Spinal Cord Contusion Research Articles

FANCC deficiency mediates microglial pyroptosis and secondary neuronal apoptosis in spinal cord contusion

BackgroundTraumatic spinal cord injury (SCI)-induced neuroinflammation results in secondary neurological destruction and functional disorder. Previous findings showed that microglial pyroptosis plays a crucial role in neuroinflammation. Thus, it is necessary to conduct a comprehensive investigation of the mechanisms associated with post-SCI microglial pyroptosis. The Fanconi Anemia Group C complementation group gene (FANCC) has been previously reported to have an anti-inflammation effect; however, whether it can regulate microglial pyroptosis remains unknown. Therefore, we probed the mechanism associated with FANCC-mediated microglial pyroptosis and neuroinflammation in vitro and in vivo in SCI mice.MethodsMicroglial pyroptosis was assessed by western blotting (WB) and immunofluorescence (IF), whereas microglial-induced neuroinflammation was evaluated by WB, Enzyme-linked immunosorbent assays and IF. Besides, flow cytometry, TdT-mediated dUTP Nick-End Labeling staining and WB were employed to examine the level of neuronal apoptosis. Morphological changes in neurons were assessed by hematoxylin–eosin and Luxol Fast Blue staining. Finally, locomotor function rehabilitation was analyzed using the Basso Mouse Scale and Louisville Swim Scale.ResultsOverexpression of FANCC suppressed microglial pyroptosis via inhibiting p38/NLRP3 expression, which in turn reduced neuronal apoptosis. By contrast, knockdown of FANCC increased the degree of neuronal apoptosis by aggravating microglial pyroptosis. Besides, increased glial scar formation, severe myelin sheath destruction and poor axon outgrowth were observed in the mice transfected with short hairpin RNA of FANCC post SCI, which caused reduced locomotor function recovery.ConclusionsTaken together, a previously unknown role of FANCC was identified in SCI, where its deficiency led to microglia pyroptosis, neuronal apoptosis and neurological damage. Mechanistically, FANCC mediated microglia pyroptosis and the inflammatory response via regulating the p38/NLRP3 pathway.

Major traumatic injury sustained during use of a virtual reality (VR) headset

We report the first documented case of significant, potentially life-changing trauma sustained during use of virtual reality (VR). A male in his fifties fell from standing whilst immersed in a VR game and sustained injuries including bilateral occipital condyle fractures, spinal cord contusion resulting in central cord syndrome, right vertebral artery occlusion, and left hypoglossal nerve palsy. His injuries were managed conservatively with a lengthy recovery period. The patient made a good recovery with full resolution of his symptoms and has returned to full-time employment. This case discusses how these injuries are commonly related and considers the implications of VR on a person’s balance and whether the headset may make neck extension injuries more likely.

Hyperbaric Oxygen Therapy Following Cervical Spinal Cord Injury Increases BDNF/TrkB Signaling in Diaphragm Muscle

Respiratory insufficiency is the leading cause of morbidity and mortality following cervical spinal cord injury (SCI). Diaphragm atrophy following SCI occurs as a result of damage to respiratory neural circuitry, which leads to impaired muscle contractility. Disruption of redox balance occurs after SCI and increased mitochondrial reactive oxygen species production is an upstream trigger for diaphragm muscle atrophy and weakness. Recently, we demonstrated that increased antioxidant capacity in response to hyperbaric oxygen (HBO) therapy may play a role in preserving mitochondrial redox homeostasis following SCI. However, a precise understanding of the mechanisms by which HBO therapy can prevent diaphragm dysfunction is lacking. In this regard, evidence indicates that the secretion of neurotrophic factors, such as brain derived neurotrophic factor (BDNF), may also contribute to enhanced functional recovery when HBO therapy is initiated acutely following cervical SCI. Through activation of the 5′ adenosine monophosphate‐activated protein kinase (AMPK) and serine‐threonine protein kinase (AKT) signaling via the phosphorylation of its receptor tropomyosin receptor kinase B (TrKb), BDNF expression has been shown to modulate mitochondrial biogenesis and skeletal muscle proteolysis. Therefore, we tested the hypothesis that 10 days of HBO therapy initiated in the acute phase following cervical SCI can increase diaphragm mitochondrial biogenesis and prevent diaphragm proteolytic signaling by activating BDNF/TrkB signaling. To test this, adult male Sprague‐Dawley rats were separated into three groups (n=8/group): (1) non‐injured, room air exposure (CON); (2) lateral‐cervical spinal cord contusion, room air exposure (SCI); and (3) lateral‐cervical spinal cord contusion, HBO therapy (SCI+HBO). Animals in the SCI and SCI+HBO groups were anesthetized, and following laminectomy at the C3–C4 level, a lateral‐cervical spinal contusion was made using a mechanical impactor. HBO therapy consisted of 10 days of exposure starting on the day of SCI, using a 40 L chamber flushed with 100% O2 and pressurized to 3 ATA. Our results indicate that HBO therapy delivered in the acute phase of SCI positively affected BDNF/TrkB signaling. Specifically, diaphragm protein expression of BDNF, p‐AKT/AKT and p‐AMPK/AMPK were increased after 10 days of HBO therapy compared to CON animals (p<0.05). Additionally, HBO therapy prevented the SCI‐induced reduction in diaphragm peroxisome proliferator‐activated receptor‐gamma coactivator (PGC1‐α) protein expression (p<0.05), as well as the caspase‐3‐specific cleavage of α‐II‐spectrin observed in the SCI group (p<0.05). In summary, these findings demonstrate that HBO therapy can induce BDNF signaling and highlight an additional mechanism for the protective effects of HBO therapy to preserve diaphragm muscle function following SCI.

Daily ketoprofen restores AIH‐induced phrenic long‐term facilitation after prolonged chronic intermittent hypoxia in spinal intact and chronically injured rats

Acute intermittent hypoxia (AIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long‐term facilitation (pLTF). In recent years, this form of plasticity has been leveraged for therapeutic benefit to restore respiratory (and non‐respiratory) motor function after spinal cord injury (SCI). While therapeutic AIH shows tremendous promise as a non‐invasive and easy‐to administer rehabilitation strategy, its efficacy may be undermined by factors such as inflammation (Huxtable et al., 2011). For example, a single night of chronic intermittent hypoxia (CIH) simulating that experienced by an individual with moderate sleep apnea (2 min hypoxic episodes; 2 min intervals; 8 hrs) abolishes AIH‐induced pLTF through a mechanism that requires systemic inflammation and spinal p38 MAP kinase activity (Huxtable et al., 2015). Similarly, prolonged CIH (7‐28 days) also abolishes pLTF (Gonzalez‐Rothi et al., 2021; El Chami and colleagues, unpublished), though the underlying mechanisms have not yet been defined. Since CIH induces time‐dependent cascades of distinct spinal inflammatory gene expression (Smith et al., 2013), we hypothesize that prolonged CIH (versus a single night) blunts pLTF through an inflammation‐dependent mechanism, although the specific pathways may differ with cumulative/repeated exposures. Further, since respiratory insufficiency is a leading cause of morbidity and mortality following cervical SCI and sleep disordered breathing/sleep apnea are highly prevalent (nearly 80% in cervical SCI), it is important to understand how untreated sleep apnea may interact with and/or undermine the efficacy of therapeutic interventions like AIH in chronic SCI. Thus, we are exploring the impact of anti‐inflammatory administration on AIH‐induced pLTF in both intact rats and rats with chronic (12 wks) C3/C4 mid‐cervical spinal cord contusion, preconditioned with 7 days of moderate CIH. In intact rats, daily administration of the non‐steroidal anti‐inflammatory drug, ketoprofen rescued expression of pLTF following prolonged CIH (70.4% increase from baseline, p=0.0039 vs. vehicle). Interestingly, a single dose of ketoprofen delivered just prior to pLTF assessments did not restore expression of plasticity (19.5% increase, p=0.8842 vs. vehicle), suggesting a shift in the underlying mechanisms by which CIH impacts phrenic motor plasticity that occurs with cumulative exposure over time. Similarly, in rats with chronic cSCI, daily ketoprofen rescued expression of pLTF following prolonged CIH (62.9% increase from baseline, p=0.0348 vs. vehicle). Upcoming experiments will explore the role of p38 MAP kinase specifically after prolonged CIH. Improved understanding of factors that undermine plasticity, (e.g., CIH, inflammation), will enable us to identify strategies to mitigate deleterious effects and optimize functional benefits, which is essential for effectively translating promising interventions like therapeutic AIH to clinical practice.

The method of quantitative assessment of posttraumatic spinal cord injury volume and structure in animal models using magnetic resonance tomography

Introduction. Spinal cord injury is a debilitating traumatic event in central nervous system resulting in tissue destruction and severe neurological deficit development. Preclinical assessment of quantitative lesion area parameters (e. g. structure and volume) is critical for subsequent evaluation of neuroprotective and / or neuroregenerative therapy efficiency. Current methods for parameter calculation require manual limitation of the interested area (region of interest, RoI). This process is tedious and often not precise enough.Study objective is to develop and implement software for automated assessment of volume and structure of posttraumatic spinal cord lesion using extra-high-field MRI 7.0 Tesla and to compare methods preciseness with the current manual techniques.Study design. Ten rat models of acute severe spinal cord contusion injury were used including female Sprague–Dawley animals weighting 250–350 gr. MRI imaging was performed in 1 day postoperative and then 4 times with interval (1 week). Study was prospective open-label uncontrolled comparative.Materials and methods. Standard spinal cord contusion injury model was used. Anesthetized animals underwent laminectomy at level Th9–Th10 vertebrae followed by “weight drop” injury technique application: 10 g weight with 2 mm pin diameter dropped from 25 mm height. Software was developed using Microsoft Visual Studio 2017 environment and programming language C#. Statistical analysis was performed using IBM SPSS Statistics 21.0 software.Results. We developed and patented specialized software Spinal cavity Searcher realizing the algorithm of T2‑weighted images (T2‑WI) analysis based on image bynarization and Freeman chain code. This algorithm supports calculation of spinal cord posttraumatic lesion parameters in a half-automatic manner. Results of this algorithm application were comparable to results of manual calculation: no statistical difference were observed between two values.Conclusion. Current method of spinal cord injury volume and structure quantitative assessment simplifies the calculation procedure due to automatization of RoI limitation comparing to manual technique. The level of preciseness is comparable in both methods.Clinical relevance. The developed algorithm optimizes the process of non-invasive control of the performed treatment efficiency according to 7.0 Tesla MRI data.

Activity dependent stimulation increases synaptic efficacy in spared pathways in an anesthetized rat model of spinal cord contusion injury.

Background:Closed-loop neuromodulation systems have received increased attention in recent years as potential therapeutic approaches for treating neurological injury and disease.Objective:The purpose of this study was to assess the ability of intraspinal microstimulation (ISMS), triggered by action potentials (spikes) recorded in motor cortex, to alter synaptic efficacy in descending motor pathways in an anesthetized rat model of spinal cord injury (SCI).Methods:Experiments were carried out in adult, male, Sprague Dawley rats with a moderate contusion injury at T8. For activity-dependent stimulation (ADS) sessions, a recording microelectrode was used to detect neuronal spikes in motor cortex that triggered ISMS in the spinal cord grey matter. SCI rats were randomly assigned to one of four experimental groups differing by: a) cortical spike-ISMS stimulus delay (10 or 25 ms) and b) number of ISMS pulses (1 or 3). Four weeks after SCI, ADS sessions were conducted in three consecutive 1-hour conditioning bouts for a total of 3 hours. At the end of each conditioning bout, changes in synaptic efficacy were assessed using intracortical microstimulation (ICMS) to examine the number of spikes evoked in spinal cord neurons during 5-minute test bouts. A multichannel microelectrode recording array was used to record cortically-evoked spike activity from multiple layers of the spinal cord.Results:The results showed that ADS resulted in an increase in cortically-evoked spikes in spinal cord neurons at specific combinations of spike-ISMS delays and numbers of pulses. Efficacy in descending motor pathways was increased throughout all dorsoventral depths of the hindlimb spinal cord.Conclusions:These results show that after an SCI, ADS can increase synaptic efficacy in spared pathways between motor cortex and spinal cord. This study provides further support for the potential of ADS therapy as an effective method for enhancing descending motor control after SCI.

Rabphilin3A reduces integrin-dependent growth cone signaling to restrict axon regeneration after trauma

Neural repair after traumatic spinal cord injury depends upon the restoration of neural networks via axonal sprouting and regeneration. Our previous genome wide loss-of-function screen identified Rab GTPases as playing a prominent role in preventing successful axon sprouting and regeneration. Here, we searched for Rab27b interactors and identified Rabphilin3A as an effector within regenerating axons. Growth cone Rabphilin3a colocalized and physically associated with integrins at puncta in the proximal body of the axonal growth cone. In regenerating axons, loss of Rabphilin3a increased integrin enrichment in the growth cone periphery, enhanced focal adhesion kinase activation, increased F-actin-rich filopodial density and stimulated axon extension. Compared to wild type, mice lacking Rabphilin3a exhibited greater regeneration of retinal ganglion cell axons after optic nerve crush as well as greater corticospinal axon regeneration after complete thoracic spinal cord crush injury. After moderate spinal cord contusion injury, there was greater corticospinal regrowth in the absence of Rph3a. Thus, an endogenous Rab27b - Raphilin3a pathway limits integrin action in the growth cone, and deletion of this monomeric GTPase pathway permits reparative axon growth in the injured adult mammalian central nervous system.

Diaphragmatic Activity and Respiratory Function Following C3 or C6 Unilateral Spinal Cord Contusion in Mice.

Simple SummaryTetraplegia is one of the most devastating conditions that an individual can sustain and affects more than 2.5 million people worldwide. Tetraplegia not only affects mobility but also impacts spontaneous breathing such that survivors can be rendered ventilator dependent and have increased mortality. Although no treatment can restore respiratory function after tetraplegia, there is a need for more exploratory studies defining therapeutic approaches to ameliorate respiratory decline in tetraplegia. Here, we studied two models of tetraplegia mimicking human forms of injury, using spinal cord contusion in mice, either above the third cervical metameric segment (C3 model) or below the sixth cervical metameric segment (C6 model) innervation of the phrenic nerves. These nerves are responsible for contraction of the diaphragm, the main inspiratory muscle. Using measurements of spontaneous breathing and muscle activity of the diaphragm, we found reduced diaphragmatic activity in both models, but only the C3 model led to reduced spontaneous breathing similar to what is seen in humans with tetraplegia. Moreover, we found a decline in basal contractility of the diaphragm in the C3 model only. We conclude that the C3 model is an appropriate model to explore interventions aimed at restoring breathing following tetraplegia.The majority of spinal cord injuries (SCIs) are cervical (cSCI), leading to a marked reduction in respiratory capacity. We aimed to investigate the effect of hemicontusion models of cSCI on both diaphragm activity and respiratory function to serve as preclinical models of cervical SCI. Since phrenic motoneuron pools are located at the C3–C5 spinal level, we investigated two models of preclinical cSCI mimicking human forms of injury, namely, one above (C3 hemicontusion—C3HC) and one below phrenic motoneuron pools (C6HC) in wild-type swiss OF-1 mice, and we compared their effects on respiratory function using whole-body plethysmography and on diaphragm activity using electromyography (EMG). At 7 days post-surgery, both C3HC and C6HC damaged spinal cord integrity above the lesion level, suggesting that C6HC potentially alters C5 motoneurons. Although both models led to decreased diaphragmatic EMG activity in the injured hemidiaphragm compared to the intact one (−46% and −26% in C3HC and C6HC, respectively, both p = 0.02), only C3HC led to a significant reduction in tidal volume and minute ventilation compared to sham surgery (−25% and −20% vs. baseline). Moreover, changes in EMG amplitude between respiratory bursts were observed post-C3HC, reflecting a change in phrenic motoneuronal excitability. Hence, C3HC and C6HC models induced alteration in respiratory function proportionally to injury level, and the C3HC model is a more appropriate model for interventional studies aiming to restore respiratory function in cSCI.

Cell-Free Extracts from Human Fat Tissue with a Hyaluronan-Based Hydrogel Attenuate Inflammation in a Spinal Cord Injury Model through M2 Microglia/Microphage Polarization.

Treatment for spinal cord injuries (SCIs) is often ineffective because SCIs result in a loss of nerve tissue, glial scar formation, local ischemia and secondary inflammation. The current promising strategy for SCI is the combination of bioactive materials and cytokines. Bioactive materials support the injured spinal cord, stabilize the morphology, and avoid excessive inflammatory responses. Fat extract (FE) is a cell-free liquid component containing a variety of cytokines extracted from human fat tissue using mechanical methods. In this research, a biocompatible HAMC (hyaluronan and methylcellulose) loaded with FE is used to treat a model of spinal cord contusion in mice. The composite not only inhibits death of neuro- and vascular cells and leads to the preservation of neural and vascular structure, but also modulates the inflammatory phenotype of macrophages in the locally injured region. Specifically, FE promotes the polarization of macrophages from an inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. During the screening of the involved pathways, it is corroborated that activation of the STAT6/Arg-1 signaling pathway is involved in macrophage M2 polarization. In summary, FE is a promising treatment for SCI, as it is easy to obtain, nonimmunogenic, and effective.

A Pinch of Salt Won’t Kill: The Role of Salt Tablets in Reversing Intractable Orthostatic Hypotension in an Incomplete Tetraplegic Patient

Background and aim: Orthostatic hypotension is a known complication of spinal cord injury, especially in individuals with tetraplegia and thoracic paraplegia. It is triggered by tilting the patient upright and often easily reversed by removing the precipitating factors. The salt tablet is an old medical supplement that works as a blood volume expander by retaining water. We present a case of intractable orthostatic hypotension in incomplete tetraplegia, which resolved after the initiation of salt tablets. Methods: The patient was a 58-year-old gentleman with background hypertension who presented with neck pain following an aggressive neck massage, associated with progressive limb weakness, for six days duration. He developed urinary and bowel retention one day before the presentation. Urgent MRI scan revealed blunt injury at cervical spine with contusion of the cervical spinal cord, epidural haematoma, and contusion of prevertebral soft tissue from C4 till C6 spinal level. He was treated conservatively with steroids. In the rehabilitation ward, the patient developed multiple syncopal episodes during tilt table exercises. The perpetuating factors were antihypertensive medications, anaemia, and urinary tract infection, which resulted in polyuria and electrolyte imbalances. The orthostatic hypotension persisted, despite optimising fluid intake, treating underlying medical issues, removing triggers, and adopting additional measures such as abdominal binder and elastic stockings. Results: Following the initiation of salt tablets 1 gram TDS, the syncopal attacks gradually resolved. The patient tolerated the tilt table exercises until 90° elevation and subsequently progressed into sitting in the wheelchair for an extended period. The patient was completely free of syncope at discharge and required minimal assistance to perform stand transfer to the wheelchair. Conclusion: Orthostatic hypotension is a treatable barrier to progress in spinal cord injury rehabilitation. Therefore, simple, cheap, and easily accessible treatment options such as salt tablets should be considered in intractable cases.

BET protein inhibition promotes non-myeloid cell mediated neuroprotection after rodent spinal cord contusion

Spinal cord injuries (SCI) often lead to multiple neurological deficits as a result from the initial trauma and also the secondary damage that follows. Despite abundant preclinical data proposing anti-inflammatory therapies to minimize secondary injury and improve functional recovery, the field still lacks an effective neuroprotective treatment. Epigenetic proteins, such as bromodomain and extraterminal domain (BET) proteins, are emerging as new targets to regulate inflammation. More importantly, pharmacological inhibition of BET proteins suppresses pro-inflammatory gene transcription after SCI. In this study, we tested the therapeutic potential of inhibiting BET proteins after SCI with clinically relevant compounds, and investigated the role of the BET protein BRD4 in macrophages during progression of SCI pathology. Systemic inhibition of BET proteins with I-BET762 significantly reduced lesion size 8 weeks after a contusion injury in rats. However, we observed no histological or locomotor improvements after SCI when we deleted Brd4 in macrophages through the use of myeloid-specific Brd4 knockout mice or after macrophage-targeted pharmacological BET inhibition. Taken together, our data indicate that systemic I-BET762 treatment is neuroprotective, and the histopathological improvement observed is likely to be a result of effects on non-macrophage targets. Expanding our understanding on the role of BET proteins after SCI is necessary to identify novel therapeutic targets that can effectively promote repair after SCI.

Ultrasound stimulation improves inflammatory resolution, neuroprotection, and functional recovery after spinal cord injury

Spinal cord injury (SCI) is associated with limited functional recovery. Despite advances in neuroscience, realistic therapeutic treatments for SCI remain unavailable. In this study, the effects of non-invasive ultrasound (US) treatment on behavior and inflammatory responses were evaluated in a rat model of SCI. Adult female Sprague–Dawley rats were subjected to spinal cord contusion injury. Two different US parameters (SCIU5: 5% and SCIU40: 40% duty cycle) were applied, and their effects on behavioral recovery after SCI were quantified. Tissue and neuronal responses were detected. Immunofluorescence was used to detect inflammatory markers. In the rat model of SCI, motor function was more effectively restored, and the lesion cavity area was smaller in the SCIU5 group. Furthermore, the SCIU5 protocol elicited an anti-inflammatory response at the injury site by reducing degenerative FJC-labeled neurons, macrophage/microglia activation, and infiltration. Thus, the lesion area decreased, and tissue density increased. Meanwhile, the SCIU40 protocol did not improve motor function or induce an anti-inflammatory response at the injury site. The SCIU5 protocol effectively accelerated the rate of improved exercise performance in the rat model while reducing inflammation. Accordingly, appropriate US stimulation may represent a promising treatment modality for SCI with beneficial anti-inflammatory effects.

Combination of repetitive transcranial magnetic stimulation and treadmill training reduces hyperreflexia by rebalancing motoneuron excitability in rats after spinal cord contusion

Spasticity commonly emerges during the process of recovery after spinal cord injury (SCI) and critically exacerbates motor dysfunction. Given insufficient effects of individual therapies, we combined repetitive transcranial magnetic stimulation (rTMS) with treadmill training (Tr) in rats with SCI to investigate potential synergistic effects on alleviating spasticity and motor dysfunction. Animals were randomized into four groups: SCI only, rTMS, Tr, and rTMS plus Tr. At the study endpoint eight weeks after the start of interventions, the rTMS plus Tr group exhibited the largest decrease in maximal H-reflex amplitude/maximal M-wave amplitude ratio (effect size (ES): −0.082, 95% confidence interval (CI): −0.118 to −0.046, p < 0.001) as well as the greatest improvement in motor function measured with the Basso, Beattie, and Bresnahan locomotor scale (ES: 1.811, 95% CI: 1.018 to 2.603, p < 0.001; significantly different from all other groups at p < 0.01) and grid-walking test (ES: −5.1, 95% CI: −7.784 to −2.416, p < 0.001, significantly different from rTMS alone at p < 0.01). Pathological analyses demonstrated that the combined treatment facilitated the growth of serotonergic axons around the lesion site, and the upregulation of 5-hydroxytryptamine, potassium-chloride cotransporter-2, and glutamic acid decarboxylases 67 in the lumbar spinal cord distal to the injury site. All effects of combined treatment of rTMS and treadmill training were enhanced compared to treadmill training or rTMS alone. Treadmill training and rTMS intervention appear to have synergistic effects on hyperreflexia and locomotion likely related to a restored balance between facilitatory and inhibitory inputs to motoneurons.

S100A9 blockade improves the functional recovery after spinal cord injury via mediating neutrophil infiltration.

Spinal cord injury (SCI) refers to damage to the spinal cord resulting from trauma, disease or degeneration. Controlling the inflammatory process and restoring neural homeostasis is hypothesized to prevent injury aggravation. S100 calcium-binding protein A9 (S100A9) is a pro-inflammatory alarm protein, which is expressed in and released by activated neutrophils. However, whether S100A9 could serve as an effective target for the treatment of SCI has not been reported to date. In the present study, a T10 spinal cord contusion injury model was established in Sprague-Dawley rats. S100A9 expression level was determined in the serum and injured spinal cord tissue via ELISA, reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The S100A9-specific blocker, ABR-238901 (ABR), was administered during the inflammatory phase of SCI, as a form of treatment. Subsequently, the morphological structure, neuronal viability and inflammatory levels of injured spinal cord were observed by histopathology, immunohistochemistry and RT-qPCR. In the obtained results, S100A9 was found to be highly expressed in the injured spinal cord and serum in the first 3 days after SCI. However, at 28 days after surgery, ABR treatment significantly improved motor function, reduced the cavity formation and neutrophil infiltration in the lesion, which was verified via H&E staining and immunohistochemistry for myeloperoxidase. Furthermore, ABR treatment was found to effectively improve the survival and viability of neurons, as shown via Nissl staining and immunofluorescence of the synaptic plasticity markers, microtubule associated protein 2 and neurofilament 200. Moreover, S100A9 blockade effectively upregulated the mRNA expression level of the anti-inflammatory genes, IL-4 and IL-10 and downregulated the mRNA expression level of the pro-inflammatory factors, IL-1β, IL-6 and TNF-α. In addition, S100A9 blockade notably alleviated the apoptosis level of the injured nerve cells. Therefore, the findings of the present study revealed that S100A9 may be a useful target for the treatment of SCI.

Cellular and Molecular Gradients in the Ventral Horns With Increasing Distance From the Injury Site After Spinal Cord Contusion.

To identify cellular and molecular gradients following spinal cord injury (SCI), a rat contusion model of severe SCI was used to investigate the expression of NG2 and molecules that identify astrocytes and axons of the ventral horns (VH) at different distances on 7 and 30 days post-injury (dpi). A gradient of expression of NG2+/Olig2+ cells was determined, with the highest concentrations focused close to the injury site. A decrease in NG2 mean intensity correlates with a decrease in the number of NG2+ cells more distally. Immunoelectron microscopy subsequently revealed the presence of NG2 in connection with the membrane and within the cytoplasm of NG2+ glial cells and in large amounts within myelin membranes. Analysis of the astrocyte marker GFAP showed increased expression local to injury site from 7 dpi, this increase in expression spread more distally from the injury site by 30 dpi. Paradoxically, astrocyte perisynaptic processes marker GLT-1 was only increased in expression in areas remote from the epicenter, which was traced both at 7 and 30 dpi. Confocal microscopy showed a significant decrease in the number of 5-HT+ axons at a distance from the epicenter in the caudal direction, which is consistent with a decrease in β3-tubulin in these areas. The results indicate significant cellular and molecular reactions not only in the area of the gray matter damage but also in adjacent and remote areas, which is important for assessing the possibility of long-distance axonal growth.

Systemic Administration of Allogeneic Cord Blood Mononuclear Cells in Adults with Severe Acute Contusion Spinal Cord Injury: Phase 1/2a Pilot Clinical Study–Safety and Primary Efficacy Evaluation

Phase 1 of the SUBSCI I/IIa (Systemic Umbilical Cord Blood Administration in Patients with Acute Severe Contusion Spinal Cord Injury) study focused on safety and primary efficacy of multiple systemic infusions of allogeneic unrelated human umbilical cord blood mononuclear cells in patients with severe acute spinal cord contusion having severe neurologic deficit. The primary end point was safety. The secondary end point was the restoration of motor and sensory function in lower limbs within a 1-year period. Ten patients with acute contusion spinal cord injury (SCI) and American Spinal Injury Association (ASIA) level A/B deficit were enrolled into phase 1. Patients were treated with 4 infusions of group-matched and rhesus-matched cord blood samples after primary surgery within 3 days after SCI. All patients were followed up for 12 months after SCI. Safety was assessed using adverse events classification depending on severity and relation to cell therapy. Primary efficacy was assessed using dynamics of deficit (ASIA scale). The overall number of adverse events reached 419 in 10 patients. Only 2 were estimated as possibly related to cell therapy, and the remaining 417 were definitely unrelated. Both adverse events were mild and clinically insignificant. No signs of immunization were found in participants. Analysis of clinical outcomes also showed that cell therapy promotes significant functional restoration of motor function. The data obtained suggest that systemic administration of allogeneic, non-human leukocyte antigen-matched human umbilical cord blood is safe and shows primary efficacy in adults with severe acute contusion SCI and ASIA level A/B deficit.

Rostral-Caudal Effect of Cervical Magnetic Stimulation on the Diaphragm Motor Evoked Potential after Cervical Spinal Cord Contusion in the Rat.

The present study was designed to investigate the rostral-caudal effect of spinal magnetic stimulation on diaphragmatic motor-evoked potentials after cervical spinal cord injury. The diaphragm electromyogram was recorded in rats that received a laminectomy or a left midcervical contusion at the acute (1 day), subchronic (2 weeks), or chronic (8 weeks) injury stages. The center of a figure-eight coil was placed at 30 mm lateral to bregma on the left side, and the effect of magnetic stimulation was evaluated by stimulating the rostral, middle, and caudal cervical regions in spontaneously breathing rats. The results demonstrated that cervical magnetic stimulation induced intensity-dependent motor-evoked potentials in the bilateral diaphragm in both uninjured and contused rats; however, the left diaphragm exhibited a higher amplitude and earlier onset than the right diaphragm. Moreover, the intensity-response curve was shifted upward in the rostral-to-caudal direction of magnetic stimulation, suggesting that caudal cervical magnetic stimulation produced more robust diaphragmatic motor-evoked potentials compared with rostral cervical magnetic stimulation. Interestingly, the diaphragmatic motor-evoked potentials were similar between uninjured and contused rats during cervical magnetic stimulation despite weaker inspiratory diaphragmatic activity in contused rats. In addition, in contused animals but not uninjured animals, diaphragmatic motor-evoked potential amplitudes were greater at the chronic stage than during earlier injury stages. These results demonstrated that cervical magnetic stimulation can excite the residual phrenic motor circuit to activate the diaphragm in the presence of a significant lesion in the cervical spinal cord. These findings indicate that this non-invasive approach is effective for modulating diaphragmatic excitability after cervical spinal cord injury.

WITHDRAWN: Physospermum cornubienseL. alleviates nociceptive and neuropathic pain: Evidences and possible mechanisms

In Iranian/Persian folkloric medicine, Physospermum cornubiense (Shokaran Baghi in Persian) is used for the treatment of pain and inflammation. This modern examination included Swiss mice to investigate the anti-neuropathic and anti-nociceptive effects of Physospermum cornubiense essential oil (PCEO). To determine PCEO 's anti-nociceptive function in formalin-induced paw licking (FML) paradigm, researchers looked at the arginine-nitric oxide and potassium channels pathway in addition to involvements of more specific examples of receptors such as adrenergic, opioid, cannabinoid, peroxisome proliferator-activated (PPA), and transient receptor potential vanilloid. The CVC or cervical spinal cord contusion exemplar has also been used to induce neuropathic pain. PCEO (450mg/kg) relative to control mice in the phase_ II of FML exemplar provided strong antinociception (p < 0.001). Furthermore, pre-treatments with arginine, glibenclamide, methylene blue, L-NAME, SNP, GW6471, naloxonazine, and GW9662 (p < 0.05) returned the PCEO antinociceptive response in the FML (inflammatory phase) model. Orally limonene administration significantly diminished (p < 0.001) acute pain in inflammatory phase of FML test. Moreover, the von Frey test indicated that both PCEO and limonene could return neuropathic pain (mechanical allodynia) in CVC mice. The results obtained from this study, together with literature, give evidence of properties of PCEO for therapy of antinociceptive and neuropathic pain.

Poly(ADP-ribose) polymerase family member 14 promotes functional recovery after spinal cord injury through regulating microglia M1/M2 polarization via STAT1/6 pathway.

Poly(ADP-ribose)polymerase family member 14 (PARP14), which is an intracellular mono(ADP-ribosyl) transferase, has been reported to promote post-stroke functional recovery, but its role in spinal cord injury (SCI) remains unclear. To investigate this, a T10 spinal cord contusion model was established in C57BL/6 mice, and immediately after the injury PARP14 shRNA-carrying lentivirus was injected 1 mm from the injury site to silence PARP14 expression. We found that PARP14 was up-regulated in the injured spinal cord and that lentivirus-mediated downregulation of PARP14 aggravated functional impairment after injury, accompanied by obvious neuronal apoptosis, severe neuroinflammation, and slight bone loss. Furthermore, PARP14 levels were elevated in microglia after SCI, PARP14 knockdown activated microglia in the spinal cord and promoted a shift from M2-polarized microglia (anti-inflammatory phenotype) to M1-polarized microglia (pro-inflammatory phenotype) that may have been mediated by the signal transducers and activators of transcription (STAT) 1/6 pathway. Next, microglia M1 and M2 polarization were induced in vitro using lipopolysaccharide/interferon-γ and interleukin-4, respectively. The results showed that PARP14 knockdown promoted microglia M1 polarization, accompanied by activation of the STAT1 pathway. In addition, PARP14 overexpression made microglia more prone to M2 polarization and further activated the STAT6 pathway. In conclusion, these findings suggest that PARP14 may improve functional recovery after SCI by regulating the phenotypic transformation of microglia via the STAT1/6 pathway.

Impairment of autophagy after spinal cord injury potentiates neuroinflammation and motor function deficit in mice.

Autophagy is a catabolic process that degrades cytoplasmic constituents and organelles in the lysosome, thus serving an important role in cellular homeostasis and protection against insults. We previously reported that defects in autophagy contribute to neuronal cell damage in traumatic spinal cord injury (SCI). Recent data from other inflammatory models implicate autophagy in regulation of immune and inflammatory responses, with low levels of autophagic flux associated with pro-inflammatory phenotypes. In the present study, we examined the effects of genetically or pharmacologically manipulating autophagy on posttraumatic neuroinflammation and motor function after SCI in mice.Methods: Young adult male C57BL/6, CX3CR1-GFP, autophagy hypomorph Becn1+/- mice, and their wildtype (WT) littermates were subjected to moderate thoracic spinal cord contusion. Neuroinflammation and autophagic flux in the injured spinal cord were assessed using flow cytometry, immunohistochemistry, and NanoString gene expression analysis. Motor function was evaluated with the Basso Mouse Scale and horizontal ladder test. Lesion volume and spared white matter were evaluated by unbiased stereology. To stimulate autophagy, disaccharide trehalose, or sucrose control, was administered in the drinking water immediately after injury and for up to 6 weeks after SCI.Results: Flow cytometry demonstrated dysregulation of autophagic function in both microglia and infiltrating myeloid cells from the injured spinal cord at 3 days post-injury. Transgenic CX3CR1-GFP mice revealed increased autophagosome formation and inhibition of autophagic flux specifically in activated microglia/macrophages. NanoString analysis using the neuroinflammation panel demonstrated increased expression of proinflammatory genes and decreased expression of genes related to neuroprotection in Becn1+/- mice as compared to WT controls at 3 days post-SCI. These findings were further validated by qPCR, wherein we observed significantly higher expression of proinflammatory cytokines. Western blot analysis confirmed higher protein expression of the microglia/macrophage marker IBA-1, inflammasome marker, NLRP3, and innate immune response markers cGAS and STING in Becn1+/- mice at 3 day after SCI. Flow cytometry demonstrated that autophagy deficit did not affect either microglial or myeloid counts at 3 days post-injury, instead resulting in increased microglial production of proinflammatory cytokines. Finally, locomotor function showed significantly worse impairments in Becn1+/- mice up to 6 weeks after SCI, which was accompanied by worsening tissue damage. Conversely, treatment with a naturally occurring autophagy inducer trehalose, reduced protein levels of p62, an adaptor protein targeting cargo to autophagosomes as well as the NLRP3, STING, and IBA-1 at 3 days post-injury. Six weeks of trehalose treatment after SCI led to improved motor function recovery as compared to control group, which was accompanied by reduced tissue damage.Conclusions: Our data indicate that inhibition of autophagy after SCI potentiates pro-inflammatory activation in microglia and is associated with worse functional outcomes. Conversely, increasing autophagy with trehalose, decreased inflammation and improved outcomes. These findings highlight the importance of autophagy in spinal cord microglia and its role in secondary injury after SCI.