Rat Model Of Spinal Cord Injury Research Articles

Characterization of Ex Vivo and In Vitro Wnt Transcriptome Induced by Spinal Cord Injury in Rat Microglial Cells.

It is well known that inflammation is crucial in the onset and progression of neurodegenerative diseases and traumatic central nervous system (CNS) injuries, and that microglia and monocyte-derived macrophages (MDMs) play a pivotal role in neuroinflammation. Therefore, the exploration of molecular signaling pathways that are involved in the microglia/macrophage response might help us to shed light on their eventual therapeutic modulation. Interestingly, there is growing evidence showing that the Wnt family of proteins is involved in different neuropathologies that are characterized by a dysregulated neuroinflammatory response, including spinal cord injury (SCI). Here, we aimed to validate a methodology with competence to assess the physiologically relevant Wnt expression patterns of active microglia and MDMs in a rat model of SCI. For that purpose, we have selected and adapted an in vitro system of primary microglia culture that were stimulated with a lesioned spinal cord extract (SCE), together with an ex vivo protocol of flow cytometry sorting of rat microglia/MDMs at different time-points after contusive SCI. Our study demonstrates that the expression profile of Wnt-related genes in microglia/MDM cells exhibit important differences between these particular scenarios which would be in line with previous studies where similar discrepancies have been described for other molecules. Moreover, our results provide for a first experimental report of the Wnt transcriptome in rat microglia and MDMs after SCI which, together with the research platform that was used in the study, and considering its limitations, we expect might contribute to foster the research on Wnt-driven immunomodulatory therapies.

Nanoformulated metformin enhanced the treatment of spinal cord injury

Spinal cord injury (SCI) leads to immediate disruption of neuronal membranes neuronal death, and inflammation, which in turn trigger secondary injury. Few therapies against SCI have been developed. Metformin displays anti-inflammatory, anti-oxidant, anti-apoptotic, and neuroprotective capacity and may therefore alleviate SCI. However, it shows a short half-life, poor water solubility, fast absorption, rapid clearance, and a lack of targeted delivery options. Here we attempt to overcome these obstacles by developing zein-based spherical nanoparticles containing the peptide CAQK for targeted delivery of metformin (CAQK-MET-NPs) in SCI. We investigated the pharamocodynamics and effects of CAQK-MET-NPs on neural recovery in cell models as well as a rat SCI model using histopathology, immunohistochemistry, behavioral tests, and various imaging techniques. CAQK-MET-NPs exerted strong anti-inflammatory and neuroprotective activities in vitro and in vivo, promoting the spinal cord repair and partial recovery of motor function after SCI. This study thus provides an important foundational step towards potential SCI therapy.

Identifying a novel KLF2/lncRNA SNHG12/miR-494-3p/RAD23B axis in Spare Nerve Injury-induced neuropathic pain

Spinal cord injury (SCI) is a devastating condition for patients, affecting nearly 2.5 million people globally. Multiple side effects of SCI have resulted in a terrible life experience for SCI patients, of which neuropathic pain has attracted the most scientific interest. Even though many efforts have been made to attenuate or eliminate neuropathic pain induced by SCI, the outcomes for patients are still poor. Therefore, identifying novel diagnosis or therapeutic targets of SCI-induced neuropathic pain is urgently needed. Recently, multiple functions of long non-coding RNA (lncRNA) have been elucidated, including those in SCI-induced neuropathic pain. In this study, lncRNA small nucleolar RNA host gene 12 (SNHG12) was found to be upregulated in the dorsal root ganglion (DRGs) of rats with spare nerve injury (SNI). By constructing SCI rat models, we found that lncRNA SNHG12 expression was increased in the DRGs, and mainly distributed in the cytoplasm of PC12 cells. Paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and enzyme linked immunosorbent assay (ELISA) results indicated that lncRNA SNHG12 knockdown attenuated SNI-induced neuropathic pain, and decreased the expression levels of interleukin (IL)−1β, IL-6, and tumour necrosis factor α (TNF-α) in the DRGs. Bioinformatics analysis, RNA pull-down, chromatin immunoprecipitation (ChIP), and luciferase reporter gene assays showed that lncRNA SNHG12 regulates the RAD23 homologue B, nucleotide excision repair protein (RAD23B) expression, through targeting micro RNA (miR)−494-3p. Furthermore, the study indicated that Kruppel-Like Factor 2 (KLF2) could regulate lncRNA SNHG12 expression in PC12 cells. This study identified a novel KLF2/lncRNA SNHG12/miR-494-3p/RAD23B axis in SNI-induced neuropathic pain, which might provide a new insight for developing novel diagnosis, or therapeutic targets of SCI-induced neuropathic pain in the future.

Macrophage Migration Inhibitory Factor (MIF) Makes Complex Contributions to Pain-Related Hyperactivity of Nociceptors after Spinal Cord Injury.

Neuropathic pain is a major, inadequately treated challenge for people with spinal cord injury (SCI). While SCI pain mechanisms are often assumed to be in the CNS, rodent studies have revealed mechanistic contributions from primary nociceptors. These neurons become chronically hyperexcitable after SCI, generating ongoing electrical activity that promotes ongoing pain. A major question is whether extrinsic chemical signals help to drive ongoing electrical activity after SCI. People living with SCI exhibit acute and chronic elevation of circulating levels of macrophage migration inhibitory factor (MIF), a cytokine implicated in preclinical pain models. Probable nociceptors isolated from male rats and exposed to an MIF concentration reported in human plasma (1 ng/ml) showed hyperactivity similar to that induced by SCI, although, surprisingly, a 10-fold higher concentration failed to increase excitability. Conditioned behavioral aversion to a chamber associated with peripheral MIF injection suggested that MIF stimulates affective pain. A MIF inhibitor, Iso-1, reversed SCI-induced hyperexcitability. Unlike chronic SCI-induced hyperexcitability, acute MIF-induced hyperexcitability was only partially abrogated by inhibiting ERK signaling. Unexpectedly, MIF concentrations that induced hyperactivity in nociceptors from naive animals, after SCI induced a long-lasting conversion from a highly excitable nonaccommodating type to a rapidly accommodating, hypoexcitable type, possibly as a homeostatic response to prolonged depolarization. Treatment with conditioned medium from cultures of DRG cells obtained after SCI was sufficient to induce MIF-dependent hyperactivity in neurons from naive rats. Thus, changes in systemic and DRG levels of MIF may help to maintain SCI-induced nociceptor hyperactivity that persistently promotes pain.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major challenge for people with spinal cord injury (SCI). Pain can drastically impair quality of life, and produces substantial economic and social burdens. Available treatments, including opioids, remain inadequate. This study shows that the cytokine macrophage migration inhibitory factor (MIF) can induce pain-like behavior and plays an important role in driving persistent ongoing electrical activity in injury-detecting sensory neurons (nociceptors) in a rat SCI model. The results indicate that SCI produces an increase in MIF release within sensory ganglia. Low MIF levels potently excite nociceptors, but higher levels trigger a long-lasting hypoexcitable state. These findings suggest that therapeutic targeting of MIF in neuropathic pain states may reduce pain and sensory dysfunction by curbing nociceptor hyperactivity.

Platelet rich plasma enhanced neuro-regeneration of human dental pulp stem cells in vitro and in rat spinal cord.

BackgroundHuman dental pulp stem cells (hDPSCs) exhibit excellent differentiation potential and are capable of differentiating into several different cellular phenotypes, including neurons. Platelet-rich plasma (PRP) contains numerous growth factors that can stimulate stem cell differentiation. In this study, we investigated the potential stimulatory effects of PRP on neurogenic differentiation and anti-apoptosis of hDPSCs in injured spinal cords.MethodsThe unipotential differentiation capacity of hDPSCs was analyzed by cell surface antigen identification and cell cycle analysis. A spinal cord injury rat model composed of 40 Sprague-Dawley (SD) rats was used to facilitate an in vivo study. Rats were divided into four groups: a double-treatment group (receiving both neurogenic-induced hDPSCs and PRP), two single-treatment groups (receiving neurogenic-induced hDPSCs or PRP) and a sham group (receiving normal saline). The Basso, Beattie, Bresnahan Locomotor Rating Scale was subsequently used to evaluate the motor function of the spinal cord. Cell viability and differentiation of hDPSCs in the damaged spinal cords were analyzed and apoptosis of neural cells was evaluated using the terminal uridine nucleotide end labeling (TUNEL) assay.ResultsGrowth pattern, cell surface marker and cell cycle analyses revealed that hDPSCs have a high degree of multi-directional differentiation potential and can be induced into neurons in vitro. In the rat spinal cord injury model, double-treatment with hDPSC/PRP or single treatment with hDPSCs or PRP significantly improved motor function compared with the sham group (P<0.05). Apoptosis of neural cells was observed to be significantly higher in the sham group compared to any of the treatment groups. Double-treatment with hDPSCs and PRP resulted in the lowest apoptotic rate among the groups analyzed.ConclusionshDPSCs exhibit differentiation potential and are capable of transforming into neural cells both in vitro and in vivo. Significantly increased inhibition of neuronal apoptosis and improved motor function recovery of the spinal cord were observed following double-treatment with hDPSCs and PRP compared with the single-treatment groups.

Injection of Cerium Oxide Nanoparticles to Treat Spinal Cord Injury in Rats.

This study investigated the effects of local injection of cerium oxide nanoparticles (CeONPs) in a rat spinal cord injury (SCI) model. Thirty-six adult male Wistar rats were divided into 4 groups: controls (healthy animals), sham (laminectomy), SCI (laminectomy+SCI induction), and treatment (laminectomy+SCI induction+intrathecal injection of CeONPs immediately after injury). SCI was induced using an aneurysm clip at the T12-T13 vertebral region. Motor performance and pain threshold tests were performed weekly; H&E staining and measurement of cavity sizes were performed 6 weeks after injury. The expression of granulocyte colony-stimulating factor (GCSF), P44/42 MAPK, P-P44/42 MAPK, Tau, myelin-associated glycoprotein(MAG) was evaluated after 6 weeks by Western blot. The Basso, Beattie, and Bresnahan locomotor scoring scales improved in animals receiving CeONPs compared with SCI animals. The cavity sizes were less in the treatment group. GCSF expression was similar in the animals receiving CeONPs compared with the SCI group but the expression of ERK1/ERK2 and phospho-ERK was lower than in the SCI group. Expression levels of Tau and MAG were significantly increased in treated animals compared to the SCI group. These data indicate that the use of CeONPs may improve motor functional recovery in SCI.

Receptor-Interacting Protein Kinase 3 Inhibition Relieves Mechanical Allodynia and Suppresses NLRP3 Inflammasome and NF-κB in a Rat Model of Spinal Cord Injury.

BackgroundNeuroinflammation is critical in developing and maintaining neuropathic pain after spinal cord injury (SCI). The receptor-interacting protein kinase 3 (RIPK3) has been shown to promote inflammatory response by exerting its non-necroptotic functions. In this study, we explored the involvement of RIPK3 in neuropathic pain after SCI.MethodsThoracic (T10) SCI rat model was conducted, and the mechanical threshold in rats was measured. The expressions of RIPK3, nod-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, and nuclear factor-κB (NF-κB) were measured with western blotting analysis or quantitative real-time polymerase chain reaction (qRT-PCR). Double immunofluorescence staining was used to explore the colabeled NLRP3 with NeuN, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (IBA1). In addition, enzyme-linked immunosorbent assay (ELISA) was applied to analyze the levels of proinflammatory factors interleukin 1 beta (IL-1β), interleukin 18 (IL-18), and tumor necrosis factor alpha (TNF-α).ResultsThe expression of RIPK3 was elevated from postoperative days 7–21, which was consistent with the development of mechanical allodynia. Intrathecal administration of RIPK3 inhibitor GSK872 could alleviate the mechanical allodynia in SCI rats and reduce the expression levels of RIPK3. The activation of NLRP3 inflammasome and NF-κB was attenuated by GSK872 treatment. Furthermore, immunofluorescence suggested that NLRP3 had colocalization with glial cells and neurons in the L4–L6 spinal dorsal horns. In addition, GSK872 treatment reduced the production of inflammatory cytokines.ConclusionOur findings indicated that RIPK3 was an important facilitated factor for SCI-induced mechanical allodynia. RIPK3 inhibition might relieve mechanical allodynia by inhibiting NLRP3 inflammasome, NF-κB, and the associated inflammation.

Identification of a circRNA-mediated comprehensive ceRNA network in spinal cord injury pathogenesis

RNAs are closely associated with human diseases; however, immune-related genes (IRGs) and their potential regulatory networks in relation to spinal cord injury (SCI) are still poorly understood. Here, we investigated the key IRGs as well as the competing endogenous RNA (ceRNA) mechanisms that are associated with SCI pathogenesis based on microarray datasets and the use of a rat SCI model. Specifically, four independent SCI microarray datasets from Gene Expression Omnibus (GEO) database were analyzed and, thereafter, differentially expressed IRGs were annotated via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Furthermore, based on the GEO datasets, differentially expressed RNAs (DERNAs), including DEcircRNAs, DEmiRNAs, and DEmRNAs were identified and interactions between them were also predicted using online databases, and to construct a circular RNA (circRNA) mediated ceRNA network, candidate RNAs were also identified. Furthermore, the support vector machine (SVM) and least absolute shrinkage and selection operator (LASSO) methods were used for the identification of critical DERNAs, while differential gene expression was validated using the GSE20907 dataset. Our results were as follows. In the SCI microarray datasets, 32, 58, and 74 DEIRGs, DEcircRNAs, and DEmiRNAs were identified, respectively. In addition, GO and KEGG analyses showed that the DEIRGs were primarily enriched in neutrophil-mediated immunity and nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor-1 (HIF-1) signaling pathways, and based on LASSO and SVM screening, PLXNB2 was identified as a DEIRG, while hsa_circ_0026646 was identified as the key circRNA, showing a higher SCI expression. Furthermore, our results proved that PLXNB2 and hsa_circ_0026646 were upregulated in SCI, whereas miR-331-3p was downregulated, and, interestingly, similar expression profiles were confirmed using the rat SCI model. Furthermore, fluorescent reporter assay indicated that both hsa_circ_0026646 and PLXNB2 have miR-331-3p target sites, and the ceRNA hypothesis suggested the dysregulation of hsa_circ_0026646, miR-331-3p, and PLXNB2 in SCI. Thus, our results suggested that in SCI pathogenesis, hsa_circ_0026646 correlates with PLXNB2 by targeting miR-331-3p.

Thrombin induces morphological and inflammatory astrocytic responses via activation of PAR1 receptor

Spinal cord injury (SCI) will result in the significant elevation of thrombin production at lesion site via either breakage of blood-spinal cord barrier or upregulated expression within nerve cells. Thrombin-induced activation of the protease activated receptors (PARs) evokes various pathological effects that deteriorate the functional outcomes of the injured cord. The cellular consequences of thrombin action on the astrocytes, as well as the underlying mechanism are not fully elucidated by far. In the present study, SCI model of rats was established by contusion, and primary astrocytes were isolated for culture from newborn rats. The expression levels of thrombin and PAR1 receptor at lesion sites of the spinal cord were determined. The primary astrocytes cultured in vitro were stimulated with different concentration of thrombin, and the resultant morphological changes, inflammatory astrocytic responses, as well as PAR1-activated signal pathway of astrocytes were accordingly examined using various agonists or antagonists of the receptor. Thrombin was found to reverse astrocytic stellation, promote proliferation but inhibit migration of astrocytes. Furthermore, the serine protease was shown to facilitate inflammatory response of astrocytes through regulation of MAPKs/NFκB pathway. Our results have provided the morphological evidence of astrocytic reactivity in response to thrombin stimulation and its neuroinflammatory effects following SCI, which will be indicative for the fundamental insights of thrombin-induced neuropathology.

Atractylenolide III ameliorates spinal cord injury in rats by modulating microglial/macrophage polarization.

BackgroundInflammatory reactions induced by spinal cord injury (SCI) are essential for recovery after SCI. Atractylenolide III (ATL‐III) is a natural monomeric herbal bioactive compound that is mainly derived in Atractylodes macrocephala Koidz and has anti‐inflammatory and neuroprotective effects.ObjectiveHere, we speculated that ATL‐III may ameliorate SCI by modulating microglial/macrophage polarization. In the present research, we focused on investigating the role of ATL‐III on SCI in rats and explored the potential mechanism.MethodsThe protective and anti‐inflammatory effects of ATL‐III on neuronal cells were examined in a rat SCI model and lipopolysaccharide (LPS)‐stimulated BV2 microglial line. The spinal cord lesion area, myelin integrity, and surviving neurons were assessed by specific staining. Locomotor function was evaluated by the Basso, Beattie, and Bresnahan (BBB) scale, grid walk test, and footprint test. The activation and polarization of microglia/macrophages were assessed by immunohistofluorescence and flow cytometry. The expression of corresponding inflammatory factors from M1/M2 and the activation of relevant signaling pathways were assessed by Western blotting.ResultsATL‐III effectively improved histological and functional recovery in SCI rats. Furthermore, ATL‐III promoted the transformation of M1 into M2 and attenuated the activation of microglia/macrophages, further suppressing the expression of corresponding inflammatory mediators. This effect may be partly mediated by inhibition of neuroinflammation through the NF‐κB, JNK MAPK, p38 MAPK, and Akt pathways.ConclusionThis study reveals a novel effect of ATL‐III in the regulation of microglial/macrophage polarization and provides initial evidence that ATL‐III has potential therapeutic benefits in SCI rats.

Targeted delivery of acrolein scavenger hydralazine in spinal cord injury using folate-linker-drug conjugation

Oxidative stress has been shown to play a critical pathogenic role in functional loss after spinal cord injury (SCI). As a direct result of oxidative stress, lipid peroxidation-derived aldehydes have emerged as key culprits that sustain secondary injury and contribute significantly to pathological outcomes. Acrolein, a neurotoxin, has been shown to be elevated in SCI and can result in post-SCI neurological deficits. Reducing acrolein has therefore emerged as a novel and effective therapeutic strategy in SCI. Previous studies have revealed that hydralazine, an FDA approved blood pressure lowering medication, when administered after SCI shows strong acrolein scavenging capabilities and significantly improves cellular and behavioral outcomes. However, while effective at scavenging acrolein, hydralazine's blood pressure lowering activity can have a detrimental impact on neurotrauma patients. Here, our goal was to preserve the acrolein scavenging capability while mitigating the effect of hydralazine on blood pressure. We accomplished this using a folate-targeted delivery system to deploy hydralazine to the folate receptor positive inflammatory site of the cord injury. Using a model of rat SCI, we found that this system is effective for targeting the injury site, and that folate targeted hydralazine can scavenge acrolein without significantly impacting blood pressure.

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.

Homeodomain Interacting Protein Kinase 2-Modified Rat Spinal Astrocytes Affect Neurofunctional Recovery After Spinal Cord Injury.

Spinal cord injury (SCI) is regarded as an acute neurological disorder, and astrocytes play a role in the progression of SCI. Herein, we investigated the roles of homeodomain-interacting protein kinase 2 (HIPK2)- modified rat spinal astrocytes in neurofunctional recovery after SCI. Rat spinal astrocytes were cultured, isolated, and then identified through microscopic observation and immunofluorescence staining. Astrocytes were infected with the adenovirus vector overexpressing HIPK2 for modification, and proliferation and apoptosis of astrocytes were examined using Cell Counting Kit-8 method and flow cytometry. SCI rat models were established and treated with astrocytes or HIPK2-modified astrocytes. Subsequently, rat motor ability was analyzed via the Basso-Beattie-Bresnahan (BBB) scoring and inclined-plane test, and the damage to spinal cord tissues and neuronal survival were observed via Hematoxylin-eosin staining and Nissl staining. The levels of HIPK2, brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and nuclear factor erythroid 2- related transcription factor 2 (Nrf2)/antioxidant response element (ARE) pathway-related proteins were detected. Rat spinal astrocytes were harvested successfully. HIPK2 overexpression accelerated the proliferation and repressed the apoptosis of rat spinal astrocytes. Rat spinal astrocytes treatment increased BBB points and the maximum angle at which SCI rats remained stable, ameliorated damage to spinal cord tissues, increased the number of neurons, and attenuated neural damage and inflammation, while the treatment of HIPK2-modified rat spinal astrocytes imparted more pronounced effects to the neurofunctional recovery of SCI rats. Meanwhile, HIPK2-modified rat spinal astrocytes further activated the Nrf2/ARE pathway. HIPK2-modified rat spinal astrocytes facilitated neurofunctional recovery and activated the Nrf2/ARE pathway after SCI.

Effects of electroacupuncture combined with hydrogel on the formation and changes in the glial scar in rats with spinal cord injury

To observe the effect of electroacupuncture (EA) combined with oriented conductive bioprotein hydrogel (OCBH) on the recovery of nerve function in rats with complete spinal cord injury (SCI) and to explore its effect and mechanism on the formation and changes of glial scars. A total of 72 female Sprague–Dawley rats were randomly divided into groups according to the treatment received. A rat model of complete SCI was constructed using a spinal cord transection. Behavioral assessments, hematoxylin-eosin (H&E) staining, immunofluorescence staining, and Western blotting were performed at a fixed period after the operation. The material group and the material + EA group obtained better results in the behavioral assessments (all P < .05) and the H&E staining. In the immunofluorescence staining and Western blotting, the GFAP protein was expressed more and denser in the material group and the material + EA group than in the model group, and the density of the GFAP expression in the material + EA group was lower at week 12 than in the material group (all P < .05). The expression of complement C3 in the model, material, and material + EA groups decreased in turn. Some inflammatory factors and the NF-κB signaling pathway showed similar results in the Western blotting (all P < .05). The expression of the GDNF protein in the material + EA group was significantly higher than that in the model group and the material group (both P < .01). EA combined with OCBH can promote the recovery of motor functions after SCI by facilitating the formation of glial scars in the early stage, preventing the further spread of an inflammatory response that would affect the activation of A1/A2 astrocytes and change the morphology of glial scars at the spinal cord–material interface in its late stage.

PBMSCs transplantation facilitates functional recovery after spinal cord injury by regulating microglia/macrophages plasticity

BackgroundStem cell therapy has been proven as one of the promising strategies for treating spinal cord injury (SCI). However, the role of peripheral blood-derived mesenchymal stem cells (PBMSCs) in animal models of SCI has not been fully uncovered. This study aimed to investigate whether transplanted PBMSCs could inhibit neuroinflammation and then promote the functional recovery by shifting the microglia/macrophages phenotype from M1 to M2 at the site of injury after SCI. MethodsPBMSCs harvested from peripheral blood were analyzed by morphology and phenotype. Rat models of SCI were administrated with PBMSCs 1 week after injury. Inclined plane test and Basso-Beattie-Bresnahan (BBB) scores were used for assessing the functional recovery. Enzyme-linked immunosorbent assay (ELISA), reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry (FCM) were performed on days 3, 7, 14, 28 and 56 after PBMSCs transplantation. ResultsPBMSCs were plastic-adherent and fibroblast-like with positive expression of cluster of differentiation (CD)29, CD90 and CD44. ELISA and RT-qPCR both showed a lower expression of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α while a higher expression of anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-β1 after PBMSCs transplantation. This was associated with increased numbers of M2 microglia/macrophages and decreased numbers of M1 microglia/macrophages. These changes taken together were associated with the functional recovery in PBMSCs groups. ConclusionsAdministration of PBMSCs following SCI may provide an anti-inflammatory and reparative micro-environment for locomotive recovery by shifting microglia/macrophages phenotype from M1 towards M2.

Effect of Sirtuin-1 and Wnt/β-Catenin Signaling Pathway in Rat Model of Spinal Cord Injury.

Sirtuin-1 (SIRT1) has anti-inflammatory and antioxidant effects and has been reported to be involved in spinal cord injury (SCI). Wnt/β-catenin signal has been shown to play a critical role in the pathogenesis of chronic diseases, and it participated in the recovery of nerve function after SCI. However, the specific link between them in SCI is unclear. In addition, targeting posttraumatic astrocyte apoptosis is crucial for improving neural degeneration and locomotor function. Therefore, in this article, we studied the relationship of β-catenin and SIRT1 using in the SCI rat model and primary astrocyte treated with hydrogen peroxide (H2O2) or lithium chloride (LiCl). Results showed that after SCI, SCI area and motor function recover over time, and β-catenin is gradually increased to the seventh day and then in turn decreases until 4 weeks, positively correlated with cell apoptosis. The expression of SIRT1 and downstream FOXO4 gradually increased, and β-catenin is negatively correlated with SIRT1 expression. Moreover, treatment with H2O2 in primary cultured astrocyte significantly increased β-catenin and Caspase-3 expression, while decreased SIRT1 and Forkhead box O- (FOXO-) 4. The immunofluorescence results are consistent with this. Administration of LiCl further aggravates the above results. These findings suggest that SIRT1 is negatively correlated with β-catenin in SCI, which promotes the apoptosis of motor neuron cells, which may be related to the participation of FOXO4.

Exosomal miR-9-5p derived from BMSCs alleviates apoptosis, inflammation and endoplasmic reticulum stress in spinal cord injury by regulating the HDAC5/FGF2 axis

Exosomes derived from human bone marrow mesenchymal stem cells (BMSCs) play potential protective roles in spinal cord injury (SCI). However, the underlying mechanisms remain not fully elucidated. Herein, we isolated exosomes from BMSCs, and exosome morphology and marker protein levels were identified by transmission electron microscopy (TEM) and Western blot, respectively. PC12 cells were treated with lipopolysaccharide (LPS) to construct an injury model, and then incubated with BMSCs-derived exosomes. We found that exosome incubation increased miR-9-5p expression, and inhibited apoptosis and the levels of inflammation cytokines and ER stress marker proteins. Moreover, histone deacetylase 5 (HDAC5) was identified as a target gene of miR-9-5p by dual-luciferase reporter gene assay. Exosomal miR-9-5p upregulated fibroblast growth factor 2 (FGF2) expression by inhibiting HDAC5-mediated FGF2 deacetylation. Then, it was observed that HDAC5 overexpression or FGF2 inhibition reversed the inhibitory effects of exosomal miR-9-5p on apoptosis, inflammation and ER stress in PC12 cells. Additionally, an SCI rat model was established and exosomes were injected for treatment. Exosomal miR-9-5p treatment alleviated locomotor ability, histopathological damage, neuronal apoptosis, inflammation and ER stress in SCI rats. In conclusion, our findings indicated that exosomal miR-9-5p derived from BMSCs promoted FGF2 expression by inhibiting HDAC5-mediated deacetylation, thus inhibiting LPS-induced apoptosis, inflammation, and ER stress in PC12 cells, and alleviating SCI in rat model. Our study may provide a therapeutic direction for SCI.

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

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

Rhoifolin Provides Neuroprotection against Spinal Cord Injury by Attenuation of Oxidative Stress, Inflammation and Apoptosis in Rats

Background: Rhoifolin is a plant flavonoid known to have antioxidant and anti-inflammatory properties. This study was taken to identify the effect of rhoifolin on Spinal cord injury (SCI) in a rat model. Methods: Motor function of SCI rats was carried out by using in Basso, Beattie and Bresnahan test. Expression levels of different proteins such as IL-1β, IL-6, TNF-α, NF-κB p65, p-IκB-α, p38MAPK, caspase-3 and caspase-9 were carried out by using western blotting analysis. The Concentration of proteins was determined by using Bradford’s method. Estimating of Glutathione (GSH), Glutathione peroxidase (GPx) and Superoxide dismutase (SOD) was carried out by using commercially available kit. Result: Rhoifolin was tested at 20 and 40 mg/kg doses and showed a significant improvement in the motor function of SCI rats in Basso, Beattie and Bresnahan test. Moreover, rhoifolin treatment also attenuated pro-inflammatory cytokine levels such as IL-1β, IL-6 and TNF-α. The NF-κB pathway showed a significant attenuation as evident in the significant downregulation of the NF-κB p65 and p-IκB-α levels. In addition, rhoifolin treatment also caused a significant reduction in oxidative stress, as evident from changes in intracellular levels of Glutathione, GPx, MDA and SOD in the articular cartilage tissue. Further, we also tested rhoifolin for its effect on the apoptotic pathway by estimating the intracellular levels of p38MAPK, caspase-3 and caspase-9. The current results show that rhoifolin has a neuroprotective in SCI rat model.

Increasing Severity of Spinal Cord Injury Results in Microglia/Macrophages With Annular-Shaped Morphology and No Change in Expression of CD40 and Tumor Growth Factor-β During the Chronic Post-injury Stage.

Determination of the quantitative composition of phenotypically and morphologically different populations of resident microglia and infiltrating macrophages in spinal cord injury (SCI) of various degrees of severity could lead to much needed novel therapeutic interventions in neurotrauma. In this regard, we investigated the CD40 and TGF-β expressing populations of microglia/macrophages and their morphological states in a rat model of SCI of varying severity. We are the first to describe the annular-shaped microglia/macrophages, the morphology of which was formed due to the spatial orientation of the processes that form round or oval micro-territories, which include disintegrating myelin fibers. This type of cell morphology was found only in the injured spinal cord and mainly in the white matter. At the same time, an assessment of the number of annular-shaped microglia/macrophages and the diameter of micro-territories formed by their processes showed an elevation in these indicators as the severity of SCI increased. While we did not find significant quantitative changes in the populations of Iba1+/CD40+ and Iba1+/TGF-β+ microglia/macrophages with increased severity of SCI in the chronic period (60 dpi), we did determine changes in the expression of cytokines and mRNAs of genes-encoding microglial marker proteins, finding the greatest changes on days 7 and 14 after SCI between experimental groups with varying severity.