Research articleDevelopment of zebrafish demyelination model for evaluation of remyelination compounds and RORγt inhibitors
Introduction
Multiple sclerosis (MS) which is characterized by the loss of oligodendrocytes and axon demyelination is a chronic, multifocal and relapsing-remitting disorder mostly in young adults (Compston, Kellarwood, & Wood, 2008). The myelin sheath is the membrane structure protecting, supporting and nourishing axons. As the multi-layered insulating structure around the axon, the myelin sheath mediates the rapid conduction of nerve impulses (Almeida, Czopka, Ffrench-Constant, & Lyons, 2011; Czopka, Ffrench-Constant, & Lyons, 2013; D'Rozario, Monk, & Petersen, 2016). Demyelination is the most common complication of MS in which the myelin sheath around the axons gets damaged (Compston et al., 2008; Paz Soldan & Rodriguez, 2002). This damage impairs the conduction of signals in the affected nerves. Then, the reduction in conduction causes deficiencies in sensation, movement, cognition, or other functions (Compston et al., 2008; Scherer & Wrabetz, 2008).
Zebrafish has emerged as a cost-efficient model to study vertebrate myelination in vivo (Buckley, Marguerie, Alderton, & Franklin, 2010; Preston & Macklin, 2015) and is particularly interesting and valuable due to its remarkable ability to regenerate injured axons in the central nerve system (CNS) (Bernhardt, Tongiorgi, Anzini, & Schachner, 1996; Stuermer, Bastmeyer, Bahr, Strobel, & Paschke, 1992; Su, Liu, Chan, & Wang, 2016). Zebrafish myelin structure, myelin synthesis and gene expression are highly conserved between zebrafish and mammals (Chung et al., 2013; Gerlai, 2011; Jung et al., 2010; Fang et al., 2015). The myelin structure is formed by oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system and surrounds the axons. In zebrafish, a non-tight structure is observed at 2 days post fertilization (dpf), and the myelin sheath forms at 4 dpf. The tunica vaginalis forms a compact myelin structure at 7 dpf (Bernhardt et al., 1996). Most myelin-associated mammalian genes have homologies with zebrafish (Emery, 2010; Farrar, Wise, Fetcho, & Schaffer, 2011; Schweitzer, Becker, Schachner, Nave, & Werner, 2006).
Chemical methods have often been used to induce demyelination in mouse models. Ethdium bromide (EB) in the white matter of the CNS is known to act like a gliotoxin, causing local oligodendroglial and astrocytic death, leading to primary demyelination, neuroinflammation, blood-brain barrier disruption and Schwann cell invasion due to the glia limitans breakdown (Graça, Bondan, Pereira, Fernandes, & Maiorka, 2001; Kuypers, James, Enzmann, Magnuson, & Whittemore, 2013; Blakemore, 1982; Bondan, Lallo, Sinhorini, Pereira, & Graça, 2000; Bondan, Lallo, Dagli, Sanchez, & Graça, 2003). EB is a simple tool for induction of neural cell degeneration and helps researchers to study the demyelination and remyelination processes (Guazzo, 2005; Bondan et al., 2000, Bondan et al., 2006). EB can also affect the motor and sensory systems (Goudarzvand et al., 2016). Thyroxine (T4) physiologically helps oligodendrocyte precursor cells to turn into myelinating oligodendrocytes, enhancing remyelination after myelin sheet damage (Pieragostino et al., 2013). Actually, it was demonstrated that T4 administration improves clinical course and the remyelination process in MS animal models (Dell'Acqua et al., 2012).
RAR-related orphan receptor-γt (ROR-γt) induces differentiation of proinflammatory T helper 17 (Th17) cells and is a potential therapeutic target for chronic autoimmune and inflammatory diseases including MS (Gaffen, Jain, Garg, & Cua, 2014; Ivanov et al., 2006; Korn, Bettelli, Oukka, & Kuchroo, 2009; Withers et al., 2016). It has been shown that the genetic deficiency of RORγt in mice severely impaired Th17 cell differentiation and conferred resistance to experimental allergic encephalomyelitis (Wang et al., 2015, Wang et al., 2012). As a nuclear receptor with a ligand-binding pocket, RORγt is considered to be an attractive pharmacologic target for the treatment of Th17-cell-mediated immune disorders. Indeed, several small molecular compounds that can inhibit the function of RORγt, including GSK805 (Wang et al., 2015; Withers et al., 2016; Xiao et al., 2014) and SR1001 (Solt et al., 2011), have been discovered.
The majority of work undertaken so far using zebrafish as a model has focused on investigating the mechanisms of remyelination and oligodendrocyte precursor cell (OPC) differentiation, using toxin-induced demyelination models and genetic cell ablation models (Burrows et al., 2019). In this study, we have taken advantage of larval zebrafish to develop a relative high-throughput demyelination model for drug screening and efficacy assessment. To validate the zebrafish demyelination models for future RORγt compounds screen and evaluation, two RORγt lead inhibitors GSK805 and SR1001 were selected for dose-response observations of remyelination, myelin basic protein (MBP) and axon regeneration, motor neuron promotion and anti-inflammation assessment. Our results indicate that zebrafish demyelination-associated motility assay in combination with quantitative myelin image assay is highly valuable for rapidly in vivo identification of remyelination compounds and RORγt inhibitors.
Section snippets
Zebrafish care and maintenance
Three lines of zebrafish were used in this study: wild-type AB line, motor neuron green-fluorescent-protein (GFP) transgenic zebrafish and neutrophil GFP transgenic zebrafish. Zebrafish were housed in a light and temperature controlled aquaculture facility with a standard 14: 10 h (h) light/dark photoperiod and fed with live brine shrimp twice daily and dry flake once a day. Four to five pairs of zebrafish were set up for natural mating every time. On average, 200–300 embryos were generated.
Zebrafish demyelination model
To develop a zebrafish demyelination model for drug screening and efficacy assessment, the optimal concentration and treatment time period of the demyelination inducer EB were first determined based on the motility assay (Fig. 1a) and FluoroMyelin staining (Fig. 1b). Zebrafish at 2 dpf were treated with 0.1, 1, 10, 25, 50, 75 and 100 μM EB for 24 h to 48 h or treated with 0.1 μM EB for 72 h did not result in the motility distance decrease and demyelination (p > .05, data not shown). As shown in
Discussion
In this study, we have optimized EB treatment concentration and treatment time period to develop a live zebrafish demyelination model with quantitative motility assay and myelin image analysis. Zebrafish at 2 dpf were treated with EB at a concentration of 75 μM for a time period of 72 h were determined as the optimum conditions for the zebrafish demyelination model development. The total distance traveled by individual zebrafish was recorded automatically by using a video-track motion detector (
Conclusions
This study developed and validated a zebrafish demyelination model that could be used for in vivo screening and efficacy assessment of remyelination compounds and RORγt inhibitors. This conventional zebrafish demyelination model could speed up therapeutic drug research and development for autoimmune diseases including multiple sclerosis.
Authors' contributions
Xiao-Yu Zhu, Yonghui Wang, Lei Wang and Chun-Qi Li designed the studies; Xiao-Yu Zhu, Sheng-Ya Guo and Bo Xia performed the experiments; Xiao-Yu Zhu and Sheng-Ya Guo analyzed the data; Xiao-Yu Zhu and Chun-Qi Li wrote the paper. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgment
This work was sponsored in part by the National Science & Technology Major Projects of China (No. 2017ZX09301-059) and National Science Foundation of China (No. 81573276). We thank Rick Li at Boston Latin School located in Boston, Massachusetts, USA, for his editing assistance.
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