Supplementary Materials2. vivo confocal microscopy at 3, 7, 21 and 60 days after injury. A subset of animals was sacrificed at each time point to further investigate cell and matrix patterning. Tissue was fixed and labeled in situ with Alexa Fluor 488 phalloidin (for F-actin), and imaged using multiphoton fluorescence and second harmonic generation (SHG) imaging (for collagen). Immediately following LK, cell death occurred in the corneal stroma directly beneath the injury. At 7 and 21 LCL-161 enzyme inhibitor days after the LCL-161 enzyme inhibitor methods, analysis of fluorescence (F-actin) and SHG results (collagen) indicated that fibroblasts were co-aligned with the collagen lamellae within this region. In contrast, stromal cells accumulating on top of the stromal wound bed were randomly arranged, contained more prominent stress fibers, and indicated alpha smooth muscle mass actin (-SMA) and fibronectin. At 60 days, cells and matrix in this region experienced become co-aligned into lamellar-like constructions; cells were elongated but did not express stress materials. Corneal haze measured using in vivo confocal microscopy peaked at 21 days after LK, and was significantly reduced by 60 days. Cell morphology and patterning observed in vivo was similar to that observed in situ. Our results suggest that the topography and alignment of the collagen lamellae direct fibroblast patterning during repopulation of the native stroma after LK injury in the rabbit. In contrast, CTSL1 stromal cells accumulating on top of the stromal wound bed initially align randomly and produce a fibrotic ECM. Remarkably, over time, these cells appear to remodel the ECM to produce a lamellar structure that is similar to the native corneal stroma. strong class=”kwd-title” Keywords: Confocal microscopy, Corneal Wound Healing, Extracellular Matrix, SHG Imaging 1. Introduction Stromal keratocytes play a central role in mediating the corneal response to injury or refractive surgery (Netto et al., 2005). During wound healing, quiescent corneal keratocytes surrounding the area of injury generally become activated, proliferate, and transform into a fibroblastic phenotype (Jester et al., 1999c; Stramer et al., 2003). In certain wound types, fibroblasts further differentiate into myofibroblasts, which generate stronger forces and synthesize a disorganized fibrotic extracellular matrix (ECM) (Blalock et al., 2003; Jester et al., 1999a). Following vision correction procedures such as photorefractive keratectomy (PRK) or laser assisted in situ keratomileusis (LASIK), cellular force generation and fibrosis can alter corneal shape and reduce corneal transparency. In addition, a decrease in the concentration of keratocyte-specific corneal crystallin proteins has been associated with an increase in cellular light scattering during wound healing, which also contributes to clinical haze (Jester et al., 2012; Jester et al., 1999b). Both PRK and LASIK result in a region of keratocyte death beneath LCL-161 enzyme inhibitor the laser-treated area (Mohan et al., 2000; M?ller-Pedersen et al., 1998; Wilson, 2002). Stromal cell death can also be induced by toxic injury (Jester et al., 1998; Maurer et al., 1997) as well as UV cross-linking of the cornea in keratoconus patients (Knappe et al., 2011; Mencucci et al., 2010; Wollensak et al., 2004). Ideally, repopulation of damaged stromal tissue following these insults should occur via intra-stromal migration of keratocytes from the surrounding stromal cells, without era of contractile makes that could disrupt the collagen structures or the creation of fibrotic ECM that may reduce transparency. Earlier work shows that myofibroblast change of corneal keratocytes during wound curing can be mediated by changing growth element beta (TGF-) in conjunction with other growth elements; (Chen et al., 2009; Etheredge et al., 2009; Funderburgh et al., 2001; Jester et al., 1999a; Jester et al., 2002; Jester et al., 1995; Jester et al., 1999c) nevertheless, less is LCL-161 enzyme inhibitor well known on the subject of the biochemical and biophysical indicators that regulate cell and matrix patterning during wound recovery We recently found in vivo confocal microscopy to assess keratocyte backscattering, positioning, connection and morphology during intra-stromal wound recovery, carrying out a full-thickness corneal freeze damage (FI) in the rabbit (Petroll et al., 2015). We also correlated these results with en bloc 3-D confocal fluorescence imaging of mobile patterning, and second harmonic era (SHG) imaging from the corneal collagen lamellae. Oddly enough, we discovered that keratocyte LCL-161 enzyme inhibitor positioning during wound repopulation was correlated with the structural corporation from the lamellae extremely, suggesting contact assistance of intra-stromal cell migration. Pursuing FI, the epithelial cellar membrane remains intact, and stromal healing involves fibroblast migration into the injured tissue, without myofibroblast transformation, fibrosis or matrix remodeling. In contrast, healing following keratectomy wounds in the rabbit has three phases: stromal repopulation (migration), fibrosis, and regeneration and/or remodeling. Specifically, using in vivo confocal microscopy, Jester and coworkers demonstrated that following PRK, corneal fibroblasts migrated into the wounded stromal tissue by 7 days after injury, without transforming into myofibroblasts (Moller-Pedersen et al., 1998). By 21 days, significant sub-epithelial haze, myofibroblast transformation and associated fibrosis.