Optimization and Pharmacological Validation of a Leukocyte Migration Assay in Zebrafish Larvae for the Rapid In Vivo Bioactivity Analysis of Anti-Inflammatory Secondary Metabolites

Abstract

Over the past decade, zebrafish (Danio rerio) have emerged as an attractive model for in vivo drug discovery. In this study, we explore the suitability of zebrafish larvae to rapidly evaluate the anti-inflammatory activity of natural products (NPs) and medicinal plants used in traditional medicine for the treatment of inflammatory disorders. First, we optimized a zebrafish assay for leukocyte migration. Inflammation was induced in four days post-fertilization (dpf) zebrafish larvae by tail transection and co-incubation with bacterial lipopolysaccharides (LPS), resulting in a robust recruitment of leukocytes to the zone of injury. Migrating zebrafish leukocytes were detected in situ by myeloperoxidase (MPO) staining, and anti-inflammatory activity was semi-quantitatively scored using a standardized scale of relative leukocyte migration (RLM). Pharmacological validation of this optimized assay was performed with a panel of anti-inflammatory drugs, demonstrating a concentration-responsive inhibition of leukocyte migration for both steroidal and non-steroidal anti-inflammatory drugs (SAIDs and NSAIDs). Subsequently, we evaluated the bioactivity of structurally diverse NPs with well-documented anti-inflammatory properties. Finally, we further used this zebrafish-based assay to quantify the anti-inflammatory activity in the aqueous and methanolic extracts of several medicinal plants. Our results indicate the suitability of this LPS-enhanced leukocyte migration assay in zebrafish larvae as a front-line screening platform in NP discovery, including for the bioassay-guided isolation of anti-inflammatory secondary metabolites from complex NP extracts.

Modeling Inflammation in Zebrafish for the Development of Anti-inflammatory Drugs

“…LPS treatment has also been used in combination with tail wounding to enhance leukocyte accumulation near the wound. This model was utilized to evaluate the bioactivity of structurally diverse natural products of an East African medicinal plant, Rhynchosia viscosa , resulting in the identification of both known and novel isoflavone derivatives with anti-inflammatory activity (Bohni et al, 2013 ; Cordero-Maldonado et al, 2013 ).…”

The value of zebrafish as an integrative model in effect-directed analysis - a review

“…Therefore, it is recommended to identify the critical aspects for endpoint assessment, to optimize bioassays accordingly and to demonstrate the validity of the bioassay by testing known bioactive compounds [61]. The endpoints and bioassays described in the different studies are summarized in Table 3 and discussed in the context of respective study objectives and outcomes.…”

Optical micromanipulation of nanoparticles and cells inside living zebrafish

“…We also tested the effects of anti-inflammatory drugs on the migration of immune cells to bacteria-invaded areas. Our preliminary experiments using fish lines with either fluorescent neutrophils or fluorescent macrophages confirmed that the anti-inflammatory drugs diclofenac and indomethacin 32 inhibited the recruitment of neutrophils (and macrophages to a lesser degree, unpublished results) to sites where bacteria had been injected 12 . However, these experiments need a systematic follow-up for their significance to be verified.…”