Both authors equally contributed to this work.
FULL-LENGTH ORIGINAL RESEARCH
Inflammatory changes during epileptogenesis and spontaneous seizures in a mouse model of mesiotemporal lobe epilepsy
Article first published online: 28 SEP 2011
Wiley Periodicals, Inc. © 2011 International League Against Epilepsy
Volume 52, Issue 12, pages 2315–2325, December 2011
How to Cite
Pernot, F., Heinrich, C., Barbier, L., Peinnequin, A., Carpentier, P., Dhote, F., Baille, V., Beaup, C., Depaulis, A. and Dorandeu, F. (2011), Inflammatory changes during epileptogenesis and spontaneous seizures in a mouse model of mesiotemporal lobe epilepsy. Epilepsia, 52: 2315–2325. doi: 10.1111/j.1528-1167.2011.03273.x
- Issue published online: 2 DEC 2011
- Article first published online: 28 SEP 2011
- Accepted August 10, 2011; Early View publication September 28, 2011.
- Temporal lobe epilepsy;
- Top of page
- Materials and Methods
- Supporting Information
Purpose: Neuroinflammation appears as a prominent feature of the mesiotemporal lobe epilepsy syndrome (MTLE) that is observed in human patients and animal models. However, the precise temporal relationship of its development during epileptogenesis remains to be determined. The aim of the present study was to investigate (1) the time course and spatial distribution of neuronal death associated with seizure development, (2) the time course of microglia and astrocyte activation, and (3) the kinetics of induction of mRNAs from neuroinflammatory-related proteins during the emergence of recurrent seizures.
Methods: Experimental MTLE was induced by the unilateral intrahippocampal injection of kainate in C57BL/6 adult mice. Microglial and astrocytic changes in both ipsilateral and contralateral hippocampi were examined by respectively analyzing griffonia simplicifolia (GSA) lectin staining and glial fibrillary acidic protein (GFAP) immunoreactivity. Changes in mRNA levels of selected genes of cytokine and cytokine regulatory proteins (interleukin-1β, IL-1β; interleukin-1 receptor antagonist, IL-1Ra; suppressor of cytokine signaling 3, SOCS3) and enzymes of the eicosanoid pathway (group IVA cytosolic phospholipase A2, cPLA2-α; cycloxygenase-2, COX-2) were studied by reverse transcription-quantitative real time polymerase chain reaction.
Key Findings: Our data show an immediate cell death occurring in the kainate-injected hippocampus during the initial status epilepticus (SE). A rapid increase of activated lectin-positive cells and GFAP-immunoreactivity was subsequently detected in the ipsilateral hippocampus. In the same structure, Il-1β, IL-1Ra, and COX-2 mRNA were specifically increased during SE and epileptogenesis with a different time course. Conversely, the expression of SOCS3 mRNA, a surrogate marker of interleukin signaling, was mainly increased in the contralateral hippocampus after SE.
Significance: Our data show that specific neuroinflammatory pathways are activated in a time- and structure-dependent manner with putative distinct roles in epileptogenesis.
The mesiotemporal lobe epilepsy (MTLE) syndrome, one of the most common forms of focal epilepsies, has been suggested to result from an initial precipitating event (e.g., febrile seizures, cerebral infection, traumatic brain injury, or stroke) (Engel, 2001; Cendes et al., 2002), followed by a “silent” period, preceding the occurrence of recurrent seizures, several years later. This period of latency appears to be associated with different cascades of molecular and cellular processes, leading to the development of seizure-generating neuronal circuits, a process called epileptogenesis. Among the different mechanisms that may be involved in epileptogenesis, neuroinflammation appears as one of the most critical (de Lanerolle & Lee, 2005; Vezzani & Granata, 2005), as suggested in animal models (Vezzani & Baram, 2007) as well as in clinical reports (Ravizza et al., 2008).
In the brain, inflammation is characterized by glial activation, edema, and synthesis of inflammatory mediators such as cytokines or enzymes of the prostaglandin pathway. Experimental evidence has demonstrated a rapid-onset inflammatory response to acute seizures in various models that largely implicates interleukin-1β (IL-1β), among others, as a prototypic inflammatory signal (Eriksson et al., 1999; Vezzani et al., 1999; De Simoni et al., 2000; Plata-Salaman et al., 2000; Turrin & Rivest, 2004; Gorter et al., 2006; Ravizza et al., 2006; Dhote et al., 2007). Some reports have demonstrated that IL-1β alters neuronal excitability and may contribute to the pathophysiologic process of epilepsy (Vezzani et al., 2000; Vezzani & Baram, 2007). Unfortunately, in the brain, IL-1 receptor antagonist (IL-1Ra), which acts by limiting IL-1β-mediated actions, is generally produced much later than IL-1β, in contrast with peripheral inflammatory reactions (Dinarello, 1996; Vezzani et al., 2008). Some other regulatory mechanisms have evolved to limit the potentially harmful consequences of cytokine signaling. A major process implicates suppressors of cytokine signaling (SOCS), a family of proteins whose expression is induced by cytokines and that, in turn, negatively regulate signaling pathways used by many cytokines, thereby modulating a wide range of inflammatory processes and considered as key regulators of inflammation (Lang et al., 2003; Yoshimura et al., 2007; Croker et al., 2008). More precisely, SOCS3 is induced by a wide range of stimuli including interleukin-6 (IL-6) family and regulates the cytokine signaling via the inhibition of the Janus kinases/signal transducers and activators of transcription (JAK/STAT) transduction pathway in a negative feedback loop (Heinrich et al., 1998; Schmitz et al., 2000; Yasukawa et al., 2000). Although to date studies of the pattern of activation of SOCS family proteins in rodent epilepsy models remains to be specified, it was demonstrated that SOCS3 can be highly regulated by neuronal and nonneuronal cells during seizure activity, with a potential implication during the remodeling process of epileptogenesis (Rosell et al., 2003). Interestingly, it was demonstrated that some of the seizure-induced proinflammatory signals remain up-regulated during epileptogenesis (Voutsinos-Porche et al., 2004; Lee et al., 2007; Ravizza et al., 2008; Maroso et al., 2010) and are likely to play a major role in the establishment of recurrent epileptic seizures (Ravizza et al., 2011).
In the present study, we aimed at further characterizing the precise and spatial temporal patterns of neuroinflammatory events that are associated with MTLE development. We thus used intrahippocampal kainate (KA) in mice, which is characterized by unilateral histologic changes reminiscent of hippocampal sclerosis often observed in human MTLE (Suzuki et al., 1995). In these mice, a single unilateral injection of KA into the hippocampus first induces status epilepticus (SE) lasting for up to 15 h (Riban et al., 2002). Then, during the next 2 weeks, recurrent spontaneous ipsilateral hippocampal discharges progressively develop to become stable for the lifetime of the animals (Heinrich et al., 2011). These recurrent seizures are largely confined to the ipsilateral, sclerotic hippocampus, but can also be detected in the contralateral hippocampus (Bouilleret et al., 1999; Riban et al., 2002; Meier et al., 2007). Here, we investigated and compared in both hippocampi (1) the time course and spatial distribution of neuronal death associated with seizure development, (2) the timing and regional activation of astrocytes and microglia, and (3) the spatiotemporal pattern of expression of the mRNAs of neuroinflammation markers, such as IL-1β, IL-1Ra, and SOCS3, as well as two enzymes of the eicosanoids pathway: group IVA cytosolic phospholipase A2 form (cPLA2-α) and cycloxygenase-2 (COX-2).
Materials and Methods
- Top of page
- Materials and Methods
- Supporting Information
Intrahippocampal injection of kainate
C57BL/6 male mice of 8–10 weeks of age were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic frame. A solution of 50 nl of KA (20 nm) in physiologic saline was injected in the dorsal hippocampus (see Text S1). Upon recovery from anesthesia, animals injected with KA displayed mild clonic movements of the forelimbs, rotations, and immobility that lasted for up to 15 h as described before (Riban et al., 2002). Because 100% of mice that experienced SE develop spontaneous recurrent seizures (Heinrich et al., 2006, 2011), only data from animals with a characteristic SE were included in the present study.
Within 2 h after the injection of KA, six mice implanted with hippocampal and cortical electrodes were placed in acrylic glass test cages and were connected to a digital video–electroencephalography (EEG) recording device (Coherence, Deltamed, France; sampling rate = 256 Hz). Continuous EEG and video acquisition was then performed for 24 h, under red light conditions during the dark period (12/12 h, light on at 7:00 a.m.), while the animals were freely moving.
Immunohistochemistry and histochemistry
Mice were deeply anesthetized with an overdose of pentobarbital (80 mg/kg, i.p.) at set times after intrahippocampal microinjection (2 and 24 h, and 3, 7, and 21 days). Tissue sections of each injected brain were analyzed after hemalun-phloxin (H&P) staining (Lillie & Fullmer, 1976) using a standard protocol (Baille et al., 2005) in order to verify (1) the correct location of the injection site, (2) the right position of the bipolar electrode, and (3) the occurrence of neuronal death in the dorsal hippocampus. Detection of DNA fragmentation was performed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique described previously (Gavrieli et al., 1992). Microglial cells were detected with peroxidase-labeled isolectin B4 according to a previously described method (Streit, 1990). Astrocytes were immunostained using an antibody directed against glial fibrillary acidic protein (GFAP).
RNA quantification of selected neuroinflammatory markers
Saline and KA-injected mice were killed by decapitation at set time points, that is, 5 h, and 1, 2, 7, and 21 days after hippocampal injection (n = 5 at each time and per condition). Because of the massive changes in the hippocampal structure of KA-injected mice (see Results), the number of animals sampled at key time points, that is, SE (5 h), epileptogenesis (7 days), and recurrence of seizures (21 days) was increased to 11 animals. Six naive animals were also sampled at 5 h (n = 6) (see Text S1 and Table S1).
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- Materials and Methods
- Supporting Information
Electroencephalographic recordings of the status epilepticus following intrahippocampal kainate injection
It was previously shown that a single unilateral injection of KA into the hippocampus of adult mice induces an initial SE, which is followed by a latent period of 2–3 weeks before the occurrence of spontaneous discharges largely confined to the injected hippocampus (Riban et al., 2002; Meier et al., 2007; Heinrich et al., 2011). To further determine the relation between EEG paroxysmal activities and the neuroinflammatory changes, we first analyzed the EEG activities occurring in both hippocampi during the initial SE. To this aim, EEG activity was continuously recorded in KA-injected mice (n = 6) in both injected and contralateral hippocampi, as well as in the ipsilateral and contralateral frontoparietal cortices, starting upon awakening from anesthesia (90–110 min after injection) and lasting for 24 h. In addition, animals were recorded at 21 days post-KA injection during the chronic phase of the disease when recurrent seizures can be observed (Riban et al., 2002; Heinrich et al., 2006).
For the entire duration of the recording, lower amplitude background activity was observed in the injected hippocampus as compared to the contralateral one. This was evidenced by a peak-to-peak amplitude analysis of the signal, representing on the ipsilateral side 35.8 ± 10.9% of the contralateral amplitude, throughout the 24 h recording (n = 6; Fig. 1A). As soon as the EEG recording was started, isolated spikes and bursts of spikes were observed regularly in both hippocampi and often in cortices at a frequency between 0.5 and 4 Hz. These spikes were observed 15–18 h post-KA and had higher amplitude in the contralateral hippocampus. Discharges of spikes and polyspikes were later observed (i.e., 3–5 h postinjection) and for up to 18 h, with a variable recurrence between animals (1–5 per hour; n = 6; Fig. 1A). They first occurred concomitantly in both hippocampi with higher amplitude in the contralateral side, and then they generally spread to the cortices (Fig. 1A). These discharges lasted about 20–40 s, during which time the animals remained immobile. In contrast, a few discharges of spikes, polyspikes, and spike-and-waves were observed in four animals, occurring concomitantly in both hippocampi and cortices, preceded by several sharp waves and lasting for up to 60 s (Fig. 1B). These discharges were associated with asymmetric clonic seizures of the forelimbs and, in some cases, rearing of the animals. Fully generalized tonic–clonic seizures were rarely observed. When recorded 21 days later, all mice displayed recurrent discharges in the injected hippocampus, as described previously (Riban et al., 2002; Heinrich et al., 2011).
Histopathologic changes associated with the initial status epilepticus, epileptogenesis, and spontaneous recurrent seizures
Neuronal cell death has been observed previously in this model in the ipsilateral hippocampus after KA injection. Indeed, H&P staining performed on hippocampal sections obtained from the first set of mice that were EEG-recorded (Fig. 1) and sacrificed at 21 days post-KA injection revealed a significant cell loss in the CA1, CA3, and hilus areas of the injected hippocampus (n = 6, Fig. 2A) as reported previously (Suzuki et al., 1995; Bouilleret et al., 1999; Riban et al., 2002; Heinrich et al., 2006). In contrast, no ultrastructural changes were detected in the contralateral hippocampus (n = 6, Fig. 2B).
To further investigate the time course and spatial distribution of this cell death, we next performed a TUNEL technique on hippocampal sections from a new set of treated mice sacrificed at 2 and 24 h and 7 days after injection (n = 3, 3, and 4, respectively). Saline-injected mice were used as control animals (n = 2). At 2 h post-KA, TUNEL positivity was observed in the injected hippocampus in the CA1 and CA3 areas (n = 3; Fig. 2C). TUNEL-positive cells were localized among pyramidal neurons in the CA1 (Fig. 2D) and CA3 areas (data not shown), whereas no labeling was observed in the CA2 area or the dentate gyrus. At 24 h (n = 3) and 7 days (n = 4) post-KA, TUNEL positivity was observed in the injected hippocampus with a similar pattern within the same areas (data not shown). However, the number of TUNEL-positive cells was reduced, suggesting the removal of the dead cells by activated microglia (see below). In contrast, in the contralateral noninjected side (Fig. 2E), no TUNEL positivity was observed in any area of the hippocampus, even at higher magnification (Fig. 2F), at any of the time points investigated.
We also assessed the extent and the time course of the cell death by H&P staining performed on hippocampal sections from mice not equipped with EEG and sacrificed at various time points after KA injection during the epileptogenesis process (3 and 7 days post-KA, n = 5 each) and during the chronic phase when stable and spontaneous recurrent seizures can be recorded (21 days post-KA, n = 5). Saline-injected mice sacrificed at similar time points were used as control animals (n = 5, each time point). No damaged cells were detected with H&P staining in saline-injected controls at any time (data not shown). In contrast, after KA injection, neurodegeneration characterized by condensed H&P-stained cells were detected in the CA1, CA3, and hilus areas at 3 days (n = 5, Fig. 2G,H) and 7 days (n = 5, Fig. 2K,L). During the chronic phase, no more injured neurons could be detected (n = 5, Fig. 2O,P). In sharp contrast, no neurodegenerative events were ever found in the contralateral hippocampus at any time point investigated (Fig. 2I,J,M,N,Q,R), in agreement with previous observations (Suzuki et al., 1995; Riban et al., 2002).
Our results confirmed the well-established pattern of neuronal damage occurring in mouse hippocampus after intrahippocampal KA injection as assessed by TUNEL and H&P staining (Suzuki et al., 1995; Bouilleret et al., 1999). Interestingly, our TUNEL data reveal that cell death, involving DNA fragmentation, occurs acutely in the ipsilateral hippocampus as soon as 2 h following KA injection.
Astrocytic changes during epileptogenesis and spontaneous recurrent seizures
Reactive astrogliosis is a ubiquitous hallmark that has been associated with neuronal loss occurring both after acute injuries and in chronic neurodegenerative disorders (Sofroniew, 2009; Robel et al., 2011). In particular, reactive astrogliosis has been associated with epilepsy (Seifert et al., 2006, 2010). Based on the evidence of cell death in the epileptic, injected hippocampus, we next investigated the time course and spatial distribution of astrocyte activation in both the ipsilateral and the contralateral noninjected hippocampus during epileptogenesis and during the chronic phase of the disease. To this aim we performed a GFAP immunohistochemistry on hippocampal sections of saline- and KA-injected mice 3, 7, and 21 days after injection (n = 5 at each time and per condition). The maximal intensity of GFAP-immunoreactivity was detected in the injected hippocampus 3 days after KA injection (n = 5, Fig. 3B) as compared to saline-injected controls (n = 5, Fig. 3A). Interestingly, at this time point, GFAP immunoreactivity was not detected in areas undergoing intensive neurodegenerative process (CA1, CA3, hilus; n = 5, Fig. 3B), whereas at 7 days (n = 5), GFAP-positive cells were observed in the whole injected dorsal hippocampus (Fig. 3D), leading to a pronounced astroglial scar at 21 days (n = 5, Fig. 3F). Despite the absence of detectable cell death in the contralateral hippocampus, numerous GFAP-stained cells were detected at 3 days (n = 5; Fig. 3C) compared to saline-injected controls (Fig. 3A). This state of activation returned to control levels at 7 and 21 days (n = 5, Fig. 3E,G).
Our data show that intrahippocampal KA injection stimulates an activation of the astroglial cells, which can be observed in both hippocampi, but more pronounced in the injected side in agreement with previous reports (Bouilleret et al., 1999; Heck et al., 2004; Heinrich et al., 2006). In addition, this reactive astrogliosis tends to decrease over time in the contralateral side, whereas it is maintained in the injected hippocampus.
Microglial changes during epileptogenesis
We next hypothesized that microglial cells could also be strongly activated, specifically in the ipsilateral hippocampus in response to cell death that we and others evidenced after KA injection. To test this hypothesis, we performed lectin staining on hippocampal sections of saline- and KA-injected mice during epileptogenesis and the chronic phase at 3, 7, and 21 days after injection, respectively (n = 5 at each time and per condition). In saline-injected controls, only a small number of lectin-positive cells with numerous and thin processes characteristic of the resting state of microglia were distributed homogeneously in the hippocampus (n = 5; Fig. 3H). In contrast, in the ipsilateral hippocampus, the lectin-positive cells at 3 days post-KA showed large and round cell bodies with short and thick processes, which are characteristic features of active microglia and suggest phagocytosis activities by these microglial cells (n = 5; Fig. 3I). Accordingly, these cells were located mainly in the neurodegenerative areas, that is, the CA1 and CA3 areas and the hilus. At 7 and 21 days after KA injection, lectin-staining persisted but round-shaped cells could no longer be observed (Fig. 3K,M). To our surprise, the lectin staining revealed in the contralateral hippocampus a transient increase in the number of positive cells at 3 days (n = 5, Fig. 3J) compared to saline-injected controls. They either exhibited a ramified shape with thin processes, characteristic of the resting state, or a denser core, characteristic of the activated state. At later time points (7 and 21 days), lectin staining in the contralateral side of KA-injected animals was not distinguishable from saline-injected mice (n = 5 per time point, Fig. 3L,N). Our data confirm that activation of microglial cells following intrahippocampal KA injection is structure dependent, reaches a maximal state of activation during the early phase of epileptogenesis, and persists in the ipsilateral hippocampus during the epileptogenesis process.
Time course of neuroinflammation marker expression during status epilepticus, epileptogenesis, and spontaneous recurrent seizures
Our study confirms the activation of astrocytes and microglial cells following intrahippocampal KA injection and gives some insight into the differential time course characteristics of these cellular events during epileptogenesis and spontaneous seizures in the two hippocampi. We next investigated whether mRNAs of cytokines and other neuroinflammation proteins could be detected in the both hippocampi during the initial SE, epileptogenesis, and the chronic phase. To this aim, we performed quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) to delineate the fine kinetic pattern of mRNA expression levels of IL-1β, IL-1Ra, SOCS3, cPLA2-α, and COX-2 on hippocampal extracts of saline- and KA-injected mice sacrificed at 5 h, and 1, 2, 7, and 21 days after injection (n = 11, 5, 5, 11, and 11, respectively). We detected a significant increase in the relative amount of IL-1β, IL-1Ra, SOCS3, and COX-2 mRNAs in the ipsilateral hippocampus between 5 and 24 h (Fig. 4A–D). For Il-1β, the maximal changes of transcript levels appeared at 5 h (×2.4, Fig. 4A), whereas the increase in the relative quantity of IL-1Ra mRNA was delayed (×3.9 at 24 h, Fig. 4B). For SOCS3 and COX-2 mRNAs, they reached a maximum at 5 h (×11.7 and ×9.1, respectively) and remained elevated at 24 h (×6.0 and ×4.5, respectively; Fig. 4C,D). At later time points, there was a trend toward a decrease of the mRNA levels of IL-1β, IL-1Ra, SOCS3, and COX-2 mRNAs (Fig. 4A–D). However, it is important to note that a delayed increase of IL-1β and IL-1Ra transcripts was also detected during epileptogenesis at 7 days (approximately ×2.5; Fig. 4A,B). All neuroinflammatory-related mRNAs returned to control level by 21 days. Some major changes were also detected in the contralateral hippocampus. A significant increase of the relative mRNA amount of COX-2 was evidenced at 5 h compared to the control levels (×3.8, Fig. 4D). It is important to note that a pronounced and delayed up-regulation of SOCS3 transcript was detected in the contralateral side at 24 and 48 h (approximately ×10, Fig. 4C). No variations of cPLA2-α mRNA amount were detected at any time points investigated following intrahippocampal KA injection (Fig. 4E).
Our results show that IL-1β and COX-2 are immediately up-regulated in the KA-injected hippocampus, suggesting that they are key mediators of neuroinflammation, whereas IL-1Ra, which shows a delayed increase, may rather contribute to the attenuation of the inflammation process. Interestingly, there was also evidence of an early and prominent increase in SOCS3 mRNA in the ipsilateral hippocampus as well as a delayed but substantial up-regulation of SOCS3 expression in the contralateral side that likely reflects an attempt to restrict neuroinflammation in this area.
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- Materials and Methods
- Supporting Information
Using the KA model for MTLE in mice, our results confirm that specific inflammatory processes take place during epileptogenesis (Ravizza et al., 2008; Auvin et al., 2010; Maroso et al., 2010). In particular, our data show that most of these changes occur in the injected hippocampus where cell loss and histologic changes occur (Suzuki et al., 1995; Bouilleret et al., 1999) and where recurrent seizures have been shown to be initiated (Meier et al., 2007). In addition, our data also reveal neuroinflammatory processes in the contralateral, presumably intact, hippocampus.
The profound astrocytic reactivity observed in the ipsilateral hippocampus in the present study confirmed previous reports in the same MTLE model (Suzuki et al., 1995; Bouilleret et al., 1999; Heinrich et al., 2006). Such a reaction is a hallmark of MTLE and is classically observed in both animal models and resected tissues from patients with MTLE (Crespel et al., 2002; Ravizza et al., 2008). We observed here a pronounced activation of astrocytes during epileptogenesis that remains during the chronic phase of the disease. An explanation for this observation could be a late proliferation of astroglial cells in agreement with previous reports (Heinrich et al., 2006; Nitta et al., 2008). Such an activation of astroglial cells may contribute to neuronal network hyperexcitability underlying recurrent seizure development in the ipsilateral hippocampus (Volterra & Meldolesi, 2005; Seifert et al., 2006).
Our study also revealed highly activated microglial cells that were associated with the presence of degenerating neurons in the injected hippocampus, as previously reported in the same MTLE model (Suzuki et al., 1995; Heinrich et al., 2006). This was also previously documented in other epilepsy models (Borges et al., 2003; Shapiro et al., 2008) as well as in human epilepsy patients (Ravizza et al., 2008). Microgliosis was essentially detected during the initial phase of epileptogenesis (i.e., the first 3 days after SE) and persists in ipsilateral hippocampus, suggesting a role in the development of recurrent seizures.
Despite their prominent function in the maintenance of neuronal function and homeostasis, the activation of astrocytes and microglial cells likely induces the secretion of cytokines, such as IL-1β (Vezzani et al., 1999, 2008) and inflammation-related proteins that may exacerbate the pathology (Vezzani & Baram, 2007). In our study, the relative quantity of IL-1β mRNA was increased in a time-specific manner in the ipsilateral hippocampus, in agreement with previous studies (Eriksson et al., 1999; Vezzani et al., 1999). The mRNA of the natural antagonist of IL-1 receptor, IL-1Ra, was increased in the ipsilateral hippocampus several hours after IL-1β, as reported previously in various models of limbic seizures (Vezzani et al., 2002) that seems to be a hallmark of epileptic process (Eriksson et al., 1999; Lehtimaki et al., 2010). In contrast, limited changes in the mRNA of key enzymes of the eicosanoid pathways were observed in our study. The bilateral increase of COX-2 observed 5 h post-KA could be secondary to the hyperactivity associated with SE on both sides, as suggested earlier (Chen et al., 1995; Adams et al., 1996). Finally, cPLA2-α mRNA levels were not changed significantly over the period of our study. This result is surprising, as the eicosanoid pathway is known to be involved in inflammation processes. To our knowledge, no study has focused on cPLA2-α mRNA changes during KA-induced seizures, but the protein was already evidenced following systemic administration of KA in rats (Sandhya et al., 1998). It is possible that other types of PLA2, such as some of the secretory enzymes, are more involved and maybe up-regulated to fuel the eicosanoid pathway known to be activated during seizures (Bazan et al., 2002).
Using this MTLE model with a well-defined, unilateral focus, it was possible to compare the sclerotic injected hippocampus with the contralateral one, where seizures may occur, but where cell loss or tissular reorganization is never observed (Suzuki et al., 1995; Bouilleret et al., 1999). In the injected hippocampus where neuronal cell death was observed in the pyramidal layer and the hilus, proinflammatory cytokines (mainly represented by IL-1β signaling), were increased and could participate in this cell injury process during SE. Indeed, previous studies have observed neurotoxic properties of IL-1β (Vezzani et al., 2000; Bernardino et al., 2005), probably by action on glutamate receptors (Viviani et al., 2003). In addition, the active contribution of neuroinflammation in cell injury processes during SE was demonstrated with KA administered intracerebroventricularly (Kwon et al., 2010) and intraperitoneally (Heida et al., 2005; Auvin et al., 2007).
By contrast, in the contralateral hippocampus, activation of microglia and astrocytes was evident only during the initial phase of epileptogenesis. SOCS is a family of intracellular proteins controlling the magnitude and/or duration of signals propagated by diverse cytokine receptors by suppressing their signal transduction process (Kovanen & Leonard, 1999) and thus represent an intracellular negative feedback loop that is essential for efficient cytokine signaling. We found a large increase in SOCS3 mRNA in contralateral hippocampus with a slight delay compared to ipsilateral structure that suggests a differential activation of cytokine signaling in the MTLE mouse model during the early phase of epileptogenesis. SOCS3 mRNA production being a surrogate marker of the presence of specific cytokines such as IL-6 (Fujimoto & Naka, 2003; Lang et al., 2003), this finding could suggest an in situ production of this cytokine. Given the major role of IL-6 in the orchestration of neuroinflammation (Spooren et al., 2011), the delayed up-regulation in the contralateral hippocampus of SOCS3, which acts by limiting IL-6 mediated actions, may indicate an attempt to limit the intensity and the duration of neuroinflammatory signals during the early phase of epileptogenesis in this area. Although this remains to be demonstrated, this massive and delayed contralateral increase of SOCS3 mRNA might be involved in the neuronal survival and/or the induction of homeostatic mechanisms against neurodegeneration by limiting cytokine signaling in this area.
Taken together, our results confirm that inflammatory events occur during MTLE epileptogenesis (Ravizza et al., 2008; Maroso et al., 2010) and precede the onset of recurrent focal seizures. Notably, the differential activation of the IL-1 system between ipsilateral and contralateral hippocampus may contribute to the development of recurrent seizures by increasing neuronal excitability and promoting cell loss in the injected hippocampus (Vezzani et al., 1999, 2000; Allan et al., 2005). The potential pathophysiologic role of IL-1β in MTLE was recently evidenced in the same mouse model of MTLE by a reduction of epileptic activity during the chronic phase of the disease following chronic inhibition of IL-1β biosynthesis (Maroso et al., 2011). Some of our results indicate equally the involvement of specific neuroinflammatory signals such as SOCS3 mRNA changes in the two hippocampi, suggesting a potential failure in the expression of regulators of inflammation during the early phase of epileptogenesis. In conclusion, our data indicate a clear difference in spatiotemporal involvement of neuroinflammatory processes during SE, epileptogenesis, and spontaneous recurrent seizures in the epileptogenic tissue and its contralateral counterpart in this unilateral mouse model of MTLE. This suggests that specific neuroinflammatory pathways could have distinct roles in epileptogenesis process and homeostatic mechanisms.
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The authors are grateful to Guy Testylier and Annie Foquin for their helpful and constructive discussions before, during, and after the experiments. This work was supported by the French Ministry of Defence/DGA: research grant 03CO011-05 and O8CO502 to F. Dorandeu and D4S/MRIS PhD grant to F. Pernot.
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None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
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Text S1. This file describes in details supporting information on Materials and Methods that was not included in the article.
Table S1. This file describes in details the mouse primers used for RT-qPCR in this study.
|EPI_3273_sm_TableS1.doc||34K||Supporting info item|
|EPI_3273_sm_TextS1.doc||68K||Supporting info item|
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