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

Abstract

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.

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

“…We previously reported that cox2b mRNA expression was upregulated after PTZ-induced seizures in 7 dpf zebrafish larva (37). To determine whether the cox1 transcription is affected following PTZ-induced seizure, we used real-time qPCR to measure the relative quantification of the cox1 gene.…”

“…Detailed experimental procedures have been described previously (37). Briefly, 7 days post fertilization (dpf) larvae were placed in a 24-well plate (one larva per well) containing 15 mM PTZ [seizure group (SG)] or PTZ-free water [control group (CG)] for 60 min.…”

“…Total RNA extraction, reverse transcription, and qPCR were performed as previously reported (37). Briefly, total RNA was extracted from each group samples (control; seizure; SC-236; vehicle (DMSO)-treated controls; and SC-560 groups) using TRIzol ® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and its concentration and quality were determined with the Epoch™ spectrophotometer (BioTek, Winooski, VT, USA) and electrophoresis using agarose gels.…”

“…Moreover, NSAIDs as indomethacin and aspirin, which inhibit the activity of both COX-1 and COX-2, reduced seizures in the absence epilepsy model, in the zebrafish seizure model, and in the pilocarpine-induced SE model (35–37). Nonetheless, the pharmacological inhibition of COX-1 isoform in acute and chronic epilepsy models remains poorly investigated.…”

Zebrafish Models of Human Disease: Gaining Insight into Human Disease at ZFIN

“…To facilitate a better understanding of epilepsy, zebrafish models of epilepsy have been created by administering pentetrazol to induce seizures (Table 1D). Studies that utilize this method have investigated potential therapeutic compounds to alleviate seizure states as well as to understand changes in gene expression due to seizure events (Barbalho et al 2016; Li et al 2015; Siebel et al 2015). PD is a neurodegenerative disease that results from the loss of dopaminergic cells in the brain (Beitz 2014; Pienaar et al 2010).…”