Sequel of spontaneous seizures after kainic acid-induced status epilepticus and associated neuropathological changes in the subiculum and entorhinal cortex

Abstract

Injection of the seaweed toxin kainic acid (KA) in rats induces a severe status epilepticus initiating complex neuropathological changes in limbic brain areas and subsequently spontaneous recurrent seizures. Although neuropathological changes have been intensively investigated in the hippocampus proper and the dentate gyrus in various seizure models, much less is known about changes in parahippocampal areas. We now established telemetric EEG recordings combined with continuous video monitoring to characterize the development of spontaneous seizures after KA-induced status epilepticus, and investigated associated neurodegenerative changes, astrocyte and microglia proliferation in the subiculum and other parahippocampal brain areas. The onset of spontaneous seizures was heterogeneous, with an average latency of 15 ± 1.4 days (range 3-36 days) to the initial status epilepticus. The frequency of late spontaneous seizures was higher in rats in which the initial status epilepticus was recurrent after its interruption with diazepam compared to rats in which this treatment was more efficient. Seizure-induced neuropathological changes were assessed in the subiculum by losses in NeuN-positive neurons and by Fluoro-Jade C staining of degenerating neurons. Neuronal loss was already prominent 24 h after KA injection and only modestly progressed at the later intervals. It was most severe in the proximal subiculum and in layer III of the medial entorhinal cortex and distinct Fluoro-Jade C labeling was observed there in 75% of rats even after 3 months. Glutamatergic neurons, labeled by in situ hybridization for the vesicular glutamate transporter 1 followed a similar pattern of cell losses, except for the medial entorhinal cortex and the proximal subiculum that appeared more vulnerable. Glutamate decarboxylase65 (GAD65) mRNA expressing neurons were generally less vulnerable than glutamate neurons. Reactive astrocytes and microglia were present after 24 h, however, became prominent only after 8 days and remained high after 30 days. In the proximal subiculum, parasubiculum and entorhinal cortex the number of microglia cells was highest after 30 days. Although numbers of reactive astrocytes and microglia were reduced again after 3 months, they were still present in most rats. The time course of astrocyte and microglia proliferation parallels that of epileptogenesis.

Fig. 1

Status epilepticus (SE) assessed by telemetric EEG- and video recording. Panel A shows a typical EEG-recording covering 20 h after KA injection (10 mg/kg). Mean latency to increased EEG activity was 15.5 ± 1.95 min after KA injection, whereas mean latency to behavioral SE was 83 ± 9.1 min. Injections of diazepam (10–20 mg/kg 120 min after first stage 3 seizure) reduced EEG activity but failed to completely stop SE, and increased EEG-activity reappeared again after 4 ± 1.1 h in average and persisted for 22 ± 2.4 h. Panels B to H show 10 s EEG traces as indicated by arrows in A. Panel B, baseline before KA injection; C, at the onset of subclinical seizures (EEG seizures only); D, at the onset of motor symptoms of SE (1 h after KA-injection); E, trace during SE (note EEG-spikes at a frequency of ∼1.6 Hz); F, reduction of EEG amplitude close to baseline after diazepam injection; G, EEG activity during the recurrent SE (around 9 h after diazepam and 12 h after KA injection), and H, activity after seizing of the recurrent SE (around 12.5 h after diazepam and 15.5 h after KA injection). Panel I depicts the development of EEG-spiking during the first 20 h after KA injection in two subpopulations of rats that experienced either few (<80; dotted line) or frequent (>140) and progressive (solid line) spontaneous recurrent seizures during the subsequent three months (see Fig. 2C and D). Note that increased spike frequency recurred in the group with subsequent progressive seizures.

Fig. 2

Development of spontaneously recurrent seizures. Panel A depicts the summative occurrence of the first spontaneous seizure in rats with initial rating 3 and 4. Three days after SE the first spontaneous EEG seizure was observed in the first rat and in 50% of rats after 12 days. After 36 days, all rats (n = 33) showed spontaneous recurrent seizures. In 77% of rats, the first spontaneous seizure was detectable in EEG only and not accompanied by motor signs. Seizures with a behavioral correlate (clinical seizures) in addition to EEG seizures were detected in average 6.4 ± 1.61 days later. B, Numbers of spontaneous seizures per day in all rats after the first spontaneous seizure (n = 19) shown as box plots. Spontaneous seizures occurred in clusters (not shown) and their frequency increased over time in some rats. Panels C and D illustrate the numbers of seizures in rats without progression of subsequent spontaneous seizures (C) and in rats with seizure progression (D), respectively. Panels E and F show 150 s of raw EEG traces of the two dominant seizure types (both seizures were spontaneous generalized stage 3 seizures). The respective spectrograms and power spectra of two representative examples of the same seizure types are shown in panels G to J. Seizure type I (E, G, I) was predominantly (69% of EEG-seizures) detected in all rats and consisted of a single period of increased EEG amplitude. Seizure type II (F, H, J) consisted of two phases of high EEG-amplitude separated by a short phase of low amplitude and accounted for 19% of all spontaneous seizures.

Fig. 3

Neurodegeneration in the parahippocampal region after KA-induced SE. NeuN-labeling in controls (A) and in rats at different time intervals after KA injection (B, C) revealed an early onset of widespread neurodegeneration in parahippocampal regions. The proximal subiculum and layer III of the medial EC were the most severely affected regions (arrows in B and C). Fluoro Jade-C (FJ-C) staining of degenerating neurons in the proximal subiculum (D–G) and layer III of the medial EC (H–K) revealed that massive neurodegeneration was present already 24 h after the initial SE (E, I). At this interval, FJ-C stained neurons, dendrites, and axons were present in virtually all subregions of the parahippocampal region and were especially dense in the proximal subiculum (E) and in layer III of the medial EC (I). In both subregions, FJ-C stained neurons were still present one (F and J) and – in 75% of rats – three months after KA-induced SE (G and K).

Fig. 4

Reactive gliosis after KA-induced SE. Activation of astrocytes (A–J) occurs slower than that of microglia. Astrocytes reveal thin processes and moderate GFAP-labeling in the parahippocampal region of controls (A, F) and 24 h after KA-induced SE (B, G; resting astrocytes). Activated astrocytes with increased GFAP-labeling and altered morphology (hypertrophic processes) were detected in the subiculum, parasubiculum, entorhinal and perirhinal cortex 8 days (C, H) and 1 month after SE (D, I). The distribution of activated astrocytes and of reactive microglia was similar at the later intervals. In the subiculum and in the EC astrocytes with increased GFAP-labeling were still present three months after KA-induced SE (E, J). Panels K–T depict changes in Ox-42 (CD11b)-labeled microglia after KA-induced seizures. Resting microglial cells are almost evenly dispersed in the parahippocampal region of controls (K) and display a highly ramified morphology (P). Activated microglia with altered morphology and intense Ox-42 labeling is present already 24 h after SE (L, Q). At this interval, activated microglial cells are still evenly distributed in all parahippocampal subregions (L). At later intervals reactive microglia accumulates in layers with severe neurodegeneration (M, N, R, S; note the cluster of reactive microglia in the parasubiculum; arrow in N) and is still present 3 months after the initial SE in the proximal subiculum and in layer III of the EC (O, T). Abbreviations: CA1, hippocampal sector CA1; DG, dentate gyrus; EC, entorhinal cortex; PaS, parasubiculum; PRC, perirhinal cortex; PrS, presubiculum; Sub, subiculum.