# Prominent astrocytic GLAST pathology occurs in newborn human and piglet hypoxic–ischemic encephalopathy: modeling relationships among laminar neuropathology, seizures, and therapeutic hypothermia

## TL;DR

Abnormal astrocyte function in newborn brains with hypoxic-ischemic encephalopathy may cause seizures, and cooling therapy can reduce these effects.

## Contribution

This study identifies astrocytic GLAST pathology as a novel contributor to seizures in neonatal HIE, and shows that therapeutic hypothermia can mitigate this.

## Key findings

- Abnormal GLAST localization in human and piglet HIE brains correlates with seizures and neuron damage.
- Therapeutic hypothermia reduces GLAST pathology and seizure burden in piglets.
- GLAST mislocalization is associated with disrupted cortical organization in HIE.

## Abstract

Neonatal hypoxic–ischemic encephalopathy (HIE) is frequently complicated by seizures that persist despite therapeutic hypothermia (HT), suggesting injury mechanisms insensitive to HT. Here, we tested the hypothesis that astrocytic glutamate–aspartate transporter (GLAST) abnormalities in the neocortex contribute to cortical hyperexcitability and seizure burden after HIE, and that HT mitigates this astrocyte-mediated mechanism. We examined the vulnerability of GLAST in the neocortex of human neonatal hypoxic–ischemic encephalopathy (HIE) and in a piglet model of hypoxia-ischemia (HI). We determined how GLAST immunoreactivity localization associates with HT outcome in clinical and experimental settings. Brain sections from postmortem human autopsy cases of term neonatal HIE and piglets (2–3 days old, n = 5–6/group) were used to localize GLAST and glial fibrillary acidic proteins (GFAP) across cortical layer I-III in the somatosensory cortex. Piglets received continuous 4-channel epidural EEG recording under normothermia (NT) or mild HT (38 °C to 34 °C with rewarming; 29 h), with hypoxic-asphyxic cardiac arrest and resuscitation, or a sham procedure. Piglet survival was assessed over 7 days. Neuropathology was identified by the number of damaged neurons and GLAST puncta metrics. EEG-seizure metrics, including ictal event frequency, duration, spike–wave events, and power spectral density (PSD), were quantified using a custom seizure classification pipeline. GLAST localization in human HIE cortex was significantly abnormal compared to non-HIE control cases, characterized by perisomatic aggregation and reduced neuropil density. HI in piglets reproduced these GLAST abnormalities, including apparent aggregation, that correlated with seizure burden and neuronal pathology. HT attenuated the GLAST pathology in HI piglets at perisomatic locations to the level of sham, particularly in layers II-III, delayed seizure onset by ~24 h, and significantly reduced ictal event frequency (to lower than 5 events per 4 h) and duration (to less than 20 s per event). These findings identify prominent GLAST pathology in newborn humans and piglet HIE. HT partially restores astrocytic GLAST localization that is temporally associated with reduced seizure burden in piglets. We conclude that astrocytic glutamate transport abnormalities contribute to cortical hyperexcitability and seizures in neonatal HIE.

Autopsy brains of newborn patients with hypoxic–ischemic encephalopathy (HIE) show abnormal astrocytic GLAST localization, characterized by perisomatic aggregation and disrupted cortical organization. Experimentally, neonatal piglet hypoxia-ischemia recapitulates a cortical GLAST pathology that correlates with seizure burden and cortical neuron injury. Therapeutic hypothermia restores GLAST distribution, delays seizure onset, reduces seizure duration, and mitigates ischemic neurodegeneration in piglets. Astrocytic glutamate transport could be a mechanism for restoring normal cortical excitability in neonatal HIE with therapeutic hypothermia.Diagram compares effects of hypoxic-ischemic insult without and with hypothermia. without hypothermia, neuronal degeneration, and GLAST mislocalization in astrocytes causes immediate, prolonged seizures. with hypothermia at 34C, GLAST is preserved, leading to delayed seizure onset, and reduced seizure duration severity.

Autopsy brains of newborn patients with hypoxic–ischemic encephalopathy (HIE) show abnormal astrocytic GLAST localization, characterized by perisomatic aggregation and disrupted cortical organization. Experimentally, neonatal piglet hypoxia-ischemia recapitulates a cortical GLAST pathology that correlates with seizure burden and cortical neuron injury. Therapeutic hypothermia restores GLAST distribution, delays seizure onset, reduces seizure duration, and mitigates ischemic neurodegeneration in piglets. Astrocytic glutamate transport could be a mechanism for restoring normal cortical excitability in neonatal HIE with therapeutic hypothermia.