Molecular Expression Profile of Changes in Rat Acute Spinal Cord Injury
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ABSTRACT: 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.
Project description:To clarify the molecular changes of sublesional muscle in the acute phase of spinal cord injury (SCI), a moderately severe injury (40 g cm) was induced in the spinal cord (T10 vertebral level) of adult male Sprague-Dawley rats (injury) and compared with sham (laminectomy only). Rats were sacrificed at 48 h (acute) post injury, and gastrocnemius muscles were excised. Morphological examination revealed no significant changes in the muscle fiber diameter between the sham and injury rats. Western blot analyses performed on the visibly red, central portion of the gastrocnemius muscle showed significantly higher expression of muscle specific E3 ubiquitin ligases (muscle ring finger-1 and muscle atrophy f-box) and significantly lower expression of phosphorylated Akt-1/2/3 in the injury group compared to the sham group. Cyclooxygenase 2, tumor necrosis factor alpha (TNF-α), and caspase-1, also had a significantly higher expression in the injury group; although, the mRNA levels of TNF-α and IL-6 did not show any significant difference between the sham and injury groups. These results suggest activation of protein degradation, deactivation of protein synthesis, and development of inflammatory reaction occurring in the sublesional muscles in the acute phase of SCI before overt muscle atrophy is seen.
Project description:Recent studies showed that RNA N6-methyladenosine (m6A) plays an important role in neurological diseases. We used methylated RNA immunoprecipitation sequencing (MeRIP-Seq) technology to generate the m6A modification map after traumatic spinal cord injury (TSCI). A total of 2,609 differential m6A peaks were identified after TSCI. Our RNA sequencing results after TSCI showed 4,206 genes with significantly altered expression. Cross-link analysis of m6A sequencing results and RNA sequencing results showed that 141 hyper-methylated genes were upregulated, 53 hyper-methylated genes were downregulated, 57 hypo-methylated genes were upregulated, and 197 hypo-methylated genes were downregulated. Among these, the important inflammatory response factor Tlr4 and the important member of the neurotrophin family Ngf were both upregulated and hyper-methylated after TSCI. This study provides that in the future, the epigenetic modifications of the genes could be used as an indicator of TSCI.
Project description:Neuropathic pain represents a primary detrimental outcome of spinal cord injury. A major challenge facing effective management is a lack of surrogate measures to examine the physiology and anatomy of neuropathic pain. To this end, we investigated the relationship between psychophysical responses to tonic heat stimulation and neuropathic pain rating after traumatic spinal cord injury. Subjects provided a continuous rating to 2 min of tonic heat at admission to rehabilitation and again at discharge. Adaptation, temporal summation of pain, and modulation profile (i.e., the relationship between adaptation and temporal summation of pain) were extracted from tonic heat curves for each subject. There was no association between any of the tonic heat outcomes and neuropathic pain severity at admission. The degree of adaptation, the degree of temporal summation of pain, and the modulation profile did not change significantly from admission to discharge. However, changes in modulation profiles between admission and discharge were significantly correlated with changes in neuropathic pain severity (p = 0.027; R 2 = 0.323). The modulation profile may represent an effective measure to track changes in neuropathic pain severity from early to later stages of spinal cord injury.
Project description:A variety of tests of sensorimotor function are used to characterize outcome after experimental spinal cord injury (SCI). These tests typically do not provide information about chemical and metabolic processes in the injured CNS. Here, we used (1) H-magnetic resonance spectroscopy (MRS) to monitor long-term and short-term chemical changes in the CNS in vivo following SCI. The investigated areas were cortex, thalamus/striatum and the spinal cord distal to injury. In cortex, glutamate (Glu) decreased 1 day after SCI and slowly returned towards normal levels. The combined glutamine (Gln) and Glu signal was similarly decreased in cortex, but increased in the distal spinal cord, suggesting opposite changes of the Glu/Gln metabolites in cortex and distal spinal cord. In lumbar spinal cord, a marked increase of myo-inositol was found 3 days, 14 days and 4 months after SCI. Changes in metabolite concentrations in the spinal cord were also found for choline and N-acetylaspartate. No significant changes in metabolite concentrations were found in thalamus/striatum. Multivariate data analysis allowed separation between rats with SCI and controls for spectra acquired in cortex and spinal cord, but not in thalamus/striatum. Our findings suggest MRS could become a helpful tool to monitor spatial and temporal alterations of metabolic conditions in vivo in the brain and spinal cord after SCI. We provide evidence for dynamic temporal changes at both ends of the neuraxis, cortex cerebri and distal spinal cord, while deep brain areas appear less affected.
Project description:Spinal cord injury (SCI) triggers the re-expression of inhibitory molecules present in early stages of development, contributing to prevention of axonal regeneration. Upregulation of EphA receptor tyrosine kinases after injury suggest their involvement in the nervous system's response to damage. However, the expression profile of their ephrinA ligands after SCI is unclear. In this study, we determined the expression of ephrinA ligands after contusive SCI. Adult Sprague-Dawley female rats were injured using the MASCIS impactor device at the T10 vertebrae, and levels of ephrinA mRNA and protein determined at different time points. Identification of the cell phenotype expressing the ephrin ligand and colocalization with Eph receptors was performed with immunohistochemistry and confocal microscopy. Behavioral studies were made, after blocking ephrinA1 expression with antisense (AS) oligonucleotides, to assess hindlimb locomotor activity. Real-time PCR demonstrated basal mRNA levels of ephrin (A1, A2, A3, and A5) in the adult spinal cord. Interestingly, ephrinA1 was the only ligand whose mRNA levels were significantly altered after SCI. Although ephrinA1 mRNA levels increased after 2 weeks and remain elevated, we did not observe this pattern at the protein level as revealed by western blot analysis. Immunohistochemical studies showed ephrinA1 expression in reactive astrocytes, axons, and neurons and also their colocalization with EphA4 and A7 receptors. Behavioral studies revealed worsening of locomotor activity when ephrinA1 expression was reduced. This study suggests that ephrinA1 ligands play a role in the pathophysiology of SCI.
Project description:Long noncoding RNAs (LncRNAs) play a crucial role in cell growth, development, and various diseases related to the central nervous system. However, LncRNA differential expression profiles in spinal cord injury are yet to be reported. In this study, we profiled the expression pattern of LncRNAs using a microarray method in a contusion spinal cord injury (SCI) mouse model. Compared with a spinal cord without injury, few changes in LncRNA expression levels were noted 1 day after injury. The differential changes in LncRNA expression peaked 1 week after SCI and subsequently declined until 3 weeks after injury. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the reliability of the microarray, demonstrating that the results were reliable. Gene ontology (GO) analysis indicated that differentially expressed mRNAs were involved in transport, cell adhesion, ion transport, and metabolic processes, among others. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the neuroactive ligand-receptor interaction, the PI3K-Akt signaling pathway, and focal adhesions were potentially implicated in SCI pathology. We constructed a dynamic LncRNA-mRNA network containing 264 LncRNAs and 949 mRNAs to elucidate the interactions between the LncRNAs and mRNAs. Overall, the results from this study indicate for the first time that LncRNAs are differentially expressed in a contusion SCI mouse model.
Project description:Hyperbaric Oxygen Therapy (HBOT) has definitive therapeutic effects on spinal cord injury (SCI), but its mechanism of action is still unclear. Here, we've conducted a systemic proteomic analysis to identify differentially expressed proteins (DEPs) between SCI rats and HBOT + SCI rats. The function clustering analysis showed that the top enriched pathways of DEPs include oxygen transport activity, oxygen binding, and regulation of T cell proliferation. The results of functional and signal pathway analyses indicated that metabolic pathways, thermogenesis, LXR/RXR activation, acute phase response signaling, and the intrinsic prothrombin pathway in the SCI + HBOT group was higher than SCI group.
Project description:Gene expression changes following spinal cord injury (SCI) are time-dependent, and an accurate understanding of these changes can be crucial in determining time-based treatment options in a clinical setting. We performed RNA sequencing of the contused spinal cord of rats at five different time points from the very acute to chronic stages (1 hour, 1 day, 1 week, 1 month, and 3 months) following SCI. We identified differentially expressed genes (DEGs) and Gene Ontology (GO) terms at each time point, and 14,257 genes were commonly expressed at all time points. The biological process of the inflammatory response was increased at 1 hour and 1 day, and the cellular component of the integral component of the synaptic membrane was increased at 1 day. DEGs associated with cell activation and the innate immune response were highly enriched at 1 week and 1 month, respectively. A total of 2841 DEGs were differentially expressed at any of the five time points, and 18 genes (17 upregulated and 1 downregulated) showed common expression differences at all time points. We found that interleukin signaling, neutrophil degranulation, eukaryotic translation, collagen degradation, LGI-ADAM interactions, GABA receptor, and L1CAM-ankyrin interactions were prominent after SCI depending on the time post injury. We also performed gene-drug network analysis and found several potential antagonists and agonists which can be used to treat SCI. We expect to discover effective treatments in the clinical field through further studies revealing the efficacy and safety of potential drugs.
Project description:Spinal cord injury (SCI) is a devastating condition that affects approximately 11,000 patients each year in the United States. Although a significant amount of research has been conducted to clarify the pathophysiology of SCI, there are limited therapeutic interventions that are currently available in the clinic. Moderate hypothermia has been used in a variety of experimental and clinical situations to target several neurological disorders, including traumatic brain and SCI. Recent studies using clinically relevant animal models of SCI have reported the efficacy of therapeutic hypothermia (TH) in terms of promoting long-term behavioral improvement and reducing histopathological damage. In addition, several clinical studies have demonstrated encouraging evidence for the use of TH in patients with a severe cervical spinal cord injury. Moderate hypothermia (33°C) introduced systemically by intravascular cooling strategies appears to be safe and provides some improvement of long-term recovery of function. TH remains an experimental clinical approach and randomized multicenter trials are needed to critically evaluate this potentially exciting therapeutic intervention targeting this patient population.