mTOR is activated in epilepsy, but the mechanisms of mTOR activation in post-traumatic epileptogenesis are unknown. It is also not clear whether mTOR inhibition has an antiepileptogenic, or merely anti-convulsive effect. The rat hippocampal organotypic culture model of post-traumatic epilepsy was used to study the effects of long term (four weeks) inhibition of signaling pathways that interact with mTOR. Ictal activity was quantified by measurement of lactate production and electrical recordings, and cell death was quantified with LDH release measurements and Nissl-stained neuron counts. Lactate and LDH measurements were well-correlated with electrographic activity and neuron counts, respectively. Inhibition of PI3K and Akt prevented activation of mTOR, and was as effective as inhibition of mTOR in reducing ictal activity and cell death. A dual inhibitor of PI3K and mTOR, NVP-BEZ235, was also effective. Inhibition of mTOR with rapamycin reduced axon sprouting. Late start of rapamycin treatment was effective in reducing epileptic activity and cell death, while early termination of rapamycin treatment did not result in increased epileptic activity or cell death. The conclusions of the study are: (1), the organotypic hippocampal culture model of posttraumatic epilepsy comprises a rapid assay of antiepileptogenic and neuroprotective activities and, in this model (2), mTOR activation depends on PI3K-Akt signaling, and (3) transient inhibition of mTOR has sustained effects on epilepsy.

Three-dimensional tissue cultures have been used as effective models for studying different diseases including epilepsy. High-throughput, non-destructive techniques are essential for rapid assessment of disease-related processes such as progressive cell death. In this study, an ultrahigh-resolution optical coherence microscopy (UHR-OCM) system with ~1.5-μm axial resolution and ~2.3-μm transverse resolution was developed to evaluate seizure-induced neuronal injury in organotypic rat hippocampal cultures. The capability of UHR-OCM to visualize cells in neural tissue was confirmed by comparison of UHR-OCM images with confocal immunostained images of the same cultures. In order to evaluate the progression of neuronal injury, UHR-OCM images were obtained from cultures on 7, 14, 21 and 28 days-in-vitro (DIVs). In comparison to DIV 7, statistically significant reductions in 3D cell count and culture thickness from UHR-OCM images were observed on subsequent time points. In cultures treated with kynurenic acid (KYNA), significantly less reduction in cell count and culture thickness was observed compared to control specimens. These results demonstrate the capability of UHR-OCM to perform rapid, label-free, and non-destructive evaluation of neuronal death in organotypic hippocampal cultures. UHR-OCM, in combination with three-dimensional tissue cultures, can potentially prove to be a promising tool for high throughput screening of drugs targeting various disorders.