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Keywords:

  • ATP ;
  • P2X7 receptor;
  • Status epilepticus;
  • Cortex;
  • Neuroprotection

Summary

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

Purpose

ATP is an essential transmitter/cotransmitter in neuron function and pathophysiology and has recently emerged as a potential contributor to prolonged seizures (status epilepticus) through the activation of the purinergic ionotropic P2X7 receptor (P2X7R). Increased P2X7R expression has been reported in the hippocampus, and P2X7R antagonists reduced seizure-induced damage to this brain region. However, status epilepticus also produces damage to the neocortex. The present study was designed to characterize P2X7R in the neocortex and assess effects of P2X7R antagonists on cortical injury after status epilepticus.

Methods

Status epilepticus was induced in mice by intraamygdala microinjection of kainic acid. Specific P2X7R inhibitors were administered into the ventricle before seizure induction, and cortical electroencephalography and behavior was recorded to assess seizure severity. P2X7R expression was examined in neocortex up to 24 h after status epilepticus, in epileptic mice, and in resected neocortex from patients with pharmacoresistent temporal lobe epilepsy (TLE). In addition, the induction of P2X7R after status epilepticus was investigated using transgenic P2X7R reporter mice, which express enhanced green fluorescent protein under the control of the p2x7r promoter.

Key Findings

Status epilepticus resulted in increased P2X7R protein levels in the neocortex of mice. Neocortical P2X7 receptor levels were also elevated in mice that developed epilepsy after status epilepticus and in resected neocortex from patients with pharmacoresistent TLE. Immunohistochemistry determined that neurons were the major cell population transcribing the P2X7R in the neocortex within the first 8 h after status epilepticus, whereas in epileptic mice, P2X7R up-regulation occurred in microglia as well as in neurons. Pretreatment of mice with the specific P2X7R inhibitor A-438079 reduced electrographic and clinical seizure severity during status epilepticus and reduced seizure-induced neuronal death in the neocortex.

Significance

Our findings identify neurons in the neocortex as an important site of P2X7R up-regulation after status epilepticus and in epilepsy, and provide support for the possible use of P2X7R antagonists for the treatment of status epilepticus and prevention of seizure-induced brain damage.

ATP is an important neurotransmitter in the peripheral and central nervous system (CNS) and has been shown to be released from neurons and astrocytes. ATP acts either as sole transmitter or as cotransmitter, and it triggers a wide array of physiologic effects (Burnstock, 2007). ATP is taken up and stored by secretory and synaptic vesicles and released into the extracellular space by exocytosis or from damaged and dying cells (Abbracchio et al., 2009). After its release, ATP and other nucleotides are rapidly degraded by ectonucleotidases into different breakdown products, including adenosine (Burnstock, 2007).

ATP-recognizing purinergic receptors are expressed in neurons and glia, including astrocytes, oligodendrocytes, and microglia, and are extensively distributed throughout the CNS (Abbracchio et al., 2009). Purinergic receptors are subdivided into P2X and P2Y receptors based on their mechanism of action, pharmacology, and molecular cloning (Burnstock, 2007). The P2X receptor subfamily consists of trimeric ligand-gated ion channels that respond solely to ATP. P2X receptors are involved in fast synaptic transmission and synaptic plasticity and have been described to be localized both presynaptically and postsynaptically in neurons. They gate rapid nonselective passage of cations across the cell membrane, resulting in depolarizing responses (Surprenant & North, 2009).

Although initially described as mainly expressed on microglia, P2X7 receptor (P2X7R) expression and function has now been reported in neurons as well as in glial cells throughout the brain including in the hippocampus, striatum, and neocortex (Deuchars et al., 2001; Sperlagh et al., 2002, 2006; Diaz-Hernandez et al., 2009; Arbeloa et al., 2012; Engel et al., 2012a). The P2X7R belongs to the slow desensitizing type with a low affinity for ATP when compared to other P2X receptor subtypes (Sperlagh et al., 2006). P2X7R inhibition has been proposed as a potential therapeutic strategy in a wide range of CNS diseases including Alzheimer's disease (Diaz-Hernandez et al., 2012), neuropathic pain (Sperlagh et al., 2006), Huntington's disease (Diaz-Hernandez et al., 2009), ischemia (Arbeloa et al., 2012), traumatic brain injury (Kimbler et al., 2012), and epilepsy (Engel et al., 2012b).

Status epilepticus (SE) is defined as a continuous seizure lasting at least 5 min and is a clinical emergency associated with potential brain injury (Wasterlain & Chen, 2008). Neuronal death and reactive gliosis has been found in brain regions such as the hippocampus, amygdala, and piriform and entorhinal cortex in humans (Fujikawa et al., 2000). Cortical damage has also been reported in different mouse models of chemically induced SE either by pilocarpine or kainic acid (KA) (Curia et al., 2008; Mouri et al., 2008) and after electrical stimulation-induced SE (Kienzler et al., 2009). Frontline treatment of SE includes benzodiazepines such as lorazepam and midazolam or barbiturates such as phenobarbitone (Wasterlain & Chen, 2008). However, SE becomes pharmacoresistant in about 30% of patients, which is thought to be due to the internalization of the γ-aminobutyric acid (GABA)A receptor and movement of N-methyl-d-aspartate (NMDA) receptors to the synapse (Wasterlain & Chen, 2008). There is therefore a need to identify new GABA-independent drug targets for the treatment of SE.

Growing evidence suggests that the P2X7R is activated during SE and contributes to seizures and their pathologic consequences. P2X7R levels increase directly after SE induced by pilocarpine and KA in hippocampus and cortex (Rappold et al., 2006; Avignone et al., 2008; Engel et al., 2012a). Increased P2X7R levels have also been found in the hippocampus of epileptic animals (Vianna et al., 2002). Immunohistochemistry and P2X7R reporter mice, which express enhanced green fluorescent protein (GFP) under the control of the p2x7r promoter, have shown increased P2X7R transcription after SE and in chronic epileptic animals in hippocampal neurons and microglia (Rappold et al., 2006; Avignone et al., 2008; Engel et al., 2012a). Critically, functional studies undertaken using specific P2X7R agonists and antagonists confirmed the recruitment of the P2X7R during seizure generation and seizure-induced cell death (Kim & Kang, 2011; Engel et al., 2012a).

Little is known about P2X7R involvement in the cortex during SE or epilepsy, or whether seizure-modulation by P2X7R-inhibiting drugs affects cortical injury. Accordingly, we assessed P2X7R expression in the cortex following SE in mice and evaluated seizure severity and damage after SE in mice given P2X7R antagonists.

Material and Methods

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

Animal model of status epilepticus

All animal experiments were performed in accordance with the principles of the European Communities Council Directive (86/609/EEC). Procedures were approved by the relevant Research Ethics Committees of the Royal College of Surgeons in Ireland and under authorization of the Universidad Complutense de Madrid bioethics committee. Procedures were undertaken as described previously (Mouri et al., 2008; Engel et al., 2013). Adult C57BL/6 mice were from Harlan (United Kingdom). P2X7R reporter mice (Tg[P2rx7-EGFP]FY174Gsat/Mmcd, stock 011959-UCD] expressing enhanced green fluorescent protein (EGFP) immediately downstream of the p2x7r promoter were obtained from U.S. National Institutes of Health Mutant Mouse Regional Resource Centers and granted by Dr. M. Nedergaard (University of Rochester, Rochester, NY, U.S.A.) as reported previously (Engel et al., 2012a). First, mice were anesthetized using isoflurane (3–5%) and maintained normothermic by means of a feedback-controlled heat blanket (Harvard Apparatus Ltd, Kent, United Kingdom). Mice were then placed in a stereotaxic frame, and three cortical skull-mounted electroencephalography (EEG) electrodes attached (Bilaney Consultants Ltd, Sevenoaks, United Kingdom). EEG was recorded using a Grass Comet XL digital EEG (Medivent Ltd, Lucan, Ireland). A guide cannula was affixed (coordinates from Bregma: AP = 0.94 mm; L = 2.85 mm) and the entire skull assembly fixed in place with dental cement. Baseline EEG was recorded for a few minutes, and an injection cannula was lowered through the guide cannula for injection of KA (Sigma-Aldrich, Arklow, Ireland) into the basolateral amygdala nucleus (0.3 μg in 0.2 μl phosphate-buffered saline). Nonseizure control mice underwent the same surgical procedure but received 0.2 μl intraamygdala vehicle. Mice received intraperitoneal lorazepam (6 mg/kg) to curtail SE 40 min following injection of KA or vehicle. Mice were killed at different time-points (1, 4, 8, or 24 h, or 14 days) later and perfused with saline to remove intravascular blood components. Brains were either flash-frozen whole in 2-methylbutane at 30°C for histopathology, perfused with 4% paraformaldehyde for confocal microscopy or dissected on ice to obtain cortex for Western blot analysis.

Quantification of EEG

EEG data was uploaded into Labchart7 software (ADInstruments Ltd, Oxford, United Kingdom) to analyze frequency, high amplitude peaks count and amplitude (power spectral density and EEG spectrogram), as described (Engel et al., 2012a; Jimenez-Mateos et al., 2012).

Behavioral assessment of seizure severity

Behavioral seizures were scored according to a modified Racine Scale as reported previously (Jimenez-Mateos et al., 2012). Score 1, immobility and freezing; Score 2, forelimb and or tail extension, rigid posture; 3, repetitive movements, head bobbing; Score 4, rearing and falling; Score 5, continuous rearing and falling; Score 6, severe tonic–clonic seizures. Mice were scored every 5 min for 40 min after KA injection. The highest score attained during each 5 min period was recorded by an observer blinded to treatment.

In vivo drug administration

The specific P2X7R inhibitors A438079 (1.75 nmol) (3-[[5-(2,3-dichlorophenyl)-1H-tetrazol-1- yl]methyl]pyridine hydrochloride) (Tocris Biosciences, Bristol, United Kingdom) and brilliant blue G (1 pmol) (BBG) (Sigma-Aldrich) were delivered intracerebroventricularly (2 μl) (coordinates from Bregma: AP = 0.4 mm; L = 0.95 mm) 10 min prior to KA injection and 60 min post-KA injection.

Human brain tissue samples

This study was approved by the Ethics (Medical Research) Committee of Beaumont Hospital, Dublin (05/18), and written informed consent was obtained from all patients. Briefly, patients (n = 3) were referred for surgical resection of the temporal lobe for the treatment of intractable temporal lobe epilepsy (TLE). After temporal lobe resection, neocortex was obtained and frozen in liquid nitrogen and stored at −70°C until use. A pathologist (Dr. Michael Farrell) assessed neocortex and confirmed the absence of significant neuronal loss. Control (autopsy) temporal cortex (n = 5; C1–C5) was obtained from five individuals from the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, Baltimore, MD, U.S.A. Samples were processed for Western blot analysis. Full details of control and patient pathology and clinical data have been reported previously (McKiernan et al., 2012).

Synaptosome preparation

Mouse neocortex was dissected on ice and tissue homogenized in 10 ml of ice cold homogenizing buffer (0.32 m sucrose, 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mg/ml bovine serum albumin, 5 mm 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH 7.4) in a glass-Teflon douncer with about 10 strokes at 4°C. Next, samples were centrifuged for 10 min at 3,000 g at 4°C and supernatant recovered (cytoplasm and synaptosomes). Samples were again centrifuged for 12 min at 14,000 g at 4°C and supernatant discarded. Pelleted synaptosomes were resuspended in 550 μl of Krebs–Ringer buffer (140 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm EDTA, 10 mm HEPES pH 7.4). Then, 450 μl of Percoll (45% v/v) was added to samples and samples were mixed inverting the tube gently. After a 2 min spin at 14,000 g at 4°C, enriched synaptosomes were recovered and re-suspended in 1 ml of Krebs–Ringer buffer. Samples were again spun for 30 s at 14,000 g and supernatant was discarded. Pellet was resuspended (the synaptosomes) in assay buffer (Hepes–Krebs buffer) and stored at −20°C.

Western blot analysis

Western blotting was performed as described previously (Engel et al., 2010b). Cortical samples were homogenized in lysis buffer containing a protease inhibitor cocktail. Protein concentration was determined, and then 25 μg (synaptosomes) or 50 μg samples were boiled in gel-loading buffer and separated by 10% or 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes. The following primary antibodies were used: P2X7R (APR-004; Alomone Labs, Jerusalem, Israel), Synaptophysin and β-Actin (Sigma-Aldrich), and α-Tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). Membranes were next incubated with horseradish peroxidase conjugated secondary antibodies (Isis Ltd, Bray, Ireland) and protein bands were visualized using Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, U.S.A.). Gel band image densities were captured using a Fuji-film LAS-3000 and analyzed using ALPHA-EASEFC4.0 software, corrected for loading (Engel et al., 2010b).

Histopathology

Histopathology was undertaken as described previously (Engel et al., 2010a). Briefly, fresh-frozen 12 μm thick coronal brain sections at the level of the dorsal hippocampus were air-dried and fixed in formalin. Neuronal cortical damage was assessed by Fluoro-Jade B (FJB) staining according to manufacturers' recommendations (Millipore, Cork, Ireland). Sections were examined and imaged using a Nikon 2000s epifluorescence microscope with a Hamamatsu Orca 285 camera (Micron-Optica, Enniscorthy, Ireland). Semi-quantification of FJB-positive cells within the neocortex was determined for vehicle and A-438079 mice 24 h after SE over an area of approximately 10 mm2 per tissue section at the level of Bregma: L = −1.94). Counts were the average of two adjacent sections assessed by an observer blind to treatment.

Confocal microscopy

For confocal microscopy, animals were transcardially perfused with 4% paraformaldehyde in Sorensen's buffer for 10 min, postfixed, and cryoprotected in sucrose before sectioning. Sections were pretreated with 1% bovine serum albumin, and 1% TTX-100 in phosphate-buffered saline (PBS), followed by incubation with the primary antibodies; anti-c-Fos (1:50; Santa Cruz Biotechnology), anti-green fluorescent protein (GFP) (1:500; Invitrogen, Dublin, Ireland), NeuN (1:400; Millipore, Billerica, MA, U.S.A.), Iba-1 (1:400; Wako Chemicals U.S.A., Richmond, VA, U.S.A.), and anti-glial fibrillary acidic protein (1:500, GFAP; Santa Cruz Biotechnology). Sections were washed again and incubated with secondary antibodies coupled to AlexaFluor488 or AlexaFluor568 (BioSciences Limited, Dublin, Ireland) and then coverslipped with Fluorosave. Confocal images were acquired with a Leica TCR 6500 microscope equipped with four laser lines (405, 488, 561, and 653 nm) using a 40× immersion oil objective (Leica Microsystems, Wetzlar, Germany).

Autopsy control experiment

Cortex was extracted from mice (adult C57Bl/6) after deep pentobarbital anesthesia and decapitation and either frozen immediately (“surgical” control) or frozen 4 or 8 h after being left at room temperature (simulated postmortem interval). Samples were then processed for Western blotting as described (Engel et al., 2013).

Data analysis

Data are presented as means ± standard error of the mean (SEM). Data were analyzed using analysis of variance (ANOVA) with post hoc Fisher's protected least significant difference test or, for two-group comparison, Student's t-test (STATVIEW software; SAS Institute, Cary, NC, U.S.A.). Racine scores were compared between groups using Harrell's C coefficient (also known as the Wilcoxon–Mann–Whitney statistic). This calculates the probability of observing a lower score when an animal selected at random from the treated group is compared with one selected at random from the control group. As such, it is a useful measure of effect size. Calculations were carried out in Stata, using Newson's command somersd and adjusted for clustering of data due to repeated measurements on each animal. This method is suited to the nature of the Racine scores, which are not based on a defined unit, and which followed a distribution that differed between the treated and control animals. Significance was accepted at p < 0.05.

Results

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

Intraamygdala KA-induced status epilepticus produces injury to the neocortex and up-regulation of the P2X7R

To study the neocortical expression and functional contribution of the P2X7R during SE we used a well-characterized model of focal-onset SE (Araki et al., 2002; Mouri et al., 2008). SE was induced by the microinjection of KA into the amygdala in mice. All mice developed SE shortly after KA injection, with typical behavior changes including freezing, immobility, Straub tail, forelimb clonus, rearing, and falling. No behavioral changes were observed in vehicle-injected animals. The anticonvulsant lorazepam was injected 40 min after KA injection to curtail seizure activity and minimize mortality. Cortical EEG recordings (Fig. 1A,B) and immunostaining for the activity-regulated immediate early gene c-Fos (Herrera & Robertson, 1996) confirmed recruitment of cortical structures during SE (Fig. 1C). C-Fos immunoreactivity was most obvious in the deep cortical layers V and VI after SE with a clear nuclear pattern (Fig. 1C). No nuclear c-Fos staining was evident in control animals (Fig. 1C).

image

Figure 1. Cell death and increased P2X7R expression in neocortex after status epilepticus. (A) Representative EEG traces during SE showing high amplitude and high frequency discharges recorded from cortical skull-mounted EEG electrodes. (B) Representative EEG recordings presented as pseudocolor heat maps of frequency and amplitude from KA injection until administration of lorazepam 40 min later. (C) Representative photomicrographs (20× lens) showing strong increase in cortical nuclear c-Fos immunostaining mainly in layers IV, V, and VI 8 h after SE. Panel to far right is a 40× view of the c-Fos staining after SE from the middle panel. (D) Representative photomicrograph showing FJB-positive cells mainly in ipsilateral cortex in layers V and VI, and the ipsilateral CA3 hippocampal subfield (see arrows) 24 h after SE. Note absence of FJB-positive cells in the contralateral side of the brain. (E) Higher magnification photomicrographs (20× lens) showing FJB-positive cells in ipsilateral cortex (layers V and VI) and CA3a/b ipsilateral hippocampal subfield. (F) Representative Western blot (n = 1 per lane) and (G) graph showing increased P2X7R protein levels in the ipsilateral cortex 24 h after SE (n = 4). (H) Representative Western blot (n = 1 per lane) showing higher P2X7R and synaptophysin (synapto) levels in the synaptosome (syn) fraction from neocortex when compared to cytoplasmic (cyt) fraction. (I) Western blot (n = 1 per lane) showing higher P2X7R levels in synaptosome fraction from neocortex 24 h after SE when compared to synaptosome fraction from neocortex from vehicle-injected control mice. CA, cornu ammonis; DG, dentate gyrus, *p < 0.05; Scale bar in C, 200 μm; D, 500 μm; E, 150 μm.

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Next, we stained coronal brain sections with the neuronal cell death marker FJB. As expected, scattered dying neurons were evident in the ipsilateral cortex as well as the hippocampus 24 h after SE (Fig. 1D,E). FJB-positive cells were most evident in cortical layer V and VI and in the CA3 subfield of the hippocampus (Fig. 1D,E). No cell death was apparent in the contralateral side of the brain of mice subjected to SE or in control animals (Fig. 1 D and data not shown).

Next, we examined P2X7R levels in the neocortex after SE. Western blot analysis showed that P2X7R protein levels were significantly increased 24 h after SE in the ipsilateral cortex (Fig. 1F,G). Because P2X7R has been reported previously to be present in the synaptosomal cellular compartment (Miras-Portugal et al., 2003), we also examined P2X7R levels in synaptosomes before and after SE. P2X7R was highly enriched in cortical synaptosomes (Fig. 1H) in basal conditions, and levels were higher in synaptosomes 24 h after SE (Fig. 1I).

Increased neuronal transcription of P2X7R in neocortex after SE

To explore where in the cortex and what cell types express the P2X7R after SE, we used a P2X7R reporter mouse that expresses GFP under the transcriptional control of the p2x7r promoter (Engel et al., 2012a). P2X7R reporter mice showed a normal response to KA, with similar behavioral changes during SE when compared to wild-type littermates (data not shown). In controls, GFP immunoreactivity was observed in cortical layers II and III only, suggesting basal P2X7R expression in these cells (Fig. S1). An increase in GFP-stained–positive cells after SE was observed in tissue sections obtained 8 h after SE mainly in cortical layers V and VI when compared to control-injected P2X7R reporter mice (Fig. S2A). To establish which cell types display P2X7R induction we used different cell type markers. Double-staining for GFP and cell-specific markers revealed a mainly neuronal GFP signal in neocortex (Fig. 2A) with no apparent costaining for microglia or astrocytes after SE (Fig. 2B,C).

image

Figure 2. Neuronal induction of P2X7R in neocortex after status epilepticus. Immunofluorescence staining showing (A) GFP fluorescence (green) and NeuN (red), (B) GFP fluorescence and Iba-1 (red), and (C) GFP fluorescence and GFAP (red) in ipsilateral neocortex from control and GFP-P2x7r reporter mice 8 h after SE. Note, colocalization between GFP- and NeuN-positive cells and absence of colocalization between GFP signal and Iba-1 and GFAP signal. Scale bar = 12 μm.

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Increased neocortical expression of P2X7R in experimental and human epilepsy

We also examined P2X7R protein levels in experimental and human epilepsy (Fig. 3A–D). To assess neocortical P2X7R levels in epileptic mice, tissue was analyzed 14 days after SE by Western blot when all mice are displaying regular spontaneous seizures (Mouri et al., 2008; Jimenez-Mateos et al., 2012). P2X7R protein levels in neocortical samples from epileptic mice were significantly higher compared to time-matched control tissue (Fig. 3A,B). In addition, P2X7R protein levels were significantly higher in human resected TLE cortex compared to matched autopsy control (Fig. 3C,D). A simulated autopsy delay using mouse brain showed P2X7R protein levels in the cortex are stable over the period corresponding to the maximal delay in the human control subjects (Fig. S2).

image

Figure 3. Increased neocortical expression of P2X7R in epilepsy. (A) Representative Western blot (n = 1 per lane) and (B) graph showing increased P2X7R levels in ipsilateral neocortex in epileptic mice. (C) Representative Western blot (n = 1 per lane) and (D) graph showing increased P2X7R levels in cortex of patients with TLE when compared to control brain. Immunofluorescence staining showing (E) GFP fluorescence (green) and NeuN (red), (F) GFP fluorescence (green) and Iba-1 (red) (upper panels [scale bar as in E]; Lower panels shown at higher magnification a GFP-positive microglia [arrow]), and (G) GFP fluorescence and GFAP (red) in ipsilateral neocortex from control and epileptic P2X7R-GFP reporter mice 14 days after KA injection. Note, colocalization between GFP and NeuN and Iba-1 positive cells and absence of colocalization between GFP signal and GFAP signal. *p < 0.05, Scale bar = 12 μm.

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To determine which cell population exhibits increased P2X7R induction, we analyzed brain sections from epileptic GFP-P2X7R reporter mice 14 days after SE, and we double stained them with GFP/NeuN, GFP/Iba-1, and GFP/GFAP. The main area of GFP-positive cells in epileptic mice was found in cortical layers V and VI (Fig. 3E). Similar to what was observed after SE, neurons were the main cell population exhibiting GFP induction (Fig. 3E). Double staining with Iba-1 and GFP revealed colocalization in some microglia, which featured large somata and thick primary processes resembling activated microglia (Fig. 3F). No colocalization of GFP-positive cells with GFAP-positive astrocytes was observed in the neocortex of epileptic mice (Fig. 3G).

P2X7R inhibition decreases seizure severity during status epilepticus and protects neocortex against damage

To test whether P2X7R antagonists could reduce seizures and neocortical damage caused by SE, we analyzed seizures and damage in animals injected with A-438079, a specific P2X7R inhibitor. EEG recordings determined that mice given A-438079 displayed reduced total seizure power, amplitude, and high amplitude peak counts during SE when compared to vehicle-treated mice (Fig. 4A-D). Behavioral analysis using a Racine scale–based scoring system determined clinical seizure behavior, including motor seizures, were reduced in mice given A-438079 before KA (Fig. 4E). Statistical analysis (see 'Material and Methods') showed that Harrell's c was 0.72 (95% CI 0.53–0.92, p = 0.034) indicating that there was a 72% probability that a treated animal would have a lower Racine score than a control.

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Figure 4. P2X7R inhibition decreases seizure severity during SE and protects against neocortical damage. (AC) Graphs showing reduced EEG total power, amplitude, and high amplitude peak counts in mice pretreated with the P2X7R inhibitor A-4387079 (A43) when compared to vehicle-injected animals (veh). (D) Representative EEG recordings presented as pseudocolor heat maps of frequency and amplitude data during SE from mice pretreated with vehicle or A43. Note, strong reduction in amplitude in mice treated with A43. (E) Graph showing Racine scores (maximum reached) per 5 min period over 40 min after injection of KA for Veh and A43 treated mice (n = 7 per group). Graph to the right shows percentage distribution of Racine scale per treatment group over the entire experiment. Graph (F) and photomicrographs (G) (10× lens) showing fewer FJB-positive cells in ipsilateral neocortex 24 h after SE in mice treated with the P2X7R inhibitor A43 when compared to vehicle-injected animals.*p < 0.05, Scale bar = 150 μm.

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Tissue sections from these animals were then stained for the neuronal cell death marker FJB. FJB-positive cell counts in the cortex at the level of the dorsal hippocampus showed A43-treated mice had significantly lower (approximately 50%) FJB-positive cells when compared to vehicle-injected mice (Fig. 4F,G). In mice pretreated with another specific P2X7R antagonist, brilliant blue G, seizure-induced cell death was also significantly reduced by 53% (p = 0.008, n = 9 per group).

Discussion

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

In the present study, we report increased expression of the ATP-gated P2X7R in the neocortex of mice after SE, and in experimental and human epilepsy. Experiments using P2X7R/GFP-reporter mice revealed that neurons were the main cell population in which P2X7R was induced. Last, inhibition of the P2X7R reduced damage to the neocortex after SE. These results extend previous hippocampal findings and suggest the P2X7R is a potential new target to protect against seizures and seizure-induced cell death.

In humans, SE is associated with significant extrahippocampal injury, including in the piriform and entorhinal cortex (Fujikawa et al., 2000). Cortical thinning has also been reported in cross-sectional and longitudinal imaging studies of patients with pharmacoresistant TLE (Lin et al., 2007; Bernhardt et al., 2009). Such injury may contribute to cognitive deficits and ictogenesis (Thompson & Duncan, 2005; Helmstaedter, 2007). Identifying molecular mechanisms that induce cerebral injury or excitotoxicity after seizures may represent important targets for the protection of these brain structures. Most common treatment options for SE involve early administration of GABA-potentiating anticonvulsants. Due to internalization of the GABAA receptor and other mechanisms, these drugs remain ineffective in approximately 30% of patients (Wasterlain & Chen, 2008). There is a need to identify new anticonvulsants that target a different mechanism. ATP may be released during seizures and contribute to seizure severity and duration (Dale & Frenguelli, 2009; Engel et al., 2012b). The ATP-gated P2X7R is an attractive new target for seizure control. Indeed, recent work showed that P2X7R antagonists significantly reduce SE and potentiate the anticonvulsive effects of lorazepam (Engel et al., 2012a). Seizure attenuation was also accompanied by potent neuroprotection in the hippocampus (Engel et al., 2012a).

The present study reports that SE results in increased levels of the P2X7R in the neocortex. Western blotting detected a low level of P2X7R protein in the neocortex of control mice, with its levels steadily increasing after SE. Previous findings in the same model showed a similar 1.5-fold up-regulation of the P2X7R in the hippocampus 24 h after SE (Engel et al., 2012a), suggesting similar responses and perhaps shared mechanisms of P2X7R up-regulation after SE between these brain regions. We also found increased P2X7R levels in the neocortex of epileptic mice and in resected neocortex from patients with TLE. These data suggest increased P2X7R levels are a common feature of seizure activity and are not restricted to SE. The mechanisms underlying P2X7R up-regulation after seizures are unknown, although recent work linked the specificity protein factor 1 transcription factor to P2X7R induction after serum deprivation in neuroblastoma cells (Garcia-Huerta et al., 2012), and this transcription factor might be active after SE (Feng et al., 1999).

Originally thought to be present exclusively on glial cells, the P2X7R has now been recognized to serve important roles in neurons (Sperlagh et al., 2006; Engel et al., 2012b). The present studies used a GFP/P2X7R reporter mouse to identify cells transcribing P2X7R. Our findings support earlier hippocampal studies showing neurons are the main cell type in mice transcribing P2X7R after SE (Engel et al., 2012a). We also show P2X7R induction in the neocortex in neurons and microglia during the chronic phase of epilepsy using the same P2X7R/GFP-reporter mouse. Therefore, neocortical findings are broadly consistent with hippocampal data. Up-regulation of the P2X7R after SE has been shown previously both in microglia and neurons in the hippocampus and cortex in different animal models of SE (Engel et al., 2012b). Our data contrast with the mainly microglial patterns reported by other groups after SE (Rappold et al., 2006). The absence of P2X7R induction in microglia shortly after SE in our model could also suggest that the reliance on immunostaining may have overlooked neuronal expression. Further support for a neuron-specific induction of the P2X7R after SE comes from the increased levels of P2X7R in the synaptosomal fraction, which suggests increased P2X7R localization in synapses after SE (Armstrong et al., 2002). The finding that P2X7R induction is elevated in microglia only during epilepsy suggests that additional mechanisms regulating the P2X7R become important in the setting of recurrent spontaneous seizures. Indeed, the expression of P2X7R in microglia may be an important mechanism of microglia activation (Monif et al., 2009). Activated microglia are thought to serve important roles in the process of epileptogenesis and during epilepsy where the cytokine interleukin-1β may play an essential role (Vezzani et al., 2011). Of interest, interleukin-1β release was blocked by P2X7R inhibitors in the hippocampus after SE (Engel et al., 2012a). Increased GFP production in activated microglia in epileptic mice is also consistent with results showing that P2X7R can drive microglial activation, and previous studies have shown increased P2X7R currents in activated microglia after SE (Avignone et al., 2008). We did not detect any transcriptional activation of P2X7R in astrocytes either after SE or in chronic epilepsy. This is perhaps unexpected because astrocytes release ATP and P2X7R have been reported to be present and functional on astrocytes (Burnstock, 2007). Therefore, seizures do not induce P2X7R in neocortical astrocytes consistent with our previous findings in the hippocampus (Engel et al., 2012a).

The present study also supports an important functional role of P2X7R in SE, since antagonists of P2X7R reduced seizure severity during SE. As shown previously, mice treated with the P2X7R inhibitor A-438079 showed reduced seizure severity during SE (Engel et al., 2012a). Seizures recorded by cortical EEG were reduced by >50%, and clinical seizures were also strongly suppressed. This is consistent with previous reports in the model (Engel et al., 2012a) and suggests P2X7R antagonists as potential anticonvulsants. Moreover, since they target an independent transmitter system, P2X7R antagonists may be suitable adjunctive drugs to potentiate GABA-based therapy (Engel et al., 2012a).

The anticonvulsant effects of A-438079 are in contrast to the reported antiseizure effects of P2X7R activation in other models. Kang and colleagues reported that pilocarpine-induced seizures were increased in mice lacking the P2X7R, although they found no influence of the receptor in a kainate model (Kim & Kang, 2011). Some in vitro studies also argue that P2X7R activation can be inhibitory, including work showing that presynaptic P2X7Rs on mossy fibers reduce neurotransmitter release in hippocampal slices (Armstrong et al., 2002). These apparently contradictory findings suggest that the contribution of P2X7R to seizures may display important model and tissue-specific differences. Whether P2X7R inhibitors protect against seizure-induced cell death by a direct mechanism has not been established here. Neuroprotective effects of P2X7R antagonists have been reported in other models (Diaz-Hernandez et al., 2009; Arbeloa et al., 2012). Most or perhaps all protection by the P2X7R antagonist in the present study is likely secondary to reduced seizure severity, but a direct neuroprotective effect remains possible.

In summary, our findings show increased P2X7R levels in the neocortex after SE and in chronic epilepsy. The strong anticonvulsant effect of P2X7R inhibitors demonstrates their efficacy in protecting the neocortex from widespread damage after SE and validate P2X7R as a new drug target during SE.

Acknowledgments

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

The authors thank Dr. Maiken Nedergaard (University of Rochester, Rochester, NY, U.S.A.) for kindly providing the P2x7r-EGFP mice. We also thank Norman Delanty, Michael Farrell, and Donncha O'Brian for the collection of TLE patient samples. We thank the University of Maryland Brain and Tissue Bank for providing the autopsy control samples.

This work was supported by grants from Health Research Board Ireland (HRA_POR/2010/123, HRA_POR/2011/41, HRA_POR/2012/56), Science Foundation Ireland (08/IN1/B1875), Comunidad de Madrid (S-SAL-0253-2006), Spanish Ministry of Science and Education (BFU2011-24743), Fundación Marcelino Botin, Consolider SICI Spanish Ion Channel Initiative (CSD2008-00005), UCM-Santander Central Hispano Bank (911585-670), and an EMBARK research fellowship from the Irish Research Council (to A.J.).

Disclosure

  1. Top of page
  2. Summary
  3. Material 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.

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  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
epi12257-sup-0001-FigureS1.tifimage/tif2454KFigure S1. Basal P2X7R induction in cortical layers II and Ill.
epi12257-sup-0002-FigureS2.tifimage/tif1165KFigure S2. Effects of simulated postmortem delay on cortical P2X7R protein levels.

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