Proteomics identify pathogenesis and novel biomarkers for monitoring and early prognostication of brain injury after cardiac arrest: a randomized animal study

Abstract

Aim: Post-cardiac arrest brain injury (PCABI) is the leading cause of death and disability after resuscitation. This study aimed to investigate the pathogenesis and novel biomarkers for monitoring the progression and early prognostication of PCABI.

Methods: Mouse model of PCABI was induced by hyperkalemia-induced asystole and successful resuscitation. Young adult male C57BL/6 mice were randomized into sham-operation or asystole/resuscitation. The sham-operated mice were selected as control. Neurological examinations were performed at 24 h after resuscitation, and three groups were set: control (n = 4), severe PCABI (n = 3), and mild PCABI (n = 3). Cerebral cortexes were collected for data-independent acquisition-proteomic analyses. The pathogenesis and potential biomarkers were identified through the pairwise comparisons of three subgroups and subsequent bioinformatics analyses. Human serum proteomes profiles, extracted from a published work of the second analysis of TTM-trial, were used for joint analyses to identify the common and clinically relevant biomarkers at the same timepoint. Experimental and external validation were performed to verify the association between novel biomarkers and neural death in PCABI.

Results: The proteomic analysis identified and quantified 7,745 proteins. The most prominent proteomic changes were related to response to external stimulus, stress response, regulation of biological and metabolic process, endomembrane system, and inflammatory response in the PCABI progression. 10 potential biomarkers were identified by the pairwise comparisons of three groups, and lipocalin-2 and angiotensinogen are common biomarkers with human studies at 24 h after resuscitation. Experimental validation verified that lipocalin-2 was closely associated with neurodegeneration in PCABI.

Conclusions: Stress, inflammatory, and metabolic responses play important roles in the progression of PCABI. Lipocalin-2 is a novel biomarker for monitoring and early neuroprognostication at 24 h after resuscitation.

Keywords: Cardiac arrest; Lipocalin-2; Post-cardiac arrest brain injury; Prognostication; Proteomics.

Graphical abstract

Fig. 1

Schematic diagram of the study design. CA indicates cardiac arrest; CPR, cardiopulmonary resuscitation; DEPs, differentially expressed proteins; LC, liquid chromatography; MS, mass spectrometry; PCABI, post-cardiac arrest brain injury; ROSC, return of spontaneous circulation.

Fig. 2

Flow diagram of the animal experiment. CA indicates cardiac arrest; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation.

Fig. 3

Proteomic profiling of cerebral cortex from mice. Three subgroups were set in this proteomic study: Control (n = 4, A1-4, normal control), CA1 (n = 3, B1, B2, B4, poor neuroprognostication after resuscitation), CA2 (n = 3, B3, B5, B6, relatively good neuroprognostication after resuscitation). Cerebral cortex was collected at 24 h after resuscitation or sham operation. (A) The numbers of quantified peptides and proteins in 10 samples. (B) Pearson's correlation of protein quantitation. (C) Principal component analysis of all samples. (D) Summary of the significantly regulated proteins in the comparison of three subgroups. (E) Heat map of all differentially expressed proteins.

Fig. 4

Bioinformatics analysis of the regulated proteins in the pairwise comparisons of three subgroups. (A-D) The volcano plots (A), GO-based enrichment analysis (B), KEGG-based enrichment analysis (C), and PPI network (D) of DEPs in the comparison between CA1 and control are shown in the left. (E-H) The volcano plots (E), GO-based enrichment analysis (F), KEGG-based enrichment analysis (G), and PPI network (H) of DEPs in the comparison between CA2 and control are shown in the middle. (I-L) The volcano plots (I), GO-based enrichment analysis (J), KEGG-based enrichment analysis (K), and PPI network (L) of DEPs in the comparison between CA2 and CA1are shown in the right.

Fig. 5

Identification of the pathogenesis and biomarkers of brain injury at 24 h after resuscitation. (A) Common DEPs and corresponding P values in the comparisons of CA1/Control and CA2/Control. These proteins may represent the common pathogenesis underlying post-cardiac arrest brain injury (PCABI) at 24 h after resuscitation. (B) Common DEPs and associated P values in the comparisons of CA1/Control, CA2/Control, and CA2/CA1. These 10 proteins, which may be closely associated with the progression of PCABI, has potential to be novel biomarkers for monitoring and early neuroprognostication at 24 h after resuscitation. Lipocalin-2 (LCN2, red color) and angiotensinogen (AGT, red color) are common markers of mouse cerebral cortex proteome profiles and human serum proteome profiles at 24 h after resuscitation, in the comparison according to neurological outcome. (C) Representative photomicrographs of LCN2 (green)/DAPI (blue)-staining brain sections after sham-operation. The expression level of LCN2 is extremely low and is almost undetectable. (D) Representative photomicrographs of LCN2 (green)/DAPI (blue)-staining and FJB (green)/DAPI (blue)-staining brain sections of severe and mild PCABI at 24 h after resuscitation. Brain regions in hippocampus (Hippo), retrosplenial cortex (Rs), somatosensory cortex (Ss), piriform cortex (Pir), amygdaloid nucleus (Amy), and hypothalamus (HTA) were examined, and there was a considerable overlap between LCN2-positive and FJB-positive areas. (E) Relative immunofluorescence signal intensity of LCN2 and percentage of FJB-positive cells in different brain regions of severe and mild PCABI. (n = 3) Data are presented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)