Application Of UPLC Q-TOF/MS-Based Metabonomics Of SD Rats With Diffuse Axonal Injury

Background Diffuse axonal injury (DAI), which manifests as diffuse injury of white matter, accounts for up to 70% of all traumatic brain injury (TBI) cases. The current golden standard for the diagnosis of DAI depends on finding of axonal retraction balls (axonal retraction ball, ARB) via histopathology. But it is difficult for clinicians to make an early diagnosis of patients with DAI. What’s more, it is almost impossible to carry out histopathology among survival patients. Biomarkers such as β-amyloid precursor protein (β-APP), myelin basic protein (MBP) reveal the defect of low specificity. It is urgent to looking for new biomarkers for the diagnosis of DAI.Objective To investigate the feasibility of the application of UPLC Q-TOF/MS-based method in studies of plasma metabonomics of Sprague-Dawley (SD) rats with diffuse axonal injury (DAI), and search for possible biomarkers for early diagnosis of DAI in SD rats.Methods Modified Marmarous method was used to establish the model of DAI in SD rats. Hematoxylin-Eosin staining and Bielschowsky staining were used to evaluate model effect. SD rats were divided into control group (n=6), sham-injured control group (time=1h,6 h,16 h. n=6, repectively), injury group (die group of injury and sacrificed at 1 h,6 h, 16h. n=6, repectively). UPLC Q-TOF/MS technique was applied for examination of plasma samples from all groups. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal signal correction-partial least squares discriminant analysis (OSC-PLS-DA) were involved to analyze the data for pattern recognition. Differences of plasma metabolites among three groups were determined. The recognition ability of PCA, PLS-DA and OSC-PLS-DA were compared. Variable importance in projection (VIP) of PLS-DA, the secondary mass spectrometry and the one-way ANOVA were used to screen possible metabolic markers for the diagnosis of DAI.Results The model of DAI in SD rats was successfully established by modified Marmarous method. There were significantly differences among the plasma metabonome of the three groups. PCA failed to identify the patterns of the three groups, while PLS-DA and OSC-PLS-DA showed significant differences among the three groups. Fourteen metabolites are concluded to be different among control groups and experimental group. Seven metabolites manifest a statistical significance up-regulation (including acetic acid, aniline, glutamine, L-lysine,6-fluoro-DL-tryptophan, myristic acid and hydrocortisone) while seven metabolites performance a statistical significance down-regulation (including L-histidine, cumic acid, pyridoxamine, arginine, lactic acid, melatonin and phosphocholine).Conclusions It is scientific to establish model of DAI in SD rats by Marmarous method. The method of plasma metabonomics of SD rats with diffuse axonal injury in early stage has been constructed using the technique of UPLC Q-TOF/MS, and PLS-DA and OSC-PLS-DA pattern recognition are superior to PCA method. Totally 14 differential metabolites are concluded to be biomarkers for the diagnosis of DAI in SD rats in early stage, which lay the foundation for the research of clinical early diagnosis, treatment and forensic identification related to diffuse axonal injury.