| Objective:The rat SCI model was established to obtain CSF samples at different time points after operation.Then the metabolomic analysis of CSF samples of rats with SCI was carried out by GC-MS.Subsequently,the changes of substance metabolism and metabolite content and types in CSF at different time points after SCI were analyzed,so as to find out the different metabolites and their changing trends and related metabolic pathways,and to find out the potential specific biomarkers that can reflect the severity of SCI and can be used to judge the prognosis,and to provide a reference for clinical diagnosis and treatment of SCI.Method of the experiment: This experiment consists of two parts1.Establishment of rat SCI model and acquisition of cerebrospinal fluid specimensMethod: 140 adult female Sprague-Dawley(SD)rats were randomly divided into 7groups: The blank control group(BCG),which is the normal control,the rats were not treated;sham control group(SCG),only laminectomy,without SCI;SCI 8 hours group(8HG),24 hours group(24HG),72 hours group(72HG),7 days group(7DG)and 14 days group(14DG),20 rats in each group.The rat SCI model(injured segment T7-8)was made by clamping method.The stable SCI model could be obtained by using special flat-headed tweezers(the tip is 3 mm wide,with 1.2 mm spacer in the middle and 1.2 mm gap in the tip after clamping).By controlling the time of clamping,a stable SCI model could be established.The basso-beattle-bresnahan(BBB)scores were scored before,after the surgery and every time the samples were taken,excluding rats with multiple of median(MOM)greater than 1.5 in each group.The CSF specimens of each group wereobtained by occipital foramen puncture,and 200μl micro-sampler was used for puncture collection.About 100μl of CSF was collected in each rat,and impurities such as inhalation of blood were avoided.The quality of CSF specimens was evaluated to exclude unqualified samples.Finally,the CSF samples were centrifuged at 4℃(10000rpm,15 min)and frozen in-80℃ refrigerator.2.GC-MS analysis of rat CSF samplesMethod: The CSF samples of SCI rats were analyzed by GC-MS.All CSF samples were pretreated and derivatized,and then scanned by GC-MS to obtain the original data such as total ion flow chromatogram of all samples.Using SIMCA-P 14.1 software,the differences of metabolites in CSF samples were investigated by unsupervised principle component analysis(PCA),and then the metabolites which contributed more to the metabolic profiles were identified by supervised orthogonal partial least squares discriminant analysis(OPLS-DA).The results of OPLS-DA were screened by variable importance in the projection(VIP)and p value of Kruskal-Wallis nonparametric test.The metabolites satisfying VIP > 1.0 and p < 0.05 were considered as differential metabolites.Finally,the identification of differential metabolites and the analysis of metabolic pathways were carried out.AMDIS,a software developed by national institute of standards and technology(NIST),was used to deconvolute the original GC-MS data and automatically match the purified mass spectra with the internal standard spectrogram library(including golm metabolome database,and agilent fiehn GC/MS metabolomics RTL library).MetaboAnalyst(https://www.metaboanalyst.ca)was used to analyze the metabolic pathways and identify the metabolic pathways that have significant correlation with SCI.Result:A stable SCI model was established by clamping.Before operation,the motor function of rats in each group was normal,BBB score was 21 points;after 8 hours of operation,the motor function of rats in blank control group and sham control group was not affected,BBB score was 21 points;after 8 hours of operation in SCI group,both hind limbs were paralysed and motor function was lost,BBB score was less than 4 points,which met the experimental requirements.By puncturing the foramen magnum of occipitale,about 100μl cerebrospinal fluid samples were obtained from each rat,whichwas enough to meet the needs of metabolomics(50μl).Metabolomic analysis of CSF samples by GC-MS showed that the metabolites in CSF in 8HG had the greatest difference compared with the SCG.With the passage of time,the difference gradually decreased and returned to the control group level at 14 DG.The metabolites with obvious changes are mainly those involved in energy metabolism,resisting oxidative stress and promoting cell repair.At the early stage of injury,the increased consumption and dysfunction of glucose led to the disorder of energy metabolism,8 hours after injury,the content of glucose was significantly lower than normal.With the activation of various energy substitution pathways(fat decomposition,amino acid participation in energy supply)and glucose supplementation pathways(gluconeogenesis,etc.),energy metabolism and glucose content gradually stabilized after24 hours of injury.The substances involved in resisting oxidative stress(e.g.2-hydroxybutyric acid,phosphorylethanolamine and degradation products of branched-chain amino acids,etc.)had basically dropped to the level of SCG at 72 HG,which indicated that oxidative stress caused by SCI mainly occurred within 72 hours of injury,especially within 8 hours.However,the metabolites involved in cell repair(such as ribose,1-Deoxypentitol,pantothenic acid,etc.)did not decrease to the level of control group until the 14 th day,which indicated that the repair process after injury lasted for a long time.The pathway enrichment analysis of metabolites in 8HG conforming to VIP >1 and p < 0.05 standard showed that the top five metabolic pathways with the greatest impact included: phosphatidylethanolamine biosynthesis,glucose-alanine cycle,phosphatidylcholine biosynthesis,citric acid cycle and β-alanine cycle.Citric acid cycle is at the center of all related metabolic pathways and is associated with other metabolic pathways.The data were analyzed by multiple linear regression to evaluate the correlation between the above metabolites concentration and the severity of SCI in rats(based on BBB score).finally,two metabolites were screened out: 3-hydroxyisovaleric acid(β=-3.173,b=-0.366,p=0.001)and pantothenic acid(β=-3.200,b=-0.257,p=0.018),which indicated that they had potential value in predicting the severity of SCI.Conclusion:By controlling the clamping time,a more stable SCI model can be established and operated more easily.About 100μl CSF specimens were obtained from each rat byoccipital foramen magnum puncture.SCI can cause disorder of substance metabolism in CSF of rats.Different metabolites in CSF can be identified by GC-MS.Some metabolites related to the degree of SCI can predict the severity of injured,which is helpful for clinical diagnosis and treatment.Among all the experimental groups,the metabolism of8 HG was most affected,mainly the substances involved in energy metabolism.The first five metabolic pathways affected were phosphatidylethanolamine biosynthesis,glucose-alanine cycle,phosphatidylcholine biosynthesis,citric acid cycle and β-alanine metabolism(related metabolites satisfied: VIP > 1,p < 0.05),these results suggest the activation of the central metabolic pathway and the resistance of the body to oxidative stress.Multivariate linear regression analysis showed that 3-Hydroxyisovaleric acid and pantothenic acid had potential value in predicting the severity of SCI and were helpful for clinical diagnosis and treatment.But the results need further clinical experiments to verify. |
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