV) and Poincaré plot analyses from 300 consecutive EMG bursts. We found that in response to hypoxia, both SCI and control rats exhibited (1) increases in burst frequency and small increases or decreases in burst amplitude for both protocols, (2) increases in frequency CV values with the largest effect being noted in protocol 2 control rats during the first hypoxic exposure; (3) sustained increases in amplitude CV values for both protocols albeit a lower magnitude effect in SCI rats; (4) increases in short‐term (SD1) and long‐term (SD2) burst‐to‐burst frequency and amplitude that were attenuated in SCI rats compared to control rats, especially in protocol 1; and (5) post hypoxic frequency and amplitude decreases that were similar between the protocols. Both the ventilatory behaviors and burst‐to‐burst variability patterns observed in response to the repeated hypoxic exposures in protocol 2 appeared to be reproducible albeit in the first hypoxic exposure, SD1 and SD2 values for burst frequency tended to be higher and exhibit greater spread across control rats. These data demonstrate that while rats with mid‐thoracic contusion SCI exhibit similar single and repeated hypoxic exposure ventilatory behaviors, the respiratory dynamics of these behaviors exhibit reduced short‐term and long‐term burst‐to‐burst variability that are minimally affected by the pattern of the 15‐min duration of acute hypoxia.Support or Funding InformationDOD CDMRP W81XWH‐17‐1‐0260; NIH NS096514

Novel Activity Detection Algorithm to Characterize Spontaneous Stepping During Multimodal Spinal Neuromodulation After Mid-Thoracic Spinal Cord Injury in Rats.

A mid-thoracic spinal cord injury (SCI) severely impairs activation of the lower limb sensorimotor spinal networks, leading to paralysis. Various neuromodulatory techniques including electrical and pharmacological activation of the spinal networks have been successful in restoring locomotor function after SCI. We hypothesized that the combination of self-training in a natural environment with epidural stimulation (ES), quipazine (Quip), and strychnine (Strych) would result in greater activity in a cage environment after paralysis compared to either intervention alone. To assess this, we developed a method measuring and characterizing the chronic EMG recordings from tibialis anterior (TA) and soleus (Sol) muscles while rats were freely moving in their home cages. We then assessed the relationship between the change in recorded activity over time and motor-evoked potentials (MEPs) in animals receiving treatments. We found that the combination of ES, Quip, and Strych (sqES) generated the greatest level of recovery followed by ES + Quip (qES) while ES + Strych (sES) and ES alone showed least improvement in recorded activity. Further, we observed an exponential relationship between late response (LR) component of the MEPs and spontaneously generated step-like activity. Our data demonstrate the feasibility and potential importance of quantitatively monitoring mechanistic factors linked to activity-dependence in response to combinatorial interventions compared to individual therapies after SCI.

Comparison of Single‐bout AIH Protocols That Differ in Timing and Number of Hypoxic Episodes on Improvement in LUT Function in Adult Rats with SCI

Exposure to acute intermittent hypoxia (AIH) has been reported to improve respiratory and non‐respiratory somatic motor function in rodents and humans, and as such has been proposed as a potential therapeutic intervention for treatment of motor incomplete spinal cord injury (SCI). In our previous studies in rats with moderate mid‐thoracic (T8) SCI, we observed that a single bout of AIH produces sustained improvements in lower urinary tract (LUT) function characterized by decreased non‐voiding bladder contractions and increased void volume. In the present study, we extend these observations on lower LUT function in SCI rats by comparing the potential therapeutic benefits of different single‐bout AIH protocols that keep the total duration of hypoxia (15 min; 12% O2) constant but differ in timing and number of hypoxic episodes. To this end, four weeks after moderate contusion (200 kilodynes) mid‐thoracic SCI, bladder intravesical pressure was recorded in urethane‐anesthetized (1.4 g/kg), spontaneously breathing, vagally intact adult female Sprague‐Dawley rats (n = 44) during continuous infusion of saline (0.04 ml/min) into the bladder to elicit reflex micturition events. Following &gt;60 min of baseline recording, rats were exposed to a single bout of AIH consisting of (1) three 5‐min episodes of hypoxia each separated by 5‐min exposures to room air, (2) five 3‐min episodes of hypoxia each separated by 3‐min exposures to room air, or (3) ten 1.5‐min episodes of hypoxia each separated by 1.5‐min exposures to room air. Following completion of the AIH exposure protocol, data acquisition continued for up to 120 minutes while the rats continued to breathe room air, and these data were quantified based on features measured in 30‐min bins (post‐AIH data normalized to pre‐AIH data). These analyses revealed that each of the single‐bout AIH protocols elicited long‐lasting improvements (up to 120 min post‐AIH) in LUT function. Moreover, each of the protocols appeared to be equally effective in reducing the ratio of non‐voiding/voiding bladder contractions and increasing bladder inter‐contraction interval and micturition volume. Single‐bout AIH exposure also produced transient increases in both bladder contraction threshold pressure and minimum bladder pressure. Modulation of LUT behavior during individual exposures to hypoxia and immediately following each hypoxic episode (“off effect”) were also observed, with the intensity of the “off effect” tending to be lower following short duration (e.g., 10 × 1.5‐min) hypoxic exposures. Our observations suggest that each single‐bout AIH protocol is effective in reducing non‐voiding bladder contractions and increasing micturition in SCI rats albeit subtle differences in magnitude and/or timing were noted across the protocols. These data also provide additional evidence that single‐bout AIH exposure reduces bladder overactivity and stabilizes bladder function to improve voiding efficiency in SCI rats.Support or Funding InformationDOD CDMRP W81XWH‐17‐1‐0260; NYS DOH SCIRB C32088GGThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

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-1β, 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.

Injury type-dependent differentiation of NG2 glia into heterogeneous astrocytes

The glial scar is comprised of a heterogeneous population of reactive astrocytes. NG2 glial cells (also known as oligodendrocyte progenitor cells or polydendrocytes) may contribute to this heterogeneity by differentiating into astrocytes in the injured CNS, but there have been conflicting reports about whether astrocytes comprise a significant portion of the NG2 cell lineage. By using genetic fate mapping after spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE) in mice, the goal of this study was to confirm and extend upon previous findings, which have shown that NG2 cell plasticity varies across CNS injuries. We generated mice that express tdTomato in NG2 lineage cells and express GFP under the Aldh1l1 or Glt1 promoter so that NG2 glia-derived astrocytes can be detected by their expression of GFAP and/or GFP. We found that astrocytes comprise approximately 25% of the total NG2 cell lineage in the glial scar by 4 weeks after mid-thoracic contusive SCI, but only 9% by the peak of functional deficit after EAE. Interestingly, a subpopulation of astrocytes expressed only GFP without co-expression of GFAP, uncovering their heterogeneity and the possibility of an underestimation of NG2 glia-derived astrocytes in previous studies. Additionally, we used high performance liquid chromatography to measure the level of tamoxifen and its metabolites in the spinal cord and show that genetic labeling of NG2 glia-derived astrocytes is not an artifact of residual tamoxifen. Overall, our data demonstrate that a heterogeneous population of astrocytes are derived from NG2 glia in an injury type-dependent manner.

Characterization of the Antibody Response after Cervical Spinal Cord Injury.

The immune system plays a critical and complex role in the pathobiology of spinal cord injury (SCI), exerting both beneficial and detrimental effects. Increasing evidence suggests that there are injury level-dependent differences in the immune response to SCI. Patients with traumatic SCI have elevated levels of circulating autoantibodies against components of the central nervous system, but the role of these antibodies in SCI outcomes remains unknown. In rodent models of mid-thoracic SCI, antibody-mediated autoimmunity appears to be detrimental to recovery. However, whether autoantibodies against the spinal cord are generated following cervical SCI (cSCI), the most common level of injury in humans, remains undetermined. To address this knowledge gap, we investigated the antibody responses following cSCI in a rat model of injury. We found increased immunoglobulin G (IgG) and IgM antibodies in the spinal cord in the subacute phase of injury (2 weeks), but not in more chronic phases (10 and 20 weeks). At 2 weeks post-cSCI, antibodies were detected at the injury epicenter and co-localized with the astroglial scar and neurons of the ventral horn. These increased levels of antibodies corresponded with enhanced activation of immune responses in the spleen. Higher counts of antibody-secreting cells were observed in the spleen of injured rats. Further, increased levels of secreted IgG antibodies and enhanced proliferation of T-cells in splenocyte cultures from injured rats were found. These findings suggest the potential development of autoantibody responses following cSCI in the rat. The impact of the post-traumatic antibody responses on functional outcomes of cSCI is a critical topic that requires further investigation.

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.

Spinal cord injury causes chronic liver pathology in rats.

Traumatic spinal cord injury (SCI) causes major disruption to peripheral organ innervation and regulation. Relatively little work has investigated these post-SCI systemic changes, however, despite considerable evidence that multiple organ system dysfunction contributes to chronic impairments in health. Because metabolic dysfunction is common after SCI and the liver is a pivotal site for metabolic homeostasis, we sought to determine if liver pathology occurs as a result of SCI in a rat spinal contusion model. Histologic evidence showed excess lipid accumulation in the liver for at least 21 days post-injury after cervical or midthoracic SCI. Lipidomic analysis revealed an acute increase in hepatic ceramides as well as chronically elevated lactosylceramide. Post-SCI hepatic changes also included increased proinflammatory gene expression, including interleukin (IL)-1α, IL-1β, chemokine ligand-2, and tumor necrosis factor-α mRNA. These were coincident with increased CD68+ macrophages in the liver through 21 days post-injury. Serum alanine transaminase, used clinically to detect liver damage, was significantly increased at 21 days post-injury, suggesting that early metabolic and inflammatory damage preceded overt liver pathology. Surprisingly, liver inflammation was even detected after lumbar SCI. Collectively, these results suggest that SCI produces chronic liver injury with symptoms strikingly similar to those of nonalcoholic steatohepatitis (fatty liver disease). These clinically significant hepatic changes after SCI are known to contribute to systemic inflammation, cardiovascular disease, and metabolic syndrome, all of which are more prevalent in persons with SCI. Targeting acute and prolonged hepatic pathology may improve recovery and reduce long-term complications after SCI.

Structural remodeling of the heart and its premotor cardioinhibitory vagal neurons following T(5) spinal cord transection.

Midthoracic spinal cord injury (SCI) is associated with enhanced cardiac sympathetic activity and reduced cardiac parasympathetic activity. The enhanced cardiac sympathetic activity is associated with sympathetic structural plasticity within the stellate ganglia, spinal cord segments T1-T4, and heart. However, changes to cardiac parasympathetic centers rostral to an experimental SCI are relatively unknown. Importantly, reduced vagal activity is a predictor of high mortality. Furthermore, this autonomic dysregulation promotes progressive left ventricular (LV) structural remodeling. Accordingly, we hypothesized that midthoracic spinal cord injury is associated with structural plasticity in premotor (preganglionic parasympathetic neurons) cardioinhibitory vagal neurons located within the nucleus ambiguus as well as LV structural remodeling. To test this hypothesis, dendritic arborization and morphology (cholera toxin B immunohistochemistry and Sholl analysis) of cardiac projecting premotor cardioinhibitory vagal neurons located within the nucleus ambiguus were determined in intact (sham transected) and thoracic level 5 transected (T5X) rats. In addition, LV chamber size, wall thickness, and collagen content (Masson trichrome stain and structural analysis) were determined. Midthoracic SCI was associated with structural changes within the nucleus ambiguus and heart. Specifically, following T5 spinal cord transection, there was a significant increase in cardiac parasympathetic preganglionic neuron dendritic arborization, soma area, maximum dendritic length, and number of intersections/animal. This parasympathetic structural remodeling was associated with a profound LV structural remodeling. Specifically, T5 spinal cord transection increased LV chamber area, reduced LV wall thickness, and increased collagen content. Accordingly, results document a dynamic interaction between the heart and its parasympathetic innervation.

Longitudinal assessment of white matter pathology in the injured mouse spinal cord through ultra-high field (16.4 T) in vivo diffusion tensor imaging

This study examined the sensitivity of ultra-high field (16.4T) diffusion tensor imaging (DTI; 70μm in-plane resolution, 1mm slice thickness) to evaluate the spatiotemporal development of severe mid-thoracic contusive spinal cord injury (SCI) in mice. In vivo imaging was performed prior to SCI, then again at 2h, 1day, 3days, 7days, and 30days post-SCI using a Bruker 16.4T small animal nuclear magnetic resonance spectrometer. Cross-sectional spinal cord areas were measured in axial slices and various DTI parameters, i.e. fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (λ||) and radial diffusivity (λ⊥), were calculated for the total spared white matter (WM), ventral funiculi (VF), lateral funiculi (LF) and dorsal columns (DCs) and then correlated with histopathology. Cross-sectional area measurements revealed significant atrophy (32% reduction) of the injured spinal cord at the lesion epicentre in the chronic phase of injury. Analysis of diffusion tensor parameters further showed that tissue integrity was most severely affected in the DCs, i.e. the site of immediate impact, which demonstrated a rapid and permanent decrease in FA and λ||. In contrast, DTI parameters for the ventrolateral white matter changed more gradually with time, suggesting that these regions are undergoing more delayed degeneration in a manner that may be amenable to therapeutic intervention. Of all the DTI parameters, λ⊥ was most closely correlated to myelin content whereas changes in FA and λ|| appeared more indicative of axonal integrity, Wallerian degeneration and associated presence of macrophages. We conclude that longitudinal DTI at 16.4T provides a clinically relevant, objective measure for assessing white matter pathology following contusive SCI in mice that may aid the translation of putative neuroprotective strategies into the clinic.

Dynamic interaction between the heart and its sympathetic innervation following T5 spinal cord transection

Midthoracic spinal cord injury (SCI) is associated with enhanced sympathetic support of heart rate as well as myocardial damage related to calcium overload. The myocardial damage may elicit an enhanced sympathetic support of contractility to maintain ventricular function. In contrast, the level of inotropic drive may be reduced to match the lower afterload that results from the injury-induced reduction in arterial pressure. Accordingly, the inotropic response to midthoracic SCI may be increased or decreased but has not been investigated and therefore remains unknown. Furthermore, the altered ventricular function may be associated with anatomical changes in cardiac sympathetic innervation. To determine the inotropic drive following midthoracic SCI, a telemetry device was used for repeated measurements of left ventricular (LV) function, with and without beta-adrenergic receptor blockade, in rats before and after midthoracic SCI or sham SCI. In addition, NGF content (ELISA) and dendritic arborization (cholera toxin B immunohistochemistry and Sholl analysis) of cardiac-projecting sympathetic postganglionic neurons in the stellate ganglia were determined. Midthoracic SCI was associated with an enhanced sympathetic support of heart rate, dP/dt(+), and dP/dt(-). Importantly, cardiac function was lower following blockade of the sympathetic nervous system in rats with midthoracic SCI compared with sham-operated rats. Finally, these functional neuroplastic changes were associated with an increased NGF content and structural neuroplasticity within the stellate ganglia. Results document impaired LV function with codirectional changes in chronotropic and inotropic responses following midthoracic SCI. These functional changes were associated with a dynamic interaction between the heart and its sympathetic innervation.