Abstract
Objective
Application of Tandem Mass Tags (TMT)-based LC–MS/MS analysis to screen for differentially expressed proteins (DEPs) in traumatic axonal injury (TAI) of the brainstem and to predict potential biomarkers and key molecular mechanisms of brainstem TAI.
Methods
A modified impact acceleration injury model was used to establish a brainstem TAI model in Sprague–Dawley rats, and the model was evaluated in terms of both functional changes (vital sign measurements) andstructural changes (HE staining, silver-plating staining and β-APP immunohistochemical staining). TMT combined with LC–MS/MS was used to analyse the DEPs in brainstem tissues from TAI and Sham groups. The biological functions of DEPs and potential molecular mechanisms in the hyperacute phase of TAI were analysed by bioinformatics techniques, and candidate biomarkers were validated using western blotting and immunohistochemistry on brainstem tissues from animal models and humans.
Results
Based on the successful establishment of the brainstem TAI model in rats, TMT-based proteomics identified 65 DEPs, and bioinformatics analysis indicated that the hyperacute phase of TAI involves multiple stages of biological processes including inflammation, oxidative stress, energy metabolism, neuronal excitotoxicity and apoptosis. Three DEPs, CBR1, EPHX2 and CYP2U1, were selected as candidate biomarkers and all three proteins were found to be significantly expressed in brainstem tissue 30 min-7 days after TAI in both animal models and humans.
Conclusion
Using TMT combined with LC–MS/MS analysis for proteomic study of early TAI in rat brainstem, we report for the first time that CBR1, EPHX2 and CYP2U1 can be used as biomarkers of early TAI in brainstem by means of western blotting and immunohistochemical staining, compensating for the limitations of silver-plating staining and β-APP immunohistochemical staining, especially in the case of very short survival time after TAI (shorter than 30 min). A number of other proteins that also have a potential marker role are also presented, providing new insights into the molecular mechanisms, therapeutic targets and forensic identification of early TAI in brainstem.
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Data Availability
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number can be found in the article/Supplementary material.
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This study is supported by the Guangzhou Science and Technology Program (Grant 2019030011).
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All authors contributed to the study conception and design. Qianling Chen, Lingyue Li and Luyao Xu contributed equally to this work. Material preparation was performed by Bin Yang and Yuebing Huang. Data collection and analysis were performed by Qianling Chen, Lingyue Li and Luyao Xu. The first draft of the manuscript was written by Qianling Chen. Dongfang Qiao and Xia Yue critically revised the work. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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414_2023_3039_Fig8_ESM.png
Fig. S1 Western blotting and immunohistochemical validation of three candidate biomarkers in pons. (A) Western blotting results. Candidate proteins were detected in triplicate and normalized to β-actin level (loading control) for quantitative analysis; (B-D) The scale bar in the Fig. is 100 μm. Quantitative scoring results of IHC analysis are shown as box plots. P < 0.05 for *, P < 0.01 for **compared with the Sham group (PNG 59680 kb)
414_2023_3039_Fig9_ESM.png
Fig. S2 Western blotting and immunohistochemical validation of three candidate biomarkers in medulla oblongata. (A) Western blotting results. Candidate proteins were detected in triplicate and normalized to β-actin level (loading control) for quantitative analysis; (B-D) The scale bar in the Fig. is 100 μm. Quantitative scoring results of IHC analysis are shown as box plots. P < 0.05 for *, P < 0.01 for **compared with the Sham group (PNG 59650 kb)
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Chen, Q., Li, L., Xu, L. et al. Proteomic analysis discovers potential biomarkers of early traumatic axonal injury in the brainstem. Int J Legal Med 138, 207–227 (2024). https://doi.org/10.1007/s00414-023-03039-5
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DOI: https://doi.org/10.1007/s00414-023-03039-5