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

Background

Neuroinflammation is a central pathological process in the progression of neurodegenerative diseases (NDs), including epilepsy, Alzheimer’s disease (AD), Parkinson’s disease (PD), and others [1–3]. These disorders are characterized by chronic activation of the innate immune system within the central nervous system (CNS), involving microglia, astrocytes, and the recruitment of peripheral immune cells [3–5]. While acute inflammatory responses may initially serve protective functions by promoting debris clearance and tissue repair, persistent and dysregulated neuroinflammation contributes to progressive neuronal damage and synaptic dysfunction [6,7]. Blood–brain barrier endothelial cells (BECs) are also disrupted during neuroinflammation through signaling from activated microglia, astrocytes, and infiltrating immune cells [8–10]. Proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, together with reactive oxygen species (ROS), impair endothelial function by disrupting tight junctions, increasing transcellular transport, and promoting enzymatic degradation of the barrier [11–14]. These changes compromise BBB integrity and facilitate additional immune cell infiltration into the central nervous system [13]. These inflammatory signals, together with matrix metalloproteinases (MMPs), compromise the integrity of the blood–brain barrier (BBB) by disrupting tight junction proteins such as occludins and claudins, leading to increased vascular permeability [15,16]. BBB dysfunction further exacerbates neuroinflammation by allowing the infiltration of circulating monocytes and lymphocytes into the brain parenchyma, creating a feed-forward inflammatory cycle [3]. Such immune cell infiltration has been reported across multiple neurodegenerative conditions. In AD, BBB disruption often precedes significant cognitive decline and facilitates the entry of peripheral immune cells and circulating Aβ into the brain [2,6,17]. In temporal lobe epilepsy (TLE), the most prevalent form of epilepsy, BBB disruption is a hallmark pathological feature that contributes to the maintenance of chronic seizures and promotes neuroinflammatory processes [18,19]. BBB dysfunction is often persistent and has been associated with increased seizure frequency, disease severity, and resistance to antiseizure medications [20]. Although infiltrating macrophages can facilitate the clearance of cellular debris and support tissue repair, they also release proinflammatory mediators that may exacerbate neuronal damage and perpetuate neuroinflammation [21,22]. Consequently, therapeutic strategies aimed at preserving BBB integrity and modulating neuroinflammatory responses—including inhibition of matrix metalloproteinase (MMP) activity, suppression of proinflammatory cytokine production, and promotion of reparative immune phenotypes—have emerged as promising approaches to limit disease progression and improve neurological outcomes [23].

Anti-inflammatory compounds that modulate endothelial activation have emerged as promising strategies to protect BBB function [24]. Both natural and synthetic molecules may regulate inflammatory signaling and immune cell interactions at the BBB. For example, a synthetic compound, edelfosine, which targets phospholipase Cβ signaling [25,26], has shown anti-inflammatory effects in BBB endothelial cells exposed to seizure-inducing stimuli such as pilocarpine [27]. Quercetin is a natural anti-inflammatory molecule that has recently been shown to promote neuroprotection by preserving BBB integrity, reducing inflammation and oxidative stress, preventing neuronal death, and enhancing synaptic function [28]. In this context, the present study investigates whether the natural flavonoid quercetin and the synthetic lysophospholipid edelfosine can attenuate macrophage adhesion under proinflammatory conditions.

Macrophage–endothelial adhesion assays are widely used to investigate leukocyte recruitment during inflammation and to evaluate the anti-inflammatory potential of therapeutic compounds. Common approaches include static adhesion assays, flow-based systems, live-cell imaging, and transwell migration assays, each providing complementary information on leukocyte–endothelial interactions [29–31]. Static assays remain the most widely used because they are simple, reproducible, and suitable for quantitative drug screening, although they do not reproduce physiological shear stress or leukocyte rolling. Despite these limitations, static assays remain the preferred approach for initial drug screening because they provide highly reproducible, quantitative measurements under controlled experimental conditions, enabling rapid evaluation of multiple therapeutic candidates before validation in more physiologically complex models. In contrast, flow-based assays better mimic the vascular microenvironment but require specialized equipment and have lower throughput. Confocal imaging provides high-resolution visualization of adhesion dynamics, whereas transwell assays combine adhesion with transmigration but are less suited for quantitative screening [29,32].