In vivo immune cell dynamics in the human cornea.

In vivo confocal microscopy (IVCM) allows the evaluation of the living human cornea at the cellular level. The non-invasive nature of this technique longitudinal, repeated examinations of the same tissue over time. Image analysis of two-dimensional time-lapse sequences of presumed immune cells with and without visible dendrites at the corneal sub-basal nerve plexus in the eyes of healthy individuals was performed.

Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear.

Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis.

Unlabelled: RPE65, an abundant membrane-associate protein in the retinal pigment epithelium (RPE), is a key retinoid isomerase of the visual cycle necessary for generating 11-cis-retinal that functions not only as a molecular switch for activating cone and rod visual pigments in response to light stimulation, but also as a chaperone for normal trafficking of cone opsins to the outer segments. Many mutations in RPE65 are associated with Leber congenital amaurosis (LCA). A R91W substitution, the most frequent LCA-associated mutation, results in a severe decrease in protein level and enzymatic activity of RPE65, causing cone opsin mislocalization and early cone degeneration in the mutation knock-in mouse model of LCA.

Functional Rescue of Retinal Degeneration-Associated Mutant RPE65 Proteins.

More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells.

The PEDF Neuroprotective Domain Plus DHA Induces Corneal Nerve Regeneration After Experimental Surgery.

Purpose: To compare a 44-mer pigment epithelial-derived factor (PEDF) peptide with neurotrophic activity, and a 34-mer PEDF with antiangiogenic properties in association with docosahexaenoic acid (DHA) in corneal nerve regeneration after experimental surgery.

Methods: A corneal stromal dissection was performed in rabbits. Treatment groups received topical 44-mer, 34-mer, or full PEDF plus DHA.

Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis.

RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood.

Rescue of enzymatic function for disease-associated RPE65 proteins containing various missense mutations in non-active sites.

Over 70 different missense mutations, including a dominant mutation, in RPE65 retinoid isomerase are associated with distinct forms of retinal degeneration; however, the disease mechanisms for most of these mutations have not been studied. Although some mutations have been shown to abolish enzyme activity, the molecular mechanisms leading to the loss of enzymatic function and retinal degeneration remain poorly understood. Here we show that the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13), a newly identified negative regulator of RPE65, plays a critical role in regulating pathogenicity of three mutations (L22P, T101I, and L408P) by mediating rapid degradation of mutated RPE65s via a ubiquitination- and proteasome-dependent non-lysosomal pathway.

Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration.

Fatty acid transport protein 4 (FATP4) prevents light-induced degeneration of cone and rod photoreceptors by inhibiting RPE65 isomerase.

Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate and photoreceptor susceptibility to light-induced degeneration. Mutations in RPE65 have been linked to blindness in affected children.

Corneal nerve architecture in a donor with unilateral epithelial basement membrane dystrophy.

Background: Epithelial basement membrane dystrophy (EBMD) is by far the most common corneal dystrophy. In this study, we used a newly developed method of immunofluorescence staining and imaging to study the entire corneal nerve architecture of a donor with unilateral EBMD.

Method: Two fresh eyes from a 56-year-old male donor were obtained; the right eye of the donor was diagnosed with EBMD and the left was normal.

EGF stimulates lipoxin A4 synthesis and modulates repair in corneal epithelial cells through ERK and p38 activation.

Purpose: To investigate the effect of epidermal growth factor (EGF) on lipoxin A4 (LXA4) synthesis and how it regulates corneal epithelial wound healing through mitogen-activated kinases, extracellular regulated kinase (ERK) 1/2, and p38.

Methods: Rabbit corneal epithelial (RCE) cells were stimulated with EGF or LXA4 at different times. In some experiments, cells were pretreated with 12/15-lipoxygenase (12/15-LOX) inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), ERK1/2 inhibitor PD98059, or p38 inhibitor SB203580.

Immunofluorescence of rabbit corneas after collagen cross-linking treatment with riboflavin and ultraviolet A.

Purpose: To assess ultrastructural modifications in keratocytes and inflammatory cell response in rabbit corneas after riboflavin and ultraviolet A exposure using immunofluorescence microscopy.

Methods: Twenty adult New Zealand albino rabbits weighing 2.0–3.

Neuroprotectin D1 synthesis and corneal nerve regeneration after experimental surgery and treatment with PEDF plus DHA.

Purpose: This study was conducted to define whether pigment epithelial-derived growth factor (PEDF), together with docosahexaenoic acid (DHA), enhances the synthesis of neuroprotectin D1 (NPD1) and the regeneration of corneal nerves damaged after surgery.

Methods: Corneal stromal dissection was performed in the left eyes of adult New Zealand rabbits treated with DHA+PEDF, PEDF, or DHA for 6 weeks. In vivo confocal images of the corneas were obtained at 2, 4, and 8 weeks, and nerve areas were quantified.

Phase resetting curves allow for simple and accurate prediction of robust N:1 phase locking for strongly coupled neural oscillators.

Existence and stability criteria for harmonic locking modes were derived for two reciprocally pulse coupled oscillators based on their first and second order phase resetting curves. Our theoretical methods are general in the sense that no assumptions about the strength of coupling, type of synaptic coupling, and model are made. These methods were then tested using two reciprocally inhibitory Wang and Buzsáki model neurons.

Epidermal growth factor synergism with TGF-beta1 via PI-3 kinase activity in corneal keratocyte differentiation.

Purpose: To investigate the action of epidermal growth factor (EGF) on corneal keratocyte differentiation and its effects in conjunction with transforming growth factor (TGF)-beta1.

Methods: Rabbit corneal keratocytes (RCKs) were treated with EGF, TGF-beta1, or EGF plus TGF-beta1 in the presence or absence of inhibitors of EGF-receptor (EGF-R), neutralizing concentrations of EGF antibody and of signaling kinases for 2 days to 1 week. RCK differentiation to myofibroblasts was identified with anti-aldehyde dehydrogenase (ALDH)-1 and alpha-smooth muscle actin (alpha-SMA) antibodies.

Association of protein tyrosine phosphatases (PTPs)-1B with c-Met receptor and modulation of corneal epithelial wound healing.

Purpose: The purpose of this study was to investigate the expression and activity of protein tyrosine phosphatases (PTPs) in epithelium during corneal wound healing and to investigate how PTPs regulate activation of the c-Met receptor and the receptor's proximal signaling.

Methods: Rabbit corneas were injured by gentle scraping of the surface, leaving the limbal epithelium intact, and epithelium was collected at 1, 2, 3, and 7 days after injury. In organ culture models, epithelium was removed and corneas were incubated with hepatocyte growth factor (HGF), with or without the PTP inhibitor bpV(phen), and the PI-3K inhibitors wortmannin and LY294002.

Protein kinase C alpha and epsilon differentially modulate hepatocyte growth factor-induced epithelial proliferation and migration.

Protein kinase C (PKC) isoenzymes require membrane translocation for physiological activation. We have recently shown that the growth factors such as epidermal growth factor and hepatocyte growth factor (HGF), but not keratinocyte growth factor (KGF), regulate PKCalpha activation to promote epithelial wound healing [Sharma, G.D.

Synergistic effect of platelet-activating factor and tumor necrosis factor-alpha on corneal myofibroblast apoptosis.

Purpose: Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-alpha in corneal myofibroblast apoptosis was explored.

Methods: Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm2).

Long-term refractive results of myopic LASIK complicated with intraoperative epithelial defects.

Purpose: To evaluate the long-term refractive results of LASIK for myopia complicated with intraoperative epithelial defects.

Methods: Twenty-six eyes with epithelial defects on the LASIK flap were compared with the contralateral eye that had no intraoperative complications. Pre- and postoperative data were compared between the two groups including 3-, 6- and 12-month postoperative spherical equivalent refraction, amount of undercorrection, and complications.

Alkali-induced corneal stromal melting prevention by a novel platelet-activating factor receptor antagonist.

Objective: To evaluate the effect of LAU0901 (2,4,6-trimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid ester), a novel platelet-activating factor (PAF) receptor antagonist, on a rabbit model of severe corneal alkali injury.

Methods: Adult New Zealand albino rabbits were anesthetized and the right eyes were injured with 2N sodium hydroxide for 60 seconds using a 12-mm plastic well, then rinsed. After the injury, 10 rabbits were treated topically with LAU0901 every 2 hours 4 times per day and received a subconjunctival injection of 200 microL of LAU0901 once per week and 10 rabbits were treated with vehicle the same way.

Use of autologous serum in corneal epithelial defects post-lamellar surgery.

Purpose: To evaluate the efficacy of an autologous serum treatment of post-LASIK (laser in situ keratomileusis) corneal epithelial defects in a rabbit model.

Methods: Five milliliters blood samples from 10 New Zealand rabbits were obtained by venepuncture. The serum was aseptically separated and diluted with saline solution to 20%.

Comparison of corneal wound-healing response in photorefractive keratectomy and laser-assisted subepithelial keratectomy.

Purpose: To evaluate in a rabbit model the differences in cellular and matrix stromal response in low and high attempted corrections between photorefractive keratectomy (PRK) and laser-assisted subepithelial keratectomy (LASEK).

Setting: Louisiana State University Health Sciences Center, Department of Ophthalmology and Neuroscience Center, New Orleans, Louisiana, USA.

Methods: Twenty-four eyes of 12 New Zealand albino rabbits were used.

Oxidative stress-induced retinal damage up-regulates DNA polymerase gamma and 8-oxoguanine-DNA-glycosylase in photoreceptor synaptic mitochondria.

Bright light triggers biphasic photoreceptor nuclear DNA fragmentation, suggesting a DNA-repair response (Invest Ophthalmol Vis Sci 43:3511; 2002; Adv Med Biol 533:229-240; Mol Neurobiol 28:111-122). Here, we demonstrate a remarkable increase in expression of the mitochondrial DNA-repair enzymes, DNA polymerase gamma and 8-oxoguanine-DNA-glycosylase, following bright light treatment in rats. DNA polymerase gamma and 8-oxoguanine, the product of guanine oxidation, were selectively localized in photoreceptor synaptic terminals only within the superior central retinal region, where most light damage occurred.

Cellular and molecular events in corneal wound healing: significance of lipid signalling.

Alterations in the normal healing process after corneal injury can produce undesirable outcomes that range from corneal haze to ulceration and perforation. Lipids play important roles in the complex inflammatory processes that occur after corneal wounding. While some lipid mediators, such as the lipoxygenase derivatives of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12[S]-HETE and 15[S]-HETE), act as second messengers to promote cell proliferation and are possibly involved in the synthesis of other molecules that suppress inflammation, others, such as platelet-activating factor (PAF), exert their actions through specific receptors, play key roles during sustained corneal inflammation (as might occur with chemical burns), and contribute to tissue destruction and neovascularization.

PAF-induced furin and MT1-MMP expression is independent of MMP-2 activation in corneal myofibroblasts.

Purpose: Corneal stromal myofibroblasts express the platelet-activating factor (PAF) receptor, but its role is unclear. In the present study, the effect of PAF on induction of metalloproteinases (MMPs) was investigated.

Methods: Rabbit corneal myofibroblasts were identified by immunodetection of alpha-smooth muscle (alpha-SM)-actin.