Cells Of Cornea Research Articles

Protective effect of lipidure on ocular surface and soft contact lens against desiccation damage

AbstractPurposeThe insertion of a contact lens (CL) disturbs the homeostasis of the tear film, favouring its evaporation and producing friction between the lens and the eyelids and the lens and the ocular surface. These events may induce or aggravate a pre‐existing condition of eye dryness. Lipidure is a biocompatible polymer of 2‐methacryloyloxi‐ethyl‐phosphorylcholine, showing a structure similar to the phosphatidylcholine polar bases; it is endowed with excellent antifouling and moisture retaining abilities. Aim of this study has been to address the protective effects of lipidure, with or without either hyaluronic acid (HA) or hydroxyl‐propyl‐methyl‐cellulose (HPMC), on desiccation of Statens Seruminstitut Rabbit Cornea (SIRC) cells and explanted porcine corneas. Moreover, the ability of lipidure to protect soft CL from desiccation damage and loss of transparency has also been evaluated.MethodsThe protective effect of lipidure was evaluated measuring viability on SIRC cells and explanted porcine corneas leaving them dried at 37°C, 5%CO2 and 95% humidity for 1 h. The ability of lipidure to protect soft CL from desiccation damage and loss of transparency has also been evaluated leaving them air‐dried at 26°C and 46% humidity.ResultsThe protective effect of 0.1% lipidure±HA (0.15%) or HPMC (0.3%), was compared to trehalose (3%), a sugar known in nature to protect cells from dessiccation damage. Results obtained indicate that lipidure either alone or in association with HA or HPMC is able to improve corneal epithelial cells survival under desiccation conditions, both in vitro and ex vivo, similar or even better than threalose alone. Moreover, lipidure in association with HPMC can better preserve morphology and transparency of CL from dehydration.ConclusionsIn conclusion, these studies support the use of lipidure both in artificial eye drops to protect the ocular surface form dryness, and as wetting agents for CL improve their tolerance over the eye surface.

Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes.

Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.

Phenylephrine induces necroptosis and apoptosis in corneal epithelial cells dose- and time-dependently

In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%–1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs.

Understanding cornea homeostasis and wound healing using a novel model of stem cell deficiency in Xenopus

Limbal Stem Cell Deficiency (LSCD) is a painful and debilitating disease that results from damage or loss of the Corneal Epithelial Stem Cells (CESCs). Therapies have been developed to treat LSCD by utilizing epithelial stem cell transplants. However, effective repair and recovery depends on many factors, such as the source and concentration of donor stem cells, and the proper conditions to support these transplanted cells. We do not yet fully understand how CESCs heal wounds or how transplanted CESCs are able to restore transparency in LSCD patients. A major hurdle has been the lack of vertebrate models to study CESCs. Here we utilized a short treatment with Psoralen AMT (a DNA cross-linker), immediately followed by UV treatment (PUV treatment), to establish a novel frog model that recapitulates the characteristics of cornea stem cell deficiency, such as pigment cell invasion from the periphery, corneal opacity, and neovascularization. These PUV treated whole corneas do not regain transparency. Moreover, PUV treatment leads to appearance of the Tcf7l2 labeled subset of apical skin cells in the cornea region. PUV treatment also results in increased cell death, immediately following treatment, with pyknosis as a primary mechanism. Furthermore, we show that PUV treatment causes depletion of p63 expressing basal epithelial cells, and can stimulate mitosis in the remaining cells in the cornea region. To study the response of CESCs, we created localized PUV damage by focusing the UV radiation on one half of the cornea. These cases initially develop localized stem cell deficiency characteristics on the treated side. The localized PUV treatment is also capable of stimulating some mitosis in the untreated (control) half of those corneas. Unlike the whole treated corneas, the treated half is ultimately able to recover and corneal transparency is restored. Our study provides insight into the response of cornea cells following stem cell depletion, and establishes Xenopus as a suitable model for studying CESCs, stem cell deficiency, and other cornea diseases. This model will also be valuable for understanding the nature of transplanted CESCs, which will lead to progress in the development of therapeutics for LSCD.

Corneal Epithelial Dendritic Cell Response as a Putative Marker of Neuro-inflammation in Small Fiber Neuropathy

ABSTRACT We report a case of a 41-year old female with systemic lupus erythematosus and Sjögren’s syndrome, who developed symptoms of painful small fiber neuropathy (SFN). Examination using in vivo confocal microscopy (IVCM) revealed dense accumulations of putative dendritic cells in the central cornea that was postulated to represent a peripheral neuro-inflammatory response. Interventions with higher dose prednisolone, and then intravenous immunoglobulin resulted in substantial, progressive improvements in her symptoms, which were paralleled by cumulative reductions in corneal dendritic cell density (DCD). This case identifies corneal DCD as a potential non-invasive marker of symptomatic SFN due to inflammatory causes.

Loss of ICP22 in HSV-1 Elicits Immune Infiltration and Maintains Stromal Keratitis Despite Reduced Primary and Latent Virus Infectivity.

PurposeWe previously have reported that ICP22, an immediate early gene of herpes simplex virus type 1 (HSV-1), binds to the CD80 promoter to suppress CD80 expression in antigen-presenting cells, leading to reduced T-cell function and protection. In contrast, overexpression of CD80 exacerbates corneal scarring (CS) in ocularly infected mice. In this study we tested the hypothesis that the absence of ICP22 could increase disease severity.MethodsTo test our hypothesis, BALB/c mice were ocularly infected after corneal scarification with a recombinant HSV-1 lacking the ICP22 gene with its parental wild-type (WT) virus (KOS) as a control. Virus replication in the eye, CS, angiogenesis, latency, and reactivation between ICP22 null virus and WT KOS were determined. In addition, expression of IL-2, IL-4, IFN-γ, IFN-α, granzyme A, granzyme B, and perforin by CD4 and CD8 T cells in corneas of infected mice on days 3, 5, 7, 10, 14, 21, and 28 postinfection were determined by flow cytometry.ResultsWe found similar levels of eye disease and angiogenesis in mice following corneal scarification and ocular infection with the ICP22 null virus or parental WT virus despite reduced virus replication in the eye and reduced latency and reactivation in mice ocularly infected with ICP22 null virus. The similar level of eye disease in ICP22 null virus– and WT virus–infected mice correlated with expression of various proinflammatory cytokines that infiltrated the eye after HSV-1 infection.ConclusionsOur study identified a critical role for ICP22 in HSV-1 pathogenicity and suggests that HSV-1–associated CS is more dependent on host immune responses to infection than to virus replication in the eye. Thus, HSV-1 as means of survival uses ICP22 as a mechanism of immune escape that protects the host from increased pathology.

In Vitro Cytotoxicity of Zinc Oxide Nanoparticles in Cultured Statens Seruminstitut Rabbit Cornea Cells

The possibility of eye exposure for workers participating in manufacturing of nanoparticles or consumers using products containing nanoparticles has been reported, but toxicity studies on the eye are scarce. In this study, cytotoxicity of five nanoparticles including silver, ceria, silica, titanium and zinc were tested using Statens Seruminstitut Rabbit Cornea (SIRC) cells. When cells were treated with nanoparticles with concentrations of 1–100 μg/mL for 24 hr, zinc oxide nanoparticles showed higher toxicity to cornea cells. LC50 of zinc oxide nanoparticles was less than 25 μg/mL but those of other nanoparticles could not be calculated in this test, which means more than 100 μg/mL. Generation of reactive oxygen species was observed, and expression of apoptosis related biomarkers including Bax and Bcl-2 were changed after treatment of zinc oxide nanoparticles, while no other significant toxicity-related changes were observed in cornea cells treated with Ag, CeO2, SiO2 and TiO2 nanoparticles.

Combined PD-1 and LAG-3 Blockade Together with HLA-A*0201 Epitopes Immunotherapy Restore the Function of CD8+ TRM Cells and Reduce UV-B Induced Recurrent Ocular Herpes in HSV-1 Infected “Humanized” HLA Transgenic Mice

Abstract Exhaustion of herpes simplex virus type 1 (HSV-1)-specific CD8+ T cells is a notable cause that contribute to the frequency and severity of recurrent herpes. In the present study, we found that functionally exhausted HSV-specific CD8+ T cells, expressing both the programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3), two immune checkpoint receptors that promotes T cell exhaustion, were frequent in symptomatic (SYMP) patients with a history of numerous episodes of recurrent corneal herpetic disease. Similarly, using a novel “humanized” HLA-A*0201 transgenic mouse model of UV-B induced recurrent corneal herpetic disease, we demonstrated that SYMP mice with increased virus shedding and severe recurrent corneal herpetic disease had more exhausted HSV-specific tissue resident PD1+LAG-3+CD8+ TRM cells in both trigeminal ganglia (TG) and cornea. Dual blockade of both PD-1 and LAG-3 pathways restored the number and function of antiviral CD8+ TRM cells and appeared to be synergistically superior to blockade of either pathway alone. Moreover, blockade of PD-1/LAG-3 immune checkpoints in combination with therapeutic vaccination with a mixture of HLA-A*0201-restricted CD8+ T cell epitopes UL9196–204, UL25572–580, and UL44400–408, recently identified from HSV-1 proteins, significantly improved protection against UV-B induced recurrent herpes. This pre-clinical study indicate that dual blockade of Immune checkpoints combined with therapeutic vaccination with human epitopes identified from HSV-1 proteins is a promising strategy to protection against recurrent herpes in human.

Role of the IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to Aspergillus fumigatus infection.

To investigate the expression of interleukin (IL)-33 in the cornea and human corneal epithelial cells (HCECs) exposed to Aspergillus fumigatus (A. fumigatus), and to determine the function of IL-33/ST2/p38 signaling pathway in the immune response of corneal epithelial cells to A. fumigatus infection. The mRNA and protein expression of IL-33 in HCECs and mice corneas were examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. IL-33 expression was also detected in cornea samples from healthy donors and patients with fungal keratitis with immunohistochemistry. The cultured HCECs were treated with inactive A. fumigatus hyphae at various concentrations with or without recombinant human IL-33 protein, soluble recombinant ST2 protein, specific ST2 neutralizing antibody, or the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 for evaluation of the expression and activation of IL-33/ST2/p38 signaling in the regulation of proinflammatory cytokines. The production levels of IL-6 and IL-1β were determined by qRT-PCR and enzyme-linked immunosorbent assay (ELISA). The proliferation of HCECs was determined by a Cell Counting Kit-8 (CCK8) assay and cell count. IL-33 expression levels increased in the corneal tissues of patients with fungal keratitis and in mice corneas of experimental A. fumigatus infection, as well as in HCECs with infection of A. fumigatus. A. fumigatus strongly stimulated HCECs-generated proinflammatory cytokine (IL-6 and IL-1β) production at both the mRNA and protein levels. This production of pro-inflammatory mediators stimulated by A. fumigatus was further stimulated by IL-33 and was prevented by soluble ST2 protein or ST2 neutralizing antibody. Moreover, IL-33 naturally promoted the p38 phosphorylation induced by A. fumigatus, which was suppressed by soluble ST2 protein. The MAPK p38 inhibitor SB203580 also inhibited the A. fumigatus-induced proinflammatory cytokine production. IL-33 administration for 48h and 72h promoted the proliferation of HCECs, which was attenuated by treatment with soluble recombinant human ST2 protein. A. fumigatus elevates IL-33 expression in human and mice corneas and HCECs. Thus, IL-33/ST2/p38 signaling may play an important role in amplifying the immune response of corneal epithelial cells to A. fumigatus infection. Besides, IL-33 promotes the cell proliferation of HCECs via its receptor ST2. These findings suggest a novel autocrine mechanism of amplification of the fungal-induced inflammatory response in the corneal epithelium, highlighting a potential therapeutic target for fungal keratitis.

The Protective Effect of Blue Light Cutting Particles Mixed Optical Material against Blue LED Light-Induced Photoreceptor and Cornea Cell Death

The Protective Effect of Blue Light Cutting Particles Mixed Optical Material against Blue LED Light-Induced Photoreceptor and Cornea Cell Death

Results of laser confocal microscopy of the cornea in viral uveitis (a preliminary report)

To evaluate the results of laser corneal confocal microscopy (CCM) in viral uveitis of varying localization. The main study group included 23 patients (23 eyes) diagnosed with unilateral herpesviral uveitis (chorioretinitis), the patients' age varied from 18 to 79 years. The control group comprised 19 healthy volunteers (38 eyes) aged 20 to 75 years. In addition, the paired eyes of the main group patients were examined. In all patients, standard ophthalmologic examination was complemented with CCM performed on the HRT III device with a corneal module, followed by analysis of the course and structure of corneal nerve fibers (CNF) using copyrighted software Liner 1.2. The preliminary results achieved in this study outline the prospects for further research on the state of cornea (in particular, changes in the course and structure of CNF, and the presence of dendritiform cells of Langerhans) with laser CCM in patients with uveitis of various etiologies. These morphological changes also has potential use as diagnostic markers of inflammation of the uveal tract. The main criteria for assessing the state of cornea in viral uveitis include the following: increased tortuosity of CNF, increase in the number and size of Langerhans cells. Further research - in particular, studying the integrated use of diagnostic methods necessary for the verification of viral uveitis, as well as detailed analysis of the history and clinical picture of the disease - is required to substantiate the inclusion of laser confocal microscopy method in the algorithm for the diagnosis of viral uveitis.

"Basal Cell Migration" in Regeneration of the Corneal Wound-Bed.

In this issue of Stem Cell Reports, Park et al. (2019) describe real-time in vivo visual monitoring of keratin-14+, Confetti-labeled limbal epithelial stem cells and their progeny as they contribute to central corneal wound-healing. The authors show that corneal wounds initially heal by “basal epithelial cell migration” into the wound-bed.

Blockade of LAG-3 Immune Checkpoint Combined With Therapeutic Vaccination Restore the Function of Tissue-Resident Anti-viral CD8+ T Cells and Protect Against Recurrent Ocular Herpes Simplex Infection and Disease.

Recurrent viral diseases often occur after the viruses evade the hosts' immune system, by inducing exhaustion of antiviral T cells. In the present study, we found that functionally exhausted herpes simplex virus type 1 (HSV-1) -specific CD8+ T cells, with elevated expression of lymphocyte activation gene-3 (LAG-3), an immune checkpoint receptor that promotes T cell exhaustion, were frequent in symptomatic (SYMP) patients with a history of numerous episodes of recurrent corneal herpetic disease. Similarly, following UV-B induced virus reactivation from latency the symptomatic wild-type (WT) B6 mice that developed increase virus shedding and severe recurrent corneal herpetic disease had more exhausted HSV-specific LAG-3+CD8+ T cells in both trigeminal ganglia (TG) and cornea. Moreover, a therapeutic blockade of LAG-3 immune checkpoint with antagonist antibodies combined with a therapeutic immunization with gB498−505 peptide immunodominant epitope of latently infected B6 mice significantly restored the quality and quantity of functional HSV-1 gB498−505 specific CD8+ T cells in both TG and cornea and protected against UV-B induced recurrent corneal herpes infection and disease. In contrast to dysfunctional HSV-specific CD8+ T cells from WT B6 mice, more functional HSV-specific CD8+ T cells were detected in LAG-3−/− deficient mice and were associated with less UV-B induced recurrent corneal herpetic disease. Thus, the LAG-3 pathway plays a fundamental role in ocular herpes T cell immunopathology and provides an important immune checkpoint target that can synergizes with T cell-based therapeutic vaccines against symptomatic recurrent ocular herpes.

Method for selective quantification of immune and inflammatory cells in the cornea using flow cytometry.

The cornea serves as a protective surface against the environment (i.e., allergens, pollutants, desiccation and microorganisms) and promotes vision, made possible by corneal transparency. This protocol describes corneal preparation for flow cytometry to assess cells localized in the cornea. Our model details the process, from determining how many corneas are needed in the experiment to corneal excision to digestion and staining of the cornea cells. The simplicity of the model allows for systematic analysis of different corneal mechanisms of immunity, inflammation, angiogenesis and wound healing. In corneal transplantation, residential immune and inflammatory cells are key to the mechanisms that underlie angiogenesis, opacity, and graft rejection. In addition, this model can also elucidate cellular mechanisms mediating corneal graft outcomes and wound healing. Lastly, this model can be used to analyze the efficacy of new medications such as instillation and subconjunctival injections and assess the potential of therapeutic molecules to enhance graft survival and wound healing in vivo.

A novel murine model for contact lens wear reveals clandestine IL-1R dependent corneal parainflammation and susceptibility to microbial keratitis upon inoculation with Pseudomonas aeruginosa

PurposeContact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. MethodsC57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. ResultsWithout deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. ConclusionsThis murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

Antiviral Activity of Geopropolis Extract from Scaptotrigona Aff. Postica against Rubella Virus

The search for functional foods, which possess bioactive substances, is a new trend for the obtention of alternative and more effective treatments of many diseases with fewer side effects. Geopropolis, elaborated by stingless bees, is a mixture of plant resin sources, wax and soil. In the geopropolis from Scaptotrigona affinis postica (Latreille, 1807), (Hymenoptera, Apidae, Meliponini) was not observed the presence of soil. In a previous study, the extract of geopropolis provided by the beekeeper, from S. postica of Barra do Corda, Maranhão State, exhibited potent antiviral activity against herpes simplex virus. In this study, the propolis extract was prepared experimentally and characterized by RP-HPLC-DAD-ESI-MS/MS. The objective of this study was to evaluate the antiviral activity of an experimentally prepared geopropolis extract from S. postica against Rubella Virus infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Rubella virus infection of susceptible women during the first trimester of pregnancy, often results in a combination of birth defects in newborns. There is not an effective treatment for rubella virus infection. Different protocols were carried out to evaluate, the antiviral effect of geopropolis extract on the viral replication of infectious RV. Cell viability and cell proliferation assays indicated that this geopropolis was not toxic to cultured SIRC cells. In the viral binding assay, antiviral assay, real-time PCR, and transmission electron microscopy, was observed that different concentrations of geopropolis (17, 34 and 68 µg/mL) was able to inhibit the binding of virions to the cell receptor and the production of infectious RV particles in post treated and pre treated infected SIRC cells. The antiviral activity could to be attributed to the high contents of the apigenin derivatives, vicenin-2 and schaftoside. As far as we know, this is the first report about the antiviral activity of geopropolis from Scaptotrigona postica against a Togaviridae virus.

Optically robust, highly permeable and elastic protein films that support dual cornea cell types

Damaged corneas can lead to blindness. Due to the worldwide shortage of donor corneas there is a tremendous unmet demand for a robust corneal replacement that supports growth of the major corneal cell types. Commercial artificial corneas comprise plastic polymers that do not adequately support diverse cell growth. We present a new class of protein elastomer-dominated synthetic corneas with attractive performance that intimately couple biologically active tropoelastin to mechanically robust and durable protein silk. Fabricated films substantially replicate the natural cornea physically and by interacting with both key cells types used in cornea repair. Performance encompasses optical clarity at high transmittance, compatible refractive index, substantial glucose permeability, compliant mechanical properties, and support of both growth and function of corneal epithelial and endothelial cells.

Epistasis between Pax6Sey and genetic background reinforces the value of defined hybrid mouse models for therapeutic trials

The small eye (Sey) mouse is a model of PAX6-aniridia syndrome (aniridia). Aniridia, a congenital ocular disorder caused by heterozygous loss-of-function mutations in PAX6, needs new vision saving therapies. However, high phenotypic variability in Sey mice makes development of such therapies challenging. We hypothesize that genetic background is a major source of undesirable variability in Sey mice. Here we performed a systematic quantitative examination of anatomical, histological, and molecular phenotypes on the inbred C57BL/6J, hybrid B6129F1, and inbred 129S1/SvImJ backgrounds. The Sey allele significantly reduced eye weight, corneal thickness, PAX6 mRNA and protein levels, and elevated blood glucose levels. Surprisingly, Pax6Sey/Sey brains had significantly elevated Pax6 transcripts compared to Pax6+/+ embryos. Genetic background significantly influenced 12/24 measurements, with inbred strains introducing severe ocular and blood sugar phenotypes not observed in hybrid mice. Additionally, significant interactions (epistasis) between Pax6 genotype and genetic background were detected in measurements of eye weight, cornea epithelial thickness and cell count, retinal mRNA levels, and blood glucose levels. The number of epistatic interactions was reduced in hybrid mice. In conclusion, severe phenotypes in the unnatural inbred strains reinforce the value of more naturalistic F1 hybrid mice for the development of therapies for aniridia and other disorders.

A Novel Murine Model of Radiation Keratopathy.

PurposeRadiation therapy results in severe chronic keratopathy and dry eye disease. We developed a novel mouse model for radiation keratopathy to allow future mechanistic studies.MethodsSix to 8-week-old BALB/c mice underwent sublethal irradiation to the head only from a Cesium-137 irradiator, 2 × 550 rad, 3-hours apart. Irradiated mice were clinically evaluated by corneal fluorescein staining (CFS) at 1, 2, and 3 months, after which corneas were excised and immunofluorescence histochemistry performed with anti-CD45, anti-MHC class II, and anti-β-tubulin antibodies.ResultsThe survival rate after irradiation was 100%. Mice demonstrated significant CFS and hair loss around the eyes. Corneal nerve density decreased in the central and peripheral corneas (P < 0.01) at 2 and 3 months, respectively. CD45+ immune cell densities increased in the central and peripheral corneas (P < 0.005, P < 0.001) at 2 and 3 months, respectively. MHC class II, a sign of antigen presenting cell activation, significantly increased after irradiation in the central and peripheral corneas at 2 and 3 months (P = 0.02). A strong inverse correlation was noted between decreased corneal nerves and increase in CD45+ cells in the central cornea at 2 (P = 0.04, r = −0.89) and 3 months (P = 0.03, r = −0.91) after irradiation.ConclusionsWe present a model of radiation keratopathy and demonstrate significant nerve loss and increase in immune cell influx and activation within months. This model will enable future investigations to understand the effects of radiation therapy on the eye, and to study mechanisms of neuro-immune crosstalk in the cornea.

Rho Kinase Type 1 (ROCK1) Promotes Lipopolysaccharide-induced Inflammation in Corneal Epithelial Cells by Activating Toll-Like Receptor 4 (TLR4)-Mediated Signaling.

BackgroundRho kinases (ROCKs) are the typical downstream effectors of RhoA, which can regulate corneal epithelial wound healing. In this study, the role of ROCK1 in lipopolysaccharide (LPS)-induced cornea inflammation was investigated.Material/MethodsThe expression of ROCK1 in human corneal epithelial cells (HCECs) was bilaterally modulated with ROCK1 expression vector and ROCK1 inhibitor Y-27632. The effects of ROCK1 modulation on the inflammatory response, cell viability, cell apoptosis, and cell cycle distribution were detected by ELISA assay, MTT assay, and flow cytometry, respectively. The pathways involved in the effect of ROCK1 in HCECs was preliminarily explained by detecting changes of TLR4-mediated NF-κB and ERK signaling using western blotting and electrophoretic mobility shift assays.ResultsOverexpression of ROCK1 promoted LPS-induced production of IL-6, IL-8, IL-1β, and TNF-α, and the apoptotic process in HCECs. Augmented inflammation and apoptosis were associated with stronger activation of TLR4-mediated signal transduction; the phosphorylation of IκBα, JNK, ERK1/2, and p38, and nuclear translocation of NF-κB p65 induced by LPS were further increased by overexpression of ROCK1. Inhibition of ROCK1 function by Y-27632 blocked the effect of LPS on HCECs; both LPS-induced inflammation and apoptosis was alleviated by Y-27632, which was associated with suppression of TLR4-mediated NF-κB and ERK signaling.ConclusionsLPS-induced inflammation and apoptosis in HCECs depended on the function of ROCK1, inhibition of which would attenuate impairments on cornea cells due to LPS.