As previously reported, all mice subject to intra-amygdala KA-induced SE go on to develop spontaneous recurrent seizures (epilepsy) after a short latent period. These epileptic seizures emerge 3 -5 days post KA-injection and occur at a frequency of 1 - 5 per day (Mouri et al., 2008a) (Figure 4.2 A). However, no previous study has presented data to support the involvement of the hippocampus during these spontaneous seizures.
As supporting evidence of recent intense neuronal firing, the expression of the activity-regulated immediate early genes C-fos and Arc was measured within microdissected ipsilateral CA1, CA3 and DG hippocampal subfields in epileptic mice (14 days after SE) using RT-qPCR. All experimental samples were assessed versus time-matched non-seizure vehicle controls. C-fos and Arc expression was significantly higher in all hippocampal subfields in epileptic animals when compared to control non-epileptic mice (Figure 4.2 B, C).
To further characterise neuropathological changes in the epileptic hippocampus, we assessed protein levels of markers of neurons and glia. Western blot analysis of hippocampal lysates showed increased levels of glial fibrillary acidic protein (GFAP) in the CA3 subfield in epileptic tissue when compared to control animals (Figure 4.2 D, F). In contrast, NeuN levels, a specific neuronal marker, were lower in the CA3 hippocampal subfield of epileptic mice compared to controls (Figure 4.2 E, G). As the CA1 and DG hippocampal subfields are spared damage in this model, neuropathological analyses were not performed in these areas (Engel et al., 2012).
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Figure 4.2 Recruitment of the ipsilateral hippocampus during epileptic seizures and presence of astrogliosis and hippocampal sclerosis
A) Cortical electrodes placement for EEG recordings. B) Typical electrographic spontaneous
seizure recorded with a telemetry device in a mouse 14 days after i.a. KA-induced SE. C) Higher resolution EEG trace during spontaneous seizure. D, E) Real-time qPCR measurement of neuronal activity regulated early-genes c-fos and Arc (normalized to actin) for CA1, CA3 and DG hippocampal subfields of control and epileptic mice (n = 9 mice per group). E, F) Western blots showing GFAP and NeuN protein levels in CA3 hippocampal subfield of control and epileptic mice (n = 5 mice per group, n = 1 sample per lane). α- Tubulin is shown as loading control. G, H) Densitometry analysis of the CA3 levels of GFAP and NeuN protein. T-test. *p < 0.05. **p < 0.01. All data are mean ± s.e.m.
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4.3.3 Increased molecular markers of inflammation in the hippocampus in epilepsy
Inflammation is a common pathophysiological response after seizures and previous studies reported that microglia and astrocytes are the primary source of cytokines (Vezzani, 2005). To explore whether inflammatory responses occur in this model of epilepsy, the levels of Iba-1, as a microglia marker, GFAP, as an astrocyte marker, and IL-1β and TNF-α as pro-inflammatory cytokines were examined. All genes analysed showed a significant increase in mRNA levels in the CA1, CA3 and DG hippocampal subfields from epileptic animals when compared to controls. (Figure 4.3 A, B, C, D).
To support these results, Western blot analysis was performed for some of these markers. Results showed an increase of the microglia marker Iba-1 in the whole hippocampus (Figure 4.3 E, G) and mature IL-1β in each hippocampal subfield of epileptic mice when compared to time-matched control mice (Figure 4.3 F, H).
In agreement with Western blot data, immunofluorescence analysis of Iba-1 showed a remarkable enhanced microglia expression in the hippocampus of epileptic mice, particularly in the CA1 and CA3 subfields when compared to control (Figure 4.4 A, B).
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Figure 4.3 Increased molecular markers of inflammation in the hippocampus in epilepsy
A, B, C, D) Real-time PCR analysis of microglia activation (Iba-1, IL-1β and TNF-α) and astroglial
(GFAP) messenger RNA levels in CA3, CA1 and dentate gyrus (DG) hippocampal subfields of control and epileptic mice (n=9 per group). E) Representative western blots (n=1 per lane) of microglia marker, Iba-1 in the whole hippocampus and F) IL-1β levels in CA1, CA3 and DG hippocampal subfields in control and epilepsy (n=4 per group). α-Tubulin is shown as loading control. G, H) Densitometry analysis of Iba-1 and IL-1β protein levels. T-test.*p < 0.05.**p < 0.01.***p<0.001.
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A
B C
Figure 4.4 Increased microglia expression in the hippocampus in epilepsy
A, B) Representative Iba-1 staining of whole hippocampus from control and epileptic mice.
Scale bar = 500 μm. White boxes show magnification views of Iba-1 staining in microglia cells in CA1 and CA3 of the hippocampus. Scale bar = 100 μm. C) Representative negative control of the CA3 area from epileptic mice. Scale bar = 100 μm.
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4.3.4 Up-regulation of P2X receptors in the hippocampus in epilepsy
To examine whether P2XRs are altered in the present model of epilepsy, qPCR was used to first examine the expression of transcripts for P2rx1, P2rx2 and P2rx4 due to their alteration after SE published in previous studies (Dona et al., 2009; Engel et al., 2012b; Ulmann et al., 2013).
P2rx1 mRNA levels were increased in all hippocampal subfields in epileptic mice (Figure 4.5 A). In contrast, P2rx2 mRNA showed a decrease in CA1 only; no differences between groups were observed for CA3 and DG hippocampal regions (Figure 4.5 B). The P2rx4 transcript showed the highest increase in the hippocampus in epilepsy, with a 4 – 7 fold increase in all hippocampal subfields when compared to levels in control animals (Figure 4.5 C).
To support these mRNA findings, protein levels of these receptors were analysed by Western blot. Protein levels of P2X1R and P2X2R were not different between epilepsy and control samples for any subfields (Figure 4.6 A, B, C, D). For the P2X4R, protein levels were higher only in the CA1 and CA3 when compared to controls (Figure 4.6 E, F).
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Figure 4.5 Altered P2XR transcript levels in the hippocampus in epilepsy
A,B,C) Real-time qPCR measurement of P2rx1, P2rx2 and P2rx4 mRNA levels for the CA1,
CA3 and DG hippocampal subfields of control and epileptic mice (n = 9 per group). T-test. *p < 0.05. **p<0.01. ns-not significant.
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Figure 4.6 Altered hippocampal P2XR levels in epilepsy
A, C, E) Western blots (n=1/lane) showing expression of different P2XR subtypes (P2X1R,
P2X2R and P2X4R) within the 3 main subfields of the hippocampus in vehicle-injected control and epileptic mice (n=9 per group). α-Tubulin is shown as guide to loading. B, D, F) Graphs showing semi-quantification analysis of each protein in all hippocampal subfields in vehicle-injected control and epileptic animals. T-test. *p< 0.05. ns-not significant.
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