Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Abstract

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Evans Blue Dye: A Revisit of Its Applications in Biomedicine

“…EB dye is a permeable dye that can be easily taken up by degenerated or damaged cells but not by cells with an intact membrane [ 14 ]. However, interestingly, EB dye and triphenyl tetrazolium chloride (TTC) ex vivo double-staining are usually used together to assess the infarct size in acute myocardial infarction in both canine and murine models, where EB is delivered to the coronary arteries and TTC is applied to stain the surface of cardiac sections [ 47 , 55 – 59 ].…”

“…Many researchers used EB dye to discriminate among injured cells in various animal models, such as the zebrafish models of neuromuscular disease [ 14 ], mouse models of muscular dystrophy [ 90 ] and experimental injury and repair [ 91 ], rat models of stroke and cardiomyocyte injury [ 92 ], myocardial infarction core in a rabbit model [ 32 ] ( Figure 1 ), and reperfused partial liver infarction model in rats [ 33 ] ( Figure 2 ).…”

“…Thus, the dye provides a simple and convenient way of identification both macroscopically and microscopically using fluorescence for both morphological investigation and quantification. The fluorescent feature of EB dye has been used in various aspects including the characterization of the lymph nodes that drain the liver [ 13 ], confirmation of a satisfactory injection into the heart chambers of a zebrafish embryo [ 14 ], and observation of the extravasation of the dye from blood vessels into tissues [ 15 – 17 ].…”

“…More recently, a series of EB derivatives have been labeled with PET isotopes and can be used as theranostics with broad potential applications [ 25 , 26 ]. EB also has a necrosis-avid feature as EB-bound albumin preferentially enters damaged cells, but not healthy cells, and persists within the damaged cells [ 14 ]. Thus, EB can be used as an agent for tissue viability assessment.…”

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy

“…Prior to injections, 2 dpf fish were anesthetized in tricaine solution and lined up on a 3% agarose gel in a Petri dish before being injected into the Duct of Cuvier (DC) with 1.5 nl (~1×10 4 EID 50 ) of wild-type A/PR/8/34 IAV or 4 nl [~6×10 2 plaque forming units (PFU) per embryo] of NS1-GFP A/PR/8/34 IAV in PBS with a final concentration of 0.25% phenol red. For experiments involving EBD, phenol red volume was replaced with EBD 2 , 22 and zebrafish were injected as before. Sterile PBS including phenol red or EBD was injected into the DC of 2 dpf control zebrafish.…”