The Heterogeneity of the Neurovascular Unit in the Optic Nerve Head During Glaucomatous Neurodegeneration Based on Single‐Cell RNA Sequencing

ABSTRACT Glaucoma is a multifactorial neurodegenerative disease, and the dysfunction of the neurovascular unit (NVU) in optic nerve head (ONH) has been gradually recognized to contribute to the neurodegeneration progression, yet its heterogeneity features remain poorly understood. Here, we established rat models of high intraocular pressure (HIOP) and lipopolysaccharide‐induced ocular inflammation (LPS), and performed single‐cell RNA sequencing (scRNA‐seq) of ONH‐centered tissues to characterize the cellular and molecular characteristics of NVU and its main cell components. Distinct alterations in the cellular composition and intercellular communication of the ONH‐NVU were observed among Ctrl, HIOP and LPS conditions. Astrocytes showed a prominent increase of relative proportion in the HIOP group, whereas microglia were preferentially expanded under LPS stimulation. Astrocytes, microglia and endothelial cells showed a firm interaction via cell–cell communication analysis, in which the interactions within astrocytes and endothelial cells were the highest. Reclustering of main NVU cell types were further identified distinct subpopulations, including Dcn + Myoc + astrocytes, S100a8 + S100a9 + microglia and Nebl + Cytl1 + endothelial cells with prominent heterogeneity features, and their cellular interactions exhibited a prominent integrin‐collagen network in orchestrating ECM restructuring under HIOP conditions. Additional histomorphological analysis further verified the early collapse of ONH astrocytic network and RGC degeneration. Therefore, these findings reveal the distinct ONH‐NVU heterogeneity under HIOP condition, the astrocyte‐mediated ECM remodeling may serve as a key early mechanobiological event for subsequent ONH cupping and glaucomatous neurodegeneration.