Fig 1.
Mild hypoxic-ischemic encephalopathy piglet model.
(A) Diagram of hypoxia-asphyxia protocol. Each piglet is intubated, then subjected to 45 minutes of 13% FiO2 at which point SaO2 reaches approximately 50%. The endotracheal tube is then occluded for 6 minutes to produce asphyxia. Piglets are then resuscitated with 50% FiO2 for 5 minutes, followed by 25% FiO2 until the piglet is fully recovered and ready for extubation. At either 24 or 72 hours post-procedure piglets were sacrificed. In total, eleven piglets were included in the study (sham = 3; 24 hour mild HIE = 4; 72 hour mild HIE = 4). (B) Biometric parameters of piglets who underwent asphyxia at baseline (white bar) compared to post hypoxia-asphyxia (gray bar). The orange lines denote the highest pH (7.15) and base excess (-10 mmol/L) that qualify for therapeutic hypothermia at our institution. All piglets undergoing hypoxia-asphyxia met at least one blood gas requirement.
Fig 2.
Untargeted high-throughput proteomics screen.
(A) Venn diagram of differentially expressed proteins compared to sham at 24 or 72 hours post-hypoxia-asphyxia using p < 0.05 and p < 0.1. (B) Principal Component (PC) Analysis of differentially expressed proteins for each condition (p < 0.1). (C) Heat map of differentially expressed proteins at 24 or 72 hours post-hypoxia-asphyxia compared to sham (p < 0.1). Green indicates lower expression, while red indicates higher expression.
Table 1.
Up-regulated proteins at both 24 and 72 hours post-hypoxia-asphyxia.
Table 2.
Down-regulated proteins at both 24 and 72 hours post-hypoxia-asphyxia.
Fig 3.
Volcano plot of all proteins at (A) 24 or (B) 72 hours post-hypoxia asphyxia. Up-regulated proteins are in red, while down-regulated proteins are in green (p < 0.1). KEGG pathways for proteins altered at (C) 24-hours or (D) 72-hours post-hypoxia asphyxia are shown. Pathways of interest are emphasized in black. For the metabolic pathways, Reactome was used to better delineate sub-pathways. The top second order and third order hierarchical pathways changed at 24 hours are shown.
Table 3.
Proteins increased at 24 hours post-hypoxia-asphyxia associated with “Metabolic Pathways” KEGG.
Table 4.
Proteins decreased at 24 hours post-hypoxia-asphyxia associated with “Metabolic Pathways” KEGG.
Table 5.
Proteins increased at 72 hours post-hypoxia-asphyxia associated with “Metabolic Pathways” KEGG.
Table 6.
Proteins decreased at 72 hours post-hypoxia-asphyxia associated with “Metabolic Pathways” KEGG.
Table 7.
Amino acids in piglet hippocampus measured by ion exchange liquid chromatography.
Fig 4.
Analysis of amino acid levels.
Select amino acid concentrations of interest corresponding to the proteomics metabolic pathways data are shown for sham, 24 hour post-hypoxia-asphyxia, and 72 hour post-hypoxia-asphyxia piglet hippocampus (mean, +/- SEM). (A) Proline. (B) Tryptophan. (C) Aspartate. (D) Asparagine. (E) Branched chain amino acids leucine, isoleucine, and valine. (F) Lysine. n = 3-4 per group, *p < 0.05 1-way ANOVA.
Table 8.
Protein and mRNA expression of canonical glucose metabolism enzymes.
Fig 5.
Canonical enzyme expression. mRNA expression relative to sham (A, E), protein abundance by LC-MS/MS (B, F), western blot expression (C, G) and quantification of western blots (D, H) for GAPDH and PDHA are shown, respectively.
N = 3-4 per group, Ordinary 1-way ANOVA, *p <0 .05, **p < 0.01.
Fig 6.
Lysine acetylation after hypoxia-asphyxia does not change.
(A) Western blot for acetylated lysine of hippocampal lysates from sham, 24 hours and 72 hours post-hypoxia asphyxia. (B) Densitometry of relative total acetylated lysine normalized to HSC70. (C) Quantification of the dominant 50 kDa band. (D) Quantification of the 14 kDa band. N = 3-4 per group, Ordinary 1-way ANOVA, *p < 0.05, **p < 0.01.
Fig 7.
Blood and brain acylcarnitines show different profiles after mild HIE.
(A) Blood acylcarnitine z-scores were calculated and presented as a heat map for each acylcarnitine species at baseline (n = 11), immediately before asphyxia (n = 8), and at sacrifice at 24 hours (n = 4) or 72 hours (n = 4). (B) Blood total acylcarnitines (AC) were calculated for each time point. (C) Post-asphyxia acylcarnitines relative to paired baseline acylcarnitines are shown and colored to parallel z-score color trends. (D) Brain acylcarnitine z-scores presented as a heat map for sham (n = 3), 24 hours post-hypoxia-asphyxia (n = 4), and 72 hours post-hypoxia-asphyxia (n = 4). (E) Brain total acylcarnitines calculated for each time point. (F) 72 hour acylcarnitines relative to sham average are shown and colored to parallel z-score trends. All calculations are ordinary one-way ANOVA, *p < 0.05, **p<0.01, and graphs show mean +/− SEM.