Browsing by Subject "Corneal Stroma"
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Item Corneal Stromal Remodeling after Photorefractive Keratectomy(2017-01-17) Su, Shan; Kivanany, Pouriska; Grose, Kyle; Petroll, W. MatthewThe cornea is an optically clear tissue that contributes 2/3 of the eye's refractive power, making it a target for vision correction procedures. A small percentage of patients who receive corneal surgery experience loss of corneal transparency (haze). Haze occurs when stromal cells in the cornea (keratocytes) become activated and transform into fibroblasts or myofibroblasts, which can generate contractile forces that may disrupt the collagen architecture. We investigated the wound healing process following photorefractive keratectomy (PRK, a clinical vision correction procedure) using 3-D imaging both in vivo and in situ. We hypothesized that following PRK in rabbits, keratocytes located within the injured stroma, where collagen is intact, would align with the collagen lamellae and will not produce fibrosis. In contrast, we expected keratocytes anterior to the wound, where collagen is disrupted, to undergo myofibroblast transformation and produce significant haze. Twelve New Zealand white rabbits were scanned one week before surgery using an in vivo confocal microscope. PRK was performed on the right eyes of the rabbits (9 diopter spherical photocorrection) with subsequent scans at 7 days, 21 days, 3 months, and 6 months. Custom software was used to build 3-D reconstructions and measure stromal thickness and haze. Some rabbits were sacrificed at each time point to obtain in situ confocal images. Thickness measurements at pre-scan, 7 days, 21 days, 3 months, and 6 months were: 335, 208, 265, 293, 313um. The decrease and subsequent increase in thickness is consistent with removal of tissue for PRK, followed by tissue regeneration. Haze measurements at the same time points were: 1797, 4453, 6906, 3212, 3433 intensity units. The increase and subsequent decrease in backscatter suggests two phases of wound healing. In situ confocal imaging showed that cells within the native stroma were aligned with collagen lamellae without prominent stress fibers at 7 and 21 days, while cells anterior to the injured stroma at 21 days aligned randomly and displayed prominent stress fibers. At 3 months, these cells aligned with correlation to the collagen and did not express stress fibers. At 6 months, the cell/collagen arrangement was similar to uninjured tissue. Our results suggest that the collagen lamellae direct fibroblast patterning during repopulation of the native stroma, without inducing fibrosis or significant haze. In contrast, cells accumulating on top of the stroma initially align randomly and produce hazy, fibrotic tissue. Remarkably, over time, cells remodel the fibrotic tissue to produce a lamellar structure that is similar to the native corneal stroma.Item Multi-Dimensional Imaging of Cell and Extracellular Matrix Interactions During Corneal Wound Healing(2018-04-04) Kivanany, Pouriska Bigvand; Xu, Chet; Petroll, W. Matthew; Eberhart, Robert C.; Mootha, Venkateswara; Schmidtke, DavidCorneal haze is a leading cause of blindness, and may occur after injury, surgery, or chemical burns. The cornea's thickest layer, the stroma, accounts for two-thirds of the eye's refractive power, and contains highly aligned collagen fibrils needed for transparency. Stromal cells, or keratocytes, help maintain the extracellular matrix (ECM). After injury or surgery, keratocytes within the wound undergo apoptosis, while keratocytes in the wound margin transform and activate into fibroblasts and myofibroblasts, leading to contraction, deposition of fibrotic ECM, and haze. Thus, spatial and temporal investigations of cell/ECM interactions are needed for further insight into this healing process. In this research, freeze-injury (FI), lamellar keratectomy (LK), and photorefractive keratectomy (PRK) in vivo rabbit models are used, and cell/ECM interactions are tracked using an in vivo confocal microscope as well as second harmonic generation (SHG) and multiphoton imaging. These in vivo models revealed that in the short term, intrastromal cells migrated in parallel to the collagen lamellae, while cells over the stromal wound bed transformed to myofibroblasts and were randomly arranged. Stromal haze initially increased after injury, but gradually decreased over time. Long term, collagen became more organized, more quiescent cells replaced myofibroblasts, and normal stromal thickness regenerated (after keratectomy) below the ablation surface. Punctate F-actin labeling was detected along cells that co-aligned with native and regenerated collagen, suggesting critical involvement of cell/ECM interactions in stromal remodeling and regeneration. Collagen lamellae appeared to provide a template for fibroblast patterning during both intrastromal migration and stromal regrowth. Overall, cells appear to regenerate and remodel the ECM to produce a lamellar structure similar to the native stroma. These results provide novel insights into the mechanisms of cell/ECM interactions involved in various stages of wound healing in the cornea, and may lead to potential therapies that prevent or reverse fibrosis in the clinic.