title: A Rapid Screenable Assay for Compounds That Protect Against Intestinal Injury in Zebrafish Larva.
abstract: 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.
authors.affiliation: Department of Internal Medicine, University of Michigan, 1500 E Medical Center Drive, Ann Arbor, 48109, MI, USA.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Goldsmith
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27713, USA.|Division of Gastroenterology, Department of Medicine, College of Medicine, University of Florida, CGRC, 2033 Mowry Rd, Office 461, P.O. Box 103633, 32610, Gainesville, FL, 32611-0882, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Tomkovich
authors.affiliation: Division of Gastroenterology, Department of Medicine, College of Medicine, University of Florida, CGRC, 2033 Mowry Rd, Office 461, P.O. Box 103633, 32610, Gainesville, FL, 32611-0882, USA. Christian.Jobin@medicine.ufl.edu.
authors.forename: Christian
authors.initials: C
authors.lastname: Jobin
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D005897:Glafenine / Q000633:toxicity; D007410:Intestinal Diseases / Q000139:chemically induced / Q000517:prevention & control; D007422:Intestines / Q000187:drug effects* / Q000293:injuries*; D007814:Larva; D020011:Protective Agents / Q000494:pharmacology*; D015027:Zebrafish / Q000254:growth & development*
chemical_list: D020011:Protective Agents; D005897:Glafenine
keywords: Drug screen; ER stress; Intestinal injury; NSAIDs; UPR; Zebrafish