Time-resolved single-nucleus profiling of inter- and intracellular signaling in optic nerve injury: From the hyperacute phase to the acute phase

Optic nerve injury induces rapid retinal neurodegeneration; however, how distinct retinal cell type responses are coordinated from the hyperacute injury phase to the early repair phase remains incompletely understood. In this study, to explore the dynamic changes in intercellular and intracellular signaling events between different cell types and elucidate their potential roles in retinal ganglion cell survival and early repair, we generated a time-resolved single-nucleus RNA sequencing atlas of adult male mouse retinas across five hyperacute-to-acute timepoints (2 hours, 8 hours, 1 day, 3 days, and 7 days) following optic nerve injury. Using computational network analysis, we reconstructed dynamic cell-to-cell communication and subsequent internal genetic responses among retinal ganglion cells, Müller glia, microglia, and endothelial cells. Distinct stage-specific intercellular communication networks were identified, including transient Itgb1-associated signaling between Müller glia and retinal ganglion cells that peaked at early timepoints, enhanced Nrxn1-Nlgn1 -mediated signaling in endothelial cells during the acute phase, and sustained Sema6a-Plxna4 interactions in microglia through day 7. Functional pathway analysis linked these signaling events to focal adhesion, energy metabolism, immune regulation, and cell adhesion pathways. Multiplex immunofluorescence further validated the temporal dynamics and spatial localization of key signaling molecules, including Itgb1, Nlgn1, and Plxna4, consistent with the transcriptomic findings. Collectively, these results delineate a coordinated hyperacute-to-acute neuro–glial–vascular signaling network that supports retinal ganglion cell survival and identify potential molecular targets for therapeutic intervention following optic nerve injury.