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.     

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.     

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.     

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.     

Posttraumatic Inflammation as a Key to Neuroregeneration after Traumatic Spinal Cord Injury

Pro- and anti-inflammatory cytokines might have a large impact on the secondary phase and on the neurological outcome of patients with acute spinal cord injury (SCI). We measured the serum levels of different cytokines (Interferon-γ, Tumor Necrosis Factor-α, Interleukin-1β, IL-6, IL-8, IL-10, and Vascular Endothelial Growth Factor) over a 12-week period in 40 acute traumatic SCI patients: at admission on average one hour after initial trauma; at four, nine, 12, and 24 h; Three, and seven days after admission; and two, four, eight, and twelve weeks after admission. This was done using a Luminex Performance Human High Sensitivity Cytokine Panel. SCI was classified using the American Spinal Injury Association (ASIA) Impairment Scale (AIS) at time of admission and after 12 weeks. TNFα, IL-1β, IL-6, IL-8, and IL-10 concentrations were significantly higher in patients without neurological remission and in patients with an initial AIS A (p < 0.05). This study shows significant differences in cytokine concentrations shown in traumatic SCI patients with different neurological impairments and within a 12-week period. IL-8 and IL-10 are potential peripheral markers for neurological remission and rehabilitation after traumatic SCI. Furthermore our cytokine expression pattern of the acute, subacute, and intermediate phase of SCI establishes a possible basis for future studies to develop standardized monitoring, prognostic, and tracking techniques.     

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.     

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1β, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).     

Comparative Analysis of Human Mesenchymal Stem Cells from Bone Marrow,Adipose Tissue,and Umbilical Cord Blood as Sources of Cell Therapy

Various source-derived mesenchymal stem cells (MSCs) have been considered for cell therapeutics in incurable diseases. To characterize MSCs from different sources, we compared human bone marrow (BM), adipose tissue (AT), and umbilical cord blood-derived MSCs (UCB-MSCs) for surface antigen expression, differentiation ability, proliferation capacity, clonality, tolerance for aging, and paracrine activity. Although MSCs from different tissues have similar levels of surface antigen expression, immunosuppressive activity, and differentiation ability, UCB-MSCs had the highest rate of cell proliferation and clonality, and significantly lower expression of p53, p21, and p16, well known markers of senescence. Since paracrine action is the main action of MSCs, we examined the anti-inflammatory activity of each MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of inflammatory cytokines including interleukin-1α (IL-1α), IL-6, and IL-8 via angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1 secretion was responsible for this beneficial effect in part by preventing inflammation. Our results demonstrate that primitive UCB-MSCs have biological advantages in comparison to adult sources, making UCB-MSCs a useful model for clinical applications of cell therapy.     

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.     

Mesenchymal Stem Cells Preserve Working Memory in the 3xTg-AD Mouse Model of Alzheimer’s Disease

The transplantation of stem cells may have a therapeutic effect on the pathogenesis and progression of neurodegenerative disorders. In the present study, we transplanted human mesenchymal stem cells (MSCs) into the lateral ventricle of a triple transgenic mouse model of Alzheimer´s disease (3xTg-AD) at the age of eight months. We evaluated spatial reference and working memory after MSC treatment and the possible underlying mechanisms, such as the influence of transplanted MSCs on neurogenesis in the subventricular zone (SVZ) and the expression levels of a 56 kDa oligomer of amyloid β (Aβ*56), glutamine synthetase (GS) and glutamate transporters (Glutamate aspartate transporter (GLAST) and Glutamate transporter-1 (GLT-1)) in the entorhinal and prefrontal cortices and the hippocampus. At 14 months of age we observed the preservation of working memory in MSC-treated 3xTg-AD mice, suggesting that such preservation might be due to the protective effect of MSCs on GS levels and the considerable downregulation of Aβ*56 levels in the entorhinal cortex. These changes were observed six months after transplantation, accompanied by clusters of proliferating cells in the SVZ. Since the grafted cells did not survive for the whole experimental period, it is likely that the observed effects could have been transiently more pronounced at earlier time points than at six months after cell application.     

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.     

Thrombin Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage Induced Neonatal Rats

Severe intraventricular hemorrhage (IVH) remains a major cause of high mortality and morbidity in extremely preterm infants. Mesenchymal stem cell (MSC) transplantation is a possible therapeutic option, and development of therapeutics with enhanced efficacy is necessary. This study investigated whether thrombin preconditioning improves the therapeutic efficacy of human Wharton’s jelly-derived MSC transplantation for severe neonatal IVH, using a rat model. Severe neonatal IVH was induced by injecting 150 μL blood into each lateral ventricle on postnatal day (P) 4 in Sprague-Dawley rats. After 2 days (P6), naïve MSCs or thrombin-preconditioned MSCs (1 × 10⁵/10 μL) were transplanted intraventricularly. After behavioral tests, brain tissues and cerebrospinal fluid of P35 rats were obtained for histological and biochemical analyses, respectively. Thrombin-preconditioned MSC transplantation significantly reduced IVH-induced ventricular dilatation on in vivo magnetic resonance imaging, which was coincident with attenuations of reactive gliosis, cell death, and the number of activated microglia and levels of inflammatory cytokines after IVH induction, compared to naïve MSC transplantation. In the behavioral tests, the sensorimotor and memory functions significantly improved after transplantation of thrombin-preconditioned MSCs, compared to naïve MSCs. Overall, thrombin preconditioning significantly improves the therapeutic potential and more effectively attenuates brain injury, including progressive ventricular dilatation, gliosis, cell death, inflammation, and neurobehavioral functional impairment, in newborn rats with induced severe IVH than does naïve MSC transplantation.     

Characterization of a Novel Aspect of Tissue Scarring Following Experimental Spinal Cord Injury and the Implantation of Bioengineered Type-I Collagen Scaffolds in the Adult Rat: Involvement of Perineurial-like Cells?

Numerous intervention strategies have been developed to promote functional tissue repair following experimental spinal cord injury (SCI), including the bridging of lesion-induced cystic cavities with bioengineered scaffolds. Integration between such implanted scaffolds and the lesioned host spinal cord is critical for supporting regenerative growth, but only moderate-to-low degrees of success have been reported. Light and electron microscopy were employed to better characterise the fibroadhesive scarring process taking place after implantation of a longitudinally microstructured type-I collagen scaffold into unilateral mid-cervical resection injuries of the adult rat spinal cord. At long survival times (10 weeks post-surgery), sheets of tightly packed cells (of uniform morphology) could be seen lining the inner surface of the repaired dura mater of lesion-only control animals, as well as forming a barrier along the implant–host interface of the scaffold-implanted animals. The highly uniform ultrastructural features of these scarring cells and their anatomical continuity with the local, reactive spinal nerve roots strongly suggest their identity to be perineurial-like cells. This novel aspect of the cellular composition of reactive spinal cord tissue highlights the increasingly complex nature of fibroadhesive scarring involved in traumatic injury, and particularly in response to the implantation of bioengineered collagen scaffolds.     

Anti-Inflammatory,Antioxidant, and Antifibrotic Effects of Gingival-Derived MSCs on Bleomycin-Induced Pulmonary Fibrosis in Mice

Background: Mesenchymal stem cell (MSC) intervention has been associated with lung protection. We attempted to determine whether mouse gingival-derived mesenchymal stem cells (GMSCs) could protect against bleomycin-induced pulmonary fibrosis. Methods: Mice were divided into three groups: control (Con), bleomycin (Bl), and bleomycin + MSCs (Bl + MSCs). Mice were treated with 5 mg/kg bleomycin via transtracheal instillation to induce pulmonary fibrosis. We assessed the following parameters: histopathological severity of injury in the lung, liver, kidney, and aortic tissues; the degree of pulmonary fibrosis; pulmonary inflammation; pulmonary oedema; profibrotic factor levels in bronchoalveolar lavage fluid (BALF) and lung tissue; oxidative stress-related indicators and apoptotic index in lung tissue; and gene expression levels of IL-1β, IL-8, TNF-α, lysophosphatidic acid (LPA), lysophosphatidic acid receptor 1 (LPA1), TGF-β, matrix metalloproteinase 9 (MMP-9), neutrophil elastase (NE), MPO, and IL-10 in lung tissue. Results: GMSC intervention attenuated bleomycin-induced pulmonary fibrosis, pulmonary inflammation, pulmonary oedema, and apoptosis. Bleomycin instillation notably increased expression levels of the IL-1β, IL-8, TNF-α, LPA, LPA1, TGF-β, MMP-9, NE, and MPO genes and attenuated expression levels of the IL-10 gene in lung tissue, and these effects were reversed by GMSC intervention. Bleomycin instillation notably upregulated MDA and MPO levels and downregulated GSH and SOD levels in lung tissue, and these effects were reversed by GMSC intervention. GMSC intervention prevented upregulation of neutrophil content in the lung, liver, and kidney tissues and the apoptotic index in lung tissue. Conclusions: GMSC intervention exhibits anti-inflammatory and antioxidant capacities. Deleterious accumulation of neutrophils, which is reduced by GMSC intervention, is a key component of bleomycin-induced pulmonary fibrosis. GMSC intervention impairs bleomycin-induced NE, MMP-9, LPA, APL1, and TGF-β release.     

Mangiferin Reduces the Inhibition of Chondrogenic Differentiation by IL-1β in Mesenchymal Stem Cells from Subchondral Bone and Targets Multiple Aspects of the Smad and SOX9 Pathways

Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs) from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β). Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y–box (SRY-box) containing gene 9 (SOX9), type 2α1 collagen (Col2α1), cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5). Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways.     

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.     

BMP3 Alone and Together with TGF-β Promote the Differentiation of Human Mesenchymal Stem Cells into a Nucleus Pulposus-Like Phenotype

Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)-β on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-β, and compared with cultures in basal and TGF-β media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF-β on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-β, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-β, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF-β induced positive effects. These actions are likely related to the activation of TGF-β signaling pathway.     

Ischemic Preconditioning Protects against Spinal Cord Ischemia-Reperfusion Injury in Rabbits by Attenuating Blood Spinal Cord Barrier Disruption

Ischemic preconditioning has been reported to protect against spinal cord ischemia-reperfusion (I-R) injury, but the underlying mechanisms are not fully understood. To investigate this, Japanese white rabbits underwent I-R (30 min aortic occlusion followed by reperfusion), ischemic preconditioning (three cycles of 5 min aortic occlusion plus 5 min reperfusion) followed by I-R, or sham surgery. At 4 and 24 h following reperfusion, neurological function was assessed using Tarlov scores, blood spinal cord barrier permeability was measured by Evan’s Blue extravasation, spinal cord edema was evaluated using the wet-dry method, and spinal cord expression of zonula occluden-1 (ZO-1), matrix metalloproteinase-9 (MMP-9), and tumor necrosis factor-α (TNF-α) were measured by Western blot and a real-time polymerase chain reaction. ZO-1 was also assessed using immunofluorescence. Spinal cord I-R injury reduced neurologic scores, and ischemic preconditioning treatment ameliorated this effect. Ischemic preconditioning inhibited I-R-induced increases in blood spinal cord barrier permeability and water content, increased ZO-1 mRNA and protein expression, and reduced MMP-9 and TNF-α mRNA and protein expression. These findings suggest that ischemic preconditioning attenuates the increase in blood spinal cord barrier permeability due to spinal cord I-R injury by preservation of tight junction protein ZO-1 and reducing MMP-9 and TNF-α expression.     

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.     

Long-Term Effects of Neural Precursor Cell Transplantation on Secondary Injury Processes and Functional Recovery after Severe Cervical Contusion-Compression Spinal Cord Injury

Cervical spinal cord injury (SCI) remains a devastating event without adequate treatment options despite decades of research. In this context, the usefulness of common preclinical SCI models has been criticized. We, therefore, aimed to use a clinically relevant animal model of severe cervical SCI to assess the long-term effects of neural precursor cell (NPC) transplantation on secondary injury processes and functional recovery. To this end, we performed a clip contusion-compression injury at the C6 level in 40 female Wistar rats and a sham surgery in 10 female Wistar rats. NPCs, isolated from the subventricular zone of green fluorescent protein (GFP) expressing transgenic rat embryos, were transplanted ten days after the injury. Functional recovery was assessed weekly, and FluoroGold (FG) retrograde fiber-labeling, as well as manganese-enhanced magnetic resonance imaging (MEMRI), were performed prior to the sacrifice of the animals eight weeks after SCI. After cryosectioning of the spinal cords, immunofluorescence staining was conducted. Results were compared between the treatment groups (NPC, Vehicle, Sham) and statistically analyzed (p < 0.05 was considered significant). Despite the severity of the injury, leading to substantial morbidity and mortality during the experiment, long-term survival of the engrafted NPCs with a predominant differentiation into oligodendrocytes could be observed after eight weeks. While myelination of the injured spinal cord was not significantly improved, NPC treated animals showed a significant increase of intact perilesional motor neurons and preserved spinal tracts compared to untreated Vehicle animals. These findings were associated with enhanced preservation of intact spinal cord tissue. However, reactive astrogliosis and inflammation where not significantly reduced by the NPC-treatment. While differences in the Basso–Beattie–Bresnahan (BBB) score and the Gridwalk test remained insignificant, animals in the NPC group performed significantly better in the more objective CatWalk XT gait analysis, suggesting some beneficial effects of the engrafted NPCs on the functional recovery after severe cervical SCI.