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Purpose: Corneal confocal microscopy is a noninvasive imaging technique to analyze corneal nerve fibers and corneal inflammatory cells (CICs). The amount of CICs is a potential biomarker of disease activity in chronic autoinflammatory diseases. To date, there are no standardized criteria for the morphological characterization of CICs. The aim was to establish a protocol for a standardized morphological classification of CICs based on a literature search and to test this protocol for applicability and reliability., Methods: A systematic review of the literature about definitions of CICs was conducted. Existing morphological descriptions were translated into a structured algorithm and applied by raters. Subsequently, the protocol was optimized by reducing and defining the criteria of the cell types. The optimized algorithm was applied by 4 raters. The interrater reliability was calculated using Fleiss kappa (K)., Results: A systematic review of the literature revealed no uniform morphological criteria for the differentiation of the individual cell types in CICs. Our first protocol achieved only a low level of agreement between 3 raters (K = 0.09; 1062 rated cells). Our revised protocol was able to achieve a higher interrater reliability with 3 (K = 0.64; 471 rated cells) and 4 (K = 0.61; 628 rated cells) raters., Conclusions: The indirect use of criteria from the literature leads to a high error rate. By clearly defining the individual cell types and standardizing the protocol, reproducible results were obtained, allowing the introduction of this protocol for the future evaluation of CICs in the corneal confocal microscopy., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: We aimed to evaluate activation of conjunctiva-associated lymphoid tissue (CALT) in patients with keratitis using in vivo confocal microscopy (IVCM) and conjunctival impression cytology (CIC)., Methods: In addition to anterior segment photography and corneal fluorescein staining, IVCM revealed the palpebral conjunctiva in all subjects, and CIC and immunofluorescence staining were performed., Results: Diffuse lymphoid tissue cell density in the eyes of patients with keratitis was significantly greater compared with healthy volunteers (P < 0.001). Similar trends were found in perifollicular lymphocyte density (P < 0.001), follicular density (P = 0.029), follicular center reflection intensity (P = 0.011), and follicular area (P < 0.001). Immunofluorescence staining showed that the proportions of CD4+ (61.7% ± 8.0% vs. 17.3% ± 10.2%, respectively, P < 0.001) and CD8+ (46.9% ± 10.0% vs. 19.6% ± 11.5%, respectively, P < 0.001) cells in patients with keratitis was greater compared with healthy volunteers. Interestingly, we also observed changes in the contralateral eye in subjects with keratitis., Conclusions: Our research suggests that CALT, as an ocular immune structure, is activated and plays an important role in the pathogenesis of keratitis. This has been overlooked previously. CALT is also active in the contralateral eye of subjects with keratitis.

The constant exposure of ocular surface to external environment and then to several microbial agents is often related to the pathogenesis of various inflammatory eye disorders. In the present study α-Smooth Muscle Actin (α-SMA) and Langerin CD/207 expression and function was investigated in a rabbit corneal keratitis. The inflammation was induced by the secreted form of glycoprotein B (gB1s) of HSV-1, in an ex vivo rabbit corneal model. α-SMA is often used as a marker for myofibroblasts. In this study, for the first time, we show α-SMA positive corneal epithelial cells, during HSV-1 cornea inflammation, demonstrating a crucial role in wound healing, especially during remodeling phase. Furthermore, we show the presence of Dendritic Cells Langerin CD/207 positive, located mainly in the basal epithelial layer and in corneal stroma during the inflammatory processes. Our result validating the ex vivo organotypic rabbit corneal model, for the study about pathogenesis of HSV-1 ocular infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Fungal keratitis (FK) is an eye disease that can lead to blindness and has a high incidence worldwide. At present, there is no effective treatment for this disease. There are innate immune response mechanisms that protect against fungal infections. One example is C-type lectin receptors (CLRs), which can identify fungal invaders and trigger signal transduction pathways and cellular responses to eliminate pathogens. However, previous studies have focused mostly on single-receptor factors, and a systematic analysis of the genetic factors underlying the pathogenesis of FK has not been conducted. This study aimed to investigate the molecular mechanisms of FK in terms of genomics and to further elucidate its pathogenesis., Methods: We performed a transcriptome analysis of a mouse model of FK using RNA sequencing to obtain the relevant gene expression profiles and to identify differentially expressed genes, signaling pathways, and regulatory networks of the key genetic factors in the pathogenesis of murine FK., Results: Several genes that are significantly associated with FK and serve as markers of FK, such as the inflammatory cytokine genes IL1B, IL6, IL10, IL23, and TNF, were identified. The mRNA and protein expression patterns of IL-1β, IL-6, and TNF-α in the corneas of mice with FK were validated by quantitative RT-PCR and Luminex multiplex assay technology. The Wnt, cGMP-PKG, and Hippo signaling pathways were significantly enriched during fungal infection of mouse corneas., Conclusions: Our study may help to elucidate the mechanisms of FK pathogenesis and to identify additional candidate drug targets for the treatment of FK.

Background and Purpose: The purpose of this study is to compare two alternative methods of collecting and transporting media for the diagnosis of corneal ulcers, as not all clinical settings have conventional culture materials and transport media available., Methods: In this open-label, prospective, comparative, and randomized study, patients with clinical suspicion of infectious keratitis with high risk of loss of vision had corneal specimens collected using two methods and transport media: Eswab scraping with Amies transport medium and 23-gauge needle scraping in BACTEC Peds broth. The order of each collection method was randomized. The samples were processed by standard methods, comparing the positivity frequencies for both by parametric and nonparametric tests, according to normality criteria., Results: Corneal infiltrates from 40 eyes of 40 patients were analyzed. Culture positivity rate was 50% for Eswab and 35% for 23-gauge needle (P=0.258). The overall growth rate of the two methods combined was not higher than with the swab alone. The results obtained with a swab were not influenced by the collection sequence (P=0.112); however, the positivity rate was significantly higher when the sample taken with the needle was performed first (P=0.046)., Conclusions: The single sample Eswab method of collection and transportation for the diagnosis of high risk corneal ulcers is a valid alternative and can be used in cases in which, for various reasons, there is no access to the full set of traditional culture materials., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The aim of this study was to elucidate the expression and functions of IL-17 in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. We found that P. aeruginosa infection induced and increased signaling of IL-23/23R/17/17R in mouse corneas. Targeting IL-17A or the IL-17A-specific receptor IL-17RA/IL-17RC with neutralizing Abs resulted in a significant decrease in the severity of P. aeruginosa keratitis, including a decrease in bacterial burden and polymorphonuclear leukocyte infiltration. IL-17A-signaling blockade also significantly reduced the expression of the proinflammatory cytokines L-1β, IL-24, and MMP-13 and increased the expression of the anti-inflammatory cytokine IL-1RA in mouse corneal epithelium. The presence of mouse IL-17A exacerbated P. aeruginosa -mediated tissue destruction. A cytokine protein array revealed that the expression of osteoprotegerin (OPG) was regulated by IL-17A, and OPG neutralization also resulted in a decrease in the severity of P. aeruginosa keratitis. Although both IL-17 and OPG affected the balanced expression of IL-1β and IL-1RA, only IL-17 inhibited the expression of TH2 cytokines. Taken together, our results revealed that IL-17A, along with its downstream factor OPG, plays a detrimental role in the pathogenesis of P. aeruginosa keratitis. Targeting IL-17A and/or the OPG/RANKL/RANK/TRAIL system is a potential therapeutic strategy in controlling the outcome of P. aeruginosa keratitis, which was demonstrated by concurrent topical application of IL-17A-neutralizing Ab and ciprofloxacin in B6 mice., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Purpose: Inflammatory corneal diseases such as bacterial keratitis provoke severe injury to the visual functions and physical structure, leading to opaqueness, wounding, damage to the cornea, and even long-lasting vision loss. Usually antioxidant substances have been of great attention as candidate therapies in the management of keratitis in both humans and animals. Based on the findings, the aim of our research was to examine the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a membrane-permeable free radical scavenger with exclusive antioxidant properties, on in vitro model of eye inflammation of rabbit corneal cells stimulated with lipopolysaccharide (LPS) (Seruminstitute Rabbit Cornea). Methods: The cells were pretreated with Tempol and incubated with LPS for 24 h. LPS stimulation triggered increased cellular mortality, oxidative stress, cytokine levels expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, and also enhanced prostaglandin E 2 (PGE 2 ) levels and cyclooxygenase-2 (COX-2) expression. Results: Pretreatment with Tempol (3 mM) significantly increased cell viability and antioxidant activity as well as decreased reactive oxygen species production, cytokines, PGE 2 levels, and COX-2 expression. Conclusions: Taken together, Tempol could be a new therapeutic strategy for management of ocular inflammatory disorders for clinical and veterinary use.

Objectives : To investigate longitudinally corneal sub-basal nerve plexus (SNP) by means of in vivo confocal microscopy (IVCM) in the contralateral eye (CE) of patients with unilateral neurotrophic keratitis (NK) secondary to central nervous system (CNS) diseases who underwent different treatments. Methods : Ten patients with NK and 10 matched controls were included. In 7 NK patients, conservative treatment maintained unchanged the clinical picture over the 1-year follow-up (Group 1), while NK progressed in 3 patients who underwent direct corneal neurotization (Group 2). IVCM scans of SNP of NK patients were acquired in CE at baseline (V0) ad after 1-year follow-up (V1). All images were analyzed with the automated software "ACCMetrics" and compared with controls. The following IVCM corneal nerve parameters were calculated at V0 and V1 with ACCMetrics: fiber density (CNFD), branch density (CNBD), fiber length (CNFL), total branch density (CTBD), fiber area (CNFA), fiber width (CNFW), and fractal dimension (CNFrD). Results : At V0, significantly lower mean values of CNFD and CNBD, and higher values of CNFW were detected in CE of NK patients compared to controls (respectively, 16.9 ± 8.7 vs 25.0 ± 8.3 n/mm 2 , P = .029; 19.3 ± 13.8 vs 33.8 ± 18.9 n/mm 2 , P = .023; 0.022 ± 0.002 vs 0.020 ± 0.001 mm/mm 2 , P < .001). From V0 to V1, all IVCM metrics of CE remained unchanged in Group 1, while they improved in Group 2. Conclusions : Contralateral eye of patients with unilateral NK secondary to CNS disease showed lower CNFD and CNBD and higher CNFW compared to controls. Unlike conservative treatment, direct corneal neurotization was able to improve SNP metrics also in CE.

Ocular glands play a critical role in eye health through the secretion of factors directly onto the ocular surface. The cornea is a normally transparent tissue necessary for visual acuity located in the anterior segment of the eye. Corneal damage can occur during microbial infection of the cornea, resulting in potentially permanent visual deficits. The involvement of ocular glands during corneal infection has been only briefly described. We hypothesized that ocular glands contribute to resistance as an arm of the eye-associated lymphoid tissue and may also be susceptible to infection secondary to microbial keratitis. Utilizing a mouse model of herpes simplex virus 1 (HSV-1) keratitis, we found that infection of corneas resulted in subsequent infection of ocular glands, including harderian glands (HGs) and extraorbital glands. Similarly, infection of corneas with Pseudomonas aeruginosa resulted in secondary infection of ocular glands. A robust immune response, characterized by increased numbers of immune cells and inflammatory mediators, occurred within ocular glands following HSV-1 keratitis. Removal of HGs altered corneal resistance to HSV-1, as measured by increased viral load, decreased corneal edema, and decreased inflammatory cell infiltration. These novel findings suggest that ocular glands are involved in microbial keratitis through their susceptibility to secondary infection and contribution to corneal resistance. IMPORTANCE Microbial keratitis accounts for up to 700,000 clinical visits annually in the United States. The involvement of ocular glands during microbial keratitis is not readily appreciated, and treatment options do not address the consequences of ocular gland dysfunction. The present study shows that ocular glands are susceptible to direct infection by common ocular pathogens, including HSV-1 and Pseudomonas aeruginosa , subsequent to microbial keratitis. Additionally, ocular glands contribute soluble factors that play a role in corneal resistance to HSV-1 and alter viral load, corneal edema, and immune cell infiltration. Further studies are needed to elucidate the mechanisms by which this occurs., (Copyright © 2019 American Society for Microbiology.)

Kodamaea ohmeri keratitis is an opportunistic pathogen seen in patients who have undergone invasive procedures and immunocompromised state. It has been identified in septicemia patients, resulting in mortality. To the best of our knowledge, we identified the first case of K. ohmeri keratitis following an injury with vegetative material. A 57-year-old woman with underlying, poorly controlled diabetes mellitus was gardening when a tree leaf accidentally poked her in the eye. Two weeks later, the patient presented with right eye pain, redness and progressive blurring of vision due to a traumatised right cornea. Slit-lamp examination showed a small inferior paracentral corneal stromal infiltrate with overlying epithelial defect. A corneal scraping sample yielded K. ohmeri from Analytical Profile Index (API) 20C yeast identification system. She was treated with intensive topical amphotericin B and fluconazole. After 6 weeks of treatment, the keratitis resolved with faint scar tissue, and her visual acuity improved., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described., Case Presentation: A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl-Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott's staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively., Conclusions: This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.

Purpose: To compare the antifungal efficacy of corneal collagen cross-linking with photoactivated riboflavin (PACK-CXL) and voriconazole in experimental Fusarium solani and Candida albicans keratitis models., Methods: Sixty-four corneas of 32 New Zealand rabbits were included and divided into two main groups. Intrastromal injection of Fusarium and Candida suspensions was performed, and it was observed that keratitis was formed on the third day. Both groups were randomly separated into the following four groups: control, PACK-CXL, voriconazole and PACK-CXL combined with voriconazole. PACK-CXL was applied using 0.25% riboflavin in an accelerated Dresden protocol (total ultraviolet A dose 5.4 J/cm²). Voriconazole was applied topically as 7x1/day with a dose of 1% (10 mg/ml). Corneal buttons were excised on the tenth day, and microbiological and pathological examinations were performed., Results: The PACK-CXL and PACK-CXL combined with voriconazole groups each had 100 colony-forming unit (CFU/ml) of reproduced micro-organisms compared with 500 CFU/ml in the voriconazole group and 1500 CFU/ml in the control group (p < 0.001) in the Fusarium keratitis model. The PACK-CXL combined with voriconazole group had 100 CFU/ml, the PACK-CXL group had 150 CFU/ml, and the voriconazole group had 200 CFU/ml of reproduced micro-organisms compared with 4000 CFU/ml in the control group (p < 0.002) in the Candida keratitis model. (p < 0.001). Fewer hyphae and non-specific stromal changes were observed in the pathological cross sections examined in subgroups that used CXL., Conclusion: There was less fungus reproduction and a lower keratitis score for Fusarium solani and Candida albicans in the treatment groups compared to the control groups, especially in groups that used PACK-CXL. These results suggest that it is useful to combine PACK-CXL treatment with medical treatment in the fungal keratitis algorithm at the early stage of the disease., (© 2018 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Purpose: Contact 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., Methods: C57BL/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., Results: Without 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., Conclusions: This 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., (Copyright © 2018. Published by Elsevier Inc.)

Purpose: To evaluate the use of spectral domain anterior segment optical coherence tomography (SD-ASOCT) in fungal keratitis., Methods: Fifty eyes of 50 patients with fungal keratitis were recruited. Serial ASOCT was performed on days 0, 7, 14, 21, 28, 42, and 56. Corneal thickness (CT) in the infiltrate area, infiltrate thickness (IT), and infiltrate width were measured at each follow-up. The presence of any specific feature on ASOCT was evaluated., Results: Mean CT and IT at presentation were 650.5 ± 108 μm and 401.1 ± 91 μm, which reduced significantly at each follow-up [on days 7, 14, 28, and 42; 626.8 ± 113 μm (P < 0.001) and 367.3 ± 94 μm (P = 0.002), 601.4 ± 109 μm and 344.7 ± 94 μm (P < 0.001), 544.8 ± 103 μm and 305.1 ± 80 μm (P < 0.001), and 522.8 ± 97 μm and 291.4 ± 79 μm (P < 0.001), respectively]. The mean CT and scar depth at complete healing were 496.3 ± 101 μm and 283.2 ± 77 μm, respectively. In 10/50 (20%) eyes, the posterior border of the cornea was not clearly visible because of posterior shadowing; therefore, IT was measured along the maximum visible area of hyperreflectivity, whereas CT was measured just adjacent to the area of shadowing. The infiltrate width was measured in 35 eyes, and the mean values at days 0, 7, 14, 28, 42, and 56 were 5.5 ± 0.8 mm, 4.6 ± 0.7 mm, 4.4 ± 0.6 mm, 4.2 ± 0.6 mm, 4.1 ± 0.6 mm, and 4.1 ± 0.6 mm, respectively. A satellite lesion and endothelial plaque were seen in 30% (15/50) and 44% (22/50) eyes, respectively., Conclusions: ASOCT is a useful adjunct in monitoring fungal keratitis especially in cases with deep stromal involvement and endothelial plaques. In addition, it also provides insight into the activity of keratitis.

Purpose: The cornea contains distinct populations of antigen-presenting cells (APCs), including conventional dendritic cells (cDCs). Little is known about the molecular mechanisms involved in cDCs homing and recruitment into the naïve and inflamed cornea. The purpose of this study was to investigate the presence of CXCR4 and its ligand CXCL12 in the murine cornea and its role in cDC migration during corneal inflammation., Methods: The expression of CXCR4 and CXCL12 in naïve and suture-inflamed murine corneas was assessed by whole-mount staining, flow cytometry, and quantitative PCR. The role of CXCR4 in recruitment into inflamed corneas was investigated using adoptive transfer of cDCs blocked with neutralizing antibody against CXCR4., Results: We show the chemokine receptor CXCR4 to be expressed on 51.7% and 64.8% of total corneal CD11c+ cDCs, equating to 98.6 ± 12.5 cells/mm2 in the peripheral and 64.7 ± 10.6 cells/mm2 in the central naïve cornea, respectively. Along with a 4.5-fold increase in CXCL12 expression during inflammation (P < 0.05), infiltrating cDCs also expressed CXCR4 in both the peripheral (222.6 ± 33.3 cells/mm2; P < 0.001) and central cornea (161.9 ± 23.8 cells/mm2; P = 0.001), representing a decrease to 31.0% and 37.3% in the cornea, respectively. Further, ex vivo blockade (390.1 ± 40.1 vs. 612.1 ± 78.3; P = 0.008) and local blockade (263.5 ± 27.1 vs. 807.5 ± 179.5, P < 0.001) with anti-CXCR4 neutralizing antibody resulted in a decrease in cDCs homing into the cornea compared with cells pretreated with isotype controls., Conclusions: Our results demonstrate that corneal CXCL12 plays a direct role in CXCR4+ cDC recruitment into the cornea. The CXCR4/CXCL12 axis is therefore a potential target to modulate corneal inflammatory responses.

The course travelled by corneal epithelial cells from their stem cell niche at the limbus toward the vertex of the cornea is normally not evident due to their transparency, but in certain conditions, the epithelial cells can be rendered visible to the clinician. In such cases, the pathway taken by epithelial cells can manifest as a whorl pattern described using a variety of terms including hurricane keratitis/keratopathy, vortex keratopathy, whorl keratopathy, cornea verticillata, and at times, named after causative agents as exemplified by amiodarone keratopathy. Here, we briefly discuss the terminology used and the spectrum of conditions that can result in keratopathies with whorl patterns in human patients. We review the manifestations of such patterns in veterinary patients and discuss the state of understanding of the underlying forces that create the whorl distribution of epithelial cells on the ocular surface., (© 2018 American College of Veterinary Ophthalmologists.)

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase β (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.

The cornea is a transparent tissue devoid of blood and lymphatic vessels. However, various inflammatory conditions can cause hemangiogenesis and lymphangiogenesis in the cornea, compromising transparency and visual acuity. Mesenchymal stem/stromal cells (MSCs) have therapeutic potentials in a variety of diseases because of anti-inflammatory properties. Herein, we investigated the effects of MSCs on corneal angiogenesis using a model of suture-induced inflammatory corneal neovascularization. Data demonstrated that an intravenous administration of MSCs suppressed corneal inflammation and neovascularization, inhibiting both hemangiogenesis and lymphangiogenesis. MSCs reduced the levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, Tek, MRC1, and MRC2 in the cornea, which are expressed by pro-angiogenic macrophages. Moreover, the number of CD11b + monocytes/macrophages in the cornea, spleen, peripheral blood, and draining lymph nodes was decreased by MSCs. Depletion of circulating CD11b + monocytes by blocking antibodies replicated the effects of MSCs. Importantly, knockdown of tumor necrosis factor alpha (TNF-α)-stimulated gene/protein 6 (TSG-6) in MSCs abrogated the effects of MSCs in inhibiting corneal hemangiogenesis and lymphangiogenesis and monocyte/macrophage infiltration. Together, the results suggest that MSCs inhibit inflammatory neovascularization in the cornea by suppressing pro-angiogenic monocyte/macrophage recruitment in a TSG-6-dependent manner., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus., Methods: Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment., Results: The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 μg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements., Conclusion: This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.

Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional electrophoresis (2DE) with subsequent western blot analysis was performed followed by mass spectrometric identification of bands or spots of interest. Indirect immunofluorescence did not reveal a difference in immune response towards corneal proteins between healthy horses and those with IMMK. Using western blot analysis some horses affected by IMMK (4/28) showed a single band (1D) or a single spot (2DE) (5/28) not detected in healthy controls. The corresponding spot was identified as maspin (SERPINB5), a protein responsible for the inhibition of corneal vascularisation, cell migration and cell adhesion to the extracellular matrix. Tests with a recombinant human protein commercially available did not verify blot findings, but the human protein may not be fully cross-reactive. Still, maspin might play a role in some cases of equine IMMK. Further research is needed to clarify the etiology of this disease., (Copyright © 2017. Published by Elsevier B.V.)

Neurotrophic keratitis (NK) is a rare degenerative disease of the cornea caused by trigeminal nerve damage, which leads to loss of corneal sensitivity, corneal epithelium breakdown, and poor healing. Though extremely uncommon, NK is increasingly recognized for its characteristics as a distinct and well-defined clinical entity rather than a rare complication of various diseases that can disrupt trigeminal innervation. Indeed, the defining feature of NK is loss of corneal sensitivity, and its clinical findings do not correlate with the wide range of systemic or ocular conditions that underlie trigeminal nerve damage. Despite increasing awareness of NK as a distinct condition, its management continues to be challenged by the lack of treatments that target nerve regeneration. This review focuses on the role of corneal nerves in maintaining ocular surface homeostasis, the consequences (such as alterations in neuromediators and corneal cell morphology/function) of impaired innervation, and advances in NK diagnosis and management. Novel therapeutic strategies should aim to improve corneal innervation in order support corneal renewal and healing. J. Cell. Physiol. 232: 717-724, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

This review summarizes the recent literature regarding corneal imaging in human subjects using in vivo confocal microscopy. It also covers the recent literature on corneal immune cells, nerves, and tear cytokine levels in ocular surface diseases as well as corneal immune privilege. The significance of interactions between corneal immune cells and nerves in health, neurotrophic keratopathy, and infectious keratitis is discussed. Furthermore, bilateral alterations of immune cells and nerves in clinically unilateral corneal diseases and the link to changes of tear cytokines or neuropeptide levels in contralateral eyes are described. Recent studies reported increased density and morphologic changes of corneal dendritic cells in ocular surface disease that correlated with a decrease in subbasal nerve and corneal nerve density, suggesting potential interactions between the immune and nervous systems in the cornea. Although the relevance of tear cytokines is poorly understood, tear cytokines might have an important role in the pathogenesis of ocular surface diseases. In humans and experimental animal models, alterations in immune cells, cytokines, and immunomodulatory neuropeptide levels in contralateral eyes might mediate the incidence of bilateral infectious keratitis and loss of immune privilege of the cornea in bilateral corneal transplantation or neurotrophic keratopathy cases. The discovery of bilateral alterations of immune cells and nerves in ocular surface diseases is considered the missing link between the immune and nervous systems in the cornea, and demonstrates how studies of animal models and humans aid our understanding of human corneal disease phenomena.

The small leucine rich repeat proteoglycans are major components of the cornea. Lumican, keratocan, decorin, biglycan and osteoglycin are present throughout the adult corneal stroma, and fibromodulin in the peripheral limbal area. In the cornea literature these proteoglycan have been reviewed as structural, collagen fibril-regulating proteins of the cornea. However, these proteoglycans are members of the leucine-rich-repeat superfamily, and share structural similarities with pathogen recognition toll-like receptors. Emerging studies are showing that these have a range of interactions with cell surface receptors, chemokines, growth factors and pathogen associated molecular patterns and are able to regulate host immune response, inflammation and wound healing. This review discusses what is known about their innate immune-related role directly in the cornea, and studies outside the field that find interesting links with innate immune and wound healing responses that are likely to be relevant to the ocular surface. In addition, the review discusses phenotypes of mice with targeted deletion of proteoglycan genes and genetic variants associated with human pathologies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: To determine the impact of contact lens wear on Langerhans cell density (LCD) in the central cornea over an 8h period., Methods: Ten participants wore a hydrogel lens in one eye (the experimental eye) for 8h. The contralateral non-lens-wearing eye served as a control. The central cornea of each eye was examined at the level of the subbasal nerve plexus using a laser scanning corneal confocal microscope, at baseline (prior to lens wear), then every 2h for 8h., Results: At baseline, LCD was 18±19 and 20±19 cells/mm(2) in the experimental and control eyes, respectively. In the experimental eye, LCD increased to 36±32 cells/mm(2) after 2h and then decreased gradually to 30±31 cells/mm(2) after 6h. LCD was greater in the experimental eye than the control eye at the 2, 4, 6 and 8h time points (p<0.05). LCD remained constant in the control eye throughout the 8h experiment., Conclusions: LCD increases two-fold within the first 2h of lens wear, indicating a rapid, sub-clinical inflammatory response to uncomplicated lens wear., (Copyright © 2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: Clarithromycin is a 14-membered ring macrolide antibiotic with anti-inflammatory as well as antibacterial activity, and has been used worldwide. Moxifloxacin is a leading fourth generation quinolone antibiotic that has been used worldwide perioperatively. We intended to evaluate whether clarithromycin can suppress angiogenesis and inflammation in the cornea, and to compare the anti-inflammatory and anti-angiogenic effects of the two antibiotics, clarithromycin and moxifloxacin., Methods: We made a murine corneal suture model and tested the anti-inflammatory and anti-angiogenic effects of clarithromycin (5 mg/ml) and moxifloxacin (5 mg/ml) in two randomly divided groups. Dexamethasone (5 mg/ml) was used as a positive control. After making two sutures on the cornea, we performed subconjunctival injections (10 μl) on each group on the day of suture, and every day thereafter until the 8th day post-suture. After harvesting corneas on the 8th post-suture day for immunohistochemical staining, we compared neovascularization (NV), lymphangiogenesis (LY) and inflammatory cell infiltration among the groups., Results: Clarithromycin suppressed NV, LY and inflammatory infiltration, compared with phosphate-buffered saline (PBS). However, moxifloxacin did not suppress NV, LY, or inflammatory infiltration, compared with PBS. Comparison between clarithromycin and moxifloxacin, clarithromycin showed a tendency of decreasing LY (p = 0.063) and had less inflammatory cell infiltration (p < 0.05) than did the moxifloxacin group. The anti-(lymph)angiogenic and anti-inflammatory effects of clarithromycin were as high as those of dexamethasone., Conclusion: Clarithromycin suppressed LY and inflammation in the cornea, and its anti-inflammatory effect was significantly superior to that of moxifloxacin.

We evaluated an anti-inflammatory effect of topical administration of tofacitinib, janus kinase (JAK) blocker, on corneal inflammation. Topical instillation of either tofacitinib or PBS was applied after wounding BALB/c mice corneas with alkali burn. Topical instillation was performed until day 14 after injury and injured eye was analyzed. The vascularized area in the alkali burned cornea was significantly reduced in the tofacitinib group compared with that in the PBS group. The immunoreactivity of Gr-1, F4/80, IFN-γ, and phosphorylated STAT(signal transducer and activator of transcription)1 in corneal stroma was diminished significantly in the tofacitinib group. Using laser capture microdissection system and quantitative PCR array analysis, the expression levels of CXCL9, CXCL5, CCL7, CCL2, MMP(matrix metalloproteinase)-9, and STAT1 in corneal stroma were down-regulated in the tofacitinib group. In in vitro study, human fibroblast pretreated by IFN-γ showed phosphorylation of STAT1, and this phosphorylation was down-regulated by adding tofacitinib to the culture medium. These results indicate the topical application of JAK inhibitor causes down-regulation of JAK- or IFN-γ-related molecules. Therefore, we deduce that application of JAK inhibitor for topical instillation may contribute to the treatment of corneal inflammation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Corneal suturing is a surgical procedure used in patients with corneal trauma or transplants. It was reported that endogenous neutrophils are brightly labelled in gene-targeted mice expressing enhanced green fluorescent protein (eGFP) under the control of the endogenous lysozyme M promoter (LysM-eGFP mice)., Methods: We applied intravital imaging methods to analyse in vivo the dynamics of LysM-positive granulocytes (neutrophils) in LysM-eGFP mice with corneal sutures and examined their role in the elicitation of neutrophil infiltration., Results: We found that in the presuturing state, neutrophils strongly positive for LysM were located in the periphery of the corneal stromal layer; none were present in the centre of the cornea. After introducing a corneal suture, neutrophils accumulated in limbal vessels and then migrated to the corneal side and the conjunctival side, suggesting that they derived from limbal vessels. Thereafter they accumulated towards the central corneal area, arriving at the suture about 7 h after its placement. Although corneal sutures may elicit the continuous infiltration of neutrophils, their number was markedly decreased by day 1 after suture removal and continued to decrease thereafter., Conclusions: Our results showed that corneal sutures may elicit the continuous infiltration of neutrophils., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Purpose: The synthesis of cytokines and adhesion molecules by corneal fibroblasts contributes to the innate immune response to corneal infection. The effects of the antimicrobial peptide LL37 on cytokine and adhesion molecule expression induced by bacterial lipopolysaccharide (LPS) in human corneal fibroblasts were examined., Methods: The release of the cytokines IL-6 and IL-8 into culture supernatants and the expression of intercellular adhesion molecule (ICAM)-1 at the cell surface were measured with ELISAs and by flow cytometry. The abundance of mRNAs was quantitated by RT and real-time PCR analysis, and the phosphorylation of signaling proteins was examined by immunoblot analysis. The subcellular localization of ICAM-1 and the transcription factor nuclear factor (NF)-κB was determined by immunofluorescence analysis. Neutrophil infiltration in a mouse model of LPS-induced keratitis was evaluated by immunohistofluorescence analysis., Results: The antimicrobial peptide LL37 inhibited the up-regulation of IL-6, IL-8, and ICAM-1 both at protein and mRNA levels in corneal fibroblasts induced by LPS without affecting those elicited by TNF-α. Furthermore, LL37 attenuated the LPS-induced phosphorylation of the NF-κB inhibitor IκBα and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun NH2-terminal kinase, as well as the translocation of NF-κB to the nucleus in corneal fibroblasts. Lipopolysaccharide-induced keratitis in mice was also suppressed by topical application of LL37., Conclusions: The inhibition of LPS-induced cytokine and adhesion molecule expression in human corneal fibroblasts by LL37 suggests that this peptide might promote the resolution of corneal inflammation associated with bacterial infection.

Purpose: To analyze the contralateral unaffected eyes of patients with microbial keratitis (MK) for any immune cell or nerve changes by laser in vivo confocal microscopy (IVCM)., Methods: A prospective study was performed on 28 patients with MK, including acute bacterial, fungal, and Acanthamoeba keratitis, as well as on their contralateral clinically unaffected eyes and on control groups, which consisted of 28 age-matched normal controls and 15 control contact lens (CL) wearers. Laser IVCM with the Heidelberg Retinal Tomograph 3/Rostock Cornea Module and Cochet-Bonnet esthesiometry of the central cornea were performed. Two masked observers assessed central corneal dendritiform cell density and subbasal corneal nerve parameters., Results: The contralateral clinically unaffected eyes of patients with MK demonstrated significant diminishment in nerve density (15,603.8 ± 1265.2 vs. 24,102.1 ± 735.6 μm/mm²), total number of nerves (11.9 ± 1.0 vs. 24.9 ± 1.2/frame), number of branches (1.7 ± 0.2 vs. 19.9 ± 1.3/frame), and branch nerve length (5775.2 ± 757.1 vs. 12,715.4 ± 648.4 μm/mm²) (P < 0.001 for all parameters) compared to normal controls and CL wearers. Further, dendritiform cell density in the contralateral unaffected eyes was significantly increased as compared to that in controls (117.5 ± 19.9 vs. 24.2 ± 3.5 cells/mm², P < 0.001)., Conclusions: We demonstrate a subclinical involvement in the contralateral clinically unaffected eyes in patients with unilateral acute MK. In vivo confocal microscopy reveals not only a diminishment of the subbasal corneal nerves and sensation, but also an increase in dendritiform cell density in the contralateral unaffected eyes of MK patients. These findings show bilateral immune alterations in a clinically unilateral disease.

Purpose: Microbial biofilms commonly comprise part of the infectious scenario, complicating the therapeutic approach. The purpose of this study was to determine in a mouse model of corneal infection if mature biofilms formed and to visualize the stages of biofilm formation., Methods: A bacterial keratitis model was established using Pseudomonas aeruginosa ATCC 9027 (1 × 10(8) CFU/ml) to infect the cornea of C57BL/6 black mouse. Eyes were examined post-infection (PI) on days 1, 2, 3, 5, and 7, and imaged by slit lamp microscopy, and light, confocal, and electron microscopy to identify the stages of biofilm formation and the time of appearance., Results: On PI day 1, Gram staining showed rod-shaped bacteria adherent on the corneal surface. On PI days 2 and 3, bacteria were seen within webs of extracellular polymeric substance (EPS) and glycocalyx secretion, imaged by confocal microscopy. Scanning electron microscopy demonstrated microcolonies of active infectious cells bound with thick fibrous material. Transmission electron microscopy substantiated the formation of classical biofilm architecture with P. aeruginosa densely packed within the extracellular polymeric substances on PI days 5 and 7., Conclusion: Direct visual evidence showed that biofilms routinely developed on the biotic surface of the mouse cornea. The mouse model can be used to develop new approaches to deal therapeutically with biofilms in corneal infections., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the role of the lipid mediator, resolvin E1 (RvE1), in corneal inflammation., Methods: The effect of RvE1 on stimulated human corneal epithelial cells (HCECs) and neutrophils, and mouse macrophage was assessed. C57BL/6 mouse corneas were abraded and treated with RvE1 either before or after stimulation with lipopolysaccharide (LPS) and antibiotic-killed Pseudomonas aeruginosa and Staphylococcus aureus. The levels of CXC chemokines in the cornea were quantified, and the presence of neutrophils in corneal infiltrates was detected by immunohistochemistry and by in vivo confocal microscopy. The effect of RvE1 on apoptosis in the corneal epithelium was assessed using the TUNEL assay., Results: RvE1 significantly inhibited cytokine production in HCECs and neutrophils, and mouse macrophages and cornea. The development of corneal infiltrates, specifically neutrophils, in response to stimulation with LPS, P. aeruginosa, and S. aureus was also significantly reduced. There was no apoptotic effect of RvE1 on mouse corneal epithelial cells., Conclusions: RvE1 inhibits corneal inflammation induced by LPS, Gram negative (P. aeruginosa) and Gram positive (S. aureus) bacteria. These findings indicate that RvE1 as a potential anti-inflammatory therapy for patients with corneal inflammation and also, when given together with antibiotics, for bacterial keratitis.

Purpose: Chemical burn in cornea may cause permanent visual problem or complete blindness. In the present study, we investigated the role of microRNA 206 (miR-206) in relieving chemical burn in mouse cornea., Method: An alkali burn model was established in C57BL/6 mice to induce chemical corneal injury. Within 72 hours, the transient inflammatory responses in alkali-treated corneas were measured by opacity and corneal neovascularization (CNV) levels, and the gene expression profile of miR-206 was measured by quantitative real-time PCR (qPCR). Inhibitory oligonucleotides of miR-206, miR-206-I, were intrastromally injected into alkali-burned corneas. The possible protective effects of down-regulating miR-206 were assessed by both in vivo measurements of inflammatory responses and in vitro histochemical examinations of corneal epithelium sections. The possible binding of miR-206 on its molecular target, connexin43 (Cx43), was assessed by luciferase reporter (LR) and western blot (WB) assays. Cx43 was silenced by siRNA to examine its effect on regulating miR-206 modulation in alkali-burned cornea., Results: Opacity and CNV levels, along with gene expression of miR-206, were all transiently elevated within 72 hours of alkali-burned mouse cornea. Intrastromal injection of miR-206-I into alkali-burned cornea down-regulated miR-206 and ameliorated inflammatory responses both in vivo and in vitro. LR and WB assays confirmed that Cx43 was directly targeted by miR-206 in mouse cornea. Genetic silencing of Cx43 reversed the protective effect of miR-206 down-regulation in alkali-burned cornea., Conclusion: miR-206, associated with Cx43, is a novel molecular modulator in alkali burn in mouse cornea.

Purpose: HLA-B27 is a major histocompatibility complex class I (MHCI) allele that has been closely associated with the development of ankylosing spondylitis and acute anterior uveitis (AAU), the most common form of uveitis worldwide. We have been characterizing the phenotypes of transgenic mice carrying a human HLA-B27 allele, but that are deficient in endogenous mouse MHCI genes (H-2K(-/-) and H-2D(-/-) double knockout, or DKO) to create the HLA-B27/DKO line. In maintaining and expanding this colony, we observed a rare sporadic severe central keratitis that developed in transgenic animals, but that was not present in wild-type (WT) animals., Methods: The corneas of affected HLA-B27/DKO and DKO mice were compared to their WT counterparts by staining with standard histological methods for markers of inflammation and neovascularization. A model of experimental corneal inflammation was subsequently used to test the responses of each genotype to insult., Results: We identified a previously unreported corneal pathology in naïve HLA-B27/DKO mice, and we describe significantly prolonged CD4(+)- and CD8(+)-associated inflammation in these animals following an experimentally induced corneal injury., Conclusions: These results demonstrate an increased T-cell response in B27/DKO corneas due to the expression of the HLA-B27 allele, suggesting that low MHCI expression in WT corneas is an important contributor to immune privilege.

Purpose: To compare corneal inflammation after syngeneic and allogeneic penetrating keratoplasty (PK) with miniature Keratoprosthesis (m-KPro) implantation in mice., Methods: BALB/C (syngeneic) or C57BL/6 (allogeneic) corneas were transplanted onto BALB/C host beds as part of PK or m-KPro implantation. Corneal inflammation was assessed by determining the frequencies of CD45(+) leukocytes, CD4(+) T cells, CD11b(+) cells, and Gr-1(+) granulocytes/monocytes by flow cytometry at 2, 4, and 8 weeks post transplantation. In addition, expression levels of the proinflammatory cytokines TNF-α and IL-1β were analyzed using real-time qPCR at 8 weeks post transplantation., Results: Cell frequencies in the syngeneic (syn) and allogeneic (allo) m-KPro groups were higher compared with the syngeneic and allogeneic PK groups, respectively, at all time points. However, after week 4, frequencies of all analyzed immune cells were higher in the alloPK group as compared with synKPro group. At 8 weeks, the expression of TNF-α was higher in synKPro, alloPK, and alloKPro groups compared with the naïve and synPK groups. The expression of IL-1β was significantly higher in both KPro groups as compared with PK groups., Conclusions: Although the m-KPro device augments the inflammatory response in the cornea after its implantation, allogenicity (of the carrier tissue) is also a significant contributor to corneal inflammation. These data suggest that using syngeneic or decellularized corneal tissue as a Boston-KPro carrier could reduce the postoperative inflammation response., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Unlabelled: Despite the clinical importance of herpes simplex virus (HSV)-induced ocular disease, the underlying pathophysiology of the disease remains poorly understood, in part due to the lack of adequate virus-natural-host models in which to study the cellular and viral factors involved in acute corneal infection. We developed an air-liquid canine corneal organ culture model and evaluated its susceptibility to canine herpesvirus type 1 (CHV-1) in order to study ocular herpes in a physiologically relevant natural host model. Canine corneas were maintained in culture at an air-liquid interface for up to 25 days, and no degenerative changes were observed in the corneal epithelium during cultivation using histology for morphometric analyses, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, and transmission electron microscopy (TEM). Next, canine corneas were inoculated with CHV-1 for 48 h, and at that time point postinfection, viral plaques could be visualized in the corneal epithelium and viral DNA copies were detected in both the infected corneas and culture supernatants. In addition, we found that canine corneas produced proinflammatory cytokines in response to CHV-1 infection similarly to what has been described for HSV-1. This emphasizes the value of our model as a virus-natural-host model to study ocular herpesvirus infections., Importance: This study is the first to describe the establishment of an air-liquid canine corneal organ culture model as a useful model to study ocular herpesvirus infections. The advantages of this physiologically relevant model include the fact that (i) it provides a system in which ocular herpes can be studied in a virus-natural-host setting and (ii) it reduces the number of experimental animals needed. In addition, this long-term explant culture model may also facilitate research in other fields where noninfectious and infectious ocular diseases of dogs and humans are being studied., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Purpose: We investigated bilateral tear cytokine levels in patients with unilateral bacterial keratitis (BK) as associated with in vivo confocal microscopic (IVCM) alterations in corneal nerves and dendritiform immune cells (DCs)., Methods: A total of 54 (13 BK, 13 contralateral, 28 healthy controls) tear samples was collected prospectively and analyzed by multiplex microbeads assay. The IVCM of the central cornea was performed on the same day, and assessed for corneal nerve and DC alterations., Results: Interleukin-1β, IL-6, and IL-8 were significantly elevated only in affected eyes (66.6 ± 26.8, 7174 ± 2430, and 810 ± 315 ρg/mL, respectively; P = 0.04, P < 0.001, and P < 0.001, respectively), compared to healthy controls (13.0 ± 4.0, 171.8 ± 32.1, and 56.5 ± 33.8 ρg/mL). Levels of chemokine ligand 2 (CCL-2), IL-10, and IL-17a were elevated only in contralateral eyes (813 ± 478, 86.7 ± 38.3, and 3350 ± 881 ρg/mL, respectively; P = 0.02, P = 0.01, and P = 0.04, respectively), compared to controls (73.7 ± 25.3, 17.5 ± 4.9, and 1350 ± 337 ρg/mL). Triggering receptor expressed on myeloid cells (TREM)-1 was significantly elevated in affected (551 ± 231 ρg/mL, P = 0.02) and contralateral unaffected (545 ± 298 ρg/mL, P = 0.03) eyes compared to controls (31.3 ± 12.4 ρg/mL). The density of DCs was significantly increased in affected (226.9 ± 37.3 cells/mm(2), P < 0.001) and unaffected (122.3 ± 23.7 cells/mm(2), P < 0.001) eyes compared to controls (22.7 ± 5.9 cells/mm(2)). Sub-basal nerve density significantly decreased in affected (3337 ± 1615 μm/mm(2), P < 0.001) and contralateral (13,230 ± 1635 μm/mm(2), P < 0.001) eyes compared to controls (21,200 ± 545 μm/mm(2)). Levels of IL-1β, IL-6, and IL-8 were significantly correlated with DC density (R = 0.40, R = 0.55, and R = 0.31, all P < 0.02) and nerve density (R = -0.30, R = -0.53, and R = -0.39, all P < 0.01)., Conclusions: Proinflammatory tear cytokines are elevated bilaterally in patients with unilateral BK, and are correlated strongly with alterations in DCs and nerve density as detected by IVCM., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

We previously showed that topical flagellin induces profound mucosal innate protection in the cornea against microbial infection, a response involving multiple genes and cell types. In this study, we used a Candida albicans (CA)-C57BL/6 mouse keratitis model to delineate the contribution of CXCL10- and CXCR3-expressing cells in flagellin-induced protection. Flagellin pretreatment markedly enhanced CXCL10 expression at 6 h post CA infection (hpi), but significantly dampened CXCL10 expression at 24 hpi. At the cellular level, CXCL10 was expressed in the epithelia at 6 hpi in flagellin-pretreated corneas, and concentrated at lesion sites 24 hpi. CXCR3-expressing cells were detected in great numbers at 24 hpi, organized within clusters at the lesion sites in CA-infected corneas. CXCL10 or CXCR3 neutralization increased keratitis severity and dampened flagellin-induced protection. CXCR3-positive cells were identified as NK cells, the depletion of which resulted in severe CA keratitis. Contributions from NK T-cells were excluded by finding no change in flagellin-induced protection in Rag1 KO mice. Recombinant CXCL10 inhibited CA growth in vitro and accelerated fungal clearance and inflammation resolution in vivo. Taken together, our data indicate that epithelium-expressed CXCL10 plays a critical role in fungal clearance and that CXCR3-expressing NK cells contribute to CA eradication in mouse corneas., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Urokinase-type plasminogen activator (u-PA) plays an important role in corneal wound healing, yet its role in corneal inflammation remains poorly understood. We investigated the role of u-PA in a murine model of lipopolysaccharide (LPS)-induced corneal inflammation., Methods: The corneal epithelium was scraped and LPS was applied to u-PA wild-type (u-PA(+/+)) and u-PA-deficient (u-PA(-/-)) mice. Corneal re-epithelialization and opacity were measured by stereomicroscopy. Fibrin zymography was performed to detect plasminogen activators in corneas from u-PA(+/+) and u-PA(-/-) mice. Neutrophil, macrophage, and u-PA receptor (u-PAR) expression were determined by immunohistochemistry. Gene expression of corneal macrophage chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 was assessed with reverse transcription-polymerase chain reaction. The in vitro effects of endogenous u-PA on MCP-1, MIP-2, matrix metalloproteinase (MMP)-2, and MMP-9 expression, and macrophage migration activity in mouse ocular fibroblasts stimulated by LPS, were examined., Results: The u-PA(+/+) mice showed enhanced corneal inflammation as compared with u-PA(-/-) mice. The u-PA expression was increased by LPS stimulation. Immunohistochemical analyses indicated that more neutrophils and macrophages were present in corneas from u-PA(+/+) mice than u-PA(-/-) mice. The u-PAR expression was detected in inflammatory cells and in the leading edges of the epithelial migrating cells. Enhanced mRNA expression of MCP-1 and MIP-2 was observed in corneas from u-PA(+/+) mice compared to u-PA(-/-) mice. Macrophage chemoattractant protein-1, MIP-2, and MMP-9, but not MMP-2, significantly increased in corneal fibroblasts from u-PA(+/+) mice compared with u-PA(-/-) mice., Conclusions: These data indicate that u-PA promotes LPS-induced leukocyte infiltration in cornea and that u-PA is an important component in LPS-induced corneal inflammatory responses., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To produce in guinea pigs a UV-induced keratitis, to analyze the effects of this pathology on corneal nerve activity., Methods: In anesthetized animals, one eye was exposed to 254 nm UV-C radiation (500-1000 mJ/cm(2)), excised 24 to 48 hours later and superfused in vitro. Nerve impulse activity was recorded in ciliary nerve filaments or in corneal sensory terminals of intact and UV-irradiated eyes. Impulse activity in response to mechanical (von Frey hairs), chemical (98.5% CO2 gas jets), and thermal stimulation (cooling from 34°C to 20°C; heating to 50°C) was analyzed. Duration of eyelid closure and blinking and tearing rates were evaluated in control and in UV-irradiated eyes, before and after application of TRPV1, TRPA1, and TRPM8 agonists (100 μM capsaicin; 10 mM AITC, and 200 μM menthol, respectively)., Results: After irradiation, mechanical threshold of mechano-nociceptor corneo-scleral fibers was reduced (0.59 ± 0.4 vs. 0.27 ± 0.07 mN; P < 0.05) while polymodal nociceptors increased their response to chemical stimulation (1.7 ± 0.2 vs. 3.4 ± 0.5 imps/s; P < 0.05). In contrast, cold thermoreceptors showed a significantly lower ongoing activity at 34°C (8.6 ± 0.5 vs. 6.1 ± 0.9 imp/s; P < 0.05) and a reduced responsiveness to cooling pulses (peak frequency = 29.8 ± 1.3 vs. 18.9 ± 1.8 imp/s; P < 0.001). Blinking but not tearing rate was significantly higher; behavioral responses to topical capsaicin and AITC, but not to menthol were enhanced in UV-irradiated animals., Conclusions: Sensitization of nociceptor and depression of cold thermoreceptor activity following UV radiation appear to result from an action of inflammatory mediators on TRP channels selectively expressed by sensory nerve terminals. Changes in nerve activity possibly underlie discomfort sensations associated with corneo-conjunctival inflammation induced by UV exposure., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To evaluate a novel bioengineered corneal endothelial cell sheet that fits the curvature of the posterior corneal surface., Methods: A spherically curved gelatin hydrogel sheet (SCGS) was prepared by the dehydrothermal cross-linking method, and its permeability to water and protein was tested. Monkey corneal endothelial cells (MCECs) were seeded onto these hydrogel sheets, and the cells were examined by immunohistochemistry. Then MCEC-SCGS constructs were transplanted in monkeys with bullous keratopathy to assess the efficacy of the hydrogel sheets as a scaffold., Results: The hydrogel sheets showed similar permeability to water and protein as that of atelocollagen and vitrigel sheets. After transplantation, the SCGS did not show wrinkling and adhered tightly to the posterior corneal surface, whereas the flat sheets developed wrinkles that inhibited tight adhesion. Monkey corneal endothelial cells grown on hydrogel sheets expressed anti-zonula occludens-1 (ZO-1), N-cadherin, and sodium, potassium, and adenosine triphosphatase (Na,K-ATPase) along the plasma membrane. In a monkey model of bullous keratopathy, transplanted MCEC-SCGS constructs showed good adhesion to the posterior corneal surface, with subsequent improvement of corneal edema and transparency., Conclusions: A novel MCEC-SCGS construct was effective in a monkey model of bullous keratopathy. The SCGS achieves close adhesion to the posterior corneal surface without wrinkling and may contribute to clinical transplantation of corneal endothelial cell sheets.

Dendritic cells (DCs) are highly potent stimulators of the immune system, and their contribution as such to the pathogenesis of corneal and ocular surface inflammatory disease has been well established. These vigorous antigen-presenting cells are reliant upon their effective migration from peripheral tissues (e.g., those of the ocular surface) to the lymphoid organs, where immune responses are triggered and can then cause disease. The chemokine receptor CCR7 expressed on DCs has emerged as the master mediator of this highly complex migratory process, and thus it is important in causing corneal and ocular surface inflammation. Furthermore, CCR7 has received considerable attention as a potential therapeutic target, as topically instilled antagonists of this receptor are quite effective therapeutically in a mouse model of ocular allergy. These findings and more are reviewed in the current article. In addition, the understanding regarding CCR7 function in mice and humans, and the biology of DCs that populate the ocular surface are also detailed herein. The involvement of DCs and their expression of CCR7 in corneal and ocular surface diseases such as in ocular allergy, dry eye disease, immune rejection and more, are also reviewed here., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Free-living amoebae of the Acanthamoeba species are the causative agent of Acanthamoeba keratitis (AK), a sight-threatening corneal infection that causes severe pain and a characteristic ring-shaped corneal infiltrate. Innate immune responses play an important role in resistance against AK. The aim of this study is to determine if Toll-like receptors (TLRs) on corneal epithelial cells are activated by Acanthamoeba, leading to initiation of inflammatory responses in the cornea. Human corneal epithelial (HCE) cells constitutively expressed TLR1, TLR2, TLR3, TLR4, and TLR9 mRNA, and A. castellanii upregulated TLR4 transcription. Expression of TLR1, TLR2, TLR3, and TLR9 was unchanged when HCE cells were exposed to A. castellanii. IL-8 mRNA expression was upregulated in HCE cells exposed to A. castellanii. A. castellanii and lipopolysaccharide (LPS) induced significant IL-8 production by HCE cells as measured by ELISA. The percentage of total cells positive for TLR4 was higher in A. castellanii stimulated HCE cells compared to unstimulated HCE cells. A. castellanii induced upregulation of IL-8 in TLR4 expressing human embryonic kidney (HEK)-293 cells, but not TLR3 expressing HEK-293 cells. TLR4 neutralizing antibody inhibited A. castellanii-induced IL-8 by HCE and HEK-293 cells. Clinical strains but not soil strains of Acanthamoeba activated TLR4 expression in Chinese hamster corneas in vivo and in vitro. Clinical isolates but not soil isolates of Acanthamoeba induced significant (P< 0.05) CXCL2 production in Chinese hamster corneas 3 and 7 days after infection, which coincided with increased inflammatory cells in the corneas. Results suggest that pathogenic species of Acanthamoeba activate TLR4 and induce production of CXCL2 in the Chinese hamster model of AK. TLR4 may be a potential target in the development of novel treatment strategies in Acanthamoeba and other microbial infections that activate TLR4 in corneal cells.

Aim: To evaluate and compare early corneal wound healing and inflammatory responses after small incision lenticule extraction (SMILE) versus femtosecond laser laser in situ keratomileusis (LASIK)., Methods: Thirty-six eyes of 36 rabbits underwent SMILE, while another 36 eyes of 36 rabbits were treated with femtosecond laser LASIK. All the eyes were subjected to the same refractive correction of -6.00 DS/-1.00 DC. Twelve eyes that had no surgery were included for control. After euthanisation, corneal tissue sections were evaluated with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis at postoperative 4 and 24 h, immunocytochemistry for Ki67 to detect keratocyte proliferation at postoperative day 3, week 1 and month 1, and immunocytochemistry for CD11b to detect inflammation at postoperative day 1, day 3 and week 1, respectively., Results: No adverse effects were noted after SMILE or LASIK. Corneal healing postoperatively was uneventful in all cases. There were significantly fewer TUNEL-positive corneal stromal cells after the SMILE procedure at 4 and 24 h postoperatively (p<0.01) compared with the LASIK procedure. In addition, immunocytochemistry showed significantly fewer Ki67-positive cells in the SMILE group than those in the femtosecond laser LASIK group at day 3 and week 1 postoperatively (p<0.05), but there was little expression of Ki67 at month 1 postoperatively in both groups. The CD11b-positive cells were significantly fewer in the SMILE group at day 1, day 3 and week 1 postoperatively (p<0.01)., Conclusions: SMILE induces less keratocyte apoptosis, proliferation and inflammation compared with femtosecond laser LASIK.

Purpose: Pseudomonas aeruginosa is a leading pathogen of blinding keratitis worldwide. In this study, the role of the serine protease in the pathogenesis of P. aeruginosa keratitis in the mouse cornea was investigated by comparing the parent and rescue strains., Methods: Cornea of C57BL/6 mice were infected with P. aeruginosa strain PAO1, serine protease (MucD protease or PA3535) mutants (ΔmucD or ΔPA3535), or a complemented strain. Corneal virulence was evaluated by determining clinical scores and bacterial enumeration. A myeloperoxidase assay was performed to determine the number of polymorphonuclear (PMN) cells infiltrating the cornea. An ELISA was used to quantify inflammatory cytokines and chemokines in the cornea., Results: The clinical score and bacterial numbers in eyes infected with ΔmucD were significantly lower than in those infected with PAO1, ΔPA3535, or the MucD rescue strain after 48 hours (P < 0.001). A larger number of infiltrating PMN cells was observed in eyes infected with ΔmucD at 12 and 24 hours, compared with eyes infected with PAO1. IL-1β, KC, and MIP2 levels were higher in eyes infected with ΔmucD than in those infected with PAO1 after 12 hours., Conclusions: The MucD protease suppressed IL-1β, KC, and MIP2 during the early stages of the infection and inhibited neutrophil recruitment in the cornea. Therefore, the MucD protease contributes significantly to the pathogenesis of keratitis. MucD protease plays a critical role in the establishment of Pseudomonas aeruginosa keratitis by facilitating evasion of the immune response.

Background: Filaggrin is expressed in the epidermis and is essential for the maintenance of the epidermal barrier. Null mutations within the filaggrin gene (FLG) lead to a disturbed epidermal barrier and are associated with a significantly increased risk of atopic dermatitis (AD). The association of AD with ocular surface disorders prompted us to speculate that common FLG mutations may be particularly prevalent in AD patients with ocular comorbidities., Methods: Corneal buttons and biopsies from AD patients with ocular involvement (n = 11) and from non-atopic patients (n = 9) with a histological diagnosis of keratitis were included in the study. DNA samples obtained from paraffin-embedded corneal specimens were genotyped for the two most common FLG mutations (R501X and 2282del4). Filaggrin protein expression was analysed by immunohistochemistry., Results: Normal skin and corneal specimens (n = 6) were positive for filaggrin, which could be detected in the stratum corneum of the skin and in the basal epithelial layer of the cornea. Interestingly, all AD corneal specimens as well as the specimens from keratitis patients without AD were negative for filaggrin expression. Genotyping of the FLG mutations R501X and 2282del4 revealed wild-type alleles in all analysed samples., Conclusions: The lack of filaggrin expression observed in the analysed corneal specimens from AD patients is not due to the two most common FLG mutations (R501X, 2282del4) but is most likely secondary to inflammation, as all keratitis specimens of non-AD patients showed lack of filaggrin expression as well., (© 2013 S. Karger AG, Basel.)

Purpose: Many studies have been conducted to elucidate the molecular consequences of ultraviolet irradiation, whereas little is known about the effect of infrared radiation on ocular disease. Heat is generated as a consequence of infrared irradiation, in addition to photons, and heat shock is widely considered to be an environmental stressor. Our previous study has demonstrated that heat shock (42 °C) induces apoptosis of Statens Seruminstitut Rabbit Cornea (SIRC) cells. However, the biological effect of mild heat shock (39 °C), which does not induce apoptosis, on SIRC cells has not been studied yet., Methods: The SIRC cells were treated with mild heat shock and the expression of inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), in the mRNA and protein levels were then assayed with reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The levels of heat shock protein 90 (HSP90), phosphorylated AKT, phosphorylated IκBα and nuclear NF-κB were assessed with immunoblot assays; the involvement of these molecules in the signaling pathway were further assessed by using specific inhibitor for HSP90, AKT or NF-κB., Results: Mild heat shock increased the production of inflammatory cytokines, IL-1β and IL-6. Mild heat shock increased expression of HSP90, which increased the phosphorylation of AKT. The activated AKT then increased the phosphorylation of IκBα, resulting in nuclear translocation of NF-κB, which up-regulated the expression of IL-1β and IL-6 in SIRC cells., Conclusions: These findings suggest a critical role for the HSP90-AKT-NF-κB signaling pathway in mild heat shock-induced inflammatory response of cornea cells.

Acute ultraviolet (UV) B exposure causes photokeratitis and induces apoptosis in corneal cells. Geranylgeranylacetone (GGA) is an acyclic polyisoprenoid that induces expression of heat shock protein (HSP)70, a soluble intracellular chaperone protein expressed in various tissues, protecting cells against stress conditions. We examined whether induction of HSP70 has therapeutic effects on UV-photokeratitis in mice. C57 BL/6 mice were divided into four groups, GGA-treated (500 mg/kg/mouse) and UVB-exposed (400 mJ/cm2), GGA-untreated UVB-exposed (400 mJ/cm2), GGA-treated (500 mg/kg/mouse) but not exposed and naive controls. Eyeballs were collected 24 h after irradiation, and corneas were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). HSP70, reactive oxygen species (ROS) production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and protein kinase B (Akt) expression were also evaluated. Irradiated corneal epithelium was significantly thicker in the eyes of mice treated with GGA compared with those given the vehicle alone (p < 0.01). Significantly fewer TUNEL-positive cells were observed in the eyes of GGA-treated mice than controls after irradiation (p < 0.01). Corneal HSP70 levels were significantly elevated in corneas of mice treated with GGA (p < 0.05). ROS signal was not affected by GGA. NF-κB activation was reduced but phospho-(Ser/Ther) Akt substrate expression was increased in corneas after irradiation when treated with GGA. GGA-treatment induced HSP70 expression and ameliorated UV-induced corneal damage through the reduced NF-κB activation and possibly increased Akt phosphorilation.

The acute clinical effect of UVR on the eye is photokeratitis, which is an inflammatory state that might be regarded as the sunburn of the eye. In this study, we used a rat model to assess the histological injuries induced in the intact rat cornea following its exposure to UVB radiation. A total of 15 two-months-old female Wistar rats were purchased from the Animal Facility of "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania. The rats were fed ad libitum and kept under standard conditions with a 12 hours light/dark cycle. The rats were randomly divided into five groups, including control group (no UVB exposure), group II (a single exposure to a dose of 45 mJ UVB/cm(2) for 47 seconds), group III (a single exposure to 90 mJ UVB/cm(2) for one minute and 57 seconds), group IV (a single exposure to 180 mJ UVB/cm(2) for three minutes and 57 seconds), and group V (a single exposure to 360 mJ UVB/cm(2)² for five minutes and 26 seconds). After 24 hours of recovery, the rats were sacrificed by cervical dislocation. The rat eyes were extracted, harvested and fixed in 10% buffered formalin. The eye samples were then processed through paraffin technique for further histological examination. We found that, following the UVB exposure, the cornea showed significant inflammatory responses (infiltration of polymorphonuclear leukocytes), hemorrhage and gross damages such as superficial and deep ulcerous keratitis and epithelial exfoliation. The severity of these findings was associated with the increase of UVB radiation intensity and exposure period.

Purpose: To investigate the biofilm-forming capacity of Fusarium solani, Cladosporium sphaerospermum, and Acremonium implicatum, and the activities of antifungal agents against the three keratitis-associated fungi., Methods: The architecture of biofilms was analyzed using scanning electron microscopy and confocal scanning laser microscopy (CSLM). Susceptibility against six antifungal drugs was measured using the CLSI M38-A method and XTT reduction assay., Results: Time course analyses of CSLM revealed that biofilm formation occurred in an organized fashion through four distinct developmental phases: adhesion, germling formation, microcolony formation, and biofilm maturation. Scanning electron microscopy revealed that mature biofilms displayed a complex three-dimensional structure, consisting of coordinated network of hyphal structures glued by the extracellular matrix (ECM). The antifungal susceptibility testing demonstrated a time-dependent decrease in efficacy for all six antifungal agents as the complexity of fungal hyphal structures developed. Natamycin (NAT), amphotericin B (AMB), and NAT were the most effective against F. solani, C. sphaerospermum, and A. implicatum biofilm, respectively., Conclusions: Corneal isolates of F. solani, C. sphaerospermum, and A. implicatum could produce biofilms that were resistant to antifungal agents in vitro.