SEARCH

SEARCH BY CITATION

Keywords:

  • Microglia;
  • Astrocytes;
  • Cytokines;
  • Temporal lobe epilepsy;
  • Mouse

Summary

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. 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

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. 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.

Video–electroencephalography recordings

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).

Results

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. 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).

image

Figure 1.   Electroencephalography (EEG) recordings during nonconvulsive status epilepticus (SE) after intrahippocampal kainate (KA) injection in mice. Panels A and B depict typical bilateral EEG recordings in the ipsilateral and contralateral hippocampi and cortices during nonconvulsive SE at 6 h (A, n = 6) and 12 h (B, n = 6) following the unilateral intrahippocampal injection of KA. (A) Example of a nonconvulsive discharge. (B) Example of a discharge associated with asymmetrical clonus of the forelimbs. HIP, hippocampus; CX, cortex. Calibration: 2 s, 500 μV.

Download figure to PowerPoint

In agreement with previous studies (Riban et al., 2002; Meier et al., 2007), these data showed that early SE after KA injection occurred in both the ipsilateral and contralateral hippocampi (<24 h post KA), whereas later, paroxysmal discharges were largely confined to the ipsilateral hippocampus.

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).

image

Figure 2.   Neuronal damage after intrahippocampal kainate (KA) injection in mice. Representative photomicrographs of ipsilateral (A) and contralateral (B) hippocampal sections of mice EEG-recorded (Fig. 1) that were sacrificed at 21 days post-KA injection and stained with H&P. A significant cell loss in the CA1, CA3, and hilus areas was observed in the injected hippocampus (A, n = 6), In contrast, no ultrastructural changes were detected in the contralateral hippocampus (B, n = 6). Panels CR depict representative photomicrographs showing neuronal damage at various time points following intrahippocampal KA injection. Neuronal damage was assessed by TUNEL technique (CF) and H&P staining (GR). At 2 h post KA, TUNEL positivity was observed in the injected hippocampus in the CA1 and hilus areas (C, n = 3). Higher power image resolution revealed that TUNEL-positive cells were localized among pyramidal neurons in CA1 (D). No neuronal damage was observed in the contralateral hippocampus (E) and CA1 area, even at a higher magnification (F). Note the increase in acidophilic cells in the ipsilateral CA1 area during epileptogenesis at 3 days (G, H; n = 5) and 7 days (K, L; n = 5). In ipsilateral hippocampus and during the chronic phase (21 days post-KA, n = 5 O, P), condensed cells were absent, suggesting cell loss. Note the massive dispersion of granule cell layer at this time point. Contralateral hippocampus displayed a normal histology of the hippocampal formation whatever the time point (I, M, Q), even at a higher magnification (J, N, R). Scale bars: 100 μm.

Download figure to PowerPoint

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).

image

Figure 3.   Temporal profile of astroglial and microglial changes following intrahippocampal kainate (KA) injection in mice. Astrocytic reactivity was revealed by glial fibrillary acidic protein (GFAP) immunohistochemistry in saline-injected (A) or in KA-injected mice at 3 days (B, C; n = 5), 7 days (D, E; n = 5), and 21 days (F, G; n = 5) in ipsilateral (B, D, F) and respective contralateral hippocampi (C, E, G). High magnification images depicting GFAP-stained cells morphology are reported on the right side of each photomicrograph. Panel A shows immunoreactivity of astrocytes with thin processes denoting their resting state. Panels B and C show enhanced GFAP immunostaining in astrocytes exhibiting hypertrophic cell bodies, and long and thick processes. Note the absence of GFAP staining in areas undergoing massive neurodegeneration in ipsilateral hippocampus (B). Astroglial activation is enhanced at 7 days in ipsilateral hippocampus (D) but decreased in contralateral hippocampus (E). Highly activated astrocytes were still present during the chronic phase in the ipsilateral hippocampus (F). Scale bar: 100 μm. Microglial cells were revealed by histochemistry using GSA-IB4 lectin in saline-injected (H, n = 5) or in KA-injected mice at 3 days (I, J; n = 5), 7 days (K, L; n = 5), and 21 days (M, N; n = 5). High magnification images depicting microglia-like cells are reported on the right side of each photomicrograph. Panel H shows resting microglia-like cells with small cell bodies and ramifications in saline-injected mice. Panel I shows strongly positive microglia-like cells with hypertrophy of processes and occasionally phagocytic features. Activated microglia-like cells were equally detected in the contralateral hippocampus (J). At longer time points (7 and 21 days), lectin-staining persisted in the ipsilateral hippocampus but the round-shaped cells of the phagocytic type were absent (K, L, M, N). Scale bar: 100 μm.

Download figure to PowerPoint

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).

image

Figure 4.   Changes in the expression of mRNAs of proinflammatory markers and associated regulatory proteins in ipsilateral and contralateral hippocampi at different time points following unilateral intrahippocampal kainate (KA) injection. RT-qPCR analysis was performed in total RNA extracts from ipsilateral (blue bars) and contralateral (yellow bars) hippocampus of mice unilaterally injected with either saline or KA in the hippocampus at 5 h (saline, n = 5; KA, n = 11), 24 h (saline, n = 5; KA, n = 5), 48 h (saline, n = 5; KA, n = 5), 7 days (saline, n = 5; KA, n = 11), and 21 days (saline, n = 5; KA, n = 11). Naive mice were only anesthetized and sampled at 5 h (n = 6). For each mouse, the expression levels were normalized relative to the geometric mean of levels of Hprt1, Ppia, Arbp, and Tbp mRNAs and fold differences were calculated relative to the control group for each marker. *p < 0.05, **p < 0.01 compared to the control group; ND, not detected, (n = 5–11 in each time group). Note the change in the scale for COX-2 and SOCS3.

Download figure to PowerPoint

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.

Discussion

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. 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.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

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.

Disclosure

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

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.

References

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
  • Adams J, Collaco-Moraes Y, de Belleroche J. (1996) Cyclooxygenase-2 induction in cerebral cortex: an intracellular response to synaptic excitation. J Neurochem 66:613.
  • Allan SM, Tyrrell PJ, Rothwell NJ. (2005) Interleukin-1 and neuronal injury. Nat Rev Immunol 5:629640.
  • Auvin S, Shin D, Mazarati A, Nakagawa J, Miyamoto J, Sankar R. (2007) Inflammation exacerbates seizure-induced injury in the immature brain. Epilepsia 48(Suppl. 5):2734.
  • Auvin S, Mazarati A, Shin D, Sankar R. (2010) Inflammation enhances epileptogenesis in the developing rat brain. Neurobiol Dis 40:303310.
  • Baille V, Clarke PG, Brochier G, Dorandeu F, Verna JM, Four E, Lallement G, Carpentier P. (2005) Soman-induced convulsions: the neuropathology revisited. Toxicology 215:124.
  • Bazan NG, Tu B, Rodriguez de Turco EB. (2002) What synaptic lipid signaling tells us about seizure-induced damage and epileptogenesis. Prog Brain Res 135:175185.
  • Bernardino L, Xapelli S, Silva AP, Jakobsen B, Poulsen FR, Oliveira CR, Vezzani A, Malva JO, Zimmer J. (2005) Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures. J Neurosci 25:67346744.
  • Borges K, Gearing M, McDermott DL, Smith AB, Almonte AG, Wainer BH, Dingledine R. (2003) Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model. Exp Neurol 182:2134.
  • Bouilleret V, Ridoux V, Depaulis A, Marescaux C, Nehlig A, Le Gal La Salle G. (1999) Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy. Neuroscience 89:717729.
  • Cendes F, Kahane P, Brodie M, Andermann F. (2002) The mesio-temporal lobe epilepsy syndrome. John Libbey and Company, Eastleigh, UK.
  • Chen J, Marsh T, Zhang JS, Graham SH. (1995) Expression of cyclo-oxygenase 2 in rat brain following kainate treatment. Neuroreport 6:245248.
  • Crespel A, Coubes P, Rousset MC, Brana C, Rougier A, Rondouin G, Bockaert J, Baldy-Moulinier M, Lerner-Natoli M. (2002) Inflammatory reactions in human medial temporal lobe epilepsy with hippocampal sclerosis. Brain Res 952:159169.
  • Croker BA, Kiu H, Nicholson SE. (2008) SOCS regulation of the JAK/STAT signalling pathway. Semin Cell Dev Biol 19:414422.
  • de Lanerolle NC, Lee TS. (2005) New facets of the neuropathology and molecular profile of human temporal lobe epilepsy. Epilepsy Behav 7:190203.
  • De Simoni MG, Perego C, Ravizza T, Moneta D, Conti M, Marchesi F, De Luigi A, Garattini S, Vezzani A. (2000) Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus. Eur J Neurosci 12:26232633.
  • Dhote F, Peinnequin A, Carpentier P, Baille V, Delacour C, Foquin A, Lallement G, Dorandeu F. (2007) Prolonged inflammatory gene response following soman-induced seizures in mice. Toxicology 238:166176.
  • Dinarello CA. (1996) Biologic basis for interleukin-1 in disease. Blood 87:20952147.
  • Engel J Jr. (2001) Mesial temporal lobe epilepsy: what have we learned? Neuroscientist 7:340352.
  • Eriksson C, Van Dam AM, Lucassen PJ, Bol JG, Winblad B, Schultzberg M. (1999) Immunohistochemical localization of interleukin-1beta, interleukin-1 receptor antagonist and interleukin-1beta converting enzyme/caspase-1 in the rat brain after peripheral administration of kainic acid. Neuroscience 93:915930.
  • Fujimoto M, Naka T. (2003) Regulation of cytokine signaling by SOCS family molecules. Trends Immunol 24:659666.
  • Gavrieli Y, Sherman Y, Ben-Sasson SA. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493501.
  • Gorter JA, van Vliet EA, Aronica E, Breit T, Rauwerda H, Lopes da Silva FH, Wadman WJ. (2006) Potential new antiepileptogenic targets indicated by microarray analysis in a rat model for temporal lobe epilepsy. J Neurosci 26:1108311110.
  • Heck N, Garwood J, Loeffler JP, Larmet Y, Faissner A. (2004) Differential upregulation of extracellular matrix molecules associated with the appearance of granule cell dispersion and mossy fiber sprouting during epileptogenesis in a murine model of temporal lobe epilepsy. Neuroscience 129:309324.
  • Heida JG, Teskey GC, Pittman QJ. (2005) Febrile convulsions induced by the combination of lipopolysaccharide and low-dose kainic acid enhance seizure susceptibility, not epileptogenesis, in rats. Epilepsia 46:18981905.
  • Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L. (1998) Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J 334 (Pt 2):297314.
  • Heinrich C, Nitta N, Flubacher A, Muller M, Fahrner A, Kirsch M, Freiman T, Suzuki F, Depaulis A, Frotscher M, Haas CA. (2006) Reelin deficiency and displacement of mature neurons, but not neurogenesis, underlie the formation of granule cell dispersion in the epileptic hippocampus. J Neurosci 26:47014713.
  • Heinrich C, Lahteinen S, Suzuki F, Anne-Marie L, Huber S, Haussler U, Haas C, Larmet Y, Castren E, Depaulis A. (2011) Increase in BDNF-mediated TrkB signaling promotes epileptogenesis in a mouse model of mesial temporal lobe epilepsy. Neurobiol Dis 42:3547.
  • Kovanen PE, Leonard WJ. (1999) Inhibitors keep cytokines in check. Curr Biol 9:R899R902.
  • Kwon MS, Seo YJ, Choi SM, Won MH, Lee JK, Park SH, Jung JS, Sim YB, Suh HW. (2010) The time-dependent effect of lipopolysaccharide on kainic acid-induced neuronal death in hippocampal CA3 region: possible involvement of cytokines via glucocorticoid. Neuroscience 165:13331344.
  • Lang R, Pauleau AL, Parganas E, Takahashi Y, Mages J, Ihle JN, Rutschman R, Murray PJ. (2003) SOCS3 regulates the plasticity of gp130 signaling. Nat Immunol 4:546550.
  • Lee B, Dziema H, Lee KH, Choi YS, Obrietan K. (2007) CRE-mediated transcription and COX-2 expression in the pilocarpine model of status epilepticus. Neurobiol Dis 25:8091.
  • Lehtimaki KA, Keranen T, Palmio J, Peltola J. (2010) Levels of IL-1beta and IL-1ra in cerebrospinal fluid of human patients after single and prolonged seizures. Neuroimmunomodulation 17:1922.
  • Lillie R, Fullmer H. (1976) Histopathologic technic and practical histochemistry. McGraw-Hill, New York.
  • Maroso M, Balosso S, Ravizza T, Liu J, Aronica E, Iyer AM, Rossetti C, Molteni M, Casalgrandi M, Manfredi AA, Bianchi ME, Vezzani A. (2010) Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures. Nat Med 16:413419.
  • Maroso M, Balosso S, Ravizza T, Iori V, Wright CI, French J, Vezzani A. (2011) Interleukin-1beta biosynthesis inhibition reduces acute seizures and drug resistant chronic epileptic activity in mice. Neurotherapeutics 8:304315.
  • Meier R, Haussler U, Aertsen A, Deransart C, Depaulis A, Egert U. (2007) Short-term changes in bilateral hippocampal coherence precede epileptiform events. Neuroimage 38:138149.
  • Nitta N, Heinrich C, Hirai H, Suzuki F. (2008) Granule cell dispersion develops without neurogenesis and does not fully depend on astroglial cell generation in a mouse model of temporal lobe epilepsy. Epilepsia 49:17111722.
  • Plata-Salaman CR, Ilyin SE, Turrin NP, Gayle D, Flynn MC, Romanovitch AE, Kelly ME, Bureau Y, Anisman H, McIntyre DC. (2000) Kindling modulates the IL-1beta system, TNF-alpha, TGF-beta1, and neuropeptide mRNAs in specific brain regions. Brain Res Mol Brain Res 75:248258.
  • Ravizza T, Lucas SM, Balosso S, Bernardino L, Ku G, Noe F, Malva J, Randle JC, Allan S, Vezzani A. (2006) Inactivation of caspase-1 in rodent brain: a novel anticonvulsive strategy. Epilepsia 47:11601168.
  • Ravizza T, Gagliardi B, Noe F, Boer K, Aronica E, Vezzani A. (2008) Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy. Neurobiol Dis 29:142160.
  • Ravizza T, Balosso S, Vezzani A. (2011) Inflammation and prevention of epileptogenesis. Neurosci Lett 497:223230.
  • Riban V, Bouilleret V, Pham-Le BT, Fritschy JM, Marescaux C, Depaulis A. (2002) Evolution of hippocampal epileptic activity during the development of hippocampal sclerosis in a mouse model of temporal lobe epilepsy. Neuroscience 112:101111.
  • Robel S, Berninger B, Gotz M. (2011) The stem cell potential of glia: lessons from reactive gliosis. Nat Rev Neurosci 12:88104.
  • Rosell DR, Akama KT, Nacher J, McEwen BS. (2003) Differential expression of suppressors of cytokine signaling-1, -2, and -3 in the rat hippocampus after seizure: implications for neuromodulation by gp130 cytokines. Neuroscience 122:349358.
  • Sandhya TL, Ong WY, Horrocks LA, Farooqui AA. (1998) A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainate lesions. Brain Res 788:223231.
  • Schmitz J, Weissenbach M, Haan S, Heinrich PC, Schaper F. (2000) SOCS3 exerts its inhibitory function on interleukin-6 signal transduction through the SHP2 recruitment site of gp130. J Biol Chem 275:1284812856.
  • Seifert G, Schilling K, Steinhauser C. (2006) Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat Rev Neurosci 7:194206.
  • Seifert G, Carmignoto G, Steinhauser C. (2010) Astrocyte dysfunction in epilepsy. Brain Res Rev 63:212221.
  • Shapiro LA, Wang L, Ribak CE. (2008) Rapid astrocyte and microglial activation following pilocarpine-induced seizures in rats. Epilepsia 49(Suppl. 2):3341.
  • Sofroniew MV. (2009) Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci 32:638647.
  • Spooren A, Kolmus K, Laureys G, Clinckers R, De Keyser J, Haegeman G, Gerlo S. (2011) Interleukin-6, a mental cytokine. Brain Res Rev 67:157183.
  • Streit WJ. (1990) An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4). J Histochem Cytochem 38:16831686.
  • Suzuki F, Junier MP, Guilhem D, Sorensen JC, Onteniente B. (1995) Morphogenetic effect of kainate on adult hippocampal neurons associated with a prolonged expression of brain-derived neurotrophic factor. Neuroscience 64:665674.
  • Turrin NP, Rivest S. (2004) Innate immune reaction in response to seizures: implications for the neuropathology associated with epilepsy. Neurobiol Dis 16:321334.
  • Vezzani A, Baram TZ. (2007) New roles for interleukin-1 Beta in the mechanisms of epilepsy. Epilepsy Curr 7:4550.
  • Vezzani A, Granata T. (2005) Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 46:17241743.
  • Vezzani A, Conti M, De Luigi A, Ravizza T, Moneta D, Marchesi F, De Simoni MG. (1999) Interleukin-1beta immunoreactivity and microglia are enhanced in the rat hippocampus by focal kainate application: functional evidence for enhancement of electrographic seizures. J Neurosci 19:50545065.
  • Vezzani A, Moneta D, Conti M, Richichi C, Ravizza T, De Luigi A, De Simoni MG, Sperk G, Andell-Jonsson S, Lundkvist J, Iverfeldt K, Bartfai T. (2000) Powerful anticonvulsant action of IL-1 receptor antagonist on intracerebral injection and astrocytic overexpression in mice. Proc Natl Acad Sci USA 97:1153411539.
  • Vezzani A, Moneta D, Richichi C, Aliprandi M, Burrows SJ, Ravizza T, Perego C, De Simoni MG. (2002) Functional role of inflammatory cytokines and antiinflammatory molecules in seizures and epileptogenesis. Epilepsia 43(Suppl. 5):3035.
  • Vezzani A, Ravizza T, Balosso S, Aronica E. (2008) Glia as a source of cytokines: implications for neuronal excitability and survival. Epilepsia 49(Suppl. 2):2432.
  • Viviani B, Bartesaghi S, Gardoni F, Vezzani A, Behrens M, Bartfai T, Binaglia M, Corsini E, Di Luca M, Galli CL, Marinovich M. (2003) Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci 23:86928700.
  • Volterra A, Meldolesi J. (2005) Astrocytes, from brain glue to communication elements: the revolution continues. Nat Rev Neurosci 6:626640.
  • Voutsinos-Porche B, Koning E, Kaplan H, Ferrandon A, Guenounou M, Nehlig A, Motte J. (2004) Temporal patterns of the cerebral inflammatory response in the rat lithium-pilocarpine model of temporal lobe epilepsy. Neurobiol Dis 17:385402.
  • Yasukawa H, Sasaki A, Yoshimura A. (2000) Negative regulation of cytokine signaling pathways. Annu Rev Immunol 18:143164.
  • Yoshimura A, Naka T, Kubo M. (2007) SOCS proteins, cytokine signalling and immune regulation. Nat Rev Immunol 7:454465.

Supporting Information

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

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.

FilenameFormatSizeDescription
EPI_3273_sm_TableS1.doc34KSupporting info item
EPI_3273_sm_TextS1.doc68KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.