Prominent Astrocytic GLAST Pathology Occurs in Newborn Human and Piglet Hypoxic-Ischemic Encephalopathy: Modeling Relationships Among Laminar Neuropathology, Seizures, and Therapeutic Hypothermia

Dongseok Park^1*Caitlin O'Brien²Jennifer K. Lee²Lee J Martin^1,2,3,4

• ¹Johns Hopkins Medicine Department of Pathology, Baltimore, United States
• ²Johns Hopkins medicine department of Anesthesiology and critical care medicine, baltimore, United States
• ³Johns Hopkins smedicien department of neuroscience, Baltimore, United States
• ⁴Johns Hopkins medicine pathology graduate training program, Baltimore, United States

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 hours) with hypoxic-asphyxic cardiac arrest and resuscitation or sham procedure were used. Piglet survival was assessed over 7 days. Neuropathology was identified by damaged neuron number 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 hours, and significantly reduced ictal event frequency (to lower than 5 events per 4 hours) and duration (to less than 20 sec 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 piglet. We conclude that astrocytic glutamate transport abnormalities contribute to cortical hyperexcitability and seizure in neonatal HIE.