Abstracts

Rationale: In temporal lobe epilepsy (TLE), a precipitating event like childhood SE, leads to spontaneous seizures after a latent phase of several years. A change in the balance between excitatory (glutamatergic) and inhibitory (GABAergic) neurotransmission is considered important in this process as well as a reduction in glutamine synthetase (GS), a glial enzyme important in the turnover of glutamate and GABA. We aim to study the development of these changes during epileptogenesis by longitudinally comparing the in vivo ¹H MRS neurochemical profile with concomitant ex vivo histological markers of neurotransmission in the latent phase of the juvenile lithium-pilocarpine model. Methods: SE was induced in 21-day old male Wistar rats by injection of lithium and pilocarpine, known to induce spontaneous seizures in 75% of animals after 3 months¹. MRS was performed at 4 weeks (n=5) and 8 weeks (n=5) after pilocarpine or saline injection on a 9.4 T MR system under halothane anesthesia. A hippocampal voxel (10x2x5 mm) was selected in EPI images (Fig 1). In this volume GABA was selectively detected using a double spin-echo sequence (TR 4 s, TE 68 ms) with editing pulses selective for GABA 3-CH2 (1.9 ppm). Spectra were quantified using a dedicated version of LCModel for both the GABA and non-edited spectra. For histology, animals were perfused with paraformaldehyde at 4 (n=6) and 8 weeks (n=6). After embedding in paraffin, brains were sectioned transversally at 7 μm. FluoroJade (FJ) staining was performed to analyze neuronal damage. Specific antibodies were used against vimentin (Vim), to detect reactive glia, against parvalbumine (PV) to detect GABAergic interneurons, and against GS. Results: Typical unedited ¹H MR spectra are shown in Figure 2. In the pilocarpine injected animals levels of NAA, glutamate (Glu) and GABA were decreased, while total choline (Cho) was increased. GABA levels decreased progressively over time and were more severely affected at 8 weeks. Glutamine (Gln) was significantly increased at 4 weeks but normalized at 8 weeks. FJ staining showed damaged neurons, with reactive glia, demonstrated by Vim immunohistochemistry. Both PV and GS staining decreased progressively with time, most prominently in the hilus of the DG. Conclusions: The decrease in NAA and increase in Cho suggests neuronal death and gliosis, confirmed by the positive FJ and Vim stain. The decrease in glutamate and GABA suggests that both glutamatergic and GABAergic neurons were affected, although the relatively greater reduction in GABA is consistent with a hyper-excitable state. We hypothesize that the rise and subsequent normalization of glutamine reflects the reduced demand for glutamine for synthesis of GABA and glutamate with subsequent adaptative changes in GS. These progressive changes were detected before spontaneous seizures occur and were validated with dedicated immunohistochemistry. We conclude that these represent early indicators of imminent epilepsy.