AMPAR dysregulation in microglia drives vascular pathology in diabetic retinopathy via the P2X7R/NLRP3/IL-1β pathway

Diabetic retinopathy (DR) is a leading cause of visual impairment in working-age adults globally, characterized by chronic retinal inflammation and inner blood-retinal barrier (iBRB) disruption. Glutamate excitotoxicity and microglial activation are key pathogenic contributors, but the molecular links from these events to vascular damage remain unclear—particularly the role of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), whose subunit composition (GluR1–4) regulates calcium permeability. Human vitreous humor and retinal tissues, streptozotocin-induced DR mice, high glucose (HG)-stimulated BV2 cells, mouse primary retinal microglia, and bEnd.3 endothelial cells were used. Glutamate levels, AMPAR subunit expression, microglial activation, calcium homeostasis, iBRB integrity and the potential mechanism were assessed via biochemical assays, immunofluorescence, transcriptomics, calcium imaging, Evans blue assays, western blot, and ELISA. Interventions included the AMPAR antagonists (perampanel and NASPM), and an IL-1β neutralizing antibody. Elevated glutamate levels were observed in the vitreous of DR patients, diabetic mouse retinas, and HG-treated BV2 cells. Critically, a consistent AMPAR subunit composition change (increased GluR1, decreased GluR2) was confirmed in human diabetic retinas, diabetic mouse retinas, HG-treated BV2 cells, and critically, in primary retinal microglia. This subunit change promoted the formation of calcium-permeable AMPARs, triggering downstream events including calcium overload, activation of the ATP/P2X7R/NLRP3 inflammasome pathway, and subsequent upregulation of IL-1β production. The direct link between AMPAR subunit remodeling, elevated intracellular calcium, and increased IL-1β was further substantiated in primary retinal microglia. Ultimately, this cascade impaired iBRB in vivo and enhanced pro-angiogenic responses in endothelial cells in vitro. Notably, both AMPAR inhibition and IL-1β neutralization effectively reversed these pathological changes. Our findings implicate microglial AMPAR subunit remodeling—favoring Ca²⁺-permeable configuration—as an early trigger of neurovascular inflammation in DR. Targeting the glutamate–AMPAR–P2X7R–IL‑1β cascade may offer a rational strategy to preserve iBRB integrity.