Epilepsy Research (2009) 86, 209—220

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Astrocyte activation and memory impairment in therepetitive febrile seizures model

Lu Yanga, Fuhai Li a, Haiju Zhangb, Wei Gea, Changrui Mia,Ruopeng Suna,∗, Chunxi Liuc

a Department of Pediatrics, Qilu Hospital, Shandong University, Jinan, Shandong Province 250012, Chinab Department of Pediatrics, Renmin Hospital, Wuhan University, Wuhan, Hubei Province, Chinac The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministryof Public Health, Shandong University Qilu Hospital, Jinan, Shandong Province, China

Received 24 March 2009; received in revised form 15 June 2009; accepted 2 July 2009Available online 29 July 2009

KEYWORDSSeizures;Febrile;Glial fibrillary acidicprotein;S100 calcium-bindingprotein beta subunit;Learning;Rats

Summary Frequently repetitive febrile seizures (FRFS) in immature brain could impair long-term memory without obvious pathological alteration. Although astrocyte activation has beenimplicated in many seizure models, it has never been examined in febrile seizure models. Weinvestigated astrocyte activation states after FRFS in postnatal-10-day (P10) rats by westernblot and immunohistochemical analysis of GFAP and S100�, two protein markers for activatedastrocytes, at three time points (P25, P35, P45). The levels of GFAP and S100� increased signif-icantly at all the time examined. Furthermore, we administered propentofylline, an astrocytemodulator, to verify the relationship between the activated astrocytes and memory injury. Afterpropentofylline treatment for 10 consecutive days following P10 frequently repetitive FS, ratsexhibited improved performances in Morris water maze at P36 and inhibitory avoidance task atP45, along with markedly suppressed overexpression of GFAP and S100�. This research suggests

that modulation of astrocyte activation might be a potential therapeutic target to improvememory outcomes after frequently repetitive febrile seizures.© 2009 Elsevier B.V. All rights re

∗ Corresponding author. Tel.: +86 13854187216.E-mail addresses: yanglukitty2002@sina.com.cn (L. Yang),

fuhaili1969@sina.com.cn (F. Li), zhjsdu2007@sina.com.cn(H. Zhang), qlgw1982@sina.com.cn (W. Ge),changruimi@sina.com.cn (C. Mi), srpyl2008@sina.com.cn (R. Sun),chunxiliu66@sina.com.cn (C. Liu).

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0920-1211/$ — see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.eplepsyres.2009.07.001

served.

ntroduction

ebrile seizure (FS) is the most common type of seizuren infants and children. About forty percent of childrenith FS experience brief and recurrent seizures (Engel and

edley, 2007). Children with multiple FS were at a greaterisk of developing epilepsy later in life (Annegers et al.,979) and performed less well in all neuropsychologicalests than controls and individuals suffering from a sin-le febrile convulsion (Kolfen et al., 1998). In a frequently

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epetitive febrile seizures (FRFS) model, Chang et al. (2003)ound that FRFS in rat pups starting at postnatal dayP) 10 impaired hippocampus-dependent long-term mem-ry despite no significant morphological changes (neuronaleath and aberrant mossy fibers) in the hippocampus. Defec-ive CREB phosphorylation, long-lasting deficits in synapticlasticity and NR2A tyrosine phosphorylation have beenuggested to be potential molecular explanations for thisippocampal dysfunction (Chang et al., 2003, 2005).

Studies about the long-term changes after FS mainlyocused on neuronal death, synaptic reorganization, neuro-ransmitter receptor regulation and ion channel modulationToth et al., 1998; Brewster et al., 2002; Bender et al.,003; Dube et al., 2004; Gonzalez Ramirez et al., 2007). Fewtudies have investigated astrocytes after FS (Lemmens etl., 2008). Astrocytes are traditionally regarded as elementsor structural support and ionic homeostasis in central ner-ous system. A study by O’Dowd et al. (1994) indicated thatstrocytic glycogenolysis might play a crucial part in consol-dation of long-term memory. Moreover, astrocytes expresslutamate receptors and could respond to neuronal gluta-ate by generating slowly propagating calcium waves and

urther releasing chemical transmitters (Cornell-Bell et al.,990; Newman, 2003; Anlauf and Derouiche, 2005). Thislia—neuron reciprocal signaling may play a role in synap-ic plasticity and eventually in information processing in therain.

We used the FRFS model developed by Chang et al. tonvestigate whether early-life FRFS could induce long-termlterations of astrocytes in the hippocampus. Propento-ytlline, a glial modulator, was administered in an efforto understand the role of astrocytes in the formation ofemory deficiency.

aterials and methods

nimals

dult pregnant Sprague—Dawley rats were purchased from Shan-ong University. Rat pups were born and housed with their damsntil weaning. Grownup rats were maintained in quiet, uncrowdedacilities and given unlimited access to food and water. All ratsere kept in a room maintained at constant temperature (23 ◦C)nd relative humidity (60%) on a 12-h light schedule.

xperimental model establishment

ccording to Chang et al. (2003), rat pups were placed into ayperthermia chamber on postnatal (P) days 10—12. An adjustabletream of heated air, coming from a hair dryer placed 50 cm abovehe chamber, was blown into the chamber. The chamber was inwater bath maintained at 37 ◦C, and the behaviors of rat pups

n the chamber were continuously monitored. The rectal tempera-ures of the rat pups were measured before and every 2 min duringyperthermia treatment. When their rectal temperatures reachedetween 40 and 43 ◦C, the pups exhibited head bobbing, clonicwitching of hindlimbs, generalized myoclonic jerk, falling, tonic-lonic seizures, etc. After 10 min in the chamber at 40—43 ◦C rectal

emperature, the pups were removed immediately and placed oncool surface until they regained posture and their core tempera-

ures returned to baseline. The rat pups were randomly divided intohe following four groups (n = 54 in each group): (1) nonhyperther-ia control (NHC); (2) hyperthermia without seizure control (HC);

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3) single FS (SFS) on P10; (4) frequently repetitive FS (FRFS), threeS daily at an interval of 4 h between seizures, starting at P10, forconsecutive days and a total of nine seizures. The NHC pups were

emoved from the cage but were not subjected to the hyperthermichamber. The HC pups were subjected to nine episodes of hyper-hermia in 3 consecutive days but seizure was prevented each timey pretreatment with pentobarbital (20 mg/kg on P10, 25 mg/kg on11, 30 mg/kg on P12, i.p.).

issue preparation

ighteen rats from each group were anesthetized and sacrificed at25, P35 and P45.

For western blot analysis, eight rats from each group wereecapitated. The hippocampi were dissected on ice and homog-nized with a glass homogenizer in a lysis buffer containing0 mM Tris—HCl, 150 mM NaCl, 0.1 mg/ml phenylmethylsulfonyl,mM ethyleneglycol tetraacetate, 1 mg/ml leupeptin, 5 mg/mlprotinin and 1 mg/ml pepstatin (Sigma Chemical Co., USA). Theomogenates were centrifuged for 15 min (12,000 rpm, 4 ◦C) andhe supernatants obtained were stored at −80 ◦C until use.

For immunohistochemistry, ten rats from each group were per-used transcardially with chilled PBS followed by 4% paraformalde-yde. The intact brains were manually dissected from the calvariumnd immersed in 4% paraformaldehyde for 24 h at 4 ◦C before paraf-n embedding. Embedded brains between 3.2 and 4.2 mm posterioro bregma were sectioned coronally with a microtome into 5 �mhick sections and collected on gelatin-coated microscope slides.

estern blot

otal protein concentrations in hippocampus supernatants wereetermined with a BCA Protein Assay Kit (Pierce Biotechnology,SA). Twenty micrograms protein of each sample was added toloading buffer and boiled for 5 min at 95 ◦C. Samples and stan-

ard protein markers were separated using SDS-PAGE (12% gels) andlectrophoretically transferred at 100 mV for 60 min onto nitrocel-ulose membranes. Nonspecific bindings were blocked by incubationith 5% nonfat milk in TBS containing 0.05% Tween 20 for 2 h.fter blocking, membranes were incubated overnight at 4 ◦C withouse anti-GFAP antibody (1:1000; Millipore Corporation, USA),ouse anti-�-actin antibody (1:5000; Abcam Inc., USA) or rabbit

nti-S100� antibody (1:1000; Epitomics Inc., USA). After incu-ation with horseradish peroxidase-conjugated goat anti-mouseZSGB-BIO, China) or goat anti-rabbit (Hangzhou HuaAn Biotech,hina) secondary antibody (1:2000) for 2 h at room temperature,

mmunoblots were detected using an ECL-Plus kit (Amersham Lifecience, USA). The mean density ratios of GFAP and S100� to �-actinere used for data analysis.

mmunohistochemistry

ippocampal sections were incubated with diluted mouse anti-FAP antibody (1:200; Millipore Corporation, USA) and rabbitnti-S100� antibody (1:200; Epitomics Inc., USA) overnight at◦C. Subsequently they were exposed to HRP-conjugated goatnti-mouse (1:200; ZSGB-BIO, China) or goat anti-rabbit (1:200;angzhou HuaAn Biotech, China) secondary antibody. After treat-ent with DAB (ZSGB-BIO, China), the slides were counterstainedith hematoxylin and mounted with permanent mounting medium.

mmunohistochemical stainings were observed under Olympusicroscope. Image-Pro Plus 6.0 was used for quantitative analysis of

FAP and S100�. Five sections (ten hippocampi) from each rat were

andomly selected for quantification. Values of background stain-ngs were subtracted from the immunoreactive intensities. Averageptical densities of GFAP and S100� in complete hippocampal areasere averaged for each rat and used for analysis (Fig. 1).

Astrocyte activation and memory impairment 211

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Propentofylline (PPF) administration

In the PPF (Sigma—Aldrich Corporation, USA) treatment experiment,rat pups were divided into three groups (n = 64 in each group): (1)saline-treated nonhyperthermia control group (S-NHC); (2) saline-treated FRFS group (S-FRFS); (3)PPF-treated FRFS group (P-FRFS),which were given PPF 10 mg/kg i.p. (Sweitzer et al., 2001; Xia etal., 2004) once a day for 10 consecutive days beginning at P14.Detection of astrocyte activation markers was performed at thetime points as mentioned in Tissue preparation section (eighteenrats from each group at each time, eight for western blot, ten forimmunohistochemistry). A separate cohort of rats from each group(n = 10) underwent Morris water maze and inhibitory avoidance taskat P36 and P45, respectively.

Behavioral test paradigm

Morris water mazeThe Morris water maze is a widely used test of visual—spatial mem-ory and hippocampal integrity.

A circular steel pool (150 cm in diameter, 70 cm in depth)was filled with water (26 ± 2 ◦C) up to 10 cm below the rim. Thewater was made opaque by covering its surface with white floatingresin beads. The pool was divided into four quadrants (northeast,northwest, southeast, southwest). The movement of the rat wasmonitored with a video and computer tracking system (HVS Image,Hampton, UK). On day 1, each rat was allowed to swim freely inthe pool for 120 s for habituation. On days 2—5, a hidden platform(10 cm × 10 cm) was submerged to 1 cm below the water surfacein the middle of the target quadrant (northeast quadrant). Duringacquisition training, six trials were conducted daily for 4 consecu-tive days. For each rat, while the target platform location remainedconstant, the rats were placed in the water facing the wall fromdifferent quadrants in a pseudorandom order. On mounting the plat-form, the rat was given a 30-s rest on the platform before the nexttrial. In case a rat failed to locate the platform after 120 s, it wasmanually placed on the platform for a 30-s rest and assigned alatency of 120 s. The total daily latency from immersion into thepool to escape onto the platform was recorded. To analyze mem-ory retention, a probe trial was conducted on day 6. Each rat wasplaced into the water diagonally from the northeast quadrant andallowed to swim freely for 120 s without the platform present. Theratio of the amount of time spent in the northeast quadrant to that

spent in the other three quadrants was measured.

Inhibitory avoidance taskThe inhibitory avoidance (IA) task, another hippocampus-dependentbehavior test, was used to measure different phases of retention

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erformance in rats on P45 (Chang et al., 2003). The apparatus con-isted of one illuminated compartment and one dark compartment.shock generator was connected to the floor of the dark compart-ent. Before the experiment, the rat was kept in a dim room forh to adjust to the environment. In the training phase, the ratas placed in the illuminated compartment facing away from theoor. As the rat turned around, the door was opened. When the ratntered the dark compartment, the door was closed, and the rat wasiven a 1.0 mA/1 second shock. The rat then was removed from thelley and returned to its home cage. The retention test was given 1,, or 24 h after training for the measurement of short-term, inter-ediate, and long-term memory, respectively. The rat was againlaced in the illuminated compartment and the latency to step intohe dark compartment was recorded as the measure of retentionerformance. Rats that did not enter the dark compartment within00 s were removed from the alley.

tatistical analysis

PSS 11.0 was used for statistical analysis. Values were expresseds mean ± SD for each group. One-Sample Kolmogorov—Smirnov Testas performed for normality of each data set. Normally distributedata were tested for homogeneity of variance and analyzed by one-ay analysis of variance (ANOVA). Non-normally distributed dataere analyzed by non-parametric Kruskal—Wallis ANOVA. One-wayNOVA with LSD post hoc tests were performed to compare multi-le groups for maximal rectal temperature, immunohistochemistry,estern blot and probe trial. The water maze escape latencyata were analyzed using ANOVA for repeated measures, with theest day as the within-subject factor. Inhibitory avoidance testsere evaluated with the non-parametric Kruskal—Wallis followedy Dunn’s multiple comparison tests. P value less than 0.05 wasonsidered statistically significant.

esults

he maximal rectal temperatures were notifferent among febrile seizure and hyperthermiaroups

he maximal rectal temperatures of rats in each episodef FS or hyperthermia were recorded and compared amongifferent groups (Table 1). There were no significant dif-erences among FS groups and hyperthermia control groupcross different episodes of hyperthermia.

212 L. Yang et al.

Table 1 Maximal rectal temperature in each episode of febrile seizure (◦C).

Group P10 P11 P12

1st FS 2nd FS 3rd FS 1st FS 2nd FS 3rd FS 1st FS 2nd FS 3rd FS

NHC — — — — — — — — —HC 42.7 ± 0.8 42.6 ± 0.7 42.6 ± 0.8 42.7 ± 0.8 42.6 ± 0.7 42.8 ± 0.8 42.5 ± 0.7 42.7 ± 0.9 42.8 ± 1.0SFS 42.7 ± 0.6 — — — — — — — —FRFS 42.5 ± 0.8 42.8 ± 0.8 42.8 ± 1.0 42.7 ± 0.7 42.7 ± 0.8 42.7 ± 0.9 42.8 ± 0.8 42.8 ± 1.0 42.6 ± 0.9S-NHC — — — — — — — — —S-FRFS 42.8 ± 0.7 42.5 ± 0.9 42.6 ± 0.7 42.8 ± 0.9 42.8 ± 0.8 42.7 ± 0.9 42.7 ± 0.7 42.8 ± 0.9 42.7 ± 0.8P-FRFS 42.6 ± 0.7 42.7 ± 0.8 42.7 ± 0.8 42.7 ± 1.0 42.7 ± 0.9 42.6 ± 0.7 42.8 ± 0.9 42.6 ± 0.8 42.8 ± 0.8

Maximal rectal temperature was expressed as mean ± SD. FS, febrile seizure; NHC, nonhyperthermia control; HC, hyperthermia withoutntlyeizur

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seizure control; SFS, single febrile seizure on P10; FRFS, frequemia control; S-FRFS, saline-treated frequently repetitive febrile sseizures.

requently repetitive febrile seizures in immaturerain led to prolonged astrocyte activation inippocampus

FAP is a cytoskeletal protein persistently expressed by

strocytes. S100B is a calcium-binding neurotrophic cytokineainly synthesized and released by astrocytes. Overexpres-

ion of these two proteins could be used as markers forstrocyte activation. Western blot analysis (Fig. 2) showedhat both GFAP/�-actin and S100�/�-actin ratios were sig-

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igure 2 Western blot analysis revealed higher expression of GFAPebrile seizures. (A) Western blot of GFAP and S100� in hippocampi frf GFAP (B) and S100� (C) were normalized against �-actin. NHC,ontrol; SFS, single febrile seizure on P10; FRFS, frequently repetitiame time point.

repetitive febrile seizures; S-NHC, saline-treated nonhyperther-es; P-FRFS, propentofylline-treated frequently repetitive febrile

ificantly increased in FRFS group compared with NHC groupt P25, P35 and P45 (GFAP, 1.76 ± 0.21 vs 0.96 ± 0.18 at P25,.86 ± 0.23 vs 0.99 ± 0.15 at P35, 2.05 ± 0.19 vs 1.04 ± 0.16t P45; S100�, 1.55 ± 0.19 vs 0.94 ± 0.18 at P25, 1.78 ± 0.18s 0.96 ± 0.17 at P35, 1.92 ± 0.19 vs 1.08 ± 0.18 at P45). The

FAP/�-actin and S100�/�-actin ratios were at compara-le basal levels among NHC, HC and SFS groups (P > 0.05).verage optical density analysis in immunohistochemistryevealed similar group differences as that in western blotFigs. 3 and 4).

and S100� in hippocampus after early-life frequently repetitiveom different groups at P25, P35 and P45. The expression levelsnonhyperthermia control; HC, hyperthermia without seizure

ve febrile seizures. *P < 0.001 compared with NHC group at the

Astrocyte activation and memory impairment 213

Figure 3 Frequently repetitive febrile seizure in early-life induced hippocampal overexpression of GFAP. (A) Representativeimmunohistochemical staining of GFAP in hippocampal CA1 region from nonhyperthermia control (NHC, a—c), hyperthermia control(HC, d—f), single FS (SFS, g—i) and frequently repetitive FS group (FRFS, j—l) at different time points (P25, P35 and P45). (B) Averageoptical density of GFAP staining in complete hippocampus in the four groups at three time points. *P < 0.001 compared with NHCgroup at the same time point.

214 L. Yang et al.

Figure 4 Frequently repetitive febrile seizure in early-life induced hippocampal overexpression of S100�. (A) Representativeimmunohistochemical staining of S100� in hippocampal CA1 region from nonhyperthermia control (NHC, a—c), hyperthermia control(HC, d—f), single FS (SFS, g—i) and frequently repetitive FS group (FRFS, j—l) at different time points (P25, P35 and P45). (B) Averageoptical density of S100� staining in complete hippocampus in the four groups at three time points. *P < 0.001 compared with NHCgroup at the same time point.

Astrocyte activation and memory impairment 215

Figure 5 Semiquantitative analysis by western blot showed that propentofylline downregulated the overexpressed GFAP andS100� in hippocampus after early-life frequently repetitive febrile seizures. (A) Western blot of GFAP and S100� in hippocampi from

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Propentofylline (PPF) administration attenuatedFRFS-induced astrocyte activation in hippocampus

The GFAP/�-actin and S100�/�-actin ratios (Fig. 5) in ratsfrom S-FRFS group were significantly elevated comparedto that from S-NHC group at P25, P35 and P45 (GFAP,1.70 ± 0.23 vs 0.96 ± 0.17 at P25, 1.69 ± 0.16 vs 1.03 ± 0.18at P35, 1.99 ± 0.18 vs 0.99 ± 0.20 at P45; S100�, 1.51 ± 0.20vs 0.95 ± 0.18 at P25, 1.74 ± 0.19 vs 0.99 ± 0.17 at P35,1.97 ± 0.25 vs 1.05 ± 0.20 at P45). Rats treated with PPFfollowing FRFS exhibited remarkably deduced ratios ofGFAP/�-actin and S100�/�-actin (GFAP, 1.24 ± 0.19 at P25,1.27 ± 0.15 at P35, 1.33 ± 0.16 at P45; S100�, 1.18 ± 0.20at P25, 1.21 ± 0.16 at P35, 1.34 ± 0.18 at P45). The sameeffects of PPF on GFAP and S100� expression could also beobserved in immunohistochemistry study (Figs. 6 and 7).

Propentofylline (PPF) administration preventedlong-term memory deficits following infant FRFS

Morris water maze was performed to assess the effects ofpropentofylline on spatial learning. As shown in Fig. 8A,mean escape latency improved in all groups over the 4acquisition training days as the subjects became more famil-

iar with the task (P < 0.001). Repeated measures ANOVArevealed differences among the S-NHC, S-FRFS and P-FRFSgroups (P = 0.003). Post hoc analysis revealed that ratsfrom S-FRFS group spent more time in finding the platformthan those from S-NHC group (P = 0.001) and PPF treatment

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) and S100� (C) were normalized against �-actin. S-NHC, saline-y repetitive febrile seizures; P-FRFS, propentofylline-treatedFS group at the same time point.

ould shorten the prolonged time in finding the platformP = 0.012). On the day of probe testing (Fig. 8B), a sig-ificant group effect was observed on the percentage ofime spent in target quadrant (P = 0.002). Rats in S-FRFSroup spent significantly less time (26.2% ± 3.7%) in the tar-et quadrant than those in S-NHC group (34.2% ± 2.6%). PPFreatment markedly increased the time ratio (31.2% ± 3.3%)ats spent in target quadrant.

In IA task (Fig. 9), S-NHC, S-FRFS and P-FRFS groupemonstrated comparable retention times at 1 h after train-ng (P = 0.296). However, at both 3 and 24 h, retentionimes of rats from S-FRFS group were significantly shorter282.3 ± 24.2 s at 3 h; 200.8 ± 20.9 s at 24 h) than those ofats from S-NHC group (527.8 ± 21.5 s at 3 h; 443.0 ± 20.8 st 24 h). PPF treatment prolonged the retention times at 3nd 24 h (474.5 ± 21.9 s at 3 h; 407.0 ± 23.2 s at 24 h).

iscussion

n this frequently repetitive febrile seizures (FRFS) model,e came to the following two conclusions. First, FRFS in

mmature brain could lead to long-term astrocyte acti-ation. Second, propentofylline could rescue the memoryeficits after FRFS by attenuating astrocyte activation,hich suggested that activated astrocytes following FRFS

articipate in the memory impairment.

The maximal rectal temperatures were not significantlyifferent among FS groups and hyperthermia group. Sostrocyte activation in FRFS group was not caused by hyper-hermia. The persistent astrocyte activation state in our

216 L. Yang et al.

Figure 6 Administration of propentofylline (PPF) reduced hippocampal overexpression of GFAP. (A) Representative immunohis-tochemical staining of GFAP in hippocampal CA1 region from saline-treated nonhyperthermia control (S-NHC, a—c), saline-treatedF —i) ad groua

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RFS group (S-FRFS, d—f) and PPF-treated FRFS group (P-FRFS, gensity of GFAP staining in complete hippocampus in the threet the same time point.

esearch is similar with that in KA-induced seizure in P15ats (Somera-Molina et al., 2007). So we believe that astro-yte activation could occur and persist in immature seizureodel without prominent neuron death and mossy fiber

prouting. Astrocytes, which make up nearly half the cellsn the central nervous system, are not only elements ofupportive structure but also active players in bidirectionalstrocyte—neuron communication. Upon neuron excitation,otassium and various transmitters released from neurons

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t different time points (P25, P35 and P45). (B) Average opticalps at three time points. *P < 0.001 compared with S-FRFS group

ould activate astrocytes (Glaum et al., 1990; Priller et al.,998a,b). Recent findings in the FS model have shed light onpotential role of interleukin-1� (IL-1�) in the epileptogenicrocess (Heida and Pittman, 2005). IL-1� is required for gen-

ration of hyperthermic seizures in the FS model becausehe temperature threshold for FS induction was significantlyncreased in mice that lack the IL-1�R1 gene (Dube et al.,005). IL-1�, which is believed to be one of the most pow-rful inducers of reactive astrogliosis (Herx and Yong, 2001;

Astrocyte activation and memory impairment 217

Figure 7 Administration of propentofylline (PPF) reduced hippocampal overexpression of S100�. (A) Representative immunohis-tochemical staining of S100� in hippocampal CA1 region from saline-treated nonhyperthermia control (S-NHC, a—c), saline-treated

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FRFS group (S-FRFS, d—f) and PPF-treated FRFS group (P-FRFS, gdensity of S100� staining in complete hippocampus in the threeat the same time point.

John et al., 2004), might also be one inducer of astrocyteactivation in this FRFS model.

Astrocytes play an important part in memory consol-

idation through communication with neurons, mainly infollowing three approaches: (1) neurons cannot synthesizeglutamate but rely on neighbouring astrocytes for the sup-ply of glutamate (Hertz and Zielke, 2004). Astrocytes carryout net synthesis of tricarboxylic acid cycle intermediates

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eeded for glutamate synthesis formed by rapid degrada-ion of glycogen (Gibbs et al., 2007). This makes learningependent on glycogenolysis. Intracerebral injection of a

lycogenolysis inhibitor, 1,4-dideoxy-1,4-imino-D-arabinitolDAB), has been proved to interrupt memory consolidationn young chickens (Gibbs et al., 2006). (2) Astrocytes couldctively take up most glutamate released by neurons, termi-ate transmitter activity and return glutamate to neurons in

218

Figure 8 Propentofylline improved rats’ performance in thewater maze task. (A) Comparison of the mean escape laten-cies to find the hidden platform at days 2—5. (B) The memoryretention ability was evaluated by the probe trial, as shown bytcg

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he percentage of time spent in the target quadrant. *P < 0.05ompared with S-FRFS group, *P < 0.01 compared with S-FRFSroup.

glutamate—glutamine cycle, which is crucial for learning.njection of a glutamine synthetase inhibitor, methionineulfoximine (MSO), in intermediate medial mesopalliumIMM)—–an area corresponding to cerebral cortex in mam-als, could prevent consolidation of memory for a passive

voidance task in day-old chicks (Gibbs et al., 1996). (3)strocytes also express glutamate receptors and respond toeuronal glutamate with a transient increase in cytosolicalcium concentration. The elevation of calcium in astro-ytes is capable of either exciting the neurons by the

igure 9 Propentofylline alleviated memory deficits in thenhibitory avoidance task. Retention time was assessed at 1,

and 24 h after training and compared among saline-treatedonhyperthermia control (S-NHC), saline-treated FRFS (S-FRFS)roup and PPF-treated FRFS (P-FRFS) group. *P < 0.01 comparedith S-FRFS group at the same time point.

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esicular release of glutamate or inhibiting neuronal activ-ty by release of ATP (Newman, 2003; Anlauf and Derouiche,005). These processes may also be of importance for mem-ry formation. Whether FRFS in immature brain alter thelutamate metabolism, neurotransmitter release or otheromeostatic function involved in memory formation in acti-ated astrocytes deserves further studies.

The two astrocyte marker proteins, GFAP and S100B, havelso been explored about their influences on neuronal phys-ology related to learning and memory.

GFAP is an intermediate filament specific to astrocytes.hibuki et al. (1996) found that mice devoid of GFAP exhib-ted deficient long-term depression (LTD) at PF-Purkinje cellynapses and significant impairment of eyeblink condition-ng. GFAP-null mice in McCall’s study displayed enhancedong-term potentiation (LTP) of both population spikemplitude and excitatory post-synaptic potential slope com-ared to control mice (McCall et al., 1996). These studiesemonstrated that GFAP play actively in astrocyte—neuronnteractions and two possible mechanisms have been sug-ested in this interaction (McCall et al., 1996): (1) GFAPight interfere with production of neurotrophic factors in

strocytes. (2) GFAP directly participates in the changes inlial processes that have been associated with LTP.

S100B, a calcium-binding protein, has multiple intra-ellular and extracellular functions, including cell growth,ell structure, energy metabolism, and calcium homeosta-is (Donato, 2001). Kubista et al. (1999) discovered that100B could affect neuronal electrical discharge activityy modulation of potassium currents. Extracellular S100Bould lead to elevation of intraneuronal calcium concen-ration which may disrupt a number of calcium-dependentrocesses implicated in cognitive function, including LTP andTD. It is noteworthy that S100B has been shown to be over-xpressed in Down’s syndrome and Alzheimer’s disease (Mraknd Griffin, 2004; Gruden et al., 2007), both of which involveompromised brain functions. Transgenic mice that overex-ressed S100B showed impaired hippocampal LTP, perturbed-maze and Morris water maze performances (Gerlai et al.,994, 1995). Mutant mice devoid of S100B had strength-ned synaptic plasticity as identified by enhanced LTP inhe hippocampal CA1 region which could be reversed byecombinant S100B proteins. Besides, the mutant mice hadnhanced spatial memory in the Morris water maze test andnhanced fear memory in the contextual fear conditioningNishiyama et al., 2002).

Propentofylline is a novel xanthine derivative withnhibitory effects on the types I, II, and IV phosphodi-sterases and adenosine transporters, therefore leadingo intracellular cyclic nucleotides (cAMP) accumulationRingheim, 2000). Elevation of cAMP by propentofyllineould downregulate activated astrocytes and restore the dif-erentiated state of astrocytes as defined by morphology andon channels. Restoration of K+ and Cl− channels is importantor stabilization of the membrane potential, effective glu-amate uptake and ion homeostasis in the neuronal synapsesSchubert et al., 1997; Schubert and Rudolphi, 1998). Astro-

ytic suppressing effects of propentofylline make for aavorable pharmacological profile in therapies designed toelay or halt disease progression by regulating astrocyteunction. For example, propentofylline could attenuateechanical allodynia in rodent models of neuropathic pain

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by suppressing astrocyte activation (Sweitzer et al., 2001,2006). In our research, attenuation of astrocyte activationalong with decreased overexpression of GFAP and S100B bypropentofylline ameliorated the memory injury after FRFS.These data support an intimate relationship between astro-cyte activation and memory dysfunction after FRFS.

Traditional xanthine derivatives, such as theophyllineand caffeine, have been shown to trigger seizures (Bannerand Czajka, 1980; Gal et al., 1980). The seizure-inducingeffects are believed to related to their adenosine A1receptor or GABA receptor antagonistic effects (Sakuraiet al., 1988; Cutrufo et al., 1992). Propentofylline andits hydroxylated metabolite A720287 are about 20 timesless potent than theophylline in displacing adenosine A1-agonist binding (Fredholm et al., 1992). Cutrufo et al.(1992) examined differential effects of various xanthineson pentylenetetrazole-induced seizures in rats and foundthat only xanthines with strong adenosine A1 receptorantagonism (theophylline and caffeine) markedly enhancedthe EEG and behavioral effects of a subconvulsive doseof pentylenetetrazole. Sakurai et al. (1988) found thatpropentofylline differed from caffeine in that it neitherantagonized diazepam nor affected the GABA system. Inthis research, during and after propentofylline adminis-tration, we did not observe spontaneous seizure in eitherpropentofylline-treated FRFS rats or saline-treated FRFS rats(data not shown). However, the safety of propentofyllineadministration after FRFS should be further studied.

Early-life FRFS is followed by long-term alteration inmemory function. Our study revealed long-term astrocyteactivation after FRFS. Propentofylline, an astrocyte modu-lator, inhibited astrocyte activation and rescued the memorydeficits. These results indicate that astrocytes are likely toprovide novel therapeutic targets after frequently repetitivefebrile seizures.

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