Mapping the Single-Cell Immune Microenvironment in Response to Different Phacoemulsification Incision Strategies

Abstract

Background: Surgical incision architecture during phacoemulsification influences postoperative inflammation, yet the cellular and molecular mechanisms underlying this response remain poorly defined. Emerging single-cell technologies enable detailed characterization of immune activation at unprecedented resolution. Objective: To delineate the incision-dependent immune microenvironment of the anterior segment following phacoemulsification and identify key signaling pathways driving postoperative inflammation. Methods: A murine model of phacoemulsification was established using three incision designs—2.2-mm single-plane, 2.2-mm bi-plane, and 3.0-mm single-plane—alongside a sham control. Aqueous humor and limbal tissues were harvested 24 hours post-surgery for single-cell RNA sequencing and flow cytometry validation. Cell clustering, differential gene expression, and pathway enrichment analyses were performed using Seurat, MAST, and clusterProfiler frameworks. Results: Single-cell profiling revealed a pronounced shift toward neutrophil and monocyte dominance in incision groups compared with sham, with neutrophils exhibiting upregulation of Cxcl2, Il1b, S100a8, and S100a9. Pathway analysis identified activation of IL6–JAK–STAT3 and NF-κB signaling networks, alongside enrichment of TGF-β and VEGF pathways indicative of concurrent inflammatory and reparative processes. Flow cytometry confirmed significant expansion of CD45⁺Ly6G⁺ and CD11b⁺Ly6C⁺ populations. Conclusion: Neutrophil-driven inflammation orchestrated through IL6–JAK–STAT3 and NF-κB axes constitutes the central immune mechanism following phacoemulsification, offering translational targets for surgical optimization and postoperative modulation.