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Background: It has been demonstrated that the zebrafish model of pentylenetetrazole (PTZ) ‑ evoked seizures and the well‑ established rodent models of epilepsy are similar pertaining to behavior, electrographic features, and c‑ fos expression. Although this zebrafish model is suitable for studying seizures, to date, inflammatory response after seizures has not been investigated using this model. Because a relationship between epilepsy and inflammation has been established, in the present study we investigated the transcript levels of the proinflammatory cytokines inter ‑ leukin‑ 1 beta (il1b) and cyclooxygenase‑ 2 (cox2a and cox2b) after PTZ‑ induced seizures in the brain of zebrafish 7 days post fertilization. Furthermore, we exposed the fish to the nonsteroidal anti ‑ inflammatory drug indomethacin prior to PTZ, and we measured its effect on seizure latency, number of seizure behaviors, and mRNA expression of il1b, cox2b, and c‑ fos. We used quantitative real‑ time PCR to assess the mRNA expression of il1b, cox2a, cox2b, and c‑ fos, and visual inspection was used to monitor seizure latency and the number of seizure‑ like behaviors. Results: We found a short‑ term upregulation of il1b, and we revealed that cox2b, but not cox2a, was induced after seizures. Indomethacin treatment prior to PTZ‑ induced seizures downregulated the mRNA expression of il1b, cox2b, and c‑ fos. Moreover, we observed that in larvae exposed to indomethacin, seizure latency increased and the number of seizure‑ like behaviors decreased. Conclusions: This is the first study showing that il1b and cox‑ 2 transcripts are upregulated following PTZ‑ induced seizures in zebrafish. In addition, we demonstrated the anticonvulsant effect of indomethacin based on (1) the inhibi‑ tion of PTZ‑ induced c‑ fos transcription, (2) increase in seizure latency, and (3) decrease in the number of seizure‑ like behaviors. Furthermore, anti‑ inflammatory effect of indomethacin is clearly demonstrated by the downregulation of the mRNA expression of il1b and cox2b. Our results are supported by previous evidences suggesting that zebrafish is a suitable alternative for studying inflammation, seizures, and the effect of anti ‑ inflammatory compounds on seizure suppression. Keywords: Seizure, Zebrafish, Pentylenetetrazol, Interleukin ‑ 1 beta, Cyclooxygenase‑ 2, Indomethacin, Inflammation *Correspondence: email@example.com Laboratory of Zebrafish, Department of Medical Genetics ‑ School of Medical Sciences, University of Campinas (UNICAMP), Tessália Vieira de Camargo, 126 Cidade Universitaria “Zeferino Vaz”, Campinas, SP 13083‑887, Brazil Full list of author information is available at the end of the article © 2016 Barbalho et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barbalho et al. BMC Neurosci (2016) 17:12 Page 2 of 9 comparing the control (CG) and seizure (SG) groups at Background each time point. We found a short-term upregulation of Zebrafish experimental models are now widely accepted i11b mRNA levels 0.05 h (p = 0.02) and 1 h (p = 0.02) for investigating human diseases, including epilepsy [1– after seizures (Fig. 1). However, no significant differences 4]. Importance of this animal model is mainly based on were found between the CG and SG 6, 12, 24, or 48 h its remarkable features combining exceptionally simple after seizures (p > 0.05) (Fig. 1). genetic manipulations, which are ideal for forward and reverse genetic investigations, and easy phenotype assess- The mRNA expression of cox2a and cox2b in the brain ment in a short period of time. Other advantages of this of zebrafish larvae after PTZ‑induced seizures sfi h species are as follows: low maintenance cost, easy Because no significant differences were found in the breeding, high fecundity, external fertilization and devel- mRNA expression of il1b pertaining to longer time peri- opment, short generation time, and transparency during ods, we chose to evaluate the temporal expression pro- embryonic and larval stage. Furthermore, their genome file of cox2a and cox2b 0.05, 1, and 6 h after PTZ-induced shares approximately 70 % homology with the human seizures. Our results showed that both cox2a and cox2b genome comprising large regions of conserved synteny . were constitutively expressed in the CG (Fig. 2a–e); how- Zebrafish exposed to chemoconvulsant drugs mimic ever, after PTZ exposure, the expression pattern of these behavior, electrographic findings, and upregulation of c- fos in brain regions related to neuronal activation [3, 4, 6]. genes showed differences. Animals in the CG and SG Zebrafish are sensitive to common anticonvulsant drugs; had similar cox2a mRNA levels after seizures (p > 0.05; therefore, they are widely used for the high throughput Fig. 2a–c). However, cox2b mRNA levels were upregu- screening of novel antiepileptic drugs (AEDs) [7–13]. lated 0.05 h (p = 0.004) and 1 h (p = 0.008) after seizures Evidence obtained using hippocampal surgical specimens in the SG compared with the corresponding data in the from patients with pharmacoresistant epilepsy and experi CGs (Fig. 2d, e). No statistical significance was found 6 h after seizures (p = 0.27; Fig. 2f ). mental rodent models demonstrated that proinflammatory cytokines and inflammatory mediators are upregulated after Eec ff t of indomethacin administered prior to PTZ on the seizures, suggesting that the inflammatory response may mRNA expression levels of il1b, cox2b, and c‑fos play an important role in the pathophysiology of epilepsy We used indomethacin at three different concentra - [14–18]. Among the proinflammatory cytokines, interleu - tions (10, 100, or 307 μM) prior to seizure-induction, kin-1 beta (IL-1β) is the most widely investigated. IL-1β and we quantified the mRNA levels of il1b, cox2b, exerts its action by binding to the IL-1 receptor, which initi- and cfos 0.05 h after PTZ-induced seizures, when the ates a downstream signaling process that activates the tran- transcript levels of il1b and cox2b are the highest. Our scription factor nuclear factor-κB (NF-κB). Activation of results revealed that indomethacin treatment prior NF-κB leads to the transcription of multiple inflammation- to PTZ-induced seizures downregulated the mRNA associated genes, including cyclooxygenase (COX)-2 [19– expression of il1b, cox2b, and c-fos. As shown in Fig. 3, 22]. COX-2 is a key enzyme responsible for the conversion expression of il1b was downregulated by indomethacin of arachidonic acid into prostaglandins, potent mediators of when we compared the SG with all indomethacin con- inflammatory signaling [19– 22]. One of these prostaglan- centration tested (p ≤ 0.001). In addition, il1b mRNA dins converted by COX-2 is prostaglandin E2 (PGE2), which levels in the indomethacin-treated groups were similar upregulates the expression of IL-1β [19–24]. to that in the CG. No significant difference was found Because a relationship between epilepsy and inflamma - between indomethacin treatment groups (Fig. 3). The tion has been established and inflammatory response in mRNA expression of cox2b was upregulated in the SG the pentylenetetrazole (PTZ)-seizure model has not been (p ≤ 0.05) and 10 μM indomethacin group (p ≤ 0.001) studied, we investigated the expression of the il1b and when both were compared to the CG (Fig. 4). Indo- cox2 transcripts in zebrafish after seizures. In addition, methacin treatment at 307 μM significantly down - we measured the effects of indomethacin, a nonsteroi - regulated the mRNA expression of cox2b compared dal antiinflammatory drug, on seizure latency, number with the SG (p ≤ 0.001; Fig. 4). Comparisons between of seizure-like behaviors, and c-fos expression used as a indomethacin treatments groups showed that cox2b marker of neuronal activity . mRNA levels were downregulated at 100 μM (p ≤ 0.05) and 307 μM (p ≤ 0.001) when compared to 10 μM Results (Fig. 4). Transcript level of c-fos was upregulated in the Temporal expression of il1b in the brain of zebrafish larvae SG (p ≤ 0.001) and 10 μM indomethacin pretreated after PTZ‑induced seizures group (p ≤ 0.05) when compared to the CG (Fig. 5). Temporal expression profile of il1b was analyzed 0.05, However, 100 μM (p ≤ 0.05) and 307 μM (p ≤ 0.001) 1, 6, 12, 24, and 48 h after PTZ-induced seizures by Barbalho et al. BMC Neurosci (2016) 17:12 Page 3 of 9 Fig. 1 Temporal expression profile of il1b in the brain of zebrafish after pentylenetetrazole ‑ evoked seizures. Relative quantification of the inter ‑ leukin‑1 beta (il1b) transcript 0.05, 1, 6, 12, 24, and 48 h after pentylenetetrazole (PTZ)‑induced seizures in the brain of zebrafish at 7 days post fertilization. Each time‑point seizure group was exposed to 15 mM PTZ for 20 min, and their time ‑matched control groups were handled identically, but included exposure to water (n = 5 per group). Data obtained from each seizure group was compared with their respective time‑point matched control group. Data are presented as mean ± SEM. Statistical analyses were performed using the Mann–Whitney test, and differences were consid‑ ered significant if p < 0.05. Asterisk (*) indicates p ≤ 0.05. CG control group, SG seizure group indomethacin treatment downregulated the cfos mRNA specimens and experimental models of epilepsy [14–18]; level when compared to the SG (Fig. 5). however, to reveal the main role of inflammatory mol - ecules in epilepsy, further investigations are necessary. Seizure onset latency and the number of seizure‑like Rodent models of epilepsy are widely used in experimen- behaviors tal research, but due to its several favorable characteris- We evaluated the effect of indomethacin administered tics, zebrafish seizure models can significantly contribute prior to PTZ-induced seizures by analyzing seizure onset to understanding the role of inflammation following sei - latency (stage 3, equivalent to the loss of body posture) zures. In addition, zebrafish models of human diseases and the number of seizure-like behaviors. Each larva was are particularly suitable for the pharmacological testing observed individually under each experimental condi- of drugs in a convenient way. Immune and inflammatory tion. In animals pretreated with 10, 100, or 307 μM indo- responses in zebrafish are comparable to those found in methacin, we found a significant increase of onset latency mammals [25–28]; however, to our knowledge, no data is (give in minutes) compared with animals in the SG available about the expression of inflammatory biomark - (p ≤ 0.001, p ≤ 0.01, and p ≤ 0.01, respectively; Fig. 6). ers such as il1b and cox2 in zebrafish seizure models. Furthermore, all indomethacin concentrations tested Our results clearly showed that the expression of il1b reduced the number of seizure-like behaviors compared is upregulated shortly after seizures in the larval brain with the PTZ treatment alone (10 μM, p ≤ 0.01; 100 μM, (Fig. 1). A previous study of Minami et al.  showed p ≤ 0.01; and 307 μM, p ≤ 0.001; Fig. 7). that the mRNA levels of IL-1β increase more rap- idly when the seizure was induced by PTZ compared Discussion with kainic acid in rodents. Recently, Järvelä et al.  It has been demonstrated previously that seizures reported that the mRNA level of IL-1β is elevated for up induce the upregulation of IL-1β and COX-2 in clinical to 24 h after status epilepticus (SE) induced by kainic acid Barbalho et al. BMC Neurosci (2016) 17:12 Page 4 of 9 Fig. 2 cox2a and cox2b expression in the brain of zebrafish after pentylenetetrazole ‑ evoked seizures. Relative quantification of cyclooxygenase 2 a and b (cox2a and cox2b) transcripts 0.05, 1, and 6 h after pentylenetetrazole (PTZ)‑induced seizures in the brain of zebrafish at 7 days post fertiliza‑ tion. Each time‑point seizure group was exposed to 15 mM PTZ for 20 min, and their time ‑matched control groups were handled identically, but included exposure to water (n = 5 per group). Data obtained from each seizure group was compared with their respective time‑point matched control group. Data are presented as mean ± SEM. Statistical analyses were performed using the Mann–Whitney, and differences were considered significant if p < 0.05. One asterisk (*) indicated that p ≤ 0.05; two asterisks (**) indicated that p ≤ 0.01. CG control group, SG seizure group in rodents. Our finding suggests that the transcription various concentrations downregulated the expression of profile of il1b after PTZ exposure in our zebrafish model il1b (Fig. 3). This result is similar to the data obtained is similar to that found in a rodent model of PTZ-induced using the pilocarpine-induced model of epilepsy, wherein seizures. indomethacin administration prior to pilocarpine injec- Because of evolutionary gene duplication, the zebrafish tion downregulates the expression of IL-1β in rats . genome contains two functional cox2 genes termed as In addition, we showed that indomethacin significantly cox2a and cox2b [31, 32]. The mRNAs of cox2a and downregulated the mRNA expression of cox2b, but only cox2b are constitutively expressed in numerous tissues, at a concentration of 307 μM. including the zebrafish brain . In this study, we inves - After showing that indomethacin was able to reduce tigated the inducible expression of both cox2 genes after the mRNA levels of il1b and cox2b, we investigated if PTZ-induced seizures. Our results revealed a character- indomethacin reduces the convulsant effect of PTZ istic transcriptional response in both genes. Expression (Fig. 4). Therefore, we assessed neuronal activity by of cox2b, but not cox2a, is upregulated immediately after measuring the mRNA expression of c-fos, and we investi- PTZ-induced seizures (0.05 h) and 1 h after the seizure in gated seizure behavior during PTZ exposure by analyzing the brain of zebrafish (Fig. 2a, b). It is important to note the latency of seizure onset and the number of seizure- that a study by Ishikawa et al.  showed that cox2b is like behaviors, which are both well-known characteristics more similar in structure to the mammalian cox2 than of seizures according to the literature . The c-fos gene cox2a, which may explain our results . is a proto–oncogene, which is expressed rapidly and tran- Because the mRNA levels of il1b and cox2b were upreg- siently in neurons following neuronal excitation such as ulated after seizures, we investigated the effect of an anti- that in seizures; therefore, expression of c-fos has been inflammatory drug administered prior to PTZ-induced widely used as a marker for neuronal activity . Bax- seizures. Our results showed that indomethacin used at endale et al.  showed that c-fos is a sensitive marker Barbalho et al. BMC Neurosci (2016) 17:12 Page 5 of 9 Fig. 3 Indomethacin, administered prior to pentylenetetrazole‑ Fig. 4 Indomethacin, administered prior to pentylenetetrazole‑ induced seizures, on il1b level in the brain of zebrafish. Relative induced seizures, on cox2b level in the brain of zebrafish. Relative quantification of interleukin‑1 beta (il1b) mRNA expression level quantification of cyclooxygenase 2 b (cox2b ) mRNA expression 0.05 h after pentylenetetrazole (PTZ)‑induced seizures in the brain of level 0.05 h after pentylenetetrazole (PTZ)‑induced seizures in the zebrafish at 7 days post fertilization. Seizure group (SG) comprised brain of zebrafish at 7 days post fertilization. Seizure group (SG) was animals exposed to 15 mM PTZ for 20 min. Indomethacin groups composed of animals exposed to 15 mM PTZ for 20 min. Indometha‑ (10, 100, and 307 μM) comprised animals that received indometha‑ cin groups (10, 100, and 307 μM) were composed of animals that cin treatment prior to PTZ. Animals in the control group (CG) were received indomethacin treatment prior to PTZ. Animals in the control handled identically, but included exposure to water (no PTZ or group (CG) were handled identically, but they were treated with indomethacin treatments; n = 5 per group). Data are presented as water (no PTZ and no indomethacin treatments; n = 5 per group). mean ± SEM. Statistical analysis was performed by one‑ way analysis Data are presented as mean ± SEM. Statistical analysis was performed of variance (ANOVA) with Bonferroni’s post hoc test. Three asterisks by one‑ way analysis of variance (ANOVA) with Bonferroni’s post hoc (***) indicate that p ≤ 0.001. CG control group, SG seizure group test. One asterisk (*) indicated that p ≤ 0.05; two asterisks (**) indicated that p ≤ 0.01; three asterisks (***) indicated that that p ≤ 0.001. CG control group, SG seizure group for investigating anticonvulsant properties of several pharmacological compounds. Our results showed that seizures had an anti-inflammatory effect by reducing indomethacin downregulated c-fos expression at 100 and the mRNA expression of il1b, cox2b, and c-fos, and by 307 μM (Fig. 5). increasing seizure latency and decreasing the number of Furthermore, indomethacin administered prior to PTZ seizure-like behaviors. reduced the behavioral signs of seizure because it both Taken together, our results demonstrate that the increased seizure latency (time to reach stage 3, which zebrafish seizure model is a valuable alternative model is characterized by the loss of body posture) (Fig. 6) and for studying the molecular mechanisms of inflammation decreased the number of seizure-like behaviors for all and seizures and for the investigation of anti-inflamma - indomethacin concentrations used in this study com- tory compounds that may have a potential therapeutic pared with the untreated group (Fig. 7). effect in seizure suppression. Although our results clearly suggest that indomethacin has significant effects on several parameters related to Methods seizure activity, we did not demonstrate that the anticon- Animals vulsant effect of indomethacin is caused exclusively by its Wild-type zebrafish (adults, larvae, and embryos) were anti-inflammatory action on il1b and cox2b; therefore, maintained according to standard procedures . Adult further studies are necessary to address this question. fish were housed in 30–50 L tanks (two animals per liter) filled with non-chlorinated water cleared with mechani - Conclusions cal and chemical filtration. Adult fish were maintained at We have shown for the first time that transcriptional lev - 26 ± 2 °C and in a simulated photoperiod cycle of 10 h els of two important inflammatory biomarkers related to dark/14 h light. Adult fish were fed twice a day with com - epilepsy, il1b and cox2, are upregulated in the brain of mercial flake fish food (Tetramin, Tetra, Blacksburg, VA) zebrafish after PTZ-induced seizures. Furthermore, we and once a day with artemia; larvae were fed with para- found that indomethacin exposure prior to PTZ-induced mecium and artemia twice a day. Fertilized eggs were Barbalho et al. BMC Neurosci (2016) 17:12 Page 6 of 9 Fig. 5 Eec ff t of indomethacin exposure, prior to pentylenetetrazole ‑ Fig. 6 Eec ff t of indomethacin treatment prior to pentylenetetrazole induced seizures, on c‑fos level of the zebrafish brain. Relative induced seizures on seizure latency. Animals were exposed to 10, 100, quantification of c‑fos transcript levels 0.05 h after pentylenetetrazole or 307 μM indomethacin for 24 h prior to pentylenetetrazole (PTZ)‑ (PTZ)‑induced seizures in the brain of zebrafish at 7 days post ‑ induced seizures, and latency of the first seizure ‑like behavior (stage fertilization. Seizure group was composed of animals exposed to 3, equivalent to loss of body posture) was evaluated following visual 15 mM PTZ for 20 min. Indomethacin groups (10, 100, and 307 μM) observation. The seizure group (SG) was composed of animals that were composed of animals that received indomethacin treatment were exposed to 15 mM PTZ (n = 6 per group). Data are presented as prior to PTZ. Animals in the control group were handled identically, mean ± SEM. Statistical analysis was performed by one‑ way analysis but included exposure to water (no PTZ or indomethacin treatments; of variance (ANOVA) with Bonferroni’s post hoc test. One asterisk (*) n = 5 per group). Data are presented as mean ± SEM. Statistical indicated that p ≤ 0.05; two asterisks (**) indicated that p ≤ 0.01; three analysis was performed by one‑ way analysis of variance (ANOVA) with asterisks (***) indicated that that p ≤ 0.001 Bonferroni’s post hoc test. One asterisk (*) indicated that p ≤ 0.05; two asterisks (**) indicated that p ≤ 0.01; three asterisks (***) indicated that that p ≤ 0.001. CG control group, SG seizure group Monitoring of seizure‑like behavior Seizure-like behavior of the larvae was monitored by visual inspection and was classified based on the crite - collected after natural spawning. Embryos and larvae ria established in a previous study . To visualize the were housed using Petri dishes filled with water in an swimming behavior of larvae, we used the Stereomaster incubator system at the same temperature and photo- microscope (Fisher Scientific, Waltham, MA). Briefly, periods that were used for maintaining the adults. Lar- classification was performed according to the following vae were staged according to the morphological criteria criteria: stage 1, increased swimming activity; stage 2, established by Kimmel et al. . All zebrafish experi - rapid circular “whirlpool-like” swimming; and stage 3, sei- ments were approved by the Ethical Committee for Ani- zure-like activity progressing to clonus-like convulsions mal Research of the University of Campinas (protocol followed by a brief loss of posture. Animals included in number 3098-1). this study exhibited these well-defined behavioral patterns during PTZ exposure. Moreover, we evaluated the latency Pentylenetetrazole treatment of seizure onset and the number of seizures during PTZ Larvae at 7 days post fertilization (dpf ) were separated in exposure. It is important to emphasize that latency was the seizure (SG) and control (CG) groups. Larvae in the determined as the period between the start of PTZ expo- SG were placed in a 24-well plate (one larvae per well) sure and the appearance of stage 3 seizure-like behavior, containing 15 mM PTZ (Sigma-Aldrich, St. Louis, MO, and the number of seizure-like behaviors was obtained by USA), a γ-aminobutyric acid (GABA) antagonist, for counting all stage 3 seizure-like behaviors displayed. 20 min . After PTZ exposure, animals were washed to eliminate the residual PTZ before being transferred into Treatment with indomethacin and its toxicological petri dishes. Fish in the CG were handled identically; evaluation however, water was used instead of PTZ. It is important Indomethacin (Sigma-Aldrich, St. Louis, MO, USA) was to highlight that each CG or SG presented in this study solubilized in 1 % Tris–HCl (pH 8.0) buffer to prepare a was composed by a different set of animals. Barbalho et al. BMC Neurosci (2016) 17:12 Page 7 of 9 the indomethacin treatment was determined based on a previous AED screening reported by Afrikanova et al. . Zebrafish larvae at six dpf were incubated in 10, 100, and 307 μM indomethacin in petri dishes for 24 h, and then at seven dpf, they were exposed to 15 mM PTZ for 20 min as described above. It is important to highlight that each indomethacin concentration group presented in this study was composed by a different set of animals. RNA isolation and reverse transcriptase‑PCR Animals were crioanesthetized and their heads were immediately isolated, quickly frozen in liquid nitrogen, and stored at −80 °C 0.05, 1, 6, 12, 24, and 48 h after PTZ treatment. We pooled 20 larval heads to obtain sufficient material for RNA extraction. A total of five samples (n = 5 for each time point) were used for each group (CG, SG, and indomethacin treatment). Total RNA Fig. 7 Eec ff t of indomethacin treatment prior to pentylenetetrazole ‑ was extracted using TRIzol (Invitrogen, Carlsbad, CA, induced seizures on the number of seizure‑like behaviors. Animals USA) according to the manufacturer’s instructions, and were exposed to 10, 100, or 307 μM indomethacin prior to pentyl‑ its concentration and quality were determined with the enetetrazole (PTZ)‑induced seizures. Number of seizure ‑like behaviors was evaluated following visual observation during the 20 min of EpochTM spectrophotometer (BioTek, Winooski, VT, PTZ (15 mM) exposure. Seizure‑like behavior was registered if the USA) and electrophoresis using agarose gels. One micro- zebrafish larvae lost their body posture (stage 3). Data are presented gram of total RNA was reverse transcribed into cDNA as mean ± SEM (n = 6 per group). Statistical analysis was performed using the High Capacity first-strand synthesis system for by one‑ way analysis of variance (ANOVA) with Bonferroni’s post hoc RT-PCR (Invitrogen, Carlsbad, CA, USA) according to test. One asterisk (*) indicated that p ≤ 0.05; two asterisks (**) indicated that p ≤ 0.01; three asterisks (***) indicated that that p ≤ 0.001. SG the manufacturer’s instructions. Reactions without RNA seizure group were run as negative controls. Real‑time quantitative PCR Quantitative PCR was performed using the ABI 7500 stock solution. A primary concentration screen assay was Real Time PCR system (Applied Biosystems, Foster set up for the toxicological evaluation of indomethacin City, CA, USA), TaqMan Universal Master Mix, and by incubating larvae at six dpf in a 96-well plate (Mil- TaqMan Gene Expression Assay (Invitrogen, Carlsbad, lipore, USA). Based on our protocol and the protocol CA, USA) for zebrafish (Table 1). All relative quantifi - described by d’Alençon et al. (2010) , indomethacin cations were performed in triplicates and were normal- was tested at seven concentrations (10, 70, 100, 140, 200, ized to the housekeeping gene eukaryotic translation 250, and 307 μM). One larva was used per well, and 12 elongation factor 1 alpha 1, like 1 (eef1a1l1) [38, 39]. larvae were used per treatment/concentration groups. The mRNA level of each target gene (il1b, cox2a, cox2b, After 24 h of incubation in indomethacin, we applied a and c-fos) was normalized to the expression level of the touch stimulus on the plate to evaluate the startle/escape housekeeping gene eef1a1l1 (Table 1). Efficiency of each response of each larva to identify any signs of locomo- quantitative real time PCR assay was assessed using tor impairment and/or toxicity. None of the tested con- standard curves, which were created by measuring five centrations impaired the startle/escape response of the serially diluted cDNA samples in triplicates. Efficiency larvae or caused body deformation, exophthalmos, or was calculated according to the following formula: death [9, 10, 37]. This assay was performed in duplicates. E = 10[−1/slope]. Relative gene expression quantifica - Then we used 10 and 100 μM indomethacin according tion (RQ) of the SG samples compared with the CG sam- to a study by d’Alençon et al. (2010)  and 307 μM ples (after normalization to the housekeeping gene) was indomethacin (the maximum dose tested in our assay) calculated according to the equation RQ = 2 − ∆∆CT before PTZ-induced seizures, and tested the effect of described by Livak and Schmittgen . Each reaction these concentrations on cox2b mRNA expression using was run without cDNA as negative control. Data were qPCR. After 24 h of indomethacin exposure, we applied a analyzed using the SDS 7500 software (Applied Biosys- touch stimulus on the plate to evaluate the startle/escape tems) to estimate qPCR efficiency and quantify the rela - response of each larva to identify any signs of locomotor tive gene expression. impairment and toxicity. The incubation period used for Barbalho et al. BMC Neurosci (2016) 17:12 Page 8 of 9 Table 1 Information about genes and qPCR assays Official full name Gene official symbol Also known as Accession Assay ID Eukaryotic translation elongation factor 1 alpha 1a eef1a1a eef1a1a NM_131263.1 Dr03432748_ml v‑fos FBJ murine osteosarcoma viral oncogene homolog Ab fosab fos, c-fos NM_205569.1 Dr03100809_gl Interleukin 1‑beta il1b il1-b NM_212844.2 Dr03114369_ml Prostaglandin‑ endoperoxide synthase 2a ptgs2a cox2a NM_153657.1 Dr03080323_ml Prostaglandin‑ endoperoxide synthase 2b ptgs2b cox2b NM_001025504.2 Dr03116323_ml 2. Shin JT, Fishman MC. From Zebrafish to human: modular medical models. Statistical analysis Annu Rev Genomics Hum Genet. 2002;3:311–40. Data are presented as mean values ± standard error of 3. Baraban SC, Taylor MR, Castro PA, Baier H. Pentylenetetrazole induced mean (SEM). Statistical analysis was performed using changes in zebrafish behavior, neural activity and c‑fos expression. Neu‑ roscience. 2005;131(3):759–68. the GraphPad Prism version 5.0 (GraphPad Software, 4. Pineda R, Beattie CE, Hall CW. Recording the adult zebrafish cerebral CA, USA). In all analyses, significance level was set at field potential during pentylenetetrazole seizures. J Neurosci Methods. p ≤ 0.05. Statistical comparisons between two groups 2011;200(1):20–8. 5. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins were performed using the Mann–Whitney test. Statis- JE, Humphray S, McLaren K, Matthews L, et al. The zebrafish reference tical comparisons between three or more groups were genome sequence and its relationship to the human genome. Nature. performed using one-way analysis of variance (ANOVA) 2013;496(7446):498–503. 6. Alfaro JM, Ripoll‑ Gómez J, Burgos JS. Kainate administered to adult with Bonferroni’s post hoc test. zebrafish causes seizures similar to those in rodent models. Eur J Neuro ‑ sci. 2011;33(7):1252–5. Abbreviations 7. Pichler FB, Laurenson S, Williams LC, Dodd A, Copp BR, Love DR. Chemical CG: control group; cox2a: cyclooxygenase‑2a; cox2b: cyclooxygenase ‑2b; dpf: discovery and global gene expression analysis in zebrafish. Nat Biotech‑ days post‑fertilization; fos: FBJ murine osteosarcoma viral oncogene homolog; nol. 2003;21(8):879–83. il1b: interleukin‑1 beta; NSAID: nonsteroidal anti‑inflammatory drugs; ptgs2: 8. Kaufman CK, White RM, Zon L. Chemical genetic screening in the prostaglandin‑ endoperoxide synthase 2; PTZ: pentylenetetrazol; qPCR: quanti‑ zebrafish embryo. Nat Protoc. 2009;4(10):1422–32. tative PCR; RT‑PCR: reverse transcription PCR; SG: seizure group. 9. Berghmans S, Hunt J, Roach A, Goldsmith P. Zebrafish offer the potential for a primary screen to identify a wide variety of potential anticonvul‑ Authors’ contributions sants. Epilepsy Res. 2007;75(1):18–28. PGB performed the experiments, participated in the design of the study, 10. Baxendale S, Holdsworth CJ, Meza Santoscoy PL, Harrison MR, Fox J, analyzed the data and wrote the paper. ILC participated in the design of the Parkin CA, Ingham PW, Cunliffe VT. Identification of compounds with study and revised the manuscript. CVMM conceived the study, participated in anti‑ convulsant properties in a zebrafish model of epileptic seizures. Dis its design and coordination and revised the manuscript. All authors read and Model Mech. 2012;5(6):773–84. approved the final manuscript. 11. Baraban SC, Löscher W. What new modeling approaches will help us identify promising drug treatments? Adv Exp Med Biol. 2014;813:283–94. Author details 12. Kwon YS, Pineda E, Auvin S, Shin D, Mazarati A, Sankar R. Neuroprotective Laboratory of Zebrafish, Department of Medical Genetics ‑ School of Medical and antiepileptogenic effects of combination of anti‑inflammatory drugs Sciences, University of Campinas (UNICAMP), Tessália Vieira de Camargo, 126 in the immature brain. J Neuroinflammation. 2013;10:30. Cidade Universitaria “Zeferino Vaz”, Campinas, SP 13083‑887, Brazil. Labora‑ 13. Gupta P, Khobragade SB, Shingatgeri VM. Eec ff t of various antie ‑ tory of Molecular Genetics, Department of Medical Genetics ‑ School of Medi‑ pileptic drugs in zebrafish PTZ ‑seizure model. Indian J Pharm Sci. cal Sciences, University of Campinas (UNICAMP), Tessália Vieira de Camargo, 2014;76(2):157–63. 126 Cidade Universitaria “Zeferino Vaz”, Campinas, SP 13083‑887, Brazil. 14. Vezzani A, Aronica E, Mazarati A, Pittman QJ. Epilepsy and brain inflamma‑ tion. Exp Neurol. 2013;244:11–21. Acknowledgements 15. Vezzani A, French J, Bartfai T, Baram TZ. The role of inflammation in epi‑ This work was supported by Brazilian Institute of Neuroscience and Neuro‑ lepsy. Nat Rev Neurol. 2011;7(1):31–40. technology BRAINN‑ CEPID (#2013/07559‑3); Fundação de Amparo a Pesquisa 16. Xu D, Miller SD, Koh S. Immune mechanisms in epileptogenesis. Front do Estado de São Paulo (FAPESP, Grant #2014/15640‑8; 2013/19151‑9); Cell Neurosci. 2013;7:195. Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Grant 17. Aronica E, Crino PB. Inflammation in epilepsy: clinical observations. #475405/2010‑2) B.G.P received fellowship from FAPESP. 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BMC Neuroscience – Springer Journals
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