Regulation of glutamate transport and neuroinflammation in a term newborn rat model of hypoxic-ischaemic brain injury

Abstract

In the newborn brain, moderate-severe hypoxia-ischaemia induces glutamate excitotoxicity and inflammation, possibly via dysregulation of candidate astrocytic glutamate transporter (Glt1) and pro-inflammatory cytokines (e.g. Tnfα, Il1β, Il6). Epigenetic mechanisms may mediate dysregulation. Hypotheses: (1) hypoxia-ischaemia dysregulates mRNA expression of these candidate genes; (2) expression changes in Glt1 are mediated by DNA methylation changes; and (3) methylation values in brain and blood are correlated. Seven-day-old rat pups (n = 42) were assigned to nine groups based on treatment (for each timepoint: naïve (n = 3), sham (n = 3), hypoxia-ischaemia (n = 8) and timepoint for tissue collection (6, 12 and 24 h post-hypoxia). Moderate hypoxic-ischemic brain injury was induced via ligation of the left common carotid artery followed by 100 min hypoxia (8% O₂, 36°C). mRNA was quantified in cortex and hippocampus for the candidate genes, myelin (Mbp), astrocytic (Gfap) and neuronal (Map2) markers (qPCR). DNA methylation was measured for Glt1 in cortex and blood (bisulphite pyrosequencing). Hypoxia-ischaemia induced pro-inflammatory cytokine upregulation in both brain regions at 6 h. This was accompanied by gene expression changes potentially indicating onset of astrogliosis and myelin injury. There were no significant changes in expression or promoter DNA methylation of Glt1. This pilot study supports accumulating evidence that hypoxia-ischaemia causes neuroinflammation in the newborn brain and prioritises further expression and DNA methylation analyses focusing on this pathway. Epigenetic blood biomarkers may facilitate identification of high-risk newborns at birth, maximising chances of neuroprotective interventions.

Keywords: Hypoxic–ischemic encephalopathy; brain; cerebral ischaemia; glutamate; hypoxia–ischaemia; neonatal; neuroinflammation; newborn.

Figure 1.

Experimental design.

Figure 2.

Diagram of the candidate promoter regions in Glt1 explored in the DNA methylation analysis.

Figure 3.

Glt1 expression following hypoxia–ischaemia in the cortex (left) and hippocampus (right).

Figure 4.

Glt1 promoter DNA methylation following HI in cortex and blood.

Figure 5.

Cytokine expression following hypoxia–ischaemia in the cortex (left) and hippocampus (right)