Cyclooxygenase-1 as a Potential Therapeutic Target for Seizure Suppression: Evidences from Zebrafish Pentylenetetrazole-Seizure Model.

Cyclooxygenases (COX)-1 and -2 are isoenzymes that catalyze the conversion of arachidonic acid into prostaglandins (PGs). COX-2 and PGs are rapidly increased following seizures and are known to play important roles in the neuroinflammatory process. COX-2 isoform has been predominantly explored as the most suitable target for pharmacological intervention in epilepsy studies, while COX-1 remains poorly investigated. In the present study, we evaluated the effects of selective COX-1 inhibitor or selective COX-2 inhibitor on seizure suppression in the zebrafish pentylenetetrazole (PTZ)-seizure model. Zebrafish larvae were incubated in 5 µM of SC-236 for 24 h or 2.8 µM of SC-560 for 30 min, followed by exposure to 15 mM PTZ for 60 min. Real-time quantitative PCR analysis was carried out to investigate transcription levels of cox1 (ptgs1), as well as to determine cfos levels, used as a marker for neuronal activity. Effects of selective COX-2 or COX-1 inhibitors on locomotor activity response (velocity and distance moved) during PTZ exposure were evaluated using the Danio Vision video-tracking system. Our results showed an inducible expression of the cox1 gene after 60 min of PTZ exposure. Cox1 mRNA levels were upregulated compared with the control group. We found that COX-2 inhibition treatment had no effect on zebrafish PTZ-induced seizures. On the other hand, COX-1 inhibition significantly attenuated PTZ-induced increase of locomotor activity and reduced the c-fos mRNA expression. These findings suggest that COX-1 inhibition rather than COX-2 has positive effects on seizure suppression in the zebrafish PTZ-seizure model.

Indomethacin treatment prior to pentylenetetrazole-induced seizures downregulates the expression of il1b and cox2 and decreases seizure-like behavior in zebrafish larvae.

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 interleukin-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 inhibition 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.

Análise Temporal dos Transcritos dos Genes bdnf e ntrk2 em Cérebro de Zebrafish Induzido à Crise Epiléptica por Pentilenotetrazol / Temporal transcript profile of bdnf, ntrk2a and ntrk2b genes in zebrafish brain after Pentylenotetrazole (PTZ)-induced seizure

Objetivo: Investigar o perfil temporal de transcritos dos genes bdnf, ntrk2a e ntrk2b em cérebro de zebrafish após crise epiléptica induzida por Pentilenotetrazol (PTZ). Metodologia: Os animais foram divididos em Grupo PTZ (induzidos à crise epiléptica com PTZ 15mM) e Grupo Controle (animais sem crise epiléptica) e seus cérebros coletados nos tempos: 0h, 12h, 24h, 48h, 72h pós-crise. Reações de transcriptase reversa-PCR quantitativa foram realizadas com os controles endógenos 18s e ef1a usando-se o sistema TaqManTM (Applied Biosystems, Foster City). A quantificação relativa foi calculada pela equação QR=2–∆∆CT e a significância estatística dada pelo teste Kruskall-Wallis (p≤0,05). Resultados: No grupo PTZ houve um aumento significativo dos niveis de RNAm do gene bdnf no tempo 0h (p=0,017). O aumento de transcritos encontrado nos outros tempos não foi significante (p>0,05). Conclusão: Nossos resultados mostraram que a indução de crise epiléptica alterou o padrão de transcrito do gene bdnf no cérebro do zebrafish como visto em outros modelos animais e em humanos, porém em um padrão temporal diferente. Este é o primeiro estudo que descreve o perfil temporal de transcritos bdnf/ntrk2 em cérebro de zebrafish após crise epiléptica e contribui para a caracterização deste pequeno peixe como modelo de estudo em epilepsias.

Objective: The main aim of this study was to investigate the transcript profile of bdnf, ntrk2a and ntrk2b genes in adult zebrafish brain after Pentylenotetrazole (PTZ)-induced seizure. Methods: Zebrafish were separated in PTZ (seizure-induced) and Control (no seizure) groups. At 0h, 12h, 24h, 48h and 72h after seizure, animals were anesthetized and their brains were immediately collected for RNA extraction. Reverse transcriptase quantitativePCRs were carried out with 18S and ef1a as endogenous control using TaqManTM System (Applied Biosystems, Foster City). The relative quantification was calculated by the equation RQ=2–∆∆CT. Statistical analysis was performed by Kruskall-Wallis test (p≤0.05). Results: Comparisons between both groups showed an increase of bdnf mRNA levels in the PTZ group at 0h after seizure (p= 0.017). No statistical significance was found in other times investigated (p>0.05). Conclusions: Our results showed an up-regulation of the transcript levels of bdnf gene in zebrafish brain after seizure as seems in other models and humans, but in a different pattern. This is the first study investigating temporal pattern of bdnf, ntrk2a and ntrk2b genes in zebrafish brain after a seizure and contributes to characterize it as a model for epilepsy studies.