Measuring blood-brain barrier dysfunction: a critical appraisal of fluid biomarkers, in vitro models, in vivo imaging, and post-mortem approaches

The blood-brain barrier (BBB) maintains central nervous system homeostasis by regulating molecular exchange between blood and brain. BBB dysfunction is associated with aging and neurological disorders such as Alzheimer's disease, stroke, and multiple sclerosis. Diverse approaches are used to study BBB structure and function, including cell-based models, imaging techniques, and fluid biomarkers. While each method has distinct strengths, inherent limitations complicate interpretation and limit comparability across studies. In addition, many methods require specialized expertise, hindering the interdisciplinary integration of findings. This review outlines commonly used methods to assess BBB dysfunction and critically evaluates their relevance, advantages, and drawbacks. It provides guidance for selecting suitable techniques, proposes guidelines, and highlights key challenges in data interpretation. Finally, the review emphasizes the need to clearly define the specific BBB aspect under investigation, calls for standardized protocols, and encourages combining approaches to improve research quality and translation into clinically meaningful insights and applications.