Spinal Cord Contusion Injury Model Research Articles

A modified impactor for establishing a graded contusion spinal cord injury model in rats.

BackgroundGiven the indispensable role of animal models in preclinical studies of spinal cord injury (SCI) and the current state of available impactors, we designed a modified impactor for establishing contusion SCI in rats. The major improvement is the replacement of the impactor rod with a weight and an impactor tip.MethodsPreoperatively, radiographs of 8-week-old female Wistar rats were taken to establish a protocol for locating the target spinal segment. A total of 72 rats were randomly divided into 4 groups: the sham, 12.5-, 25- and 50-mm groups. Within 35 days postinjury (dpi), the Basso, Beattie, and Bresnahan locomotor rating scale (BBB) was used to evaluate the hindlimb motor function of the rats. At 7 dpi, the rats were sacrificed, and the spinal cord tissue was fixed. Hematoxylin-eosin (HE) staining was used to assess histological changes. Subsequently, immunofluorescence staining was performed to visualize the expression and distribution of GFAP, CD68, MBP, and NeuN. Additionally, rats were sacrificed, and their tissues were extracted for relevant protein assays. At 3 and 7 dpi, electrophysiological function was evaluated by measuring motor evoked potentials (MEPs) and sensory evoked potentials (SEPs).ResultsThe behavioral results revealed that higher strike heights were associated with lower BBB scores. Over time, the BBB scores of the SCI rats exhibited an improving trend. Quantitative analysis of the lesions indicated that as the impact height increased, the area of histological destruction, GFAP-negative area, CD68-positive cell count, and MBP-positive destruction area increased, and the number of NeuN-positive cells decreased. Western blot analysis further verified relevant protein changes. Electrophysiology confirmed that the MEP and SEP amplitudes decreased as the strike height increased.ConclusionsThus, these results confirm that this modified impactor can be used to establish a graded SCI model in rats. The model is clinically relevant, reproducible, stable, accessible, and affordable, providing a practical tool with which to elucidate the pathophysiological mechanisms and potential therapies for contusive SCI.

Beneficial Effects of Ethanolic Extract of the Medicinal Herb Mucuna Pruriens Against Oxidative Stress and Inflammation Might be Limited in Contusive Spinal Cord Injury

Experimental studies found extracts of Mucuna pruriens (L.) DC, a plant used in the traditional medical systems to possess strong anti-inflammatory and anti-oxidant properties among a wide range of other beneficial effects. Hypothesizing the possibility for a multifaceted beneficial effect, the therapeutic potential of the ethanolic extract of Mucuna pruriens (MP) in treating spinal cord injury (SCI) was tested using the highly standardized Multicenter Animal Spinal Cord Injury Study (MASCIS) animal model of contusive SCI. Male Wister rats with SCI were treated with the ethanolic extract of MP at a dose of 200 mg/kg/day for 10 weeks. The outcome was assessed through molecular, biochemical, histological, and behavioral scoring parameters. Results indicated anti-apoptotic, anti-necrotic, anti-inflammatory, and neuroprotective effects of MP. Nevertheless, many of these beneficial effects were not statistically significant and there was no functional improvement due to MP treatment. MP at comparable doses was reported to be significantly effective in mitigating oxidative stress and/or inflammation under various other pathological conditions. Lack of significance in the present study could be due to the presence of blood-spinal cord-barrier; that might have prevented the components of MP from reaching spinal cord tissue in adequate quantities. Additionally, heterogeneity in the function of the cells typical to the CNS environment could be the reason for the failure of recovery. To conclude, the limitations posed by the structural and functional distinctions in the spinal cord environment in comparison to other non-CNS tissue environments deserve attention while adjudicating the efficacies of herbal remedies for SCI.

Therapeutic induction of energy metabolism reduces neural tissue damage and increases microglia activation in severe spinal cord injury

Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament-positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI.

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.

Comparison of the Regenerative Effect of Adipose Tissue Mesenchymal Stem Cell Encapsulated into Two Hydrogel Scaffolds on Spinal Cord Injury

Background: Spinal cord injury (SCI) is a severe neurological disease leading to poor quality of life. Objectives: The regenerative effect of adipose-derived mesenchymal stem cells (AD-MSCs) encapsulated into fibrin, and collagen hydrogel scaffolds on a rat model of SCI was investigated using clinical and histopathological examinations. Methods: A total of 18 adult male Wistar rats (250 - 300 g) were prepared and randomly divided into three equal groups, each with six rats, including the control or SCI group (SCI contusion model without treatment), SCI contusion model treated with AD-MSCs encapsulated in fibrin hydrogel, and SCI contusion model treated with AD-MSCs encapsulated in collagen hydrogel groups. Clinically, functional recovery or hindlimb locomotor activity was assessed using Basso, Beattie, and Bresnahan's (BBB) scoring system four weeks post-treatment. The rats were sacrificed at week four post-treatment, and their spinal cords were examined histopathologically. Results: Faster functional recovery indicated with hindlimb locomotor activity was seen in both treatment groups compared to the control group. Severe polio and leuko-myelomalacia associated with disruption of spinal cord structure were identified in the control group. Mild polio and leuko-myelomalacia associated with mild to moderate disruption of spinal cord structure were seen in the collagen hydrogel + AD-MSCs and fibrin hydrogel + AD-MSCs groups. Conclusions: AD-MSCs encapsulated into fibrin and collagen hydrogels, as two of the most promising ECM-based or natural scaffolds have the potential to be developed in neural tissue engineering (NTE), such as for the treatment of SCI.

Human Umbilical Cord Mesenchymal Stem Cells Derived Exosomes Promote Neurological Function Recovery in a Rat Spinal Cord Injury Model.

Spinal cord injury (SCI) often leads to personal and social-economic consequences with limited therapeutic options. Exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSC) have been explored as a promising alternative to cell therapies. In the current study, we explored the mechanism of hUC-MSC derived exosome's ameliorative effect on the spinal cord injury by combining data from in vivo contusion SCI model and in vitro cell viability of PC12 cell line stimulated with lipopolysaccharide. Intravenous administration of hUC-MSC derived exosomes dramatically improved motor function of Sprague-Dawley rats after SCI, with reduced apoptosis demonstrated by increased expression of B-cell lymphoma 2 (BCL2), decreased BCL2 associated X, apoptosis regulator (Bax), and reduced cleaved caspase 9. Conversely, exosome treatment reduced the transcription levels of astrocytes marker GFAP and microglia marker IBA1, suggesting a reduced inflammatory state from SCI injury. Furthermore, exosome treatment in vitro increased the cell viability of PC12 cells. Exosome application activated the Wnt/β-Catenin signaling in the spinal cord. Our study demonstrated that hUC-MSC derived exosomes could improve motor function through anti-apoptosis and anti-inflammatory effects. BCL2/Bax and Wnt/β-catenin signaling pathways were involved in the SCI process and could potentially mediate the protective effect of hUC-MSC derived exosomes.

Deletion or Inhibition of Astrocytic Transglutaminase 2 Promotes Functional Recovery after Spinal Cord Injury.

Following CNS injury, astrocytes become “reactive” and exhibit pro-regenerative or harmful properties. However, the molecular mechanisms that cause astrocytes to adopt either phenotype are not well understood. Transglutaminase 2 (TG2) plays a key role in regulating the response of astrocytes to insults. Here, we used mice in which TG2 was specifically deleted in astrocytes (Gfap-Cre+/− TG2fl/fl, referred to here as TG2-A-cKO) in a spinal cord contusion injury (SCI) model. Deletion of TG2 from astrocytes resulted in a significant improvement in motor function following SCI. GFAP and NG2 immunoreactivity, as well as number of SOX9 positive cells, were significantly reduced in TG2-A-cKO mice. RNA-seq analysis of spinal cords from TG2-A-cKO and control mice 3 days post-injury identified thirty-seven differentially expressed genes, all of which were increased in TG2-A-cKO mice. Pathway analysis revealed a prevalence for fatty acid metabolism, lipid storage and energy pathways, which play essential roles in neuron–astrocyte metabolic coupling. Excitingly, treatment of wild type mice with the selective TG2 inhibitor VA4 significantly improved functional recovery after SCI, similar to what was observed using the genetic model. These findings indicate the use of TG2 inhibitors as a novel strategy for the treatment of SCI and other CNS injuries.

Local delivery of fingolimod through PLGA nanoparticles and PuraMatrix-embedded neural precursor cells promote motor function recovery and tissue repair in spinal cordinjury.

Spinal cord injury (SCI) is a devastating clinical problem that can lead to permanent motor dysfunction. Fingolimod (FTY720) is a sphingosine structural analogue, and recently, its therapeutic benefits in SCI have been reported. The present study aimed to evaluate the therapeutic efficacy of fingolimod-incorporated poly lactic-co-glycolic acid (PLGA) nanoparticles (nanofingolimod) delivered locally together with neural stem/progenitor cells (NS/PCs) transplantation in a mouse model of contusive acute SCI. Fingolimod was encapsulated in PLGA nanoparticles by the emulsion-evaporation method. Mouse NS/PCs were harvested and cultured from embryonic Day 14 (E14) ganglionic eminences. Induction of SCI was followed by the intrathecal delivery of nanofingolimod with and without intralesional transplantation of PuraMatrix-encapsulated NS/PCs. Functional recovery, injury size and the fate of the transplanted cells were evaluated after 28 days. The nanofingolimod particles represented spherical morphology. The entrapment efficiency determined by UV-visible spectroscopy was approximately 90%, and the drug content of fingolimod loaded nanoparticles was 13%. About 68% of encapsulated fingolimod was slowly released within 10 days. Local delivery of nanofingolimod in combination with NS/PCs transplantation led to a stronger improvement in neurological functions and minimized tissue damage. Furthermore, co-administration of nanofingolimod and NS/PCs not only increased the survival of transplanted cells but also promoted their fate towards more oligodendrocytic phenotype. Our data suggest that local release of nanofingolimod in combination with three-dimensional (3D) transplantation of NS/PCs in the acute phase of SCI could be a promising approach to restore the damaged tissues and improve neurological functions.

Neutrophil Decoys with Anti‐Inflammatory and Anti‐Oxidative Properties Reduce Secondary Spinal Cord Injury and Improve Neurological Functional Recovery

AbstractFollowing spinal cord injury (SCI), immune cell infiltration creates an inflammatory and oxidative microenvironment (known as the secondary injury), which causes neuron death and spinal cord damage, and dramatically hinders neurological functional recovery. Strategies that inhibit the infiltration and/or function of neutrophils offer promises for SCI treatment because they can reduce the secondary injury; however, such strategies remain largely unexplored. Herein, a strategy using neutrophil membrane‐coated nanoparticles (NPs) as decoys (neutrophil decoy, ND) is presented to reduce local neutrophil infiltration and relieve oxidative stress in the injured spinal cord after SCI. Coated with membranes of activated neutrophils, the NDs inherit multiple receptors from the “parent” neutrophils, which can adsorb and neutralize the elevated neutrophil‐related cytokines. In addition, polydopamine NPs with multi‐antioxidative properties (selected as the core for ND) scavenge excessive reactive oxygen and nitrogen species. In a contusion model of SCI, ND treatment significantly reduces neutrophil infiltration and reprograms the inflammatory and oxidative microenvironment in injured spinal cords. Importantly, ND treatment significantly improves neural regeneration and functional recovery in rats. Such a nano‐decoy platform opens up new approaches for efficiently treating SCI.

Human-Induced Neural and Mesenchymal Stem Cell Therapy Combined with a Curcumin Nanoconjugate as a Spinal Cord Injury Treatment.

We currently lack effective treatments for the devastating loss of neural function associated with spinal cord injury (SCI). In this study, we evaluated a combination therapy comprising human neural stem cells derived from induced pluripotent stem cells (iPSC-NSC), human mesenchymal stem cells (MSC), and a pH-responsive polyacetal–curcumin nanoconjugate (PA-C) that allows the sustained release of curcumin. In vitro analysis demonstrated that PA-C treatment protected iPSC-NSC from oxidative damage in vitro, while MSC co-culture prevented lipopolysaccharide-induced activation of nuclear factor-κB (NF-κB) in iPSC-NSC. Then, we evaluated the combination of PA-C delivery into the intrathecal space in a rat model of contusive SCI with stem cell transplantation. While we failed to observe significant improvements in locomotor function (BBB scale) in treated animals, histological analysis revealed that PA-C-treated or PA-C and iPSC-NSC + MSC-treated animals displayed significantly smaller scars, while PA-C and iPSC-NSC + MSC treatment induced the preservation of β-III Tubulin-positive axons. iPSC-NSC + MSC transplantation fostered the preservation of motoneurons and myelinated tracts, while PA-C treatment polarized microglia into an anti-inflammatory phenotype. Overall, the combination of stem cell transplantation and PA-C treatment confers higher neuroprotective effects compared to individual treatments.

Whole body measurements using near-infrared spectroscopy in a rat spinal cord contusion injury model

Background Spinal cord injuries cause great damage to the central nervous system as well as the peripheral vasculature. While treatments for spinal cord injury typically focus on the spine itself, improvements in the function of the peripheral vasculature after spinal cord injury have shown to improve overall neurological recovery. Objective This study focused on the use of near-infrared spectroscopy (NIRS) as a mode to monitor cerebral and peripheral vascular condition non-invasively during the recovery process. Design Animal research study. Methods Rats underwent spinal contusion or sham injury and relative concentrations of de-/oxyhemoglobin (Δ[HbO]/Δ[Hb]) over time were measured over the cerebral, spinal, and pedal regions via NIRS. Correlational relationships across the body were determined. Rats received 1 NIRS measurement before injury and 3 after injury: 4, 7, and 14 days post. Results Correlational relationships between signals across the body, between animals with and without spinal cord injury, indicate that NIRS was able to detect patterns of vascular change in the spine and the periphery occurring secondary to spinal cord injury and evolving during subsequent recovery. Additionally, NIRS determined an overall correlational decrease within the central nervous system, between spinal and cerebral measurements. Conclusion NIRS was able to closely reflect physiologic changes in the rat during recovery, demonstrating a promising method to monitor whole body hemodynamics after spinal cord injury.

Hypoxia-inducible factor prolyl hydroxylase domain (PHD) inhibition after contusive spinal cord injury does not improve locomotor recovery

Traumatic spinal cord injury (SCI) is a devastating neurological condition that involves both primary and secondary tissue loss. Various cytotoxic events including hypoxia, hemorrhage and blood lysis, bioenergetic failure, oxidative stress, endoplasmic reticulum (ER) stress, and neuroinflammation contribute to secondary injury. The HIF prolyl hydroxylase domain (PHD/EGLN) family of proteins are iron-dependent, oxygen-sensing enzymes that regulate the stability of hypoxia inducible factor-1α (HIF-1α) and also mediate oxidative stress caused by free iron liberated from the lysis of blood. PHD inhibition improves outcome after experimental intracerebral hemorrhage (ICH) by reducing activating transcription factor 4 (ATF4)-driven neuronal death. As the ATF4-CHOP (CCAAT-enhancer-binding protein homologous protein) pathway plays a role in the pathogenesis of contusive SCI, we examined the effects of PHD inhibition in a mouse model of moderate T9 contusive SCI in which white matter damage is the primary driver of locomotor dysfunction. Pharmacological inhibition of PHDs using adaptaquin (AQ) moderately lowers acute induction of Atf4 and Chop mRNAs and prevents the acute decline of oligodendrocyte (OL) lineage mRNAs, but does not improve long-term recovery of hindlimb locomotion or increase chronic white matter sparing. Conditional genetic ablation of all three PHD isoenzymes in OLs did not affect Atf4, Chop or OL mRNAs expression levels, locomotor recovery, and white matter sparing after SCI. Hence, PHDs may not be suitable targets to improve outcomes in traumatic CNS pathologies that involve acute white matter injury.

Early escitalopram administration as a preemptive treatment strategy against spasticity after contusive spinal cord injury in rats

In the majority of spinal cord injury (SCI) patients, spasticity develops in the subacute phase and chronically persists with muscle hypertonia. Among various pathological conditions underlying spasticity, upregulated expression of 5-HT receptors (5-HTR) on the spinal motor neurons due to 5-HT denervation is considered one of crucial factors for hyperexcitability of the spinal circuit. As a 5-HT signal modulator, selective serotonin re-uptake inhibitors (SSRIs) are ordinarily prescribed for diseases associated with 5-HT in the CNS, and are known for their ability to increase 5-HT levels as well as to desensitize 5-HTR. Here, we hypothesized that early SSRI administration as a preemptive treatment strategy would effectively prevent the onset of spasticity. We used a rat model of contusive SCI and administered escitalopram during the first 4 weeks after injury, which is the period required for spasticity development in rodent models. We performed a swimming test to quantify spastic behaviors and conducted the Hoffman reflex test as well as histological analyses for 5-HT2AR and KCC2 expressions. Four weeks of escitalopram administration suppressed spastic behaviors during the swimming test and reduced the population of spasticity-strong rats. Moreover, the treatment resulted in decreased immunoreactivity of 5-HT2AR in the spinal motor neurons. Result of the H-reflex test and membrane expression of KCC2 were not significantly altered. In summary, early escitalopram administration could prevent the onset of spastic behaviors via regulation of 5-HT system after SCI, but could not modulate exaggerated spinal reflex. Our results suggest a novel application of SSRIs for preventative treatment of spasticity.

Use of a Self-Delivering Anti-CCL3 FANA Oligonucleotide as an Innovative Approach to Target Inflammation after Spinal Cord Injury.

Secondary damage after spinal cord injury (SCI) occurs because of a sequence of events after the initial injury, including exacerbated inflammation that contributes to increased lesion size and poor locomotor recovery. Thus, mitigating secondary damage is critical to preserve neural tissue and improve neurologic outcome. In this work, we examined the therapeutic potential of a novel antisense oligonucleotide (ASO) with special chemical modifications [2′-deoxy-2-fluoro-D-arabinonucleic acid (FANA) ASO] for specifically inhibiting an inflammatory molecule in the injured spinal cord. The chemokine CCL3 plays a complex role in the activation and attraction of immune cells and is upregulated in the injured tissue after SCI. We used specific FANA ASO to inhibit CCL3 in a contusive mouse model of murine SCI. Our results show that self-delivering FANA ASO molecules targeting the chemokine CCL3 penetrate the spinal cord lesion site and suppress the expression of CCL3 transcripts. Furthermore, they reduce other proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1β after SCI. In summary, we demonstrate for the first time the potential of FANA ASO molecules to penetrate the spinal cord lesion site to specifically inhibit CCL3, reducing proinflammatory cytokines and improve functional recovery after SCI. This novel approach may be used in new treatment strategies for SCI and other pathologic conditions of the CNS.

GIT1 protects traumatically injured spinal cord by prompting microvascular endothelial cells to clear myelin debris.

The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). As phagocytes do, microvascular endothelial cells (MECs) participate in myelin debris clearance at the injury site within one week. Our group has verified that G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) is essential in autophagy and angiogenesis, both of which are tightly related to the uptake and degradation of myelin debris by MECs. Here, we analyzed the performance and mechanism of GIT1 in myelin debris clearance after SCI. The SCI contusion model was established and in vitro MECs were treated with myelin debris. Better recovery from traumatic SCI was observed in the GIT1 WT mice than in the GIT1 KO mice. More importantly, we found that GIT1 prompted MECs to clear myelin debris and further enhanced MECs angiogenesis in vivo and in vitro. Mechanistically, GIT1-mediated autophagy contributed to the clearance of myelin debris by MECs. In this study, we demonstrated that GIT1 may prompt MECs to clear myelin debris via autophagy and further stimulate MECs angiogenesis via upregulating VEGF. Our results indicate that GITI may serve as a promising target for accelerating myelin debris clearance and improving SCI recovery.

Therapeutics effects of [Pyr1] apelin-13 on rat contusion model of spinal cord injury: An experimental study

Spinal cord injury (SCI) can cause various symptoms, including pain, complete or incomplete loss of autonomic, sensory, motor and functions inferior to the site of the damage. Despite wondrous advances in medicine, treating spinal cord injuries remains a thorny issue yet. Recently, the control of inflammatory processes after damage to the nervous system has been noticed as a promising therapeutic target. The goal of the present experiment was to identify the effects of apelin-13 on the histological outcome, inflammatory factors, and functional recovery in the animal contusion model of SCI were analyzed. 40 Female Wistar rats were randomly but equally assigned in laminectomy, contusion, PBS (1 mL PBS, i.p), control group which received apelin-13 (control + apelin, 100 μg/kg, i.p), and apelin-13 treatment groups. In the treatment group, apelin-13 (100 μg/kg) was injected intraperitoneally 30 min after injury. The weight-dropping contusion model was used for inducing SCI. The Basso, Beattie, and Bresnahan scale (BBB), narrow beam test (NBT), rotarod test, and the open-field test was applied to evaluate locomotor and behavioral activity. Real-time polymerase chain reaction (PCR) and ELISA technique was accomplished eight weeks after inducing SCI to measure the level of fibroblast growth factor FGF-1, FGFR1 and the inflammatory factors including interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-10. Furthermore, histological change was estimated by H&E staining. Our results showed that apelin-13 treatment after SCI led to a significant increase in functional recovery and behavioral tests. Stereological estimation illustrated that apelin-13 could reduce significantly central cavity volume and number of glial cells, and also increase significantly spinal cord volume and number of neural cells. PCR and ELISA evaluation shows a significant increase in IL-10 level and decrease in levels of FGF-1, FGF-R1, and pro-inflammatory cytokines (PIC). This study suggested that apelin-13 has neuroprotective effects by regulating the inflammatory process after SCI.

Relationships between spinal cord blood flow measured with flow-sensitive alternating inversion recovery (FAIR) and neurobehavioral outcomes in rat spinal cord injury

In the traumatically injured spinal cord, decreased perfusion is believed to contribute to secondary tissue damage beyond the primary mechanical impact, and restoration of perfusion is believed to be a promising therapeutic target. However, methods to monitor spinal cord perfusion non-invasively are limited. Perfusion magnetic resonance imaging (MRI) techniques established for the brain have not been routinely adopted to the spinal cord. The purpose of this study was to examine the relationship between spinal cord blood flow (SCBF) and injury severity in a rat thoracic spinal cord contusion injury (SCI) model using flow-sensitive alternating inversion recovery (FAIR) with two variants of the label position. SCBF as a marker of severity was compared to T1 mapping and to spinal cord-optimized diffusion weighted imaging (DWI) with filtered parallel apparent diffusion coefficient. Thirty-eight rats underwent a T10 contusion injury with varying severities (8 sham; 10 mild; 10 moderate; 10 severe) with MRI performed at 1 day post injury at the lesion site and follow-up neurological assessments using the Basso, Beattie, Bresnahan (BBB) locomotor scoring up to 28 days post injury. Using whole-cord regions of interest at the lesion epicenter, SCBF was decreased with injury severity and had a significant correlation with BBB scores at 28 days post injury. Importantly, estimates of arterial transit times (ATT) in the injured spinal cord were not altered after injury, which suggests that FAIR protocols optimized to measure SCBF provide more value in the context of acute traumatic injury to the cord. T1-relaxation time constants were strongly related to injury severity and had a larger extent of changes than either SCBF or DWI measures. These findings suggest that perfusion decreases in the spinal cord can be monitored non-invasively after injury, and multi-parametric MRI assessments of perfusion, diffusion, and relaxation capture unique features of the pathophysiology of preclinical injury.

Improving Culture Conditions, Proliferation, and Migration of Porcine Mesenchymal Stem Cells on Spinal Cord Contusion Injury Model in vitro

Adipose tissue-derived mesenchymal stem cells (AD-MSCs) are promising for cell therapy in spinal cord injury (SCI). The pig is one of the most approximate models of many human diseases, including SCI. In our study, we selected the optimal conditions for the culture of porcine AD-MSCs and developed an in vitro SCI model based on the culture of cells in injured spinal cord extracts (SCE) 3 days and 6 weeks after SCI. We show that Dulbecco’s Modified Eagle Medium (DMEM) with 20% serum content, supplemented with a combination of 5 mM L-ascorbate-2-phosphate and nonessential amino acids, stimulated a typical fibroblast-like morphology and high proliferation of porcine AD-MSCs. SCE caused a higher proliferation of porcine AD-MSCs compared with extracts from an intact spinal cord. The optimal proliferating effect was achieved using rostral 3 days SCE, and proliferation was lower in caudal and central SCE. Porcine AD-MSCs migration to the 3 days and 6 weeks SCE was higher than to an intact one and preferred the rostral SCE, avoiding central and caudal SCE. We also studied 13 cytokines contained in SCE but did not observe any definite relationship between some analyte concentrations and a change in the behavior of AD-MSCs.

SARM1 promotes neuroinflammation and inhibits neural regeneration after spinal cord injury through NF-κB signaling.

Axonal degeneration is a common pathological feature in many acute and chronic neurological diseases such as spinal cord injury (SCI). SARM1 (sterile alpha and TIR motif-containing 1), the fifth TLR (Toll-like receptor) adaptor, has diverse functions in the immune and nervous systems, and recently has been identified as a key mediator of Wallerian degeneration (WD). However, the detailed functions of SARM1 after SCI still remain unclear.Methods: Modified Allen's method was used to establish a contusion model of SCI in mice. Furthermore, to address the function of SARM1 after SCI, conditional knockout (CKO) mice in the central nervous system (CNS), SARM1Nestin-CKO mice, and SARM1GFAP-CKO mice were successfully generated by Nestin-Cre and GFAP-Cre transgenic mice crossed with SARM1flox/flox mice, respectively. Immunostaining, Hematoxylin-Eosin (HE) staining, Nissl staining and behavioral test assays such as footprint and Basso Mouse Scale (BMS) scoring were used to examine the roles of SARM1 pathway in SCI based on these conditional knockout mice. Drugs such as FK866, an inhibitor of SARM1, and apoptozole, an inhibitor of heat shock protein 70 (HSP70), were used to further explore the molecular mechanism of SARM1 in neural regeneration after SCI.Results: We found that SARM1 was upregulated in neurons and astrocytes at early stage after SCI. SARM1Nestin-CKO and SARM1GFAP-CKO mice displayed normal development of the spinal cords and motor function. Interestingly, conditional deletion of SARM1 in neurons and astrocytes promoted the functional recovery of behavior performance after SCI. Mechanistically, conditional deletion of SARM1 in neurons and astrocytes promoted neuronal regeneration at intermediate phase after SCI, and reduced neuroinflammation at SCI early phase through downregulation of NF-κB signaling after SCI, which may be due to upregulation of HSP70. Finally, FK866, an inhibitor of SARM1, reduced the neuroinflammation and promoted the neuronal regeneration after SCI.Conclusion: Our results indicate that SARM1-mediated prodegenerative pathway and neuroinflammation promotes the pathological progress of SCI and anti-SARM1 therapeutics are viable and promising approaches for preserving neuronal function after SCI.

Regeneration of Functional Neurons After Spinal Cord Injury via in situ NeuroD1-Mediated Astrocyte-to-Neuron Conversion.

Spinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimer's disease by overexpressing a single neural transcription factor NeuroD1. Here we demonstrate regeneration of spinal cord neurons from reactive astrocytes after SCI through AAV NeuroD1-based gene therapy. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (~95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate after conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model with a long delay after injury, allowing future studies to further evaluate this in vivo reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift from classical axonal regeneration to neuronal regeneration for spinal cord repair, using in vivo astrocyte-to-neuron conversion technology to regenerate functional new neurons in the gray matter.