Measurement of Blood-Brain Barrier Disruption in Mice Following Ozone Exposure Using Highly Sensitive Radiotracer Assays.

Ozone is a widespread air toxicant. Although its primary target organ is the lungs, emerging evidence suggests that ozone also has harmful effects on the brain. The vascular blood-brain barrier (BBB), an endothelial interface that regulates passage of substances between the brain and peripheral tissues, is a likely mediator of ozone's adverse effects on the brain. Ozone can cause BBB disruption, a pathological state in which the BBB becomes leaky, resulting in the unregulated entry of circulating substances into the brain. BBB disruption can be detected using many methods, which each have their strengths and limitations. Recent data suggest that BBB disruption can occur in mice following ozone exposures, albeit at a low level. Therefore, robust and highly sensitive assays for BBB disruption are needed. Assays commonly used to detect BBB disruption, however, can be time consuming, lack sensitivity, and can be vulnerable to artifacts that are typically not addressed in the experimental design. Radiochemical assays are among the most sensitive and specific for detecting subtle disruptions of the BBB and require minimal sample processing for detection. Radiochemical assays can also be multiplexed to include radiotracer conjugates of large and small molecular weights, and the uptake of each of them can provide information about the severity and mechanism of BBB disruption. Here, we describe a protocol to use two of these radiotracer conjugates, 14 C-sucrose and 99m Tc- albumin, to measure BBB disruption following an acute exposure to ozone in mice. We provide the steps to expose mice acutely to ozone, to label albumin with 99m Tc-pertechnetate, and to measure BBB disruption by evaluating permeability to 99m Tc-albumin and 14 C-sucrose after ozone exposure. These methods can be adapted to different ozone exposure paradigms and to different rodent species/strains, allowing for the sensitive and rapid assessment of BBB disruption that is detectable in whole brains or in brain regions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Ozone exposures in mice Basic Protocol 2: Measurement of blood-brain barrier disruption by evaluating permeability to 14 C-sucrose and 99m Tc-albumin Support Protocol: Labeling of bovine serum albumin with 99m Tc.