Abstract

Background and study goal

Glial glutamate transporter GLT1 plays a key role in the maintenance of extracellular glutamate homeostasis. Recent human genetic studies have suggested that de novo mutations in GLT1 (EAAT2) cause early-onset epilepsy with multiple seizure types. Consistent with these findings, global GLT1 null mice show lethal spontaneous seizures. The consequences of GLT1 dysfunction vary between different brain regions, suggesting that the role of GLT1 dysfunction in epilepsy may also vary with brain regions. In this study, we aim to investigate the correlation of astrocytic GLT1 expression with physiopathological phenomena, including neuronal morphology, and hyperexcitation/inflammation-related astrocytes and microglia activation in the epileptogenic zone via resected human specimen, which proved by MR images, PET, vEEG, or SEEG.

Materials and methods

The human specimen was collected from 30 patients with drug-resistant epilepsy (DRE), who underwent epilepsy surgeries for lesion resection. These patients underwent comprehensive presurgical evaluation, including each patient"s clinical and noninvasive study such as semiology, scalp EEG, neuropychiatric test, PET, MEG, and SPECT. We will examine the human specimen using immunostaining analysis for GLT1, neuron marker NeuN, dendritic marker MAP2, reactive astrocyte marker GFAP, and microglia marker Iba1 to analyze the relationship between GLT1 expression and neuronal damage and gliosis. TUNEL and Fluoro-Jade C stain will be performed to determine the cell apoptosis and neuronal degeneration, respectively. The brain imaging, PET, EEG, neuropsychiatric test, and intraoperative ECoG will be interpreted and cross-correlated to the stain result. The brain connectivity study will also be discussed.

Results

By using AI to develop a module for automated counting, enabling the quantification of meaningful specific primary antibodies (neuronal markers: anti MAP-2, anti NeuN; astrocytic markers: anti GLT-1, anti GFAP; microglial marker: anti Iba-1, etc.). Since we have already recruited 30 patients, we are now able to make clinical correlations. This includes comparing glutamate buffering, neuro-astrocyte coupling, and hippocampal volume. We have observed significant trends in seizure onset time and frequency. In terms of cognitive function and staining of GLT-1, there is a positive trend in neuro-astrocyte coupling, and the study of functional connectivity also indicates a clear decline in patients with hippocampal atrophy.

Discussion

The results show that the smaller hippocampus volumes were tend to have poor glutamate buffering and neuro-astrocyte coupling, which also related to longer seizure onset period, duration and frequency. In neuro-cognitive study, some dominant side cognitive function (e.g. language function or naming function) were tend to have poor neuro-astrocyte coupling. Finally the functional connectivity of MR (+) and MR(-) patients were different, and this section are preparing to correlate with the sprouting of neuron or the function of glutamate buffering. However, we still need complete the patient collection and perform comprehensive statistics to prove the micro- and macro- relationship.