Inflammation in the developing rat modulates astroglial reactivity to seizures in the mature brain

Abstract

Astrocytes participate in neuronal development and excitability, and produce factors enhancing or suppressing inflammatory processes occurring due to neurodegenerative diseases, such as epilepsy. Seizures, in turn, trigger the release of inflammatory mediators, causing structural and functional changes in the brain. Therefore, it appears reasonable to determine whether generalized inflammation at developmental periods can affect astrocyte reactivity to epileptic seizures occurring in the adult brain. Lipopolysaccharide (LPS) was injected in 6- or 30-day-old rats (P6 or P30, respectively). At the age of 2 months, seizures were induced, and pilocarpine and morphological changes of astrocytes located within the hippocampal formation were assessed. Additionally, expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), aquaporin 4 (AQP4), and inwardly rectifying potassium channel Kir 4.1 (Kir4.1) was determined using Western blots. The animal group given LPS on P6 displayed maximal susceptibility to pilocarpine-induced seizures, significantly higher than the group that received LPS on P30. In the immunohistologically examined hippocampal formation, the GFAP-immunoreactive area was not affected by LPS alone. However, it was reduced following seizures in naïve controls but not in LPS-pretreated rats. Increases in the ramification of astrocytic processes were detected only in adult rats given LPS on P30, not on P6. Seizures abolished the effects. Following seizures, the process ramification showed no significant change in the two LPS-treated rat groups, whereas it was significantly reduced in the dentate gyrus of LPS-untreated controls. Glial fibrillary acidic protein (GFAP) expression showed no changes induced with LPS alone and rose slightly after seizures. AQP4 content was lower in rats given LPS on P6 and was seizure-resistant in the two LPS-treated groups, contrary to a decrease in untreated controls. GS expression was not affected by LPS treatments and was reduced after seizures without an intergroup difference. Kir4.1 underwent highly significant increases in all groups experiencing seizures, but LPS alone had no effect. It can be concluded that the generalized inflammatory status led to some important changes in astrocytes reflected, in part at least by permanent modifications of their morphology and molecular profile. Moreover, the previously experienced inflammation prevented the cells from much stronger changes in response to seizures observed in adult untreated controls. The obtained results point to a link between the activation of astrocytes by transient systemic inflammation occurring during the developmental period and their subsequent reactivity to seizures, which may play an important role in the functional features of the brain, including its susceptibility to seizures.

Keywords: GFAP; astrocyte; cell morphology; epilepsy; lipopolysaccharide.

Figure 1

Frontal sections of the hippocampal formation immunostained for glial fibrillary acidic protein (GFAP) to visualize astrocytes (Bregma − 3.8 mm, Paxinos & Watson, 1986). The two examples showing the range of differences in GFAP immunostaining are taken from rats given LPS on P06 which were not subjected to seizure induction (A) and from rats given LPS on P30 and experiencing seizures induced in adulthood (B). Scale bar: 1 mm.

Figure 2

Assessment of glial fibrillary acidic protein (GFAP)‐immunoreactive area fraction (GFAP⁺AF) illustrated on examples of two images taken from the dentate gyrus region of the rat treated on P6 with LPS alone (left column) and of the control one (right column). Initially, RGB images were taken with a digital camera (A, B). Then, an 8‐bit green channel was extracted (C, D) with subsequent background subtraction (E, F). After contrast enhancement (G, H), a thresholding method (Korzynska et al. 2013) was applied to obtain binary images (I, J). Finally, GFAP⁺AF was measured. For the two samples represented by the left and right columns, values of GFAP⁺AF are 0.25 and 0.39, respectively.

Figure 3

Assessment of astrocyte morphology in the CA3 area. (A, E) Enlarged segments of Fig. 1A and E were taken, respectively, from rats given LPS on P06 which were not subjected to seizure induction and rats given LPS on P30 and experiencing seizures induced in adulthood. Scale bar: 500 μm. Examples of GFAP‐immunopositive astrocytes indicated by arrows in (A) and (E) are shown in (B–D) and (F–H), respectively. The degree of ramification of astrocyte processes (branching index) was defined according to Garcia‐Segura & Perez‐Marquez (2014). Two circles of 25  and 50 μm diameter (B and F, respectively) were centered on the astrocyte cell body and intersections between the circles; the astrocyte processes were counted, and the ratio between number of intersections with the outer and inner circles was calculated.

Figure 4

Assessment of astrocyte morphology in the DG area. (A, E) Enlarged segments of Fig. 1A and B taken, respectively, from rats given LPS on P06 which were not subjected to seizure induction and from rats given LPS on P30 and experiencing seizures induced in adulthood. Scale bar: 500 μm. Examples of GFAP‐immunopositive astrocytes indicated by arrows in (A) and (E) are shown in (B–D) and (F‐H), respectively. The degree of ramification of astrocyte processes (branching index) was defined according to Garcia‐Segura & Perez‐Marquez (2014). Two circles of 25  and 50 μm diameter (B and F) were centered on the astrocyte cell body and intersections between the circles; the astrocyte processes were counted, and the ratio between number of intersections with the outer and inner circles was calculated.

Figure 5

Effects of LPS injections on the seizure latency (A) maximal seizure intensity (B) and sum of scores of maximal seizures for each of subsequent 10‐min periods during 6 h observation (C). Each graph shows the median (small black square), the 25–75% variability range (large box), and maximal and minimal values (whiskers). Decimal indexes over double‐headed arrows show statistical significance of differences between two animal groups (non‐parametric Mann–Whitney U‐test). Indexes in right top corners present levels of statistical significance of intergroup differences (non‐parametric Kruskal–Wallis test). An index in square brackets indicates statistical significance lower than 0.05. NSE, normal rats, L06 SE and L30 SE rats, injected with LPS alone on postnatal days 6 or 30, respectively, then subjected to pilocarpine‐induced seizures at the age of 2 months.

Figure 6

Glial fibrillary acidic protein (GFAP)‐immunopositive area fraction in the hippocampal formation of rats treated on P06 or P30 with LPS alone (A) or additionally injected with pilocarpine at the age of 2 months (B). The diagrams show mean values (small squares), standard error of mean (large boxes), and standard deviation (whiskers). A decimal index at the upper right corner shows one‐way anova P‐value. Indexes located over double‐headed arrows show statistical significance of differences between two groups (Spjotvoll/Stoline post hoc test). A P‐value within the range 0.05–0.1 is enclosed in brackets. N, normal rats, L06 and L30, rats injected with LPS on postnatal days 6 or 30, respectively. SE added to each of the symbols indicates animal groups which additionally experienced seizures induced with pilocarpine at the age of 2 months.

Figure 7

Morphological changes of glial fibrillary acidic protein (GFAP)‐immunopositive astrocytes in the CA3 (left column diagrams) and dentate gyrus (DG, right column diagrams) areas in 60‐day‐old rats. The diagrams show mean values (small squares), standard error of mean (large boxes) and standard deviation (whiskers). Decimal indexes at upper right corners show one‐way anova P‐values. Decimal indexes located over double‐headed arrows show statistical significance of differences between two animal groups (Spjotvoll/Stoline post hoc test). N, normal, untreated rats, L06 and L30, rats injected with LPS on postnatal days 6 or 30, respectively; L06 SE and L30 SE, rats injected with LPS on postnatal days 6 or 30, respectively, and experiencing seizures evoked on postnatal day 60. N SE, rats without an LPS pretreatment but experiencing seizures at the age of 2 months.

Figure 8

Expression of glial fibrillary acidic protein (GFAP), aquaporin 4 (AQP4), glutamine synthetase (GS) and Kir4.1 in the hippocampal formation of 60‐day‐old rats. (A‐D) Rats treated with LPS alone on postnatal days 6 (P6) or 30 (P30) and perfused at the age of 60 days (P60). (E‐H) Rats treated with LPS on P6 or P30 and, additionally, experiencing seizures evoked with pilocarpine on P60 then subjected to perfusion‐fixation. Western blotting data on protein expression levels are shown under the abscissa of each diagram and correspond with symbols of examined animal groups. The data are shown as means (small squares) with standard error of mean (large boxes) and standard deviation (whiskers). Values are given in percentages with 100% set as a mean value for the control group. Indexes at upper right corners show one‐way anova P‐values. Indexes located over double‐headed arrows show statistical significance of differences between two groups (Spjotvoll/Stoline post hoc test). A P‐value within the range 0.05–0.1 is shown in brackets. N, normal, untreated rats, L06 and L30, rats injected with LPS on postnatal days 6 or 30, respectively; L06 SE and L30 SE, rats injected with LPS on P6 or P30, respectively, and experiencing seizures evoked on P60. N SE, rats without an LPS pretreatment but experiencing seizures on P60.