Epilepsy Research (2011) 97, 6472

journal homepage: www.elsevier.com/locate/epilepsyres

Protective effect of the KCNQ activator upirtine on a model of repetitive febrile seizures
Fang Yu a,1, Yanlan Liu a,b,1, Yuncui Wang a,c, Jun Yin a, Hui Wang a, Wanhong Liu a, Biwen Peng a,, Xiaohua He a,
a

Wuhan University School of Basic Medical Sciences, Wuhan 430071, China Shanghai Prison General Hospital, Shanghai 21000, China c Nursing College, Hubei University of Chinese Medicine, Wuhan 430000, China
b

Received 5 April 2011; received in revised form 7 July 2011; accepted 12 July 2011 Available online 9 August 2011

KEYWORDS
KCNQ channel; Repetitive febrile seizures; Flupirtine; Phenobarbital

Summary Activation of KCNQ-channels has been shown to decrease or reduce the propagation of neuronal excitation in the immature central nervous system, and KCNQ activators represent a new class of anticonvulsant compounds. Their effectiveness has been demonstrated in many seizure models but not in repetitive febrile seizures (RFS) models. This study aimed to test whether the KCNQ channel activator upirtine is also effective for RFS in rats. RFS were induced in Sprague-Dawley (SD) rats at postnatal day 10 (P10) in a warm water bath for eight consecutive days with or without the pre-administration of upirtine or phenobarbital. As results, both drugs signicantly increased the latency and decreased the rate of febrile seizures. Furthermore, seizures in the upirtine group had a signicantly shorter duration and were less severe compared with the phenobarbital group. The upirtine-treated group showed less impairment in learning and memory and less obvious pathological changes in the brain following RFS compared with the phenobarbital-treated group. In summary, upirtine appears to be effective in RFS prophylaxis and may merit further study as a candidate for the treatment of RFS in infants and children. 2011 Elsevier B.V. All rights reserved.

Introduction
Febrile seizures (FS) are the most common type of convulsive events in infants and young children (Hauser, 1994). They are prolonged in 9% of cases and recur in 2342%

Corresponding authors. E-mail addresses: pengbiwen@whu.edu.cn (B. Peng), hexiaohua@whu.edu.cn (X. He). 1 These authors contributed equally to this work.

cases (Annegers et al., 1990; Offringa et al., 1994; Tosun et al., 2010). Recurrent FS may be a risk factor that predicts a greater likelihood of later epilepsy (Annegers et al., 1979, 1987; Berg and Shinnar, 1996). Furthermore, impaired hippocampus-dependent long-term memory has been reported in a frequently repetitive febrile seizures (FRFS) model starting at postnatal day 10 (Chang et al., 2005; Yang et al., 2009). FS are correlated with an elevation in body temperature. It is recommended that antipyretic or physical methods should be applied to suppress fever and that anti-infection

0920-1211/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2011.07.005

Protective effect of the KCNQ activator upirtine interventions should be used to control primary diseases (Van Esch et al., 1995). Although these treatments effectively control symptoms in most cases, they do not reduce recurrences of febrile seizures (Lux, 2010a; Strengell et al., 2009). Antiepileptic drugs, including carbamazepine, diazepam, phenobarbital and sodium valproate, have been used as prophylactics in FS treatment trials (Lux, 2010b) and have proven to be effective for preventing or reducing the recurrence of FS. However, routine prophylactic treatment is not recommended due to the considerable potential side effects and ineffectiveness in reducing the risk of developing epilepsy (Wolf and Forsythe, 1989). KCNQ channels are an attractive pharmacological target for the treatment of neonatal seizures (Brown and Passmore, 2009; Cooper and Jan, 2003). KCNQ2 and KCNQ3 subunits have been proposed to constitute the neuronal Mcurrent (Jentsch, 2000; Wang et al., 1998), which are critical determinants of cellular excitability, and contribute functionally to the regulation of neuronal networks, postnatal brain development, and cognitive performance (Brown and Passmore, 2009; Peters et al., 2005). Mutations of the KCNQ2 and KCNQ3 genes cause benign familial neonatal seizures (BFNC), a dominant inherited syndrome (Castaldo et al., 2002; Singh et al., 2003). Pharmacological and genetic methods have been used successfully to suppress KCNQ channels in rodents, thus to promote neuronal and network hyperexcitability and induce spontaneous seizures, behavioral hyperactivity and morphological changes in the hippocampus (Peters et al., 2005). Flupirtine is clinically used as a non-opioid analgesic. In addition to its well-established direct effects as a KCNQ activator, upirtine also shows anticonvulsant activity in the Antiepileptic Drug Development program (Miceli et al., 2008). Several studies also have demonstrated signicant neuroprotective actions of upirtine in both in vitro and in vivo experimental models (Boscia et al., 2006; Klawe and Maschke, 2009; Sattler et al., 2008). Recently, upirtine has been shown to be effective in the treatment of neonatal seizures in rats (Raol et al., 2009), but its role in febrile seizures is unclear. This study examined the possible preventative effects of upirtine on a model of RFS. Chemicals and drugs

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Flupirtine maleate and phenobarbital were purchased from the Huazhong University of Science and Technology and were freshly dissolved in a 1:2 (v/v) mixture of dimethylsulfoxide and saline before intraperitoneal (IP) injection. A dose of 25 mg/kg was used (Raol et al., 2009) and the injection volume was 0.05 ml/10 g body weight.

Establishment of the experimental model

Forty-ve young, male, 10-day-old SD rats were used in the experiments. The experimental protocols and procedures listed were approved by the Ethics Committee at Wuhan University and conformed to the 1996 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences). The method to create the hyperthermia-induced seizure model was described previously with minor modications (Palmer et al., 1998). Briey, hyperthermia was induced by placing the animals in a 20 cm 20 cm 40 cm water bath lled with 45 0.1 C water. An adjustable perforated base was placed in the bath for hind limb support to prevent forced swimming. The water depth allowed the rat to have its head above water surface by standing on its hind legs and leaning against the container wall. On P11, rats were put into water for a maximum of 4 min or until convulsions appeared. At the end of immersion in the water bath and the seizure observation period (30 min), the animals were dried off in a temperature controlled incubator (28 C) and subsequently returned to the home cages. The rectal temperatures of the rat pups were measured during the course of hyperthermia treatment. FS Latency (time to seizure onset) was determined from the time the rats were put into the water bath to the onset of seizures. Seizures were scored according to Racines scale (Racine, 1972). Maximum seizure grade or score, seizure duration (min), and time to seizure onset (min) were scored by two observers blind to the treatment. In order to determine whether the electrographic correlates with the hyperthermic seizures, 10 days old rat pups were implanted with electrodes for EEG recording. Briey, pups were anesthetized with 10% chloral hydrate and then were implanted the electrodes with sterile surgical procedures. All the electrodes were attached to the skull and proofed from water with dental acrylic. Pups were recovered from surgery and anesthesia for 1 h. Electrical seizure activity was recorded between two hippocampal electrodes and a reference electrode attached to the skull. EEG was recorded using RM6240BD biological signal acquisition and processing system (ChengDu Instruments, Sichuan, China). Baseline EEG was recorded for approximately 30 min before hot water bath. The onsets of hyperthermia seizure were determined by both behavioral and electrographic criteria. Here, an example was showed for typical EEG under seizure, with a baseline EEG. The onset of behavioral seizures of stage 4 or above was well correlated with EEG showing continuous seizure activity (Fig. 1).

Materials and methods

Animals
Adult Sprague-Dawley female rats and their 67-days old male pups (n = 45) were maintained at a constant temperature (22 C) and relative humidity (60%) on a 12-h light schedule.

Figure 1 EEG correlates of hyperthermia seizures in a 10-day-old rat. (A) The baseline recorded before hot water bath. (B) The onset of a behavioral seizure. The arrow points that stage 4 seizures were observed at the same time.

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All the following experiments were based on the validity of this model conrmed by behavioral observation and EEG recording. The animals were divided into 4 groups. In the drug groups, rats were administered upirtine or phenobarbital (25 mg/kg body mass) 90 min before being placed into the water for eight consecutive days. Rats in the control and non-treatment groups received DMSO/saline. Control group rats were put into 33 C water for 4 min, while the other groups endured 45 C water. On P28, rats from the upirtine, phenobarbital and nontreatment groups were put into 45 C water for 4 min or until convulsions appeared without any drugs or vehicle-treated. The seizure activity was graded.

F. Yu et al.
Hippocampal samples taken from 4 rats from each group were dissected and immersed in 2.5% glutaraldehyde for 24 h at 4 C. Samples were then xed with 1% osmium tetroxide (OsO4 ), dehydrated, and embedded in Epon 812 and acetone solution (1:1). Semithin sections were stained with methylene blue. Ultrathin sections (60 nm) were double stained with uranyl acetate and lead citrate and examined with a transmission electron microscope (EM). Three independent persons who were blind to treatments performed the morphological analysis.

Data analysis
Data were analyzed with SPSS 13.0 software to compare the differences between the groups. The data are reported as the mean SEM, and statistical signicance was dened as p < 0.05. Normally distributed data were tested by one-way analysis of variance (ANOVA) and where signicant differences were recorded, the post hoc NewmanKeuls analysis was applied. Non-normally distributed data were analyzed by non-parametric KruskalWallis test. The MannWhitney test was used to analyze the difference between groups in maximal seizure score, apoptosis rate and percentage of time spent in the target quadrant.

Morris water maze test

On P30, the Morris water maze was used to test hippocampusdependent spatial memory. The method was a modication of the model described previously (Peters et al., 2005). On days 1 and 2, pre-training sessions consisting of four trials per day were performed using a visible platform (10 cm 10 cm). On days 36, the platform was placed at a xed location equidistant from the sidewall and the middle of the pool and submerged 1.0 cm below the waters surface. Rats from each group were placed in a random quadrant and were allowed to explore the maze to escape. The escape latency was monitored by a video and computer tracking system (EthoVision 3.0). On day 7, the platform was removed and the rats were placed into the opposite quadrant of the previous target quadrant.

Results
Flupirtine signicantly attenuated RFS
To assess the effect of upirtine on RFS, rats at P10 were used to establish the animal model of RFS. The neurological maturity of newborn rats (P10) roughly corresponds to that of human neonates (Jensen and Baram, 2000). Rectal temperature, which was 38.0 0.3 C before exposure to hyperthermia, increased to a mean of 43.0 0.4 C after exposure to warm water and at the beginning of the seizure. There were no signicant differences among non-treatment group and other groups on rectal temperature. During the eightday hyperthermia period, one rat from the non-treatment group (1/12) died. No deaths were seen in the other groups. Data from the dead rat were not included in the analysis. In this model of RFS, all non-treatment rats (n = 11) endured clinical seizures and developed progressively longer duration, and 80% progressed to stage 4 or above. To identify the different preventative effects of upirtine and phenobarbital on FS, a pre-treatment protocol was used. Animals received 25 mg/kg of one of the two antiepileptic drugs. As illustrated in Table 1, upirtine signicantly

Morphological analysis
On P36, 8 rats from each group were deeply anesthetized and perfused transcardially with a normal saline solution followed by 4% paraformaldehyde for the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay (Roche, Mannheim, Germany) (n = 4) and Nissl staining (n = 4). The intact brains were immersed in 4% paraformaldehyde for 24 h at 4 C before parafn embedding. The analysis was performed on 4 animals from each group. Three different visual elds were chosen randomly from various regions of the brain for each animal. One frame (200 m 200 m) was used for counting and measuring. TUNEL positive cells were counted on images from one of every ten coronal sections. For Nissl staining, the slides were treated with 0.1 M sodium acetate buffer at pH 4, stained in 0.2% Cresyl Violet in sodium acetate buffer at 45 C for 7 min, treated with 4% ammonium molybdate aqueous solution for 5 min, and then differentiated and dehydrated. Nissl bodies of neurons in the hippocampal CA1 region were observed using an optical microscope.

Table 1 Groups

Parameters of the four groups during the RFS. Number 10 11 12 12 Latency (min) Not applicable 6.15 0.25 8.07 0.27a 7.76 0.30a Duration (min) Not applicable 12.91 0.28 10.03 0.56b 5.73 0.69b,* Maximum seizure grade percentage (%, >3) Not applicable 80 50 22.9c,*

Control Non-treatment Phenobarbital Flupirtine

a Compare with non-treatment group: ANOVA with post hoc NewmanKeuls analyses, upirtine group vs non-treatment group p < 0.001; phenobarbital group vs non-treatment group p < 0.001. b Compare with non-treatment group: KruskalWallis H test, upirtine group vs non-treatment group p < 0.001; phenobarbital group vs non-treatment group p = 0.013. c Compare with non-treatment group: MannWhitney U test, upirtine group vs non-treatment group p < 0.001; phenobarbital group vs non-treatment group p < 0.001. * Compare with phenobarbital group p < 0.001.

Protective effect of the KCNQ activator upirtine increased the latency (p < 0.001) and decreased the duration (p < 0.001) of hyperthermic seizures compared to the non-treatment group. In the upirtine group, the proportion of animals with the maximal seizure score over 3 was only 22.9%, which is lower than both the phenobarbital group (50%, p < 0.001, MannWhitney U test) and non-treatment group (80%, p < 0.001, MannWhitney U test). Although phenobarbital also increased the latency (p = 0.013, ANOVA with post hoc NewmanKeuls analyses) and decreased the maximal seizure score (p < 0.001, MannWhitney U test) of hyperthermic seizures compared to the non-treatment group, its effect was not as robust as upirtine (p < 0.001, Table 1).
Stage 5% seizing

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Stage 4% seizing Stage 3% seizing Stage 2% seizing

Both upirtine and phenobarbital reduced the risk of further hyperthermic seizures
On P28, all rats from different group were exposed to hyperthermic conditions again to detect their susceptibility to further FS. Rats in the non-treatment group presented myoclonic, clonic and tonic seizures, with 7/12 progressing to stage 5 (Table 2). In contrast, the degree of convulsion in the upirtine and phenobarbital groups decreased signicantly when hyperthermia was induced again (p < 0.001, MannWhitney U test, Table 2). Compared to the nontreatment group, both upirtine and phenobarbital were able to delay the onset of seizures in rat pups and reduce their susceptibility to the recurrence of FS.
Convulsion degree of the three groups during the second period of hyperthermia.

0 25 (3/12) 17 (2/12) Non-treatment Phenobarbital* Flupirtine* 11 12 12 0 17 (2/12) 17 (2/12) 0 42 (5/12) 67 (8/12)

0 17 (2/12) 0
* Phenobarbital group or upirtine group vs non-treatment group: p < 0.001, MannWhitney U test. Non-convulsion % seizing is referencing animals that the observer scored as not having a seizure.

To examine the protective effects of upirtine on the impairment of spatial learning and memory introduced by RFS, all rats from each group were tested for their performance ability in the Morris water maze at P30. Non-treatment group and drugs-treatment groups showed normal swimming speed compared to controls (data not shown). As shown in Fig. 2A, student NewmanKeuls post hoc ANOVA test revealed that rats from upirtine group spent less time in nding the platform than those from non-treatment group (p = 0.001) and phenobarbital group (p = 0.006) but not more than those from the control group (p = 0.42). No signicant differences of the escape latency were found between the non-treatment and phenobarbital groups (p = 0.49). In the probe trial (day 7), rats were placed in the pool without the platform to test spatial memory. The performance of rats in the non-treatment group was indicative of impaired memory, as they spent signicantly less time (23.6% 1.4%) than the control (36.6% 2.9%, p < 0.001) and upirtine groups (32.1% 2.4%, p = 0.008) in the platform quadrant. When searching for the missing platform, the percentage of time spent in target quadrant was increased in the upirtine group compared to the phenobarbital group (26.1% 1.86%, p = 0.024), indicating a clear preference for the correct position. We did not nd a difference between the non-treatment group and the phenobarbital group (p = 0.31, Fig. 2B).

Table 2

Group

Number

Non-convulsion % seizing

Stage 1% seizing

Flupirtine prevented learning and memory impairment caused by repetitive hyperthermic seizures

36 (4/11) 0 0

64 (7/11) 0 0

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F. Yu et al. EM was used to examine the ultrastructural features of neurons in the hippocampal CA1. In the control group, mitochondria were oval-shaped with a normal arrangement of cristae (Fig. 5A). In the upirtine group, only a few mitochondria were swollen with blurred cristae or devoid of all cristae (Fig. 5B). However, a large number of mitochondria were also swollen with blurred cristae or devoid of all cristae in the non-treatment and phenobarbital groups (Fig. 5C and D).

Discussion
The primary ndings of the current study were that upirtine, a KCNQ channel activator, can not only signicantly suppress seizures but also reduce the risk of further hyperthermic seizures. Moreover, upirtine can signicantly prevent memory impairment and neuronal morphological changes in neonatal rats caused by repetitive hyperthermic seizures. In a previous study, the rats aged 2021 days was immersed in a 45 C water bath to elicited seizures as an animal model of infantile febrile seizures (Palmer et al., 1998). The current study used animals at P10 because the development of the rats brain development during the period of 1017 postnatal day best corresponds to the stage of brain development at which human infants and young children are most susceptible to febrile seizures (Baram and Shinnar, 2001; Dobbing and Sands, 1973; Gottlieb et al., 1977). This minor modication did not appear to impact the validity this febrile seizure model. Both behavior observation (Table 1) and electroencephalogram (EEG, Fig. 1) examination have shown that all non-treated rats endured clinical seizures in this model. Under the present experimental conditions, the vehicle-treated rats did develop progressively longer duration of seizures and 80% of rats progressed to stage 4 or above over the 8 consecutive days. Because phenobarbital has long been used as a prophylactic drug for febrile seizure, we chose it as a positive control to test the effectiveness of upirtine in a prophylaxis treatment of RFS. Pre-treatment with either upirtine or phenobarbital prolonged the latency of seizures and reduced the stage of seizures in the rats. Although both drugs were effective in suppressing repetitive febrile seizure, animal treated with phenobarbital had a relatively higher average seizure grade (behavior score > 3) and did not signicantly reduce the seizure durations as compared to upirtine treated animals. The incidence of FS decreases with age in humans. Interestingly, the expression level the Kv7.2 subunit is low in neonatal neurons and markedly increases after the rst postnatal week (Saulina et al., 2008). Moreover, during the early stages of the brain development, the expression level of the KCNQ channels is critically involved in the neuronal excitability. As a result, even a relatively small decrease in the M-current amplitude could cause epileptic seizures (Weber et al., 2006). Previous studies have also indicated that KCNQ channels prevent the propagation of neuronal excitability in the immature CNS (Okada et al., 2003). Taken together, these suggest that the efcacy of upirtine for the treatment of RFS may be due to the increased KCNQ channel function.

Figure 2 Water maze performance in the four groups following febrile seizures. (A) Rats from the non-treatment and phenobarbital groups showed greater performance latencies to the escape platform during the four testing days than the control and upirtine groups. Error bars, SEM, * and ** indicate p < 0.05 and p < 0.001, NewmanKeuls test. (B) Rats from the phenobarbital and non-treatment groups differed signicantly from the control rats when the platform was not visible. The phenobarbital group also had a decreased preference for passing through the platform quadrant than the upirtine group. No differences were noted between the phenobarbital group and the non-treatment group. * and ** indicate p < 0.05 and p < 0.001, MannWhitney test.

Flupirtine reduced the biomarkers of neuronal degeneration associated with RFS

To examine whether repeated convulsions induced by hyperthermia leads to neuronal death, we assessed the neuronal damage in brain sections using the TUNEL assay. It is notable that neuronal death was prominent in the non-treatment and phenobarbital groups (Fig. 3C and D). The non-treatment group showed the greatest amount of apoptosis in the cerebral cortex. There were signicant differences in the rate of apoptosis between the non-treatment group and control (p < 0.001) or upirtine groups (p = 0.002) (Fig. 3A and B), while a similar apoptosis rate was found in the non-treatment and phenobarbital groups (p = 0.5, Fig. 3E). Nissl-stained hippocampal sections showed that the cytoplasm of neurons in the control and upirtine groups was rich in granular Nissl bodies, and no signicant differences were seen in neuronal distribution (Fig. 4A and B). Compared to the control group, the non-treatment and phenobarbital groups showed a sparse distribution of hippocampal neurons, fewer Nissl bodies and more lightly stained cytoplasm (Fig. 4C and D).

Protective effect of the KCNQ activator upirtine

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Figure 3 TUNEL assay for neuronal damage in brain sections. (A) Control group, (B) upirtine group, (C) phenobarbital group, (D) non-treatment group, and (E) apoptosis rates of the four groups. * and ** indicate p < 0.05 and p < 0.001, MannWhitney test.

Remarkably, upirtine had a signicant effect against the learning and memory impairment induced by RFS, which conrms the results of an earlier study (Otto et al., 2004). Several studies have demonstrated that febrile seizure in rat pups results in hippocampus-dependent long-term memory decits (Chang et al., 2005; Dube et al., 2009; Yang et al., 2009). In this study, rats from non-treatment group also showed long-term memory decits in the test of Morris water maze. Although drug treatment in early life did not alter the sensorimotor function as evidenced by the fact that the visual-motor (swim speeds or total distance) performance in the water maze was similar between control and FS groups, the learning and memory abilities in the upirtine-treated group were much improved than

that of the phenobarbital-treatment and non-treatment groups. Morphological analysis revealed that upirtine also reduced neuronal degeneration caused by repetitive hyperthermic seizures. The effects of FS on neuronal degeneration are mixed in the literature. FS did not induce signicant morphological changes (neuronal death and aberrant mossy bers) in the hippocampus in one study (Chang et al., 2003), but lead to neuronal apoptosis in cortical elds (Chen et al., 2008) and ultrastructural changes in the CA1 neurons of the hippocampus (Toth et al., 1998) in other studies. This study showed that neuronal death was prominent in the non-treatment group, supporting the notion that hyperthermic seizures can produce a strong, deleterious effect on

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F. Yu et al.

Figure 4 Nissl staining of neurons in the CA1 region of the hippocampus. (A) Control group, (B) upirtine group, (C) phenobarbital group, and (D) non-treatment group; indicated that Nissl bodies were stained with blue colour.

Figure 5 Electron micrographs of neurons in the hippocampal CA1 region. There are no obvious differences between the ultrastructures of neurons in the control and upirtine groups. (A) Control group, (B) upirtine group, (C) phenobarbital group, and (D) non-treatment group. indicated a normal arrangement of cristae, indicated swollen.

Protective effect of the KCNQ activator upirtine the neurons of immature rats. The irregular Nissl body and the ultrastructure changes in the hippocampus of RFS rats suggested that the long term alterations in the neuronal plasticity may account for the memory decits. Possible interpretation for these changes includes abnormal ring rate and poor stability of hippocampal CA1 neurons, which is involved in the encoding and retrieval of spatial information. This study demonstrated that upirtine was effective at modifying seizures induced by hyperthermia and preventing the consequential pathological processes. The present study also found that urpirtine had benecial effects on hippocampal and cortical neurons in young rats. Whether this is a specic effect of urpirtine or a secondary effect due to the shorter seizure duration, or a combination of both is unclear. Taken together, this study suggests that upirtine can prevent the brain damage caused by RFS in neonatal rats, which appears to be advantageous over some currently available drugs. The current treatment urpirtine regimen (25 mg/kg) did not show measurable unwanted effects. Specic KCNQ activators may warrant further studies as candidate treatments for RFS, which may eventually expand the list of currently available pharmacotherapeutics.

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Acknowledgments
We thank Dr. Jun-Xu Li, Department of Pharmacology and Toxicology, University at Buffalo, for his critical reading of the manuscript. This work was supported by National Natural Sciences Foundation of China (No. 30770734, No. 30970994), Program for New Century Excellent Talents (No. NCET-07-0630), National Basic Research Program of China (No. 2010CB529803) and the Fundamental Research Funds for the Central Universities.

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