Quantitative Proteomics Reveals the Dynamic Pathophysiology Across Different Stages in a Rat Model of Severe Traumatic Brain Injury

Abstract

BackgroundSevere traumatic brain injury (TBI) has become a global health problem and causes a vast worldwide societal burden. However, distinct mechanisms between acute and subacute stages have not been systemically revealed. The present study aimed to identify differentially expressed proteins in severe TBI from the acute to subacute phase.MethodsSixty Sprague Dawley (SD) rats were randomly divided into sham surgery and model groups. The severe TBI models were induced by the controlled cortical impact (CCI) method. We evaluated the neurological deficits through the modified neurological severity score (NSS). Meanwhile, H&E staining and immunofluorescence were performed to assess the injured brain tissues. The protein expressions of the hippocampus on the wounded side of CCI groups and the same side of Sham groups were analyzed by the tandem mass tag-based (TMT) quantitative proteomics on the third and fourteenth days. Then, using the gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and protein–protein interaction (PPI), the shared and stage-specific differentially expressed proteins (DEPs) were screened, analyzed, and visualized. Eventually, target proteins were further verified by Western blotting (WB).ResultsIn the severe TBI, the neurological deficits always exist from the acute stage to the subacute stage, and brain parenchyma was dramatically impaired in either period. Of the significant DEPs identified, 312 were unique to the acute phase, 76 were specific to the subacute phase, and 63 were shared in both. Of the 375 DEPs between Sham-a and CCI-a, 240 and 135 proteins were up-regulated and down-regulated, respectively. Of 139 DEPs, 84 proteins were upregulated, and 55 were downregulated in the Sham-s and CCI-s. Bioinformatics analysis revealed that the differential pathophysiology across both stages. One of the most critical shared pathways is the complement and coagulation cascades. Notably, three pathways associated with gastric acid secretion, insulin secretion, and thyroid hormone synthesis were only enriched in the acute phase. Amyotrophic lateral sclerosis (ALS) was significantly enriched in the subacute stage. WB experiments confirmed the reliability of the TMT quantitative proteomics results.ConclusionOur findings highlight the same and different pathological processes in the acute and subacute phases of severe TBI at the proteomic level. The results of potential protein biomarkers might facilitate the design of novel strategies to treat TBI.

Identification of Key Genes and Pathways in the Hippocampus after Traumatic Brain Injury: Bioinformatics Analysis and Experimental Validation

“…The staining results suggested that the level of microglia activation was markedly elevated in the hippocampus of rats in the TBI group when compared with the sham group. Through immunofluorescence staining analysis, Luo et al [52] also found that the number of microglia was significantly increased in the hippocampus during the subacute phase of TBI, which was consistent with our results. Similarly, the immune-related DEGs identified by bioinformatic analyses were significantly enriched in BP terms associated with the activation of microglia and macrophages.…”

“…Current evidence indicates extensive crosstalk between the complement and coagulation systems, which have fundamental clinical implications for inflammation, immunity, and tissue damage [51]. A previous proteomics study suggested that complement and coagulation cascades were activated during the acute and subacute phases of TBI, which was consistent with our findings [52].…”

“…For instance, Saber et al [65] found that both the protein and mRNA expressions of Trem2 were significantly increased in the ipsilateral cortex in mice on the third day after experimental TBI. The study by Luo and colleagues [52] revealed that the multiple proteins (e.g., C1qa, Pycard, Aif1, Ptpn6, and Fcer1g) in the ipsilateral hippocampus were markedly upregulated in a rat model of TBI on the third day. In addition, Wang et al [54] reported that the gene expression levels of C1qa, Aif1, and Csf1r were prominently increased in the perilesional area on the fifth day post injury in TBI mice.…”

Temporal changes in the microglial proteome of male and female mice after a diffuse brain injury using label‐free quantitative proteomics

“…Inflammatory proteins are typically secreted from microglia to act upon other cells such as neurons and astrocytes (reviewed in Matejuk & Ransohoff, 2020 ; reviewed in Gao et al, 2013 ). Other proteomic studies post‐TBI use whole brain homogenate which contains extracellular components such as effector proteins (Abu Hamdeh et al, 2018 ; Luo et al, 2021 ; Zhang et al, 2021 ). In this study, we isolated microglia from whole brain homogenate, thus, we cannot detect proteins that have been secreted from microglia.…”

Research progress in the application of proteomics technology in brain injury

“…Oxygen transport and acute phase response may be the focus of further research on TBI in the future. Luo et al (2021) combined bioinformatics techniques to discover that in the acute phase of sTBI, in addition to platelet activation and neural‐related pathways, endocrine‐related pathways also undergo changes, including gastric acid secretion, thyroid hormone synthesis, and insulin secretion. This provides proteomic clues for the different pathogenesis of sTBI in the acute and subacute phases and also provides potential signaling pathways and targets for future research.…”