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The impact of UV cross-linking on corneal stromal cell migration, differentiation and patterning

间质细胞 基质 肌成纤维细胞 基底膜 细胞外基质 共焦 病理 成纤维细胞 共焦显微镜 光折变性角膜切除术 化学 光疗性角膜切除术 体内 角膜 纤维化 解剖 眼科 细胞生物学 医学 生物 体外 光学 免疫组织化学 生物化学 物理 生物技术
作者
W. Matthew Petroll,Miguel Miron-Mendoza,Yukta Sunkara,Hikaru R. Ikebe,Nishith R. Sripathi,Hajar Hassaniardekani
出处
期刊:Experimental Eye Research [Elsevier BV]
卷期号:233: 109523-109523
标识
DOI:10.1016/j.exer.2023.109523
摘要

Previous studies have demonstrated that UV cross-linking (CXL) increases stromal stiffness and produces alterations in extracellular matrix (ECM) microstructure. In order to investigate how CXL impacts both keratocyte differentiation and patterning within the stroma, and fibroblast migration and myofibroblast differentiation on top of the stroma, we combined CXL with superficial phototherapeutic keratectomy (PTK) in a rabbit model. Twenty-six rabbits underwent a 6 mm diameter, 70 μm deep phototherapeutic keratectomy (PTK) with an excimer laser to remove the epithelium and anterior basement membrane. In 14 rabbits, standard CXL was performed in the same eye immediately after PTK. Contralateral eyes served as controls. In vivo confocal microscopy through focusing (CMTF) was used to analyze corneal epithelial and stromal thickness, as well as stromal keratocyte activation and corneal haze. CMTF scans were collected pre-operatively, and from 7 to 120 days after the procedure. A subset of rabbits was sacrificed at each time point, and corneas were fixed and labeled in situ for multiphoton fluorescence microscopy and second harmonic generation imaging. In vivo and in situ imaging demonstrated that haze after PTK was primarily derived from a layer of myofibroblasts that formed on top of the native stroma. Over time, this fibrotic layer was remodeled into more transparent stromal lamellae, and quiescent cells replaced myofibroblasts. Migrating cells within the native stroma underneath the photoablated area were elongated, co-aligned with collagen, and lacked stress fibers. In contrast, following PTK + CXL, haze was derived primarily from highly reflective necrotic "ghost cells" in the anterior stroma, and fibrosis on top of the photoablated stroma was not observed at any time point evaluated. Cells formed clusters as they migrated into the cross-linked stromal tissue and expressed stress fibers; some cells at the edge of the CXL area also expressed α-SM actin, suggesting myofibroblast transformation. Stromal thickness increased significantly between 21 and 90 days after PTK + CXL (P < 0.001) and was over 35 μm higher than baseline at Day 90 (P < 0.05). Overall, these data suggest that cross-linking inhibits interlamellar cell movement, and that these changes lead to a disruption of normal keratocyte patterning and increased activation during stromal repopulation. Interestingly, CXL also prevents PTK-induced fibrosis on top of the stroma, and results in long term increases in stromal thickness in the rabbit model.
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