A Rapid Screenable Assay for Compounds That Protect Against Intestinal Injury in Zebrafish Larva

This chapter describes a method to assay compounds modulating NSAID-induced intestinal injury in zebrafish larvae. The assay employs the NSAID glafenine, which causes intestinal epithelial cell damage and death by inducing organelle stress responses (endoplasmic reticulum and mitochondrial) and blocking the unfolded protein response pathway. This epithelial damage includes sloughing of intestinal cells into the lumen and out the cloaca of the zebrafish larvae. Exposing larvae to acridine orange highlights this injury when visualized under fluorescence microscope; injured fish develop intensely red-staining intestines, as well as a "tube" or cord of red color extending through the intestine and out the cloaca. Using this rapid visually screenable method, various candidate compounds were successfully tested for their ability to prevent glafenine-induced intestinal injury. Because this assay involves examination of larval zebrafish intestinal pathology, we have also included our protocol for preparation and analysis of zebrafish histology. The protocol includes numerous steps to generate high-quality zebrafish histology slides, as well as protocols to establish accurate anatomic localization of any given tissue cross-section-processes that are made technically difficult by the small size of zebrafish larvae.