| Spinal Cord Injury(SCI)is a high disability rate disease,involving cell toxicity,oxidative stress and immune endocrine multiple pathological mechanism.The complexity of the pathological process depends not only on the initial mechanical injury,but also on the secondary damage,including ischemia,hypoxia,the formation of free radicals and excitotoxicity,etc.At present,there are few therapeutic interventions that limit the extent of tissue damage following SCI.The mechanisms of secondary injury have not been fully discovered.Although many components are altered after spinal cord injury,it is difficult to find characteristic biomarkers to evaluate and predict the prognosis.Metabolomics is a new approach that involves the determination of changes in the levels of endogenous or exogenous metabolites in biological samples,owing to physiological stimuli of genetic modification.It is an important part of Systems Biology.The method of high-throughput identification and quantification of metabolites provides an effective means for the study of a large number of metabolites and complex metabolic pathways involved in Spinal Cord Injury.Recently,it has been shown that metabolomics screening of plasma or CSF obtaining from GC-MS?1H NMR or LC-MS can be used to establish an injury severity evaluation model based on the identified metabolomics fingerprints.Though the metabolic profile in blood and CSF has previously been studied,the relationship between metabolic changes in CSF,blood and spinal cord tissue remains to be investigated.OBJECTIVEDetection and analysis of plasma,CSF and spinal cord tissue metabolite changes after acute spinal cord injury in rats,and discussed its relationship between three types of tissues.Verify the reliability and stability of metabolomics profiling of serum after experimental spinal cord injury in Macaca Mulatt.METHODS 1.Establishment of Spinal Cord Injury model in rats 60 adult male healthy SD rats were randomly assigned to 4 groups: normal control group,6-,24-and the 72-hour post SCI group,with 15 rats in each group.In the injury group,the laminectomy was performed at the 9 thoracic vertebras.The spinal cord injury models were established by using NYU impactor.The weight of the striking rod was 10 g,and the initial height was 25 mm.The quality of the models was accurately controlled.2.Biological specimen acquisition After anesthesia,the rat was fixed on the stereotactic frame.The CSF samples were collected by cisternal puncture and stored in the EP tube.The blood samples were collected by intracardiac puncture and heparinized.10 minutes after the operation,the blood samples were centrifuged.The plasma samples were collected and stored.The spinal cord was exposed,and the spinal cord tissues close to the epicenter were obtained.All specimens stored at-80 ? and the quality was controlled.3.Metabonomics Analysis Characterized the metabolic features of plasma,CSF and spinal cord tissues using a non-targeted metabolic profiling strategy based on gas chromatography time-of-flight mass spectrometry profiling method.Only those peak-pair features shared by more than 50% of the samples were retained for statistical analysis.Multivariate statistical analysis including Principle Component Analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)was carried out using SIMCA-P software.The threshold for selection of significant features in OPLS-DA was VIP > 1.Metabolites with p < 0.05 from the analysis of variance or Rank sum test and VIP > 1 were identified as differential metabolites.Pathway analysis was performed by Metaboanalyst(www.metaboanalyst.ca).4.Metabolic correlation analysis among different biological specimens The spectral peak data in the laboratory standard material library was searched to identify metabolites which allow us to perform targeted metabolic profiling and evaluation of perturbed pathways triggered by SCI among different biological specimens.Correlation analysis of key differential metabolites in the pathway was carried out to investigate the reliability and correlation of body fluid as a specimen of spinal cord injury.5.Metabolomic Profiling of Serum after SCI in Macaca Mulatta Characterized the metabolic features of serum using a non-targeted metabolic profiling strategy based on gas chromatography time-of-flight mass spectrometry profiling method.Three time points(-1day,3 hours and 3 days)during the acute complete spinal cord injury processes in the Macaca Mulatta were evaluated.Parallel analyzed the plasma(serum)metabonomics data in rats and rhesus monkeys after spinal cord injury.Evaluated the potential and reliability of plasma(serum)as the biological sample to perform metabonomics study.RESULTS Total 48 sets of biological specimens,12 sets per group,were randomly selected for performing GC-MS analysis.478 chromatographic peaks were detected from the plasma TICs.Three abnormal specimens were excluded by PCA model.34 metabolites were confirmed to be differential metabolites based on the OPLS-DA model and analysis of variance.6 pathways were significant altered after SCI.We detected 359 peaks from CSF TICs,excluded 2 abnormal specimens,and 20 differential metabolites and 2 significant altered pathways were identified.There were 673 peaks that were detected from spinal cord tissue TICs.40 metabolites and 14 pathways were identified involved in the processors post SCI.Parallel metabolomic profiling of CSF,plasma and spinal cord tissue show the common metabolites of plasma and spinal cord accounted for 88.5% of spinal cord metabolites.CSF and spinal cord tissue's common metabolites accounted for 75.4% of the total spinal cord metabolites.The altered metabolic pathways shared by plasm and spinal cord tissue account for 42.9% and the altered metabolic pathways shared by CSF and spinal cord tissue account for 23.8% of the significant altered pathways in the spinal cord tissue after SCI.Alanine,aspartate and glutamate metabolism,Glycine,serine and threonine metabolism were significantly altered in all type of tissues and blood samples of the rat and rhesus monkey SCI models.Alanine,aspartate,glutamate,glutamine,threonine,glycine and serine are significant differences in most specimens.The Metabolomics Profiling was performed on the serum of the Macaca Mulatta.There were 14 pathways identified as significantly altered pathways.When comparing the pathway analysis results of the Macaca Mulatta to those of the Rat,all of the rats significantly altered pathways were involved.CONCLUSIONS Both biofluid and spinal cord tissue can be applied to the metabolomics research.Because of the metabolic specificity of different tissue,the quantity and species of the altered metabolic pathways were different.The metabolic changes in spinal cord tissue more directly reflect the local pathological processes.The change of humoral metabolism can reflect the local metabolic characteristics of spinal cord injury in part.At the same time,it can reflect the metabolic changes of the whole nervous system and even the organism.The metabolism of Plasma(serum)and spinal cord tissue is more relevant and responsive.Although susceptible to other factors,Plasma(serum)still showed a high stability as the potential biological samples applied for spinal cord injury metabolic biomarkers screening.The relationship between CSF and spinal cord tissue metabolism is relatively lower.Due to its relatively closed,conservative and stable features,combined with the accurate reflection of the metabolic changes of the spinal cord tissue during a long period of spinal cord injury,CSF should be used to screen the biomarkers for prognosis of SCI.The metabolic changes of injured spinal cord tissue directly reflect the local metabolic characteristics post injury.It's suitable for pathophysiology and injury mechanism research.In this study,we also found that Alanine,aspartate and glutamate metabolism,Glycine,serine and threonine metabolism were significantly altered after spinal cord injury.The role and mechanism of which were still not clear.It's a potential research direction.In summary,Plasma(serum)and cerebrospinal fluid can reflect the local metabolic characteristics of spinal cord injury.Both are suitable for spinal cord injury metabolomics research.We should select the samples according to the purpose of the study to obtain more reliable conclusions.In the screening of biomarkers of spinal cord injury,the combination of plasma(serum)and CSF for metabolomics analysis is an advantageous strategy. |
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