Spinal Cord T-Cell Infiltration in the Rat Spared Nerve Injury Model: A Time Course Study

The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.     

Premarin Reduces Neurodegeneration and Promotes Improvement of Function in an Animal Model of Spinal Cord Injury

Spinal cord injury (SCI) causes significant mortality and morbidity. Currently, no FDA-approved pharmacotherapy is available for treating SCI. Previously, low doses of estrogen (17β-estradiol, E2) were shown to improve the post-injury outcome in a rat SCI model. However, the range of associated side effects makes advocating its therapeutic use difficult. Therefore, this study aimed at investigating the therapeutic efficacy of Premarin (PRM) in SCI. PRM is an FDA-approved E2 (10%) formulation, which is used for hormone replacement therapy with minimal risk of serious side effects. The effects of PRM on SCI were examined by magnetic resonance imaging, immunofluorescent staining, and western blot analysis in a rat model. SCI animals treated with vehicle alone, PRM, E2 receptor antagonist (ICI), or PRM + ICI were graded in a blinded way for locomotor function by using the Basso–Beattie–Bresnahan (BBB) locomotor scale. PRM treatment for 7 days decreased post-SCI lesion volume and attenuated neuronal cell death, inflammation, and axonal damage. PRM also altered the balance of pro- and anti-apoptotic proteins in favor of cell survival and improved angiogenesis and microvascular growth. Increased expression of estrogen receptors (ERs) ERα and ERβ following PRM treatment and their inhibition by ER inhibitor indicated that the neuroprotection associated with PRM treatment might be E2-receptor mediated. The attenuation of glial activation with decreased inflammation and cell death, and increased angiogenesis by PRM led to improved functional outcome as determined by the BBB locomotor scale. These results suggest that PRM treatment has significant therapeutic implications for the improvement of post-SCI outcome.     

The Application of an Omentum Graft or Flap in Spinal Cord Injury

Background: Spinal cord injury (SCI) causes a primary injury at the lesion site and triggers a secondary injury and prolonged inflammation. There has been no definitive treatment till now. Promoting angiogenesis is one of the most important strategies for functional recovery after SCI. The omentum, abundant in blood and lymph vessels, possesses the potent ability of tissue regeneration. Methods: The present work examines the efficacy of autologous omentum, either as a flap (with vascular connection intact) or graft (severed vascular connection), on spinal nerve regeneration. After contusive SCI in rats, a thin sheath of omentum was grafted to the injured spinal cord. Results: Omental graft improved behavior scores significantly from the 3rd to 6th week after injury (6th week, 5.5 ± 0.5 vs. 8.6 ± 1.3, p < 0.05). Furthermore, the reduction in cavity and the preservation of class III β-tubulin-positive nerve fibers in the injury area was noted. Next, the free omental flap was transposed to a completely transected SCI in rats through a pre-implanted tunnel. The flap remained vascularized and survived well several weeks after the operation. At 16 weeks post-treatment, SCI rats with omentum flap treatment displayed the preservation of significantly more nerve fibers (p < 0.05) and a reduced injured cavity, though locomotor scores were similar. Conclusions: Taken together, the findings of this study indicate that treatment with an omental graft or transposition of an omental flap on an injured spinal cord has a positive effect on nerve protection and tissue preservation in SCI rats. The current data highlight the importance of omentum in clinical applications.     

The Role of Microglia in Modulating Neuroinflammation after Spinal Cord Injury

The pathobiology of traumatic and nontraumatic spinal cord injury (SCI), including degenerative myelopathy, is influenced by neuroinflammation. The neuroinflammatory response is initiated by a multitude of injury signals emanating from necrotic and apoptotic cells at the lesion site, recruiting local and infiltrating immune cells that modulate inflammatory cascades to aid in the protection of the lesion site and encourage regenerative processes. While peripheral immune cells are involved, microglia, the resident immune cells of the central nervous system (CNS), are known to play a central role in modulating this response. Microglia are armed with numerous cell surface receptors that interact with neurons, astrocytes, infiltrating monocytes, and endothelial cells to facilitate a dynamic, multi-faceted injury response. While their origin and essential nature are understood, their mechanisms of action and spatial and temporal profiles warrant extensive additional research. In this review, we describe the role of microglia and the cellular network in SCI, discuss tools for their investigation, outline their spatiotemporal profile, and propose translationally-relevant therapeutic targets to modulate neuroinflammation in the setting of SCI.     

大鼠急性脊髓损伤后热休克蛋白47基因mRNA的转录水平

目的探讨大鼠急性脊髓损伤(Spinal cord injury,SCI)后热休克蛋白47(HSP47)基因mRNA的转录水平。方法将12只Wistar大鼠随机分为1周SCI组、3周SCI组、5周SCI组和8周SCI组,复制钳夹型SCI模型,另设正常对照组和假手术组。分别在SCI后3d及每周进行BBB评分,并在1周、3周、5周和8周采用半定量RT-PCR法检测各组大鼠脊髓组织中HSP47基因mRNA的转录水平。结果SCI后,BBB评分低,随着时间的延长,评分逐渐上升;HSP47基因mRNA的转录水平明显升高,且在第8周升高更为明显。结论Wistar大鼠SCI后,HSP47基因mRNA的转录水平升高,提示其很可能参与了SCI的病理改变过程。     

Chrysin Suppressed Inflammatory Responses and the Inducible Nitric Oxide Synthase Pathway after Spinal Cord Injury in Rats

Chrysin (CH), a natural plant flavonoid, has shown a variety of beneficial effects. Our present study was conducted to evaluate the therapeutic potential of CH three days after spinal cord injury (SCI) in rats and to probe the underlying neuroprotective mechanisms. SCI was induced using the modified weight-drop method in Wistar rats. Then, they were treated with saline or CH by doses of 30 and 100 mg/kg for 26 days. Neuronal function was assessed with the Basso Beattle Bresnahan locomotor rating scale (BBB). The water content of spinal cord was determined after traumatic SCI. The NF-κB p65 unit, TNF-α, IL-1β and IL-6 in serums, as well as the apoptotic marker, caspase-3, of spinal cord tissues were measured using commercial kits. The protein level and activity of inducible nitric oxide synthase (iNOS) were detected by western blot and a commercial kit, respectively. NO (nitric oxide) production was evaluated by the determination of nitrite concentration. The rats with SCI showed marked reductions in BBB scores, coupled with increases in the water content of spinal cord, the NF-κB p65 unit, TNF-α, IL-1β, IL-6, iNOS, NO production and caspase-3. However, a CH supplement dramatically promoted the recovery of neuronal function and suppressed the inflammatory factors, as well as the iNOS pathway in rats with SCI. Our findings disclose that CH improved neural function after SCI in rats, which might be linked with suppressing inflammation and the iNOS pathway.     

The Role of Biomaterials in Peripheral Nerve and Spinal Cord Injury: A Review

Peripheral nerve and spinal cord injuries are potentially devastating traumatic conditions with major consequences for patients’ lives. Severe cases of these conditions are currently incurable. In both the peripheral nerves and the spinal cord, disruption and degeneration of axons is the main cause of neurological deficits. Biomaterials offer experimental solutions to improve these conditions. They can be engineered as scaffolds that mimic the nerve tissue extracellular matrix and, upon implantation, encourage axonal regeneration. Furthermore, biomaterial scaffolds can be designed to deliver therapeutic agents to the lesion site. This article presents the principles and recent advances in the use of biomaterials for axonal regeneration and nervous system repair.     

Upregulation of TRESK Channels Contributes to Motor and Sensory Recovery after Spinal Cord Injury

TWIK (tandem-pore domain weak inward rectifying K⁺)-related spinal cord K⁺ channel (TRESK), a member of the two-pore domain K⁺ channel family, is abundantly expressed in dorsal root ganglion (DRG) neurons. It is well documented that TRESK expression is changed in several models of peripheral nerve injury, resulting in a shift in sensory neuron excitability. However, the role of TRESK in the model of spinal cord injury (SCI) has not been fully understood. This study investigates the role of TRESK in a thoracic spinal cord contusion model, and in transgenic mice overexpressed with the TRESK gene (TG_TRESK). Immunostaining analysis showed that TRESK was expressed in the dorsal and ventral neurons of the spinal cord. The TRESK expression was increased by SCI in both dorsal and ventral neurons. TRESK mRNA expression was upregulated in the spinal cord and DRG isolated from the ninth thoracic (T9) spinal cord contusion rats. The expression was significantly upregulated in the spinal cord below the injury site at acute time points (6, 24, and 48 h) after SCI (p < 0.05). In addition, TRESK expression was markedly increased in DRGs below and adjacent to the injury site. TRESK was expressed in inflammatory cells. In addition, the number and fluorescence intensity of TRESK-positive neurons increased in the dorsal and ventral horns of the spinal cord after SCI. TG_TRESK SCI mice showed faster paralysis recovery and higher mechanical threshold compared to wild-type (WT)-SCI mice. TG_TRESK mice showed lower TNF-α concentrations in the blood than WT mice. In addition, IL-1β concentration and apoptotic signals in the caudal spinal cord and DRG were significantly decreased in TG_TRESK SCI mice compared to WT-SCI mice (p < 0.05). These results indicate that TRESK upregulated following SCI contributes to the recovery of paralysis and mechanical pain threshold by suppressing the excitability of motor and sensory neurons and inflammatory and apoptotic processes.     

Multifaceted Benefits of GDF11 Treatment in Spinal Cord Injury: In Vitro and In Vivo Studies

Traumatic spinal cord injury (SCI) initiates a series of cellular and molecular events that include both primary and secondary injury cascades. This secondary cascade provides opportunities for the delivery of therapeutic intervention. Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β (TGF-β) superfamily, regulates various biological processes in mammals. The effects of GDF11 in the nervous system were not fully elucidated. Here, we perform extensive in vitro and in vivo studies to unravel the effects of GDF11 on spinal cord after injury. In vitro culture studies showed that GDF11 increased the survival of both neuronal and oligodendroglial cells but decreased microglial cells. In stressed cultures, GDF11 effectively inhibited LPS stimulation and also protected neurons from ischemic damage. Intravenous GDF11 administration to rat after eliciting SCI significantly improved hindlimb functional restoration of SCI rats. Reduced neuronal connectivity was evident at 6 weeks post-injury and these deficits were markedly attenuated by GDF11 treatment. Furthermore, SCI-associated oligodendroglial alteration were more preserved by GDF11 treatment. Taken together, GDF11 infusion via intravenous route to SCI rats is beneficial, facilitating its therapeutic application in the future.     

Anti-Inflammatory Mechanism of Neural Stem Cell Transplantation in Spinal Cord Injury

Neural stem cell (NSC) transplantation has been proposed to promote functional recovery after spinal cord injury. However, a detailed understanding of the mechanisms of how NSCs exert their therapeutic plasticity is lacking. We transplanted mouse NSCs into the injured spinal cord seven days after SCI, and the Basso Mouse Scale (BMS) score was performed to assess locomotor function. The anti-inflammatory effects of NSC transplantation was analyzed by immunofluorescence staining of neutrophil and macrophages and the detection of mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-12 (IL-12). Furthermore, bone marrow-derived macrophages (BMDMs) were co-cultured with NSCs and followed by analyzing the mRNA levels of inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-6 and IL-10 with quantitative real-time PCR. The production of TNF-α and IL-1β by BMDMs was examined using the enzyme-linked immunosorbent assay (ELISA). Transplanted NSCs had significantly increased BMS scores (p < 0.05). Histological results showed that the grafted NSCs migrated from the injection site toward the injured area. NSCs transplantation significantly reduced the number of neutrophils and iNOS+/Mac-2+ cells at the epicenter of the injured area (p < 0.05). Meanwhile, mRNA levels of TNF-α, IL-1β, IL-6 and IL-12 in the NSCs transplantation group were significantly decreased compared to the control group. Furthermore, NSCs inhibited the iNOS expression of BMDMs and the release of inflammatory factors by macrophages in vitro (p < 0.05). These results suggest that NSC transplantation could modulate SCI-induced inflammatory responses and enhance neurological function after SCI via reducing M1 macrophage activation and infiltrating neutrophils. Thus, this study provides a new insight into the mechanisms responsible for the anti-inflammatory effect of NSC transplantation after SCI.     

Systemic Inflammation and the Breakdown of Intestinal Homeostasis Are Key Events in Chronic Spinal Cord Injury Patients

Our aim was to investigate the subset distribution and function of circulating monocytes, proinflammatory cytokine levels, gut barrier damage, and bacterial translocation in chronic spinal cord injury (SCI) patients. Thus, 56 SCI patients and 28 healthy donors were studied. The levels of circulating CD14^+highCD16⁻, CD14^+highCD16⁺, and CD14^+lowCD16⁺ monocytes, membrane TLR2, TLR4, and TLR9, phagocytic activity, ROS generation, and intracytoplasmic TNF-α, IL-1, IL-6, and IL-10 after lipopolysaccharide (LPS) stimulation were analyzed by polychromatic flow cytometry. Serum TNF-α, IL-1, IL-6 and IL-10 levels were measured by Luminex and LPS-binding protein (LBP), intestinal fatty acid-binding protein (I-FABP) and zonulin by ELISA. SCI patients had normal monocyte counts and subset distribution. CD14^+highCD16⁻ and CD14^+highCD16⁺ monocytes exhibited decreased TLR4, normal TLR2 and increased TLR9 expression. CD14^+highCD16⁻ monocytes had increased LPS-induced TNF-α but normal IL-1, IL-6, and IL-10 production. Monocytes exhibited defective phagocytosis but normal ROS production. Patients had enhanced serum TNF-α and IL-6 levels, normal IL-1 and IL-10 levels, and increased circulating LBP, I-FABP, and zonulin levels. Chronic SCI patients displayed impaired circulating monocyte function. These patients exhibited a systemic proinflammatory state characterized by enhanced serum TNF-α and IL-6 levels. These patients also had increased bacterial translocation and gut barrier damage.     

Therapeutic Hypothermia in Spinal Cord Injury: The Status of Its Use and Open Questions

Spinal cord injury (SCI) is a major health problem and is associated with a diversity of neurological symptoms. Pathophysiologically, dysfunction after SCI results from the culmination of tissue damage produced both by the primary insult and a range of secondary injury mechanisms. The application of hypothermia has been demonstrated to be neuroprotective after SCI in both experimental and human studies. The myriad of protective mechanisms of hypothermia include the slowing down of metabolism, decreasing free radical generation, inhibiting excitotoxicity and apoptosis, ameliorating inflammation, preserving the blood spinal cord barrier, inhibiting astrogliosis, promoting angiogenesis, as well as decreasing axonal damage and encouraging neurogenesis. Hypothermia has also been combined with other interventions, such as antioxidants, anesthetics, alkalinization and cell transplantation for additional benefit. Although a large body of work has reported on the effectiveness of hypothermia as a neuroprotective approach after SCI and its application has been translated to the clinic, a number of questions still remain regarding its use, including the identification of hypothermia’s therapeutic window, optimal duration and the most appropriate rewarming rate. In addition, it is necessary to investigate the neuroprotective effect of combining therapeutic hypothermia with other treatment strategies for putative synergies, particularly those involving neurorepair.     

Spinal Cord Injury Causes Marked Tissue Rearrangement in the Urethra—Experimental Study in the Rat

Traumatic spinal cord injury (SCI) results in the time-dependent development of urinary impairment due to neurogenic detrusor overactivity (NDO) and detrusor-sphincter-dyssynergia (DSD). This is known to be accompanied by massive changes in the bladder wall. It is presently less clear if the urethra wall also undergoes remodelling. To investigate this issue, female rats were submitted to complete spinal transection at the T8/T9 level and left to recover for 1 week and 4 weeks. To confirm the presence of SCI-induced NDO, bladder function was assessed by cystometry under urethane anesthesia before euthanasia. Spinal intact animals were used as controls. Urethras were collected and processed for further analysis. Following thoracic SCI, time-dependent changes in the urethra wall were observed. Histological assessment revealed marked urethral epithelium reorganization in response to SCI, as evidenced by an increase in epithelial thickness. At the muscular layer, SCI resulted in strong atrophy of the smooth muscle present in the urethral sphincter. Innervation was also affected, as evidenced by a pronounced decrease in the expression of markers of general innervation, particularly those present in sensory and sympathetic nerve fibres. The present data show an evident impact of SCI on the urethra, with significant histological rearrangement, accompanied by sensory and sympathetic denervation. It is likely that these changes will affect urethral function and contribute to SCI-induced urinary dysfunction, and they deserve further investigation.     

Rho/ROCK Pathway and Noncoding RNAs: Implications in Ischemic Stroke and Spinal Cord Injury

Ischemic strokes (IS) and spinal cord injuries (SCI) are major causes of disability. RhoA is a small GTPase protein that activates a downstream effector, ROCK. The up-regulation of the RhoA/ROCK pathway contributes to neuronal apoptosis, neuroinflammation, blood-brain barrier dysfunction, astrogliosis, and axon growth inhibition in IS and SCI. Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), were previously considered to be non-functional. However, they have attracted much attention because they play an essential role in regulating gene expression in physiological and pathological conditions. There is growing evidence that ROCK inhibitors, such as fasudil and VX-210, can reduce injury in IS and SCI in animal models and clinical trials. Recently, it has been reported that miRNAs are decreased in IS and SCI, while lncRNAs are increased. Inhibiting the Rho/ROCK pathway with miRNAs alleviates apoptosis, neuroinflammation, oxidative stress, and axon growth inhibition in IS and SCI. Further studies are required to explore the significance of ncRNAs in IS and SCI and to establish new strategies for preventing and treating these devastating diseases.     

Evaluation of Injured Axons Using Two-Photon Excited Fluorescence Microscopy after Spinal Cord Contusion Injury in YFP-H Line Mice

Elucidation of the process of degeneration of injured axons is important for the development of therapeutic modules for the treatment of spinal cord injuries. The aim of this study was to establish a method for time-lapse observation of injured axons in living animals after spinal cord contusion injury. YFP (yellow fluorescent protein)-H transgenic mice, which we used in this study, express fluorescence in their nerve fibers. Contusion damage to the spinal cord at the 11th vertebra was performed by IH (Infinite Horizon) impactor, which applied a pressure of 50 kdyn. The damaged spinal cords were re-exposed during the observation period under anesthesia, and then observed by two-photon excited fluorescence microscopy, which can observe deep regions of tissues including spinal cord axons. No significant morphological change of injured axons was observed immediately after injury. Three days after injury, the number of axons decreased, and residual axons were fragmented. Seven days after injury, only fragments were present in the damaged tissue. No hind-limb movement was observed during the observation period after injury. Despite the immediate paresis of hind-limbs following the contusion injury, the morphological degeneration of injured axons was delayed. This method may help clarification of pathophysiology of axon degeneration and development of therapeutic modules for the treatment of spinal cord injury.     

Therapeutic Potential of Mesenchymal Stem Cells (MSCs) and MSC-Derived Extracellular Vesicles for the Treatment of Spinal Cord Injury

Spinal cord injury (SCI) is a life-threatening condition that leads to permanent disability with partial or complete loss of motor, sensory, and autonomic functions. SCI is usually caused by initial mechanical insult, followed by a cascade of several neuroinflammation and structural changes. For ameliorating the neuroinflammatory cascades, MSC has been regarded as a therapeutic agent. The animal SCI research has demonstrated that MSC can be a valuable therapeutic agent with several growth factors and cytokines that may induce anti-inflammatory and regenerative effects. However, the therapeutic efficacy of MSCs in animal SCI models is inconsistent, and the optimal method of MSCs remains debatable. Moreover, there are several limitations to developing these therapeutic agents for humans. Therefore, identifying novel agents for regenerative medicine is necessary. Extracellular vesicles are a novel source for regenerative medicine; they possess nucleic acids, functional proteins, and bioactive lipids and perform various functions, including damaged tissue repair, immune response regulation, and reduction of inflammation. MSC-derived exosomes have advantages over MSCs, including small dimensions, low immunogenicity, and no need for additional procedures for culture expansion or delivery. Certain studies have demonstrated that MSC-derived extracellular vesicles (EVs), including exosomes, exhibit outstanding chondroprotective and anti-inflammatory effects. Therefore, we reviewed the principles and patho-mechanisms and summarized the research outcomes of MSCs and MSC-derived EVs for SCI, reported to date.     

Optimal Preclinical Conditions for Using Adult Human Multipotent Neural Cells in the Treatment of Spinal Cord Injury

Stem cell-based therapeutics are amongst the most promising next-generation therapeutic approaches for the treatment of spinal cord injury (SCI), as they may promote the repair or regeneration of damaged spinal cord tissues. However, preclinical optimization should be performed before clinical application to guarantee safety and therapeutic effect. Here, we investigated the optimal injection route and dose for adult human multipotent neural cells (ahMNCs) from patients with hemorrhagic stroke using an SCI animal model. ahMNCs demonstrate several characteristics associated with neural stem cells (NSCs), including the expression of NSC-specific markers, self-renewal, and multi neural cell lineage differentiation potential. When ahMNCs were transplanted into the lateral ventricle of the SCI animal model, they specifically migrated within 24 h of injection to the damaged spinal cord, where they survived for at least 5 weeks after injection. Although ahMNC transplantation promoted significant locomotor recovery, the injection dose was shown to influence treatment outcomes, with a 1 × 10⁶ (medium) dose of ahMNCs producing significantly better functional recovery than a 3 × 10⁵ (low) dose. There was no significant gain in effect with the 3 × 10⁶ ahMNCs dose. Histological analysis suggested that ahMNCs exert their effects by modulating glial scar formation, neuroprotection, and/or angiogenesis. These data indicate that ahMNCs from patients with hemorrhagic stroke could be used to develop stem cell therapies for SCI and that the indirect injection route could be clinically relevant. Moreover, the optimal transplantation dose of ahMNCs defined in this preclinical study might be helpful in calculating its optimal injection dose for patients with SCI in the future.     

Time-Course Changes of Extracellular Matrix Encoding Genes Expression Level in the Spinal Cord Following Contusion Injury—A Data-Driven Approach

The involvement of the extracellular matrix (ECM) in lesion evolution and functional outcome is well recognized in spinal cord injury. Most attention has been dedicated to the “core” area of the lesion and scar formation, while only scattered reports consider ECM modification based on the temporal evolution and the segments adjacent to the lesion. In this study, we investigated the expression profile of 100 genes encoding for ECM proteins at 1, 8 and 45 days post-injury, in the spinal cord segments rostral and caudal to the lesion and in the scar segment, in a rat model. During both the active lesion phases and the lesion stabilization, we observed an asymmetric gene expression induced by the injury, with a higher regulation in the rostral segment of genes involved in ECM remodeling, adhesion and cell migration. Using bioinformatic approaches, the metalloproteases inhibitor Timp1 and the hyaluronan receptor Cd44 emerged as the hub genes at all post-lesion times. Results from the bioinformatic gene expression analysis were then confirmed at protein level by tissue analysis and by cell culture using primary astrocytes. These results indicated that ECM regulation also takes place outside of the lesion area in spinal cord injury.