Acute Spinal Cord Injury In Rats Research Articles

Sonic Hedgehog reduces inflammatory response, decreases blood-spinal cord barrier permeability, and improves locomotor function recovery in an acute spinal cord injury rat model

BackgroundSonic Hedgehog (Shh), extensively researched for its role in early neurogenesis and brain development, has recently been recognized for its neuroprotective potential following neuronal injuries. This study examines the immediate impact of early administered Shh on the local inflammatory response post-acute spinal cord injury in rats.MethodsThirty-four female Wistar rats underwent either sham surgery (laminectomy; n = 10) or clip compression/contusion spinal cord injury (SCI) at the T9 level. This was followed by implantation of an osmotic pump and a subdural catheter for continuous intrathecal delivery of Shh (n = 12) or placebo (NaCl; n = 12). Locomotor function was assessed at 3- and 7-days post-injury (dpi) using the Basso, Beattie, and Bresnahan (BBB) score and the Gridwalk test. Animals were euthanized after 3 or 7 days for immunohistochemical analysis of the local inflammatory reaction and immune cell migration.ResultsShh-treated rats demonstrated significant hindlimb movement and coordination improvements at 7 days post-injury, compared to controls. This enhancement was accompanied by a significant reduction in both immune cell presence and blood plasma products within spinal cord lesions, suggesting Shh’s dual role in modulating immune cell migration and maintaining the integrity of the blood-spinal cord barrier. Separately, these Shh-treated rats also showed an increase in M(IL-4) polarization of macrophages, further underlining the potential therapeutic impact of Shh in post-injury recovery. Notably, these effects were not evident at three days post-injury.ConclusionShh application at 7 days post-injury showed immunomodulatory effects, possibly via enhanced blood-spinal cord barrier integrity, reduced immune cell migration, and increased anti-inflammatory immune cell differentiation. These mechanisms collectively contribute to enhanced locomotor recovery.

Targeted-delivery of nanomedicine-enabled methylprednisolone to injured spinal cord promotes neuroprotection and functional recovery after acute spinal cord injury in rats

To date, no therapy has been proven to be efficacious in fully restoring neurological functions after spinal cord injury (SCI). Systemic high-dose methylprednisolone (MP) improves neurological recovery after acute SCI in both animal and human. MP therapy remains controversial due to its modest effect on functional recovery and significant adverse effects. To overcome the limitation of MP therapy, we have developed a N-(2-hydroxypropyl) methacrylamide copolymer-based MP prodrug nanomedicine (Nano-MP) that can selectively deliver MP to the SCI lesion when administered systemically in a rat model of acute SCI. Our in vivo data reveal that Nano-MP is significantly more effective than free MP in attenuating secondary injuries and neuronal apoptosis. Nano-MP is superior to free MP in improving functional recovery after acute SCI in rats. These data support Nano-MP as a promising neurotherapeutic candidate, which may provide potent neuroprotection and accelerate functional recovery with improved safety for patients with acute SCI.

Sovateltide (ILR-1620) Improves Motor Function and Reduces Hyperalgesia in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) presents a major global health challenge, with rising incidence rates and substantial disability. Although progress has been made in understanding SCI's pathophysiology and early management, there is still a lack of effective treatments to mitigate long-term consequences. This study investigates the potential of sovateltide, a selective endothelin B receptor agonist, in improving clinical outcomes in an acute SCI rat model. Thirty male Sprague-Dawley rats underwent sham surgery (group A) or SCI and treated with vehicle (group B) or sovateltide (group C). Clinical tests, including Basso, Beattie, and Bresnahan scoring, inclined plane, and allodynia testing with von Frey hair, were performed at various time points. Statistical analyses assessed treatment effects. Sovateltide administration significantly improved motor function, reducing neurological deficits and enhancing locomotor recovery compared with vehicle-treated rats, starting from day 7 post injury. Additionally, the allodynic threshold improved, suggesting antinociceptive properties. Notably, the sovateltide group demonstrated sustained recovery, and even reached preinjury performance levels, whereas the vehicle group plateaued. This study suggests that sovateltide may offer neuroprotective effects, enhancing neurogenesis and angiogenesis. Furthermore, it may possess anti-inflammatory and antinociceptive properties. Future clinical trials are needed to validate these findings, but sovateltide shows promise as a potential therapeutic strategy to improve functional outcomes in SCI. Sovateltide, an endothelin B receptor agonist, exhibits neuroprotective properties, enhancing motor recovery and ameliorating hyperalgesia in a rat SCI model. These findings could pave the way for innovative pharmacological interventions for SCI in clinical settings.

Mechanism of Shikonin on spinal cord injury in rats based on TNFR/RIPK1 pathway

To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI) in rats. 96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group (Group A), sham operation+shikonin group (Group B), SCI+ DMSO(Group C), SCI+shikonin group (Group D).The acute SCI model of rats was made by clamp method in groups C and D . After subdural catheterization, no drug was given in group A. rats in groups B and D were injected with 100 mg·kg-1 of shikonin through catheter 30 min after modeling, and rats in group C were given with the same amount of DMSO, once a day until the time point of collection tissue. Basso-Beattie-Bresnahan(BBB) scores were performed on 8 rats in each group at 6, 12, and 3 d after moneling, and oblique plate tests were performed on 1, 3, 7 and 14 d after modeling, and then spinal cord tissues were collected. Eight rats were intraperitoneally injected with propidine iodide(PI) 1 h before sacrificed to detection PI positive cells at 24 h in each group. Eight rats were sacrificed in each group at 24 h after modeling, the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells. Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1. After modeling, BBB scores were normal in group A and B, but in group C and D were significantly higher than those in group A and B. And the scores in group D were higher than those in group C in each time point (P<0.05). At 12 h after modeling, the PI red stained cells in group D were significantly reduced compared with that in group C, and the disintegration of neurons was alleviated(P<0.05). HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation. The degree of SCI and the number of neuronal survival in group D were better than those in group C, the difference was statistically significant at 24h (P<0.05). The expression of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D, with a statistically significant difference (P<0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C (P<0.05). Shikonin can alleviate the pathological changes after acute SCI in rats, improve the behavioral score, and promote the recovery of spinal nerve function. The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.

Protective effect of bone morphogenetic protein-7 induced differentiation of bone marrow mesenchymal stem cells in rat with acute spinal cord injury.

The principal aim of present study was to assess the therapeutic efficacy of bone morphogenetic protein-7 (BMP-7) induced differentiation of bone marrow mesenchymal stem cells (BMSCs) in a rat acute spinal cord injury (SCI) model. BMSCs were isolated from rats, and then divided into a control and a BMP-7 induction groups. The proliferation ability of BMSCs and glial cell markers were determined. Forty Sprague-Dawley (SD) rats were randomly divided into sham, SCI, BMSC, and BMP7 + BMSC groups (n = 10). Among these rats, the recovery of hind limb motor function, the pathological related markers, and motor evoked potentials (MEP) were identified. BMSCs differentiated into neuron-like cells after the introduction of exogenous BMP-7. Interestingly, the expression levels of MAP-2 and Nestin increased, whereas the expression level of GFAP decreased after the treatment with exogenous BMP-7. Furthermore, the Basso, Beattie, and Bresnahan (BBB) score reached 19.33 ± 0.58 in the BMP-7 + BMSC group at day 42. Nissl bodies in the model group were reduced compared to the sham group. After 42days, in both the BMSC and BMP-7 + BMSC groups, the number of Nissl bodies increased. This is especially so for the number of Nissl bodies in the BMP-7 + BMSC group, which was more than that in the BMSC group. The expression of Tuj-1 and MBP in BMP-7 + BMSC group increased, whereas the expression of GFAP decreased. Moreover, the MEP waveform decreased significantly after surgery. Furthermore, the waveform was wider and the amplitude was higher in BMP-7 + BMSC group than that in BMSC group. BMP-7 promotes BMSC proliferation, induces the differentiation of BMSCsinto neuron-like cells, and inhibits the formation of glial scar. BMP-7 plays a confident role in the recovery of SCI rats.

Adipose-derived mesenchymal stem cells overexpressing prion improve outcomes via the NLRP3 inflammasome/DAMP signalling after spinal cord injury in rat.

Traumatic spinal cord injury (SCI) is a highly destructive disease in human neurological functions. Adipose-derived mesenchymal stem cells (ADMSCs) have tissue regenerations and anti-inflammations, especially with prion protein overexpression (PrPcOE ). Therefore, this study tested whether PrPcOE -ADMSCs therapy offered benefits in improving outcomes via regulating nod-like-receptor-protein-3 (NLRP3) inflammasome/DAMP signalling after acute SCI in rats. Compared with ADMSCs only, the capabilities of PrPcOE -ADMSCs were significantly enhanced in cellular viability, anti-oxidative stress and migration against H2 O2 and lipopolysaccharide damages. Similarly, PrPcOE -ADMSCs significantly inhibited the inflammatory patterns of Raw264.7 cells. The SD rats (n=32) were categorized into group 1 (Sham-operated-control), group 2 (SCI), group 3 (SCI + ADMSCs) and group 4 (SCI + PrPcOE -ADMSCs). Compared with SCI group 2, both ADMSCs and PrPcOE -ADMSCs significantly improved neurological functions. Additionally, the circulatory inflammatory cytokines levels (TNF-α/IL-6) and inflammatory cells (CD11b/c+/MPO+/Ly6G+) were highest in group 2, lowest in group 1, and significantly higher in group 3 than in group 4. By Day 3 after SCI induction, the protein expressions of inflammasome signalling (HGMB1/TLR4/MyD88/TRIF/c-caspase8/FADD/p-NF-κB/NEK7/NRLP3/ASC/c-caspase1/IL-ß) and by Day 42 the protein expressions of DAMP-inflammatory signalling (HGMB1/TLR-4/MyD88/TRIF/TRAF6/p-NF-κB/TNF-α/IL-1ß) in spinal cord tissues displayed an identical pattern as the inflammatory patterns. In conclusion, PrPcOE -ADMSCs significantly attenuated SCI in rodents that could be through suppressing the inflammatory signalling.

Triad1 Promotes the Inflammatory Response and Neuronal Apoptosis to Aggravate Acute Spinal Cord Injury in Rats.

Objective Spinal cord injury (SCI) is one of the most devastating central injuries, resulting in serious locomotor deficits. Triad1 is known to play an important role in SCI, but its effects on the inflammatory response and physiological behavior have not been thoroughly studied. This study is aimed at examining the effects of Triad1 on the inflammatory response and neuronal injury in acute SCI in rats. Methods Twenty-four male Sprague–Dawley (SD) rats were randomly divided into a control group, SCI group, sh-NC group, and Triad1 knockout group (sh-Triad1). The Basso Beattie Bresnahan locomotor rating scale was utilized for the assessment of the motor ability of rats. Hematoxylin and eosin (H&E), Luxol fast blue (LFB), and TUNEL staining were used to assess the pathological injury, demyelination, and neuronal apoptosis, respectively. ELISA was used to detect the levels of IL-1β, IL-10, and TNF-α, and qRT-PCR was used to examine the expression level of Triad1. Furthermore, the protein levels of Triad1, Bax, Bcl-2, and cleaved caspase-3 were determined using western blotting. Results The Triad1 expression level was upregulated in damaged spinal cord tissue. Knockdown of Triad1 improved motor function and reduced SCI as well as apoptosis of spinal cord neurons. In addition, the knockdown of Triad1 inhibited the inflammatory response caused by SCI. Conclusion Knockdown of Triad1 can reduce SCI in rats with acute SCI by inhibiting the inflammatory response and apoptosis.

Molecular Expression Profile of Changes in Rat Acute Spinal Cord Injury.

Background: Spinal cord injury (SCI) is a highly lethal and debilitating disease with a variety of etiologies. To date, there is no effective therapeutic modality for a complete cure. The pathological mechanisms of spinal cord injury at the molecular gene and protein expression levels remain unclear.Methods: This study used single-cell transcriptomic analysis and protein microarray analysis to analyzes changes in the gene expression profiles of cells and secretion of inflammatory factors respectively, around the lesion site in a rat SCI model.Results: Single-cell transcriptomic analysis found that three types of glial cells (microglia, astrocyte, and oligodendrocyte) becomes activated after acute injury, with GO exhibiting a variety of inflammatory-related terms after injury, such as metabolic processes, immune regulation, and antigen presentation. Protein microarray results showed that the levels of four inflammatory cytokines favoring SCI repair decreased while the levels of nine inflammatory cytokines hindering SCI repair increased after injury.Conclusion: These findings thus reveal the changes in cellular state from homeostatic to reactive cell type after SCI, which contribute to understand the pathology process of SCI, and the potential relationship between glial cells and inflammatory factors after SCI, and provides new theoretical foundation for further elucidating the molecular mechanisms of secondary SCI.

EZH2 Mediates miR-146a-5p/HIF-1α to Alleviate Inflammation and Glycolysis after Acute Spinal Cord Injury.

Acute spinal cord injury (ASCI) is a severe traumatic disease of the central nervous system, the underlying mechanism of which is unclear. This study was intended to study the role of EZH2 and miR-146a-5p/HIF-1α in inflammation and glycolysis after ASCI, providing reference and basis for the clinical treatment and prognosis of ASCI injury. We used lipopolysaccharide (LPS) to induce inflammation of microglia, and we constructed the ASCI animal model. qRT-PCR detected the relative expression levels of EZH2, HIF-1α, miR-146a-5p, IL-6, TNF-α, IL-17, PKM2, GLUT1, and HK2 in cells and tissues. Western blot was performed to detect the expression levels of EZH2, HIF-1α, H3K27me3, IL-6, TNF-α, IL-17, PKM2, GLUT1, and HK2. ChIP verified the enrichment of H3K27me3 in the miR-146a-5p promoter region. Bioinformatics predicted the binding sites of HIF-1α and miR-146a-5p, and dual-luciferase reporter assay verified the binding of HIF-1α and miR-146a-5p. ELISA detects the levels of inflammatory factors IL-6, TNF-α, and IL-17 in the cerebrospinal fluid of rats. The GC-TOFMS was used to detect the changes of glycolytic metabolites in the cerebrospinal fluid of rats. EZH2 could mediate inflammation and glycolysis of microglia. EZH2 regulates inflammation and glycolysis through HIF-1α. EZH2 indirectly regulated the HIF-1α expression by mediating miR-146a-5p. EZH2 mediates miR-146a-5p/HIF-1α to alleviate inflammation and glycolysis in ASCI rats. In the present study, our results demonstrated that EZH2 could mediate miR-146a-5p/HIF-1α to alleviate the inflammation and glycolysis after ASCI. Therefore, EZH2/miR-146a-5p/HIF-1α might be a novel potential target for treating ASCI.

PU.1 interaction with p50 promotes microglial-mediated inflammation in secondary spinal cord injury in SCI rats

Purpose/aim of the study Secondary spinal cord injury is the inflammatory damage to surrounding tissues caused by activated microglial-mediated neuroinflammatory responses. The nuclear factor-κB (p65/p50) pathway and PU.1 are closely correlated with inflammatory responses; thus, we examined the relationship and function between PU.1 and p50 in secondary spinal cord injury.Materials and methods In this study, we established an adult rat acute spinal cord injury model to simulate the pathological process of spinal cord injury.Results: We found that the expression of PU.1 was significantly increased at three days after spinal cord injury and mainly expressed in activated microglia. Moreover, p-p50 expression was increased in SCI rats and the protein interacted with PU.1. Lipopolysaccharide was used to induce microglia activation in vitro.Conclusions: The results showed that PU.1 and p-p50 expression was significantly increased and PU.1 interacted with p50 in the nucleus. The levels of tumor necrosis factor-α and interleukin-1β secreted by microglia were detected by enzyme-linked immunosorbent assay. The results showed that when both PU.1 and p50 were overexpressed, tumor necrosis factor-α and interleukin-1β secretion was significantly increased to levels higher than in cells overexpressing PU.1 or p50 alone. These results suggest that PU.1 and p50 interact to promote p65 transcription and the expression of inflammatory factors, which is an important mechanism of the microglial-mediated inflammatory response to secondary injury after spinal cord injury.

Identification of four genes and biological characteristics associated with acute spinal cord injury in rats integrated bioinformatics analysis.

Spinal cord injury (SCI) is a serious condition that can cause physical disability and sensory dysfunction. Cytokines play an extremely important role in the acute phase of SCI. Clarifying the cytokine expression profile is of great importance. Cytokine array analysis was used to explore the changes in 67 different proteins at 0 hours, 2 hours, 1 day, 3 days, and 7 days after acute SCI in rats. The differentially expressed cytokines in the various periods were analyzed and compared. The biological processes related to the differentially expressed proteins were examined using Gene Ontology (GO) analysis. Immediately after SCI (0 hours), only ciliary neurotrophic factor (CNTF) was slightly up-regulated, while 23 other proteins were down-regulated. At 2 hours after SCI, there were 3 upregulated and 21 downregulated proteins. At 1 day after SCI, there were 5 upregulated and 6 downregulated proteins. At 3 days after SCI, there were 6 upregulated and 4 downregulated proteins. At 7 days after SCI, there were 4 upregulated and 9 downregulated proteins. Erythropoietin (EPO) and Fms related tyrosine kinase 3 ligand (Flt-3L) were downregulated at all time points. CD48 was decreased at 2 hours to 7 days after SCI. Monocyte chemotactic protein-1 (MCP-1) was the only protein that was upregulated at 2 hours to 7 days. The GO and pathway analyses revealed that the cytokine-related pathways, cell death, and proliferation might play a key role during secondary SCI. This study identified 3 downregulated proteins during SCI, that being EPO, Flt-3L, and CD48. MCP-1 was the only upregulated protein, and its expression was upregulated till day 7 following SCI. These 4 identified genes may be potential therapeutic targets for the treatment of SCI.

LncRNA MIAT activates vascular endothelial growth factor A through RAD21 to promote nerve injury repair in acute spinal cord injury

BackgroundThe main pathological feature of acute spinal cord injury (ASCI) is neuronal apoptosis and Long non-coding RNA (lncRNA) myocardial infarction-related transcript (MIAT) is involved in the regulation of neuronal apoptosis. This study aimed to investigate the role and potential mechanism of LncRNA MIAT in neuronal apoptosis induced by ASCI. MethodsAfter Lenti-MIAT lentivirus was microinjected into ASCI rats, Basso, Beattie and Bresnahan Score, Hematoxylin-eosin staining, TUNEL staining, immunohistochemical, immunofluorescence, quantitative real-time PCR and Western blot were used to observe the effect of LncRNA MIAT on the nerve function of ASCI rats. MTT and flow cytometry assays were used to identify the in vitro function of LncRNA MIAT. RNA immunoprecipitation, RNA pull-down, Cycloheximide chase and Chromatin immunoprecipitation combined with qPCR experiments were used to study the mechanism. ResultsThe overexpression of LncRNA MIAT was conducive to the recovery of motor function in ASCI rats and repressed neuronal cell apoptosis and increased neuronal cell viability. Furthermore, the overexpression of LncRNA MIAT in PC12 cells upregulated RAD21 expression by repressing RAD21 protein degradation and further promoted VEGFA transcription to inhibit neuronal cell apoptosis, ultimately improved ASCI. ConclusionOur data indicated that the overexpression of LncRNA MIAT activated VEGFA through RAD21 to inhibit neuronal cell apoptosis in ASCI.

Long non-coding RNA TUG1 knockdown prevents neurons from death to alleviate acute spinal cord injury via the microRNA-338/BIK axis

ABSTRACT Taurine up-regulated gene 1 (TUG1) is a cancer-associated long noncoding RNA (lncRNA) and engages in the development of spinal cord injury (SCI), a suffering neuropathological disorder. However, the regulatory role of TUG1 in acute SCI (ASCI) is still underdetermined. RT-qPCR and western blot analysis were applied to measure the expression of TUG1, microRNA-338 (miR-338), Bcl2-interacting killer (BIK), cleaved caspase 3 (c-caspase 3) and hypoxia-inducible factor-1 alpha (HIF-1α) in ASCI rats and hypoxic cells. Cell death was evaluated using flow cytometric analysis. The relationships among miR-338, TUG1 or BIK were confirmed by luciferase reporter assay, RNA immunoprecipitation and RNA pull-down. Accordingly, we monitored higher expression of TUG1 and BIK, but lower expression of miR-338 in ASCI rats and hypoxic cells. In vitro, hypoxia expedited cell death and c-caspase 3 levels. In vivo, ASCI rats were successfully developed as evidenced by diminished Basso-Beattie-Bresnahan (BBB) locomotor score and enhanced c-caspase 3 and HIF-1α expression. Nevertheless, TUG1 knockdown mitigated the cell death in ASCI rats and hypoxic cells. Mechanically, TUG1 interacted with miR-338 to regulate the BIK expression. Together, TUG1 silencing could alleviate the death in neurons and ASCI models via modulating the miR-338/BIK axis.

Autophagy Induction Contributes to the Neuroprotective Impact of Intermittent Fasting on the Acutely Injured Spinal Cord.

Spinal cord injury (SCI) is one of the leading causes of neurological disability and death. So far, there is no satisfactory treatment for SCI, because of its complex and ill-defined pathophysiology. Recently, autophagy has been implicated as protective in acute SCI rat models. Here, we investigated the therapeutic value of a dietary intervention, namely, intermittent fasting (IF), on neuronal survival after acute SCI in rats, and its underlying mechanism related to autophagy regulation. We found remarkable improvement in both behavioral performance and neuronal survival at the injured segment of the spinal cord of animals previously subjected to IF. Western blotting revealed a marked decrease in apoptosis-related markers such as cleaved caspase 3 levels and the bax/bcl-2 ratio in the IF group, which suggested an inhibition of the intrinsic apoptosis pathway. In addition, the expression of the autophagy markers LC3-II and beclin 1 was also increased in the IF group compared with ad libitum fed animals. In parallel, IF decreased the levels of the substrate protein of autophagy, p62, indicative of an upregulation of the autophagic processes. Treatment with 3-methyladenine (3-MA), a selective inhibitor of autophagy, reversed the downregulated apoptosis-related markers by IF. Finally, IF could activate the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway and enhance lysosome function by upregulating transcription factor (TF)EB expression. Altogether, the present findings suggest that IF exerts a neuroprotective effect after acute SCI via the upregulation of autophagy, and further points to dietary interventions as a promising combinatorial treatment for SCI.

Microenvironment-responsive immunoregulatory electrospun fibers for promoting nerve function recovery

The strategies concerning modification of the complex immune pathological inflammatory environment during acute spinal cord injury remain oversimplified and superficial. Inspired by the acidic microenvironment at acute injury sites, a functional pH-responsive immunoregulation-assisted neural regeneration strategy was constructed. With the capability of directly responding to the acidic microenvironment at focal areas followed by triggered release of the IL-4 plasmid-loaded liposomes within a few hours to suppress the release of inflammatory cytokines and promote neural differentiation of mesenchymal stem cells in vitro, the microenvironment-responsive immunoregulatory electrospun fibers were implanted into acute spinal cord injury rats. Together with sustained release of nerve growth factor (NGF) achieved by microsol core-shell structure, the immunological fiber scaffolds were revealed to bring significantly shifted immune cells subtype to down-regulate the acute inflammation response, reduce scar tissue formation, promote angiogenesis as well as neural differentiation at the injury site, and enhance functional recovery in vivo. Overall, this strategy provided a delivery system through microenvironment-responsive immunological regulation effect so as to break through the current dilemma from the contradiction between immune response and nerve regeneration, providing an alternative for the treatment of acute spinal cord injury.

Effects of sacral nerve electrical stimulation on 5‑HT and 5‑HT3AR/5‑HT4R levels in the colon and sacral cord of acute spinal cord injury rat models.

Spinal cord injury (SCI) often leads to defecation dysfunction. Sacral nerve electrical stimulation (SNS) therapy could improve defecation function. The present study aimed to assess SNS therapy, with regard to the levels of serotonin (5-HT) and its receptors (5-HT3AR and 5-HT4R) in the colon and sacral cord, a rat model of acute severe SCI was used. This rat model was made using the New York University Impactor device. Model rats were randomized to the SCI and SNS (electrical stimulation on the S3 nerve) groups. After 14 days of treatment, enteric transmission function was assessed. 5-HT and 5-HT3AR/5-HT4R were measured by ELISA, quantitative PCR, immunohistochemistry and western blotting. In SCI rats, SNS significantly increased the quantity of feces, shortened the time to the first fecal passage, and improved fecal texture and colon histology. SNS elevated 5-HT contents in the colon and spinal cord, and enhanced 5-HT3AR/5-HT4R protein expression and distribution in the colonic myenteric plexus and mucosa, sacral intermediolateral nucleus and dorsal horn. SNS upregulated the relative expression levels of 5-HT3AR/5-HT4R mRNA and protein in the colon and spinal cord. SNS can improve defecation and accelerate the recovery of colonic transmission functions in rat models of acute SCI. These effects involved upregulation of the 5-HT/5-HT3AR/5-HT4R axes.

A novel CX3CR1 inhibitor AZD8797 facilitates early recovery of rat acute spinal cord injury by inhibiting inflammation and apoptosis.

The present study aimed to evaluate the effect of the CX3CR1 inhibitor AZD8797 in early recovery after acute SCI and elucidate its potential mechanism in blocking inflammation and apoptosis. Adult rats were sacrificed after 3, 7, 10, or 14 days of SCI. The injured spinal tissues were collected for assessing C-X3-C motif chemokine ligand 1(CX3CL1)/C-X3-C motif chemokine receptor 1 (CX3CR1) expression at each time point via western blotting (WB) and quantitative PCR. The cellular localization of the proteins was detected by immunofluorescence. Another batch of rats (subdivided into sham, injury model, AZD8797 and methylprednisolone groups) were used to evaluate locomotive recovery with a Basso Beattie Bresnahan score. Based on the expression level of CX3CR1, these rats were sacrificed at the most prominent stage of CX3CR1 expression (10 days after SCI), for assessing the serum levels of tumor necrosis factor-α/interleukin (IL)-6/IL-1β and the expression of CX3CL1/CX3CR1/caspase 3/Bcl-2/Bax in the spinal cord tissues through WB and ELISA. Additionally, apoptosis and necrosis in the injured spinal cord were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining/fluoro-jade B staining. Expression levels of both CX3CR1 and CX3CL1 reached their peak 10 days after the injury, followed by a dramatic downward trend at 14 days. The enhanced expression of CX3CR1 was detected in astrocytes and microglia of the injured spinal cord. AZD8797 improved locomotive recovery after 10 days of SCI and was as effective as methylprednisolone. The effect of AZD8797 was mediated by suppressing apoptosis, necrosis and inflammatory responses, as assessed by WB/ELISA and morphological examinations. The current study has demonstrated that AZD8797 can effectively block overwhelming inflammation, apoptosis and necrosis after SCI and facilitate early recovery of locomotive function.

Acute upregulation of bone morphogenetic protein-4 regulates endogenous cell response and promotes cell death in spinal cord injury

Traumatic spinal cord injury (SCI) elicits a cascade of secondary injury mechanisms that induce profound changes in glia and neurons resulting in their activation, injury or cell death. The resultant imbalanced microenvironment of acute SCI also negatively impacts regenerative processes in the injured spinal cord. Thus, it is imperative to uncover endogenous mechanisms that drive these acute injury events. Here, we demonstrate that the active form of bone morphogenetic protein-4 (BMP4) is robustly and transiently upregulated in acute SCI in rats. BMP4 is a key morphogen in neurodevelopment; however, its role in SCI is not fully defined. Thus, we elucidated the ramification of BMP4 upregulation in a preclinical model of compressive/contusive SCI in the rat by employing noggin, an endogenous antagonist of BMP ligands, and LDN193189, an intracellular inhibitor of BMP signaling. In parallel, we studied cell-specific effects of BMP4 on neural precursor cells (NPCs), oligodendrocyte precursor cells (OPCs), neurons and astrocytes in vitro. We demonstrate that activation of BMP4 inhibits differentiation of spinal cord NPCs and OPCs into mature myelin-expressing oligodendrocytes, and acute blockade of BMPs promotes oligodendrogenesis, oligodendrocyte preservation and remyelination after SCI. Importantly, we report for the first time that BMP4 directly induces caspase-3 mediated apoptosis in neurons and oligodendrocytes in vitro, and noggin and LDN193189 remarkably attenuate caspase-3 activation and lipid peroxidation in acute SCI. BMP4 also enhances the production of inhibitory chondroitin sulfate proteoglycans (CSPGs) in activated astrocytes in vitro and after SCI. Interestingly, our work reveals that despite the beneficial effects of BMP inhibition in acute SCI, neither noggin nor LDN193189 treatment resulted in long-term functional recovery. Collectively, our findings suggest a role for BMP4 in regulating acute secondary injury mechanisms following SCI, and a potential target for combinatorial approaches to improve endogenous cell response and remyelination.

Protective Effects of MiR-129-5p on Acute Spinal Cord Injury Rats.

BackgroundSpinal cord injury (SCI) is a severe devastating condition associated with serious disability and neurologic deficits. Aberrant micro RNA (miRNA) expression has been related to a variety of central nervous system diseases including SCI. In the present study, we aimed to discover the role of miR-129-5p on SCI.Material/MethodsAn acute SCI rat model was induced, following the modified Allen method. A total of 36 rats were randomly assigned into 4 groups (n=9 in every group): Sham group; Model group (SCI+saline); SCI+NC group; and SCI+miR-129-5p group (100 nm solution, every 2 days). Basso-Beattie-Bresnahan (BBB) locomotor rating score was carried out to determine functional recovery. TUNEL (terminal dUTP nick-end labeling) staining was used to evaluate cell apoptosis. Hematoxylin and eosin staining was performed to assess the pathological state of spinal cord. Furthermore, western blot assay was conducted to measure the calpain1 and calpain2 expression.ResultsOur data suggested that the expression level of miR-129-5p was markedly reduced in rats after SCI. Then miR-129-5p mimic was injected into the vertebral canal. We found that the SCI+miR-129-5p group had a high score in the BBB test compared with the SCI+NC group and the Model group. The overexpression of miR-129-5p obviously reduced tissue loss, damaged cells, and the number of TUNEL positive cells. Moreover, western blot assay exhibited that overexpression of miR-129-5p decreased calpain1, calpain2, and cleaved caspase-3 expression.ConclusionsOur findings suggested that overexpression of miR-129-5p improved neurological function by promoting functional recovery, reducing tissue loss and cell apoptosis in rats in an SCI model, possibly through downregulation of calpain1 and calpain2.

Identification of serum exosomal microRNAs in acute spinal cord injured rats

It is important to find specific and easily detectable diagnostic markers in acute stage of spinal cord injury for guiding treatment and estimating prognosis. Although, microRNAs are attractive biomarkers, there is still no uniform standard for clinical evaluation of spinal cord injury based on “free circulation” miRNA spectrum. The reason may be that miRNA analysis from biological fluids is influenced by many pre-analysis variables. Exosome miRNAs are widely distributed in body fluids and have many advantages comparing with free miRNAs. The specific miRNAs in the central nervous system can be transported to the peripheral circulation and concentrated in exosomes. Therefore, we hypothesized that there might be some physiological changes associated with spinal cord injury in serum exosomal miRNAs. Using next-generation sequencing, miRNA profiles in serum exosomes of sham and acute spinal cord injury rats were analyzed, and integrative bioinformatics were used to analyze the function and regulation of putative target genes. The results showed that acute spinal cord injury can lead to changes in miRNA expression in the circulating exosomes. The changed miRNAs and their associated pathways may explain the pathology of acute spinal cord injury. More importantly, we determined serum exosomal miR-125b-5p, miR-152-3p, and miR-130a-3p are specific and easily detectable diagnostic markers in acute spinal cord injury. More interestingly, we also found some valuable known and novel miRNAs. Further bioinformatics analysis and functional research will be of great help to make clear their role in the pathological process of spinal cord injury and judging whether they can be used as diagnostic markers. Impact statement This research hypothesized that there might be some physiological changes associated with SCI in serum exosomal miRNAs. Using next-generation sequencing, miRNA profiles in serum exosomes of sham and acute SCI rats were analyzed, and integrative bioinformatics were used to analyze the function and regulation of putative target genes. The results showed that acute SCI can lead to changes in miRNA expression in the circulating exosomes. The changed miRNAs and their associated pathways may explain the pathology of acute SCI. More importantly, we determined serum exosomal miR-125b-5p, miR-152-3p, and miR-130a-3p are specific and easily detectable diagnostic markers in acute SCI.