Abstract
The blood-brain barrier (BBB) is an indispensable, selectively permeable interface that controls the entry and exit of nutrients, ions and waste products into the brain. Despite its biological importance, most measurements of BBB permeability rely on dyes that suffer from nonspecific signals, lack of spatial fidelity, and incompatibility with longitudinal or repeated measurements. Here we present HaloTrace: a method which leverages the HaloTag ligand-receptor tool to generate a precise spatiotemporal readout of BBB integrity that avoids major pitfalls of existing methods. We present evidence that the fluorescent HaloTag ligand has minimal interactions with blood contents but can enter the brain specifically at sites of BBB dysfunction, where it covalently binds to nearby HaloTag receptors. The ligand accumulates in the brain during its short lifetime in circulation and is stably anchored in place for at least 24 hours. Unlike existing tracers, free ligand is not retained in the blood vessels at detectable levels, so the entirety of ligand fluorescence represents true BBB leakage. Furthermore, we demonstrate that HaloTrace can quantify BBB permeability at multiple discrete timepoints prior to the experiment endpoint. This offers researchers the ability to study the progression or resolution of BBB permeability in a way current methods cannot. HaloTrace is thus uniquely poised to characterize the spatiotemporal dynamics of BBB leakage in mouse models.