Anxious Profile Influences Behavioral and Immunohistological Findings in the Pilocarpine Model of Epilepsy

Abstract

Anxiety and epilepsy have a complex bidirectional relationship, where a depressive/anxious condition is a factor that can trigger seizures which in turn can aggravate the depressive/anxious condition. In addition, brain structures such as the hippocampus and amygdala might have a critical relevance in both epilepsy and anxiety. The aim of the present work was to investigate the influence of different anxious profiles to epileptogenesis. Initially, animals were screened through the elevated plus-maze anxiety test, and then seizure development was evaluated using the pilocarpine model of epilepsy. There were no differences in the susceptibility to status epilepticus, mortality rate or frequency of spontaneous recurrent seizures between animals characterized as anxious as compared to the non-anxious animals. Next, we evaluated immunohistological patterns related to seizures and anxiety in various related brain areas. Despite a decrease in the density of neuropeptide Y and parvalbumin expression in epileptic animals, those presenting greater neuropeptide Y immunoreactivity in various brain regions, also showed higher spontaneous recurrent seizures frequency. Differences on the anxious profile showed to interfere with some of these findings in some regions. In addition, animals that were injected with pilocarpine, but did not develop status epilepticus, had behavioral and neuroanatomical alterations as compared to control animals, indicating its importance as an additional tool for investigating the heterogeneity of the epileptogenic response after an initial insult. This study allowed to better understand the association between anxiety and temporal lobe epilepsy and might allow for therapeutic targets to be developed to minimize the negative impacts associated with it.

Keywords: epileptogenesis; neuropeptide y; parvalbumin; seizure; temporal lobe epilepsy.

FIGURE 1

Schematic representation of the experimental timeline. Five days prior the pilocarpine-induced SE (day 0), animals were subjected to the behavior test. Thirty days after the SE, animals were video monitored. Sixty days after SE induction, a behavior retest was performed (day 60), and ninety days after SE, animals were perfused and their brains processed for Nissl and neo-Timm staining, and for immunohistochemistry against NPY and PV. EPM, elevated plus maze; OF, open field; NPY, neuropeptide Y; PV, parvalbumin.

FIGURE 2

Density of neuronal cells in the hilus 90 days after pilocarpine injection. SE animals presented decreased neuronal density as compared to controls and NoSE animals in both Anx and Non-Anx groups. Control animals and NoSE animals, in both Anx and Non-Anx groups, showed similar amounts of cells. Values: mean ± SEM. Two-Way ANOVA, Bonferroni post hoc test, Seizure p < 0.05 *vs Controls, #vs NoSE; Anxiety p>0.05; Interaction p>0.05.

FIGURE 3

Photomicrographs of Nissl staining in the hilus of the hippocampus 90 days after pilocarpine injection. (A) Control (B) NoSE (C) SE animal. Note intense neuronal loss in SE group as compared to controls and NoSE groups. Scale bar: 100 μm.

FIGURE 4

Density of NPY + cells in several regions 90 days after pilocarpine injection. (A) Anxious group. (B) Non-Anxious group. The NPY + cell density in the hilus and CA3 hippocampal regions was influenced by anxiety (^&A vs B). Most differences occurred within groups, where pilocarpine injection and SE development influenced the NPY + cell density as compared to controls (except for the dentate gyrus, and the piriform and entorhinal cortices in Non-Anx animals, A and B). Values: mean ± SEM. Two-Way ANOVA, Bonferroni post hoc test, ^& p < 0.05 Anxious vs Non-Anxious; SE group vs Controls (**p < 0.01, ***p < 0.001) and SE group vs NoSE (^# p < 0.05, ##p < 0.01, ###p < 0.001). NPY, neuropeptide Y; DG, dentate gyrus; BL, basolateral amygdala; BM, basomedial amygdala; Pir, piriform; Ent, entorhinal cortex.

FIGURE 5

Photomicrographs of NPY + cells in hippocampal regions. (1) Dentate gyrus and hilus, (2) CA3 and (3) CA1. (a, b and c) Anx (a`, b` and c`) Non-Anx (a, a`) Controls (b, b`) NoSE (c, c`) SE. Note intense (darker) and widespread staining in regions which coincide with mossy fiber sprouting in the dentate gyrus, hilus and CA3, exclusively in SE animals (arrow heads). Scale bar: 250 μm.

FIGURE 6

Photomicrographs of NPY + cells in different brain regions. (1) Amygdala, (2) Piriform cortex, (3) Entorhinal cortex (a, b and c) Anx (a`, b` and c`) Non-Anx (a, a`) Controls (b, b`) NoSE (c, c`) SE. Scale bars: 500 μm.

FIGURE 7

Density of PV in several regions 90 days after pilocarpine injection. (A) Anxious group (B) Non-Anxious group. The PV density in the dentate gyrus and hilus was influenced by the anxiety (&A vs B). Also, pilocarpine injection and SE development influenced PV density in the basolateral amygdala and piriform cortex, but only in Non-Anx group (B). Values: mean ± SEM. Two-Way ANOVA, Bonferroni post hoc test, p < 0 .05 &Anxious vs Non-Anxious; SE group vs Controls (*p < 0.05; ***p < 0.001) and SE group vs NoSE (^# p < 0.05); Interaction p > 0.05. Parvalbumin, PV; DG, dentate gyrus; BL, basolateral amygdala; BM, basomedial amygdala; Pir, piriform; Ent, entorhinal cortex.

FIGURE 8

Photomicrographs of PV + cells in the different brain regions. Hippocampal dentate gyrus and hilus in Non-Anxious (A) and Anxious (B) animals. Piriform cortex in Control (C), NoSE (D) and SE (E) animals. Note increased number of PV + cells in the animal of the Anx group as compared to Non-Anx group and decreased PV density in NoSE and SE animals as compared to Controls. Scale bar: 100 µm.

FIGURE 9

Photomicrographs of Neo-Timm staining in the hippocampal dentate gyrus. (A) Control (B) NoSE (C) SE animal. Note slightly increase of Timm staining in NoSE animals and evident darker staining, especially at the inner molecular layer of the dentate gyrus in SE animals. (D) Densitometry analysis of the inner molecular layer confirmed increased Neo-Timm intensity in SE animals and no influence of the anxiety level on mossy fiber sprouting. Values: mean ± SEM. Two-Way ANOVA, Bonferroni post hoc test, p < 0.05 SE groups *vs Controls, ^#vs NoSE. Scale bar: 50 μm.