Development and validation of chemical kindling in adult zebrafish: A simple and improved chronic model for screening of antiepileptic agents

Highlights

• Exposure of 1.25 mM PTZ for 22 days induced kindling-like state in adult zebrafish.

• The expression of c-fos, crebbpa, and crebbpb increased in kindled fish brain.

• The kindled fish brain showed an increased level of glutamate/GABA.

• Valproic acid treatment inhibited the development of the kindling-like state in fish.

• Valproic acid reduced the severity of seizures mediated by PTZ in kindled fish.

Abstract

Background

Zebrafish has emerged as a potential animal model of acute convulsion for early screening of antiepileptic agents. There is a need for alternative chronic zebrafish models of epilepsy with more correlation to the clinical condition.

New Method

Adult zebrafish were repeatedly exposed to subeffective concentrations of pentylenetetrazole (PTZ), until appearance to tonic-clonic seizures, considered as kindled. Valproic acid (VPA) exposure was given during kindling and in kindled fish in 2 different groups. The neurotransmitters level and expression of the genes associated with kindling were studied in the fish brain.

Results

There was an increase in seizure severity score at 1.25 mM concentration of PTZ, and 66.66 % of fish achieved kindling after 22 days’ exposure. A marked increase in c-fos, crebbpa and crebbpbexpression, and glutamate/GABA level was observed in the brain of kindled fish. VPA inhibited the induction of PTZ-mediated kindling and reduced seizure severity in kindled fish.

Comparison with existing method

In contrast to an existing adult zebrafish kindling method, the present protocol is of longer duration, with more similarity to clinical epilepsy. Moreover, the induction of kindling involves a simple non-invasive technique without the use of anesthesia. The protocol can be used for evaluation of both antiepileptic and antiepileptogenic agents.

Conclusion

Repeated exposure of 1.25 mM PTZ induced kindling in zebrafish, altering the brain neurotransmitter levels and gene expression. Inhibition of kindling induction and decrease in seizures in normal and kindled fish, respectively by VPA validated application of the model for preclinical testing of agents against epilepsy.

Introduction

Epilepsy is a chronic condition that results in unusual seizure episodes in the brain, and brutally affects patients' quality of life. There are around 5 million people annually identified with epilepsy, and it accounts for 0.5 percent of the global burden of the disease (WHO, 2019). Despite of available nonpharmacotherapeutic strategies, the majority of the epileptic population depends on antiepileptic drugs (AEDs) for its management (Mitchell et al., 2012; Campos et al., 2018). In the past 25 years, several new AEDs have developed, but despite of this, around 25% of patients remain unresponsive to current therapies and presents a need to develop more effective drugs (Dalkara and Karakurt, 2012; Golyala and Kwan, 2017). In many instances, it has been observed that preclinical lead antiepileptic molecules show little efficacy during clinical practice. The variation has been suggested due to the use of traditional pentylenetetrazole (PTZ) and maximal electroshock acute tests as a primary preclinical drug screening tool (Bialer and White, 2010; Löscher, 2011). Additionally, comprehensive studies involving seizure generation and understanding the complex mechanisms underlying epileptogenesis are challenging to conduct in human beings due to ethical constraints (Kandratavicius et al., 2014). Thus, suggesting a need for new animal models with a better pathogenic similarity with human epilepsy for a successful AEDs development program.

Since epilepsy is a chronic condition, hence chronic models are more appropriate supportive aid for its study, as they closely mimic clinical conditions. Rodent kindling is a well-established chronic model that involves repeated electrical stimuli of subeffective intensity, or repeated low dose administration of a chemoconvulsant, till induction of full tonic-clonic seizures. The model shares many pathogenic features of human partial seizures and serves as an important AEDs screening tool (Löscher, 2011; Singh et al., 2013; Grone and Baraban, 2015). Although rodents play a crucial role in AEDs development, there are several limitations to their use (Kandratavicius et al., 2014). Moreover, mammalian epilepsy models require high maintenance costs and are difficult to modify genetically, making them unsuitable for large-scale therapeutic screening. In this regard, the zebrafish (Danio rerio) model has emerged to be a less expensive and potential tool for drug screening (Hortopan et al., 2010).

Zebrafish is a small, freshwater fish with a high anatomical and genetic homology with mammals (Sharma et al., 2014). Zebrafish presents a tight junction-based blood-brain barrier similar to higher vertebrates, with substantial macromolecule permeability, making it an ideal model organism for CNS drug discovery. The application of zebrafish in epilepsy research has made feasible by the adaptation of Racine scale stages similar to rodents to a measurable equivalent behavior, for both larvae and adult zebrafish (Baraban et al., 2005; Mussulini et al., 2013). It has become a popular acute convulsion model for early screening of antiepileptic agents against different chemoconvulsants (Gupta et al., 2014; Winter et al., 2017; Tanwar et al., 2019). However, there is a need to develop chronic epilepsy models of zebrafish with more correlation to human epilepsy. Limited work has been carried out in this regard. In a recent study the effect of embelin was investigated on seizure development in an adult zebrafish model. A daily intraperitoneal injection of PTZ at a subeffective dose of 80 mg/kg was given following anesthesia with benzocaine (30 mg/L) for 10 days for the seizure induction (Kundap et al., 2019). In the present study, a simple, non-invasive kindling method of longer duration in adult zebrafish was developed. Moreover, kindling was achieved without anesthetics, as they are reported to interact with antiepileptic agents and epilepsy processes (Taberner, 1976; Modica et al., 1990), thus leading to variation in results. Further, gene expression and neurotransmitters quantification studies in the brain tissue were performed to ensure kindling. Valproic acid (VPA) was tested as a standard in separate protocols to validate the application of the developed model for antiepileptic and antiepileptogenic studies.