An In Vitro Model to Study Drugs That Affect Macrophage Adhesion to Murine Brain Endothelial Cells After Proinflammatory Insults of LPS and Pilocarpine.

Neuroinflammation disrupts blood-brain barrier (BBB) integrity, promoting leukocyte recruitment into the central nervous system and contributing to the progression of neurological disorders. This protocol describes a reproducible macrophage adhesion assay to evaluate interactions between immune cells and brain endothelial cells and to screen compounds with potential anti-inflammatory activity. Murine brain endothelial cells (bEnd.3) were cultured to confluency and exposed to inflammatory stimuli, such as lipopolysaccharide (LPS) or pilocarpine, a cholinergic muscarinic receptor agonist reported to induce inflammatory responses through seizure-associated neuroinflammatory mechanisms, in the presence or absence of candidate therapeutic compounds. In these studies, the natural flavonoid quercetin and the synthetic alkyl-lysophospholipid edelfosine were tested for their effects on macrophage adhesion. After 48 h of treatment, fluorescently labeled murine macrophages (RAW 264.7) were added to the endothelial monolayer, and adherent cells were quantified by fluorescence microscopy. The assay was validated using dexamethasone as an anti-inflammatory control and inflammatory stimulation with LPS or pilocarpine. As expected, dexamethasone reduced macrophage adhesion, whereas both LPS and pilocarpine significantly increased adhesion, demonstrating the assay's sensitivity to changes in endothelial inflammatory status. Overall, this protocol provides a reliable and accessible platform for investigating endothelial-immune cell interactions under neuroinflammatory conditions and for evaluating therapeutic compounds that may preserve BBB function and reduce inflammatory cell recruitment in neurological disease models. Key features • This protocol creates an in vitro model that simulates the luminal interface of the blood-brain barrier (BBB). • The protocol accounts for the 48-h interval required for maximal physiological expression of adhesion molecules at the cell surface. • The protocol quantifies macrophage adhesion to brain endothelial cells following inflammatory stimulation with lipopolysaccharide (LPS) or pilocarpine. • It enables evaluation of therapeutic candidates for their ability to modulate macrophage-brain endothelial cell adhesion under neuroinflammatory conditions.