A zebrafish multimodal toolbox to study the blood-brain barrier in health and disease

Vasogenic edema, due to inner blood-retinal barrier (BRB) disruption, is a major cause of vision loss across various ocular diseases. Similarly, blood-brain barrier (BBB) breakdown is a hallmark of many neurological disorders. However, the precise molecular and cellular mechanisms underlying barrier dysfunction under pathological conditions remain poorly understood. In this study, we aimed to develop a pathological model to investigate the integrity of the inner BRB and BBB using in vivo live imaging in zebrafish larvae, employing hyperglycemia-induced leakage as a proof of concept. Our results show that external glucose exposure elevates internal glucose levels, inducing hyperglycemia. Hyperglycemia increased midbrain blood vessel diameter and tracer leakage, indicative of barrier dysfunction, without affecting overall larval survival. Using fluorescent reporters, we found that hyperglycemia concurrently reduced claudin-5 and increased PLVAP, suggesting compromised tight junction integrity and impeded BBB maturation. Preliminary studies using transmission electron microscopy further revealed ultrastructural defects of junctions. However, further optimization is needed to reliably assess differences in caveolae between the experimental groups. This proof of concept study establishes an in vivo protocol for visualizing and quantifying barrier permeability, protein expression, and ultrastructure. It provides a valuable foundation for a zebrafish model to dissect the molecular mechanisms of blood-barrier breakdown, with direct implications for investigations of diabetic macular edema and other neurovascular diseases.