J Mol Neurosci DOI 10.1007/s12031-017-0968-z

Inhibition of P2X7 Receptor Ameliorates Nuclear Factor-Kappa B Mediated Neuroinflammation Induced by Status Epilepticus in Rat Hippocampus Cheng Huang 1,2 & Xiao-sa Chi 2,3 & Rui Li 4 & Xin Hu 5 & Hai-xia Xu 6 & Jin-mei Li 2 & Dong Zhou 2

Received: 3 June 2017 / Accepted: 21 August 2017 # Springer Science+Business Media, LLC 2017

Abstract P2X7 receptor (P2X7R) has been reported participating in neuroinflammation in multiple neurological diseases. We explored the role of P2X7R in a rat status epilepticus (SE) model induced by coriaria lactone (CL) and its association with neuroinflammation. Thirty minutes after intracerebroventricular infusion with P2X7R antagonists Brilliant blue G (BBG), A-438079, A-740003, or agonists 2′,3′-O-(4benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP), SE was induced by intramuscular injection of CL in Sprague-Dawley rats. Seizures severity was recorded according to the Racine scale and Morris water maze test was performed. P2X7R expression was measured by western blotting. Immunohistochemical staining was performed to assess proinflammation cytokines expression, neuronal loss, and astrocyte activation. The results showed P2X7R level began to increase at 1 day, peaked at 2 days, and gradually decreased

to baseline by 2 weeks in rat hippocampus after SE. P2X7R activation induced NF-κB phosphorylation, along with increased IL-1β and IL-6 expression. Pretreatment with P2X7R antagonists ameliorated SE-induced neuroinflammation, neuronal damage, and astroglial and microglial activation to variable extent. In addition, these antagonists ameliorated seizure severity and improved cognitive function. These findings suggest P2X7R activation plays a critical role in epileptogenesis via regulation of neuroinflammation and blocking P2X7R may be a novel therapeutic strategy for epilepsy. Keywords P2X7 receptor . Neuroinflammation . Epilepsy . Hippocampus . Status epilepticus

Introduction Cheng Huang and Xiao-sa Chi contributed equally to this work. * Dong Zhou [email protected] 1

Rehabilitation Medicine Center, West China Hospital of Sichuan University, Chengdu 610041, China

2

Department of Neurology, West China Hospital, Sichuan University, Chengdu 610041, China

3

Department of Geriatrics, Affiliated Hospital of Qingdao University, Qingdao, Shandong 266021, China

4

Key Laboratory of Transplant Engineering and Immunology, MOH, West China Hospital, Sichuan University, Chengdu 610041, China

5

Department of Neurosurgery, West China Hospital, Sichuan University, Chengdu 610041, China

6

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China

Epilepsy is a common chronic neurologic disorder, which is characterized by abnormal hypersynchronized neuronal discharge (Sander and Shorvon 1996). The prevalence of epilepsy is approximately 0.9%, affecting around 65 million people worldwide. Temporal lobe epilepsy is the most common syndrome in adult patients, around two thirds of which are resistant to most anti-epileptic drugs (AEDs). It is now accepted that epilepsy is a result of comprehensive factors, including congenital susceptibility and post-natal environmental insults. However, the specific pathophysiologic mechanism of epilepsy remains poorly understood. Existing AEDs target a relatively limited number of proteins and ion channels. Although the introduction of newer generation of AEDs has largely reduced side effects and improved drug tolerance, the overall effectiveness of AEDs has not changed significantly (Wiebe and Jette 2012). Therefore, the invention of new AEDs that act through alternative mechanism is needed.

J Mol Neurosci

triphosphate (BzATP) to investigate the role of P2X7R in a coriaria lactone (CL) induced SE rat models of epilepsy. We also explored the association between the P2X7R and neuroinflammation after SE insults.

Neuroinflammation, characterized by astroglial and microglial activation, as well as release of various cytotoxic agents, is found to play an crucial role in the process of epileptogenesis (Vezzani 2005), during which the aberrant neuronal circuits and hyperexcitability are formed. The P2X7 receptor (P2X7R) belongs to the family of purinergic receptors, which is found predominantly expressed in the microglia as well as neurons and astrocytes in the central nervous system (CNS) (Burnstock 2008). The P2X7R can be activated by high concentration of extracellular ATP, leading to the microglia activation and release of various pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The P2X7R has been reported participating in many CNS disorders, including ischemia (Yu et al. 2013), spinal cord injury (Peng et al. 2009), Alzheimer’s disease (Wei et al. 2008), Huntington’s disease, and multiple sclerosis, and may serve as a novel therapeutic target of these diseases. In the recent years, several studies suggest that altered extracellular ATP levels and P2X7R dysfunction might be involved in the epileptogenesis (Kim et al. 2010). Application of P2X7R antagonist on epileptic animal models was found to inhibit microglial activation, reduce IL-1β production, and protect against cell death in some brain regions (Kim et al. 2009, 2011b). However, other studies reported contradict results that P2X7R antagonists would increase the seizure time during status epilepticus (SE) and aggravate cell damage (Kim and Kang 2011). Considering the fact that paradoxical performance of P2X7R antagonists on seizure is found in the pilocarpine and kainite epilepsy models, further investigations using different animal models are needed to confirm the role of P2X7R in epilepsy. Here, we used P2X7R antagonists, Brilliant blue G (BBG), selective P2X7R antagonists A-438079 and A74003, and P2X7R agonist 2′,3′-O-(4-benzoylbenzoyl)-adenosine 5′-

Rats were divided into eight groups (Fig. 1): (1) control group (sham operated); (2) vehicle group (saline 2 μL intracerebroventricular (i.c.v.)); (3) Brilliant blue G (BBG) 1 μg, 5 μg, and 10 μg groups (Sigma-Aldrich, St. Louis, MO, BBG 1 μg/ 5 μg/10 μg i.c.v.); (4) A-438079 group (Tocris Bioscience, Ellis-ville, MO, A-438079 10 μM in saline i.c.v.); (5) A-740003 group (Tocris Bioscience, Ellis-ville, MO, A-740003 10 μM in 0.001% saline i.c.v.); (6) 2′,3′-O-(4benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP) group (Sigma-Aldrich, St. Louis, MO, BzATP 5 mM in saline i.c.v.).

Fig. 1 The flow charts of the study. Rats (n = 240) were divided into eight groups (n = 30/group) and administered with corresponding drugs or vehicle 30 min prior to and 60 min post CL injection by intracerebroventricular injection. Thirty minutes after

intracerebroventricular drug injection, rats were treated with CL. Behavioral seizure attacks were scored according to the Racine scale. Rats were sacrificed at designated time points for western blotting and immunohistochemistry

Methods and Materials Experimental Animals Male Sprague-Dawley (SD) rats weighing 280–320 g were provided by West China Center for Experimental Animals, Sichuan University. The experiment strictly complied with the Laboratory Animal Welfare Protection Law of China and has been approved by the Experimental Animal Management Institute of Sichuan University. Before the experiment, it was ensured that rats adapted to the environment for more than 72 h. The animals were provided with commercial diet and free drinking water under controlled room temperature, humidity and lighting conditions (22 ± 2 °C, 55 ± 5% and 12:12 light/dark cycle with lights). Intracerebroventricular Drug Pretreatment

J Mol Neurosci The Racine scale for rat epileptic seizures

Animals were anesthetized with 4% chloral hydrate (400 mg/kg, intraperitoneally (i.p.)) and placed in stereotaxic frame. Cannulas for intracerebroventricular injection were fixed with dental cement (AP 1 mm, ML 1.5 mm, DV 3.5 mm, with the bregma as reference) (Paxinos and Watson 2007). Postoperative rats were allowed to rest for 24 h. Drugs or vehicle were injected into the third ventricle using a microinjector through cannulas. Drugs or vehicle were administered 30 min prior to and 60 min post CL injection with a total volume of 2 μL at a speed of 0.5 μL/min (Jimenez-Pacheco et al. 2013).

Table 1

Status Epilepticus Induction

hematoxylin and eosin (H&E) staining or immunohistochemistry. The hippocampus was separated and stored in liquid nitrogen for the western blot.

Thirty minutes after intracerebroventricular drug injection, rats were treated with CL (40 mg/kg, i.m.). Behavioral seizure attacks were scored according to the Racine scale (Table 1) (Racine 1972). Approximately 85% of CL-induced rats experienced acute stage 5 seizures that were maintained for at least 40– 50 min. Chloral hydrate (400 mg/kg, i.p.) was administered 1 h after onset of SE and repeated, if needed. Age-matched normal rats treated with equivalent saline (7.5 mL/kg) were used as controls (n = 6). Rats were scored every 5 min for 40 min after CL injection. The highest score obtained during each 5 min period and total seizure numbers were recorded by two observers blinded to treatment respectively. The seizure latency was defined as time from CL injection to the first behavioral seizure attack.

Stage

Presentation

0

No seizure

1

Immobility, eye closure, twitching of vibrissae, facial clonus

2 3

Mild head nodding Clonus of one forelimb

4 5

Rearing, often accompanied by bilateral forelimb clonus Rearing and falling with forelimb clonus

Western Blotting

At designated time points (non-SE, 0 h, 8 h, 24 h, 2 days, 4 days, 1 week, and 2 weeks after the onset of SE, n = 4, respectively), animals were intracardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS (pH = 7.4). The entire brains were removed and fixed overnight in 4% paraformaldehyde. Brains were then embedded in paraffin and cut into 4 μm coronal sections at the level of the bregma for

For western blotting (WB), rats were killed at designated time point and the hippocampus were removed immediately and lysed with 20 mM Tris-HCl buffer (pH = 8.0), containing 1% NP-40, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Lmercaptoethanol, 0.5 mM dithiothreitol, and a mixture of proteinase and phosphatase inhibitors (Sigma-Aldrich). BCA protein assay method was applied to measure the protein concentration using bovine serum albumin (BSA) as standard. One hundred micrograms of protein samples from hippocampus was loaded per lane, separated by SDS-PAGE (12% polyacrylamide gels) and electrotransferred onto nitrocellulose membranes. The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline for 1 h and then incubated at 4 °C overnight with either antibodies against P2X7R (1:500, APR-004, Alomone Labs), GAPDH (1:800, 5174, Cell Signaling Technology), Iba1 (0.5 μg/mL, 016-20001, Wako, Richmond, VA, USA), diluted in 2% BSA in PBS. The membrane were then incubated with alkaline phosphatase-conjuncted goat antirabbit IgG (Sigma-Aldrich) or goat anti-mouse IgG (Sigma-

Fig. 2 The expression of P2X7R following SE induced by CL. a Scheme showing experimental protocol. Surgery for cannulas was performed 24 h before SE. Drugs or vehicle were intracerebroventricular injected 10 min prior to and 1 h after CL injection. b The expression of P2X7R after SE at

different time points. c The P2X7R levels after SE compared with normal control. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; the 0 h group was sacrificed immediately 1 h after the onset of SE)

Sample Preparation

J Mol Neurosci

Aldrich) diluted 1:5000 in 2% BSA in PBS at room temperature for 2 h. Bands were visualized by enhanced chemiluminescence (Amersham, Brauschweig, Germany). The levels of tested proteins were evaluated by measuring the optical density of the protein bands using the Quantity One v4.62 software. Expression of GAPDH as internal controls was analyzed in the same protein extracts.

Immunohistochemistry H&E staining was carried out under standard method to observe hippocampus and cortical pathological changes. Immunohistochemistry was performed as previously described (Li et al. 2014a). Briefly, sections were dewaxed with xylene and dehydrated by ethanol at graded concentrations and distilled water. Sections were then incubated for 10 min in 3% hydrogen peroxide to block endogenous peroxidase activity. Hightemperature antigen retrieval was performed in 0.01 M citrate buffer (pH = 6.0) for 20 min. The brain sections were incubated at 4 °C overnight with mouse anti-GFAP (glia fibrillary acidic protein) IgG (1:200, PA5-16291, Invitrogen, ThermoFisher Scientific), NeuN (neuronal nuclei) (1:400, ABN78, Chemicon-Merck, Millipore), IL-1β (1:200, SC-7884, SantaCruz Biotechnology), IL-6 (1:200, sc-1265, Santa-Cruz

Fig. 3 The effect of P2X7R on seizure severity and behavioral deficits after status epilepticus. a The effects of P2X7R agonist and antagonists on seizure latency during status epilepticus. The seizure latency was defined as the latency to the first behavioral seizure activity. b Mean seizure stage during status epilepticus. c Mean seizure numbers during status

Biotechnology) and phospho-NF-κB p65 (phospho-nuclear factor-kappa B, Ser536) (1:200, ab86299, Abcam). The sections were then washed in 0.5 M Tris-HCl (pH = 7.6) containing 0.15 M NaCl and incubated for 1 h with appropriate biotinylated secondary antibody diluted in PBS (1:200). Photomicrographs of hippocampal region were taken using a digital camera (Olympus DP20, Tokyo, Japan) connected to an inverted microscope (Olympus BX51, Tokyo, Japan). Neuronal damage and astrogial activation were quantified by measuring the value of the integrated optical density (IOD) stained with antibodies against NeuN and GFAP, respectively, in the hippocampus at high magnification (×400). The expressions of IL-1, IL-6, and NF-κB were also evaluated by IOD value. The IOD value was calculated with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA) (Chu et al. 2012). Three high-power (×400) images were randomly selected for each animal, and the mean IOD of these six fields was considered as the IOD of the animal.

Morris Water Maze At day 7 after SE, spatial learning and memory were tested with Morris water maze, a circular black tank of 130 cm in diameter and 60 cm in height. The tank was filled with a depth

epilepticus. d The effects of P2X7R agonist and antagonists on Morris water maze performance after status epilepticus. (n = 30/group; a–c, Mann-Whitney U test; d, e, two-way ANOVA;*P < 0.05, **P < 0.005, ***P < 0.001 versus normal control)

J Mol Neurosci

Fig. 4 The NF-κB phosphorylation in the hippocampal CA3 region following CL-induced SE. (a) NF-κB phosphorylation in different groups at 2 days after SE. (b) NF-κB phosphorylation in different groups at 2 weeks after SE. (c) Quantitative analysis of NF-κB IOD value at 2 days after SE. (d) Quantitative analysis of p-P65 IOD value

at 2 weeks after SE. The arrows indicate NF-κB immunopositive stained cell. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; # P < 0.05, ## P < 0.005, ### P < 0.001 versus vehicle control. All photomicrographs are ×400. Bar = 50 μm)

of 30 cm water at 25 ± 1 °C. The maze was divided into four equal quadrants. The trials were performed according to Vorhees’ method. Spatial acquisition: all rats received a training trial consisting of daily session of four consecutive trials for 5 days. The hidden platform (diameter 10 cm, 1.5 cm below the water surface) was positioned in the middle of the southwest (SW) quadrant for all rats. The rats were released into the tank facing the maze wall at north (N), west (W), south (S), or east (E) quadrants in a predetermined pseudorandom order. A trial was terminated as soon as the rat found the platform. If the rat did not succeed within 120 s, it was guided onto the platform with a stick. The rat was allowed to stay on the platform for 20 s before being removed. Probe trial: immediately after the final training trial, the platform was removed. Rats were allowed into the pool at NE position and allowed to swim freely for 2 min. The time needed to find the platform (escape latency) in the training trial and time spent in the SW quadrant in the probe trial were recorded. The mean value of four escape latencies in the daily four training trials was taken as the escape latency for the rat. Values from eight rats in the same group were averaged to generate mean escape latency for that day.

Statistical Analysis Values were expressed as the mean ± SEM. For analysis of escape latency in the behavioral study, a two-way analysis of variance (ANOVA) was performed. Seizure number, seizure latency, and seizure stage were analyzed by the MannWhitney U test. Other data were subjected to one-way ANOVA and post hoc comparisons were carried out using Bonferroni’s test. P value < 0.05 was considered to be statistically significant.

Results P2X7R Is Upregulated in the Hippocampus of CL-Induced SE Rat Model The overall working flow of this study is shown in Fig. 1. Expressions of P2X7R in rat hippocampus at different time points after SE were evaluated by WB. P2X7R expression began to increase at 1 day, peaked at 2 days, and gradually decreased to baseline by 2 weeks following CL-induced SE (Fig. 2b). Specifically, expressions of P2X7R at 1 day, 2 day,

J Mol Neurosci

Fig. 5 The expression of IL-1 in the hippocampal CA3 region following CL-induced SE. (a) The expression of IL-1 in different groups at 2 days after SE. (b) The expression of IL-1 in different groups at 2 weeks after SE. (c) Quantitative analysis of IL-1 IOD value at 2 days after SE. (d) Quantitative analysis of IL-1 IOD value at 2 weeks after SE. The arrows

indicate IL-1 immunopositive stained cell. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; # P < 0.05, ##P < 0.005, ###P < 0.001 versus vehicle control. All photomicrographs are ×400. Bar = 50 μm)

4 day, and 1 week after SE were significantly higher than that of controls (P < 0.05).

At 2 days after SE, BzATP significantly increased the NF-κB phosphorylation while BBG 10 μg, A740003, and A438079 greatly decreased NF-κB phosphorylation compared to the vehicle group, but still higher than that of the normal control group (Fig. 4 (c)). At 2 weeks after SE, only the A438079 group significantly lowered NF-κB phosphorylation while no significant effect of BBG 10 μg and A740003 was detected (Fig. 4 (d)).

P2X7R Is Associated with Seizure Severity and Behavioral Deficits After SE The Racine scale was applied to monitor the motor seizure severity. Pretreatment with P2X7R antagonists including BBG 10 μg, A740003, and A438079 significantly decreased the occurrence of severe seizure attacks (seizure scored ≥ 4) at each time point (Fig. 3a). No significant difference was detected between the vehicle group and agonist group. The spatial memory was analyzed by Morris water maze. The escape latency of all groups decreased in a day-dependent pattern during the 5-day platform trial (Fig. 3b). Rats in the antagonist groups containing BBG 10 μg and A438079 needed shorter time to escape than the vehicle group. No significant difference was detected between the vehicle group, BzATP group, and A740003 group. P2X7R Is Associated with NF-κB Phosphorylation After SE NF-κB phosphorylation increased in the nuclei of CA3 pyramidal cells at both 2 days and 2 weeks after SE (Fig. 4 (c, d)).

P2X7R Antagonists Decrease Cytokine Overexpression After SE In the vehicle group, robust increase of IL-1β and IL-6 were observed compared with the control group (Figs. 5 and 6). At both 2 days and 2 weeks after SE, BzATP significantly increased IL-1β (Fig. 4 (c, d)). Three antagonists decreased IL-1β at 2 days after SE (Fig. 5 (c)) while only A438079 significantly attenuated the SEinduced IL-1β expression at 2 weeks (Fig. 5 (d)). As for IL-6 expression at 2 days after SE, no significant difference was observed between BBG 10 μg, A740003, A438079 group, and the vehicle group, respectively (Fig. 6 (c)). The IL-6 expression at 2 weeks was increased in the BzATP group, and decreased in the A438079 group, compared to the vehicle group (Fig. 6 (d)). No

J Mol Neurosci

Fig. 6 The expression of IL-6 in the hippocampal CA3 region following CL-induced SE. (a) The expression of IL-6 in different groups at 2 days after SE. (b) The expression of IL-6 in different groups at 2 weeks after SE. (c) Quantitative analysis of IL-6 IOD value at 2 days after SE. (d) Quantitative analysis of IL-6 IOD value at 2 weeks after SE. The arrows

indicate IL-6 immunopositive stained cell. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; # P < 0.05, ##P < 0.005, ###P < 0.001 versus vehicle control. All photomicrographs are ×400. Bar = 50 μm)

significant inhibition on IL-6 was found in the BBG 10 μg and A740003 group.

significantly lower at 2 weeks, but higher at 2 days compared to the vehicle group.

P2X7R Antagonists Inhibit Astroglial Activation After SE The GFAP-positive cells were shin and few in normal controls and showed hypertrophic and significantly increased in the hippocampal CA1 region of the vehicle group after induction of SE (Fig. 7 (a, c)). Compared with the vehicle group, the GFAP IOD value was significantly increased in the BzATP group and decreased in the BBG 10 μg, A740003, and A438079 groups at both 2 days and 2 weeks after SE (Fig. 7 (c, d)).

P2X7R Antagonists Inhibit Microglia Activation After SE At both 2 days and 2 week after SE, the expressions of Iba-1 in the vehicle group were significantly higher than those of the control group (P < 0.05; Fig. 9a, b). Pretreatment with BzATP significantly increased Iba-1 expression, compared with the vehicle group (P < 0.05; Fig. 9a, b). Meanwhile, pretreatment with BBG 10 μg and A438079 significantly decreased the expression of Iba-1 after SE (P < 0.05; Fig. 9a, b). Besides, BBG showed a dose-dependent inhibitory effect on Iba-1 expression at 2 days after SE.

P2X7R Antagonists Prevent Neuron Damage After SE

Discussion Compared to normal control, the NeuN-positive cells significantly decreased in the hippocampal CA3 region at 2 days and 2 weeks after SE (Fig. 8 (c, d)). The antagonists BBG 10 μg, A740003, and especially A438079 significantly ameliorated the SE-induced neuronal damage at 2 days and 2 weeks (Fig. 8 (c, d)). As for the BzATP group, the NeuN IOD value was

The key finding of our study is that the P2X7R was upregulated and involved in the SE-induced inflammatory response. Pretreatment with P2X7R antagonists including BBG, A740003, and A438079 decreased the overexpression of variant cytokines, as well as ameliorated astrogial activation,

J Mol Neurosci

Fig. 7 The expression of GFAP in the hippocampal CA1 region following CL-induced SE. (a) The expression of GFAP in different groups at 2 days after SE. (b) The expression of GFAP in different groups at 2 weeks after SE. (c) Quantitative analysis of GFAP IOD value at 2 days after SE. (d) Quantitative analysis of GFAP IOD value

at 2 weeks after SE. The arrows indicate GFAP immunopositive stained cell. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; # P < 0.05, ## P < 0.005, ### P < 0.001 versus vehicle control. All photomicrographs are ×400. Bar = 50 μm)

microglial activation, and neuron loss to different extent. In addition, SE severity was also alleviated by antagonists with improvement of spatial memory. These findings strongly suggest P2X7R play a critical role in epilepsy and may serve as a potential therapeutic target. P2X7Rs was located in both presynaptic and postsynaptic sites, serving as an ATP-gated ion channels (Gever et al. 2006). Since ATP acts as transmitter or cotransmitter in CNS, P2X7R may affect the release and transmission of neurotransmitter (Sperlagh et al. 2006) and thus involved in epilepsy through modulating neuronal excitability (Henshall et al. 2013). The present study showed pretreatment with antagonist including BBG, A740003, and A438079 reduced seizure severity during the status epilepticus. Similarly, several previous studies also found A438079 and BBG could produce anticonvulsant and neuroprotection effects in both mice and rat SE models induced by kainic acid (Engel et al. 2012; Jimenez-Pacheco et al. 2013). However, another study reported contradict results that administration of P2X7R antagonists or P2X7R knockout mice showed increased susceptibility to seizures induced by pilocarpine (Kim and Kang 2011). The paradoxical results of P2X7R inhibition on epilepsy might attribute to application of different animal models and drug administration methods. Although these epilepsy models have

long been used to explore the underlying mechanism of epilepsy and discover novel therapeutic compounds, distribution, intensity, and time course of vulnerable temporal lobe neurons as well as cell damage and neurogenesis extent were found different in kainic acid and pilocarpine-induced status epilepticus (Covolan and Mello 2000; Covolan et al. 2000). Coriaria lactone is extracted from Chinese traditional herb coriaria and has long been used as an epileptogeneic agent for establishment of epilepsy models in China. Its specific epileptogenic property is still under investigation and several preliminary studies showed it may be associated with altered glutamate receptor 5 (Li et al. 2010), ATP-sensitive potassium channels (Zou et al. 2003), and hypoxia-inducible factor-1 (Li et al. 2014b). Although most studies including the present research supported the antagonist of P2X7R as an anticonvulsant effect, further studies on both animal models and human cases are needed to confirm the effect of P2X7R in treatment of epilepsy. In contrast to our historical concept that the CNS is immune privileged owing to strict constriction of the bloodbrain barrier (BBB), neuroinflammation has emerged as a prominent feature in virtually all neurological process and been proved to participated in pathological progression in epileptogenesis (Aronica and Crino 2011; Vezzani et al.

J Mol Neurosci

Fig. 8 The expression of NeuN in the hippocampal CA3 region following CL-induced SE. (a) The expression of NeuN in different groups at 2 days after SE. (b) The expression of NeuN in different groups at 2 weeks after SE. (c) Quantitative analysis of NeuN IOD value at 2 days after SE. (d) Quantitative analysis of NeuN IOD value

at 2 weeks after SE. The arrows indicate NeuN immunopositive stained cell. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; # P < 0.05, ## P < 0.005, ### P < 0.001 versus vehicle control. All photomicrographs are ×400. Bar = 50 μm)

2011). Neuroinflammation is mainly characterized by activation of astroglia, microglia, and endothelial cells of BBB, as well as infiltration of various immune cells and inflammatory molecules. Pro-inflammatory cytokines such as IL-1β, IL-6,

and TNF-α normally express at low basal levels in the brain and are rapidly upregulated in both glia and neurons in human epilepsy foci and in experimental epilepsy animal models (Ravizza et al. 2008; Ravizza and Vezzani 2006). These

Fig. 9 a The expression of Iba-1 in the hippocampus following CLinduced SE. The expression and quantitative analysis of Iba-1in different groups at 2 days after SE. b The expression and quantitative

analysis of Iba-1 in different groups at 2 weeks after SE. (n = 3/group, one-way ANOVA; *P < 0.05, **P < 0.005, ***P < 0.001 versus normal control; #P < 0.05, ##P < 0.005, ###P < 0.001 versus vehicle control)

J Mol Neurosci

elevated cytokines are demonstrated to increase neuronal hyperexcitability and contribute to epileptogenesis through multiple mechanisms, such as blocking astrocyte-mediated reuptake of glutamate from synaptic space (Hu et al. 2000), altering GABAergic neurotransmission (Roseti et al. 2015), and modulating voltage-gated ion channels (Vezzani and Viviani 2015). The P2X7R can be activated by high concentration of extracellular ATP in reaction to variant stimuli. It has been reported to participate in CNS inflammation by activating microglia and releasing of various pro-inflammatory cytokines, thus promoting brain injury or excitotoxicity (Bernardino et al. 2008; Chu et al. 2012; Friedle et al. 2011). In our study, the level of P2X7R increased after SE accompanied with increase of IL-1β and IL-6 at 2 days and 2 weeks after SE. Pretreatment with P2X7R antagonists, especially A438079, significantly reduced expression of these cytokines while the agonist BzATP increased their expression. Comparing with other two antagonists, in this study, A438079 showed prominent inhibition on SE-induced neuroinflammation, which may attribute to different effect sites of different antagonists. Extensive evidence from clinical and experimental data suggests that neuroinflammation is associated with seizure severity and recurrence (Auvin et al. 2010a, b). Accordingly, we found that pretreatment with P2X7R agonist exacerbate seizure severity and cognitive performance while P2X7 antagonists improved neurological behavior in SE rats. NF-κB is a dimeric transcription factor, which presents in cytoplasm and serves as a critical regulator of inducible expression of genes involved in immunity, inflammation, and cell death. NF-κB phosphorylation contributes to neuronal vulnerability in response to different pathophysiological conditions. In this study, we found that P2X7R activation modulated neuroinflammation via targeting NF-κB p65 in the hippocampus. In accordance with previous study, P2X7R selectively targets NF-κB p65 in CA1 and CA3 after SE (Kim et al. 2013) while P2X7R antagonists including OxATP, A438079, and A740003 reduced NF-κB p65 expression (Kim et al. 2011a). In patients with epilepsy, neuronal death and gliosis in the hippocampus are main pathological changes (Fujikawa et al. 2000) and may be involved with memory deficits and epileptogenesis. P2X7R was reported expressed in neuron and astrocyte, contributing to cell death by modulating cytokine overexpression (Matute et al. 2007; Sperlagh and Illes 2014), superoxide or nitric oxide production (Codocedo et al. 2013; Parvathenani et al. 2003), and morphological transformation (Monif et al. 2009). In accordance with previous studies in pilocarpine-induced epilepsy (Kim et al. 2011b, 2015), we found that CL-induced SE caused prominent astroglial activation and neuron loss in hippocampal CA1 and CA3 region respectively, which could be lightened by P2X7R antagonists. Similar to findings in pilocarpine model, A438079 showed prominent neuroprotective effects and inhibit the seizure-

induced cell death (Kim et al. 2011a; Mesuret et al. 2014). In cerebral ischemia/reperfusion injury model, the P2X7R antagonists protected hippocampal neurons from damage (Chu et al. 2012). Taking together, our findings suggest that P2X7R antagonists may serve as potential neuroprotector in epileptogenesis. There are several limitations of this study. Firstly, the study was mainly conducted on animal model. It might need further consolidation on a larger group of patients with epilepsy to confirm the association between P2X7R and epileptogenesis. Secondly, the seizure severity and manifestation were evaluated in the acute phase of epileptogenesis after SE. Development of spontaneous recurrent seizure was not studied in this study, leading to the lack of long-term effect of P2X7R on epileptogenesis. Thirdly, epilepsy is commonly regarded as a result of comprehensive factors and neuroinflammation contains various pathways and molecules besides NF-κB pathway. Other mechanism and molecules involved in this process might need further investigations in the future.

Conclusions In conclusion, the present study describes the role of P2X7R in NF-κB mediated neuroinflammation in response to CLinduced SE in rat hippocampus and suggests that P2X7R activation may induce cytokine overexpression, astrogial activation, and neural loss in epileptogenesis. Antagonists of P2X7R could robustly inhibit neuroinflammation and neuronal damage in rat hippocampus, as well as reduce seizure severity and ameliorate memory deficits caused by SE. Thus, blocking P2X7R may be a novel therapeutic strategy for epilepsy and neuroinflammation. Acknowledgements This study was supported by the National Natural Science Foundation of China (Item Number: 81501127).

Author Contributions D.Z. and J.-M.L. conceived and designed the experiments; C.H. and X.-S.C. performed the experiments and wrote the paper; and X.H. and H.-X.X. contributed materials and analysis tools and protocols.

Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interest.

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