The pilocarpine model of temporal lobe epilepsy

Abstract

Understanding the pathophysiogenesis of temporal lobe epilepsy (TLE) largely rests on the use of models of status epilepticus (SE), as in the case of the pilocarpine model. The main features of TLE are: (i) epileptic foci in the limbic system; (ii) an "initial precipitating injury"; (iii) the so-called "latent period"; and (iv) the presence of hippocampal sclerosis leading to reorganization of neuronal networks. Many of these characteristics can be reproduced in rodents by systemic injection of pilocarpine; in this animal model, SE is followed by a latent period and later by the appearance of spontaneous recurrent seizures (SRSs). These processes are, however, influenced by experimental conditions such as rodent species, strain, gender, age, doses and routes of pilocarpine administration, as well as combinations with other drugs administered before and/or after SE. In the attempt to limit these sources of variability, we evaluated the methodological procedures used by several investigators in the pilocarpine model; in particular, we have focused on the behavioural, electrophysiological and histopathological findings obtained with different protocols. We addressed the various experimental approaches published to date, by comparing mortality rates, onset of SRSs, neuronal damage, and network reorganization. Based on the evidence reviewed here, we propose that the pilocarpine model can be a valuable tool to investigate the mechanisms involved in TLE, and even more so when standardized to reduce mortality at the time of pilocarpine injection, differences in latent period duration, variability in the lesion extent, and SRS frequency.

Fig. 1

Histogram showing the changes in mortality as function of status epilepticus (SE) duration. Values represent the means of three to four different experiments (n = 15–20 rats for the different time intervals) in which seizures were quelled by injecting diazepam (20 mg/kg, i.p.) 30, 60, 120 or 180 min after pilocarpine-induced SE. Pilocarpine was used at 380 mg/kg after a scopolamine methylnitrate injection (1 mg/kg, 30 min before pilocarpine) in Sprague–Dawley rats at 8 weeks of age (cf. Biagini et al., 2006, 2008). Note that a 180-min SE causes a significantly (p < 0.05) higher mortality when compared with all the other time intervals (analysis of variance followed by Games–Howell test for multiple comparisons).

Fig. 2

EEG recordings obtained after pilocarpine (400 mg/kg, i.p.) administration. Note that 5 min after injection, low voltage fast activity appears in amygdala (Amy) and neocortex (Ctx), while theta rhythm is evident in the hippocampus (Hippo). Twenty minutes after, high-voltage fast activity is seen in amygdala and neocortex, while spikes superpose in the hippocampus. In the 30 min traces, high voltage spikes are detected first in the hippocampus while at 40 min, high voltage spikes are recorded from all the fields. After 50 min from the injection, electrographic seizures are seen and followed by post-ictal depression (60 min sample). At 120 min, the EEG corresponds to status epilepticus (modified from Turski et al., 1983a).

Fig. 3

Kaplan–Meier analysis of the time of spontaneous seizure appearance after status epilepticus (SE) induced by injecting Sprague–Dawley rats with pilocarpine (380 mg/kg, i.p.) after a scopolamine methylnitrate injection (1 mg/kg, 30 min before pilocarpine). Note that the spontaneous seizure onset is progressively delayed by increasing SE duration (seizures were quelled by injecting 20 mg/kg diazepam at different time intervals from the pilocarpine injection, cf. Biagini et al., 2006). The log rank test revealed a significant (**p < 0.01) difference between the 180-min SE group and the others (n = 9–14/group).

Fig. 4

Grading of neuronal damage in the hippocampal formation of pilocarpine-treated rats performed in toluidine blue-stained sections that were cut 3 weeks after the pilocarpine injection. (A) grading of the lesion (n = 18) from non-detectable (ND) to almost complete lesion (grade IV) is shown for different areas (see Biagini et al., 2005 for the complete description of the method). Sections of an intact subiculum (B) and of a grade II-damaged subiculum (C). Non-epileptic control (NEC) (D) and pilocarpine-treated (E) dentate gyrus; note that the section in (E) presents a grade IV lesion in the hilus, but a well-preserved granule cell layer. Medial entorhinal cortex close to the boundary with the lateral entorhinal cortex in control (F) and pilocarpine-treated rats (G). Note in (G) the reduced thickness of layer III, in which neurons are replaced by a glial infiltrate (arrow). In (F) and (G), apart the sparse layer IV located immediately above the lamina dissecans (asterisk), the other cortical layers are indicated at the boundary with the parasubiculum. Scale bars for panels (B) and (C), (F) and (G) are 250 μm; for (D) and (E) is 200 μm. Abbreviations in this figure: CA1–2, CA3: Cornu Ammonis hippocampal subfields; DG: dentate gyrus; DH: dentate hilus; medEC: medial entorhinal cortex; latEC: lateral entorhinal cortex; Sub: subiculum.