Effect of a protein kinase B (Akt) inhibitor on the angiogenesis of HUVECs and corneal neovascularization.

BACKGROUND: Corneal neovascularization (CNV) is a vision-threatening disease and an increasing public health concern. It was found that administering an Akt inhibitor in the second phase of retinopathy significantly decreased retinal neovascularization. METHODS: This study investigated the effect of an Akt inhibitor on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and its impacts on the degree of CNV and corneal opacity in a rat keratoplasty model. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays, tube formation assays, cell scratch experiments, and a fully allogeneic corneal transplant model were performed. RESULTS: It was found that an Akt inhibitor inhibited the proliferation, angiogenesis, and migration of HUVECs induced by vascular endothelial growth factor (VEGF). The results showed that both CNV and corneal opacity were decreased in rats after Akt inhibitor administration. CONCLUSION: The research illustrates the vital role of Akt inhibitors in mediating CNV. The analysis shows that the Akt inhibitor may provide a novel and feasible therapeutic approach to prevent CNV, but its mechanism needs further investigation.

Topical Losartan Decreases Myofibroblast Generation But Not Corneal Opacity After Surface Blast-Simulating Irregular PTK in Rabbits.

Purpose: To evaluate the efficacy of topical losartan after blast injury-simulating irregular phototherapeutic keratectomy (PTK) in rabbits. Methods: Twelve NZW rabbits underwent 100 pulse 6.5 mm diameter PTK over a metal screen to generate severe surface irregularity and inhibit epithelial basement membrane regeneration. Corneas were treated with 0.8 mg/mL losartan in balanced salt solution (BSS) or BSS 50 µL six times per day for six weeks after PTK. All corneas had slit lamp photography, with and without 1% fluorescein at two, four, and six weeks after PTK, and were analyzed using immunohistochemistry for the myofibroblast marker α-smooth muscle actin (α-SMA), keratocyte marker keratocan, mesenchymal cell marker vimentin, transforming growth factor (TGF)-ß1, and collagen type IV. Results: Topical 0.8 mg/mL losartan six times a day significantly decreased anterior stromal α-SMA intensity units compared to BSS at six weeks after anterior stromal irregularity-inducing screened PTK (P = 0.009). Central corneal opacity, however, was not significantly different between the two groups. Keratocan, vimentin, TGF-ß1, or collagen type IV levels in the anterior stroma were not significantly different between the two groups. Conclusions: Topical losartan effectively decreased myofibroblast generation after surface blast simulation irregular PTK. However, these results suggest initial masking-smoothing PTK, along with adjuvant topical losartan therapy, may be needed to decrease corneal stromal opacity after traumatic injuries that produce severe surface irregularity. Translational Relevance: Topical losartan decreased scar-producing stromal myofibroblasts after irregular PTK over a metal screen but early smoothing of irregularity would also likely be needed to significantly decrease corneal opacity.

Lipoxin A4 (LXA4) Reduces Alkali-Induced Corneal Inflammation and Neovascularization and Upregulates a Repair Transcriptome.

PURPOSE: To investigate the anti-inflammatory and anti-angiogenic effects of the bioactive lipid mediator LXA4 on a rat model of severe corneal alkali injury. METHODS: To induce a corneal alkali injury in the right eyes of anesthetized Sprague Dawley rats. They were injured with a Φ 4 mm filter paper disc soaked in 1 N NaOH placed on the center of the cornea. After injury, the rats were treated topically with LXA4 (65 ng/20 µL) or vehicle three times a day for 14 days. Corneal opacity, neovascularization (NV), and hyphema were recorded and evaluated in a blind manner. Pro-inflammatory cytokine expression and genes involved in cornel repair were assayed by RNA sequencing and capillary Western blot. Cornea cell infiltration and monocytes isolated from the blood were analyzed by immunofluorescence and by flow cytometry. RESULTS: Topical treatment with LXA4 for two weeks significantly reduced corneal opacity, NV, and hyphema compared to the vehicle treatment. RNA-seq and Western blot results showed that LXA4 decreased the gene and protein expression of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 and pro-angiogenic mediators matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGFA). It also induces genes involved in keratinization and ErbB signaling and downregulates immune pathways to stimulate wound healing. Flow cytometry and immunohistochemistry showed significantly less infiltration of neutrophils in the corneas treated with LXA4 compared to vehicle treatment. It also revealed that LXA4 treatment increases the proportion of type 2 macrophages (M2) compared to M1 in blood-isolated monocytes. CONCLUSIONS: LXA4 decreases corneal inflammation and NV induced by a strong alkali burn. Its mechanism of action includes inhibition of inflammatory leukocyte infiltration, reduction in cytokine release, suppression of angiogenic factors, and promotion of corneal repair gene expression and macrophage polarization in blood from alkali burn corneas. LXA4 has potential as a therapeutic candidate for severe corneal chemical injuries.

Acute central corneal haze and thinning with atypical keratitis presentation.

A 24-year-old woman presented with a 7-day history of blurry vision, redness, and extreme pain in her right eye. She had no pertinent medical or ocular history and did not use spectacles or contacts. Uncorrected distance visual acuity (UDVA) was 20/40 in the right eye and could not be improved with refraction. Slitlamp examination revealed a 1.5 × 1.5 mm central epithelial defect with surrounding white blood cell recruitment. Confocal microscopy (Figure 1JOURNAL/jcrs/04.03/02158034-202304000-00020/figure1/v/2023-03-24T200747Z/r/image-tiff) was performed, and she was treated with chlorhexidine 0.02% drops every hour in the right eye. 2 weeks later, the cornea had completely re-epithelialized; however, persistent corneal haze, decreased visual acuity, and corneal thinning and flattening was noted. Pachymetry was 484 µm in the right eye and UDVA was 20/40 (Supplemental Figure 1, available at http://links.lww.com/JRS/A836). In the following 2 weeks, UDVA improved to 20/25. 6 months after the initial presentation, UDVA was unchanged and faint central corneal haze was noted on examination (Figure 2JOURNAL/jcrs/04.03/02158034-202304000-00020/figure2/v/2023-03-24T200747Z/r/image-tiff). Of interest, her family history is significant for her younger 16-year-old brother with 3 prior episodes of a similar type of keratitis/keratopathy over the course of 2 years in both eyes with similar central paracentral corneal haze, thinning, and flattening and similar confocal findings (Figure 3JOURNAL/jcrs/04.03/02158034-202304000-00020/figure3/v/2023-03-24T200747Z/r/image-tiff). He also was unresponsive to topical antibiotics and antivirals except topical chlorhexidine. Her brother has been our patient for the last several years prior to her first visit to our clinic. What is your diagnosis? What medical diagnostic tests, if any, would you recommend? Is this an infectious or simply an inflammatory response? Is there any genetic or familial predisposition?

Suppression of lipopolysaccharide-induced corneal opacity by hepatocyte growth factor.

Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity.

Pediatric Use of Recombinant Human Nerve Growth Factor 20 µg/mL Eye Drops (Cenegermin) for Bilateral Neurotrophic Keratopathy in Congenital Corneal Anesthesia.

PURPOSE: This study aimed to present the efficacy and safety of cenegermin eye drop (Oxervate; Dompè Farmaceutici, Milan, Italy) treatment in a pediatric patient affected by neurotrophic keratopathy (NK) with Goldenhar syndrome. METHODS: This case reports an infant presenting ulceration and a small central opacity in the cornea of the right and left eyes, respectively. The NK bilaterally worsened despite the use of therapeutic contact lenses and temporary partial tarsorrhaphy. Magnetic resonance imaging showed absence and hypoplasia of the right and left trigeminal nerves, respectively. Cenegermin eye drops were administered 1 drop/each eye, 6 times daily for 8 weeks to promote corneal healing. RESULTS: Complete healing was achieved in both eyes after treatment. During the 16-month follow-up period, no epithelial defect, recurrence, or complications were noticed, whereas corneal opacities progressively became clearer, although insignificant improvements in corneal sensitivity or in the reflex tearing were observed. CONCLUSIONS: Cenegermin was effective in treating NK in an infant with Goldenhar syndrome.

Cucurbita argyrosperma Seed Extracts Attenuate Angiogenesis in a Corneal Chemical Burn Model.

Severe corneal inflammation produces opacity or even perforation, scarring, and angiogenesis, resulting in blindness. In this study, we used the cornea to examine the effect of new anti-angiogenic chemopreventive agents. We researched the anti-angiogenic effect of two extracts, methanol (Met) and hexane (Hex), from the seed of Cucurbita argyrosperma, on inflamed corneas. The corneas of Wistar rats were alkali-injured and treated intragastrically for seven successive days. We evaluated: opacity score, corneal neovascularization (CNV) area, re-epithelialization percentage, and histological changes. Also, we assessed the inflammatory (cyclooxigenase-2, nuclear factor-kappaB, and interleukin-1ß) and angiogenic (vascular endothelial growth factor A, VEGF-A; -receptor 1, VEGFR1; and -receptor 2, VEGFR2) markers. Levels of Cox-2, Il-1ß, and Vegf-a mRNA were also determined. After treatment, we observed a reduction in corneal edema, with lower opacity scores and cell infiltration compared to untreated rats. Treatment also accelerated wound healing and decreased the CNV area. The staining of inflammatory and angiogenic factors was significantly decreased and related to a down-expression of Cox-2, Il-1ß, and Vegf. These results suggest that intake of C. argyrosperma seed has the potential to attenuate the angiogenesis secondary to inflammation in corneal chemical damage.

Nanostructured lipid carriers containing rapamycin for prevention of corneal fibroblasts proliferation and haze propagation after burn injuries: In vitro and in vivo.

Chemical burns are a major cause of corneal haze and blindness. Corticosteroids are commonly used after corneal burns to attenuate the severity of the inflammation-related fibrosis. While research efforts have been aimed toward application of novel therapeutics. In the current study, a novel drug delivery system based nanostructured lipid carriers (NLCs) were designed to treat corneal alkaline burn injury. Rapamycin, a potent inhibitor of mammalian target of rapamycin pathway, was loaded in NLCs (rapa-NLCs), and the NLCs were characterized. Cell viability assay, cellular uptake of NLCs, and in vitro evaluation of the fibrotic/angiogenic genes suppression by rapa-NLCs were carried out on human isolated corneal fibroblasts. Immunohistochemistry (IHC) assays were also performed after treatment of murine model of corneal alkaline burn with rapa-NLCs. According to the results, rapamycin was efficiently loaded in NLCs. NLCs could enhance coumarin-6 fibroblast uptake by 1.5 times. Rapa-NLCs efficiently downregulated platelet-derived growth factor and transforming growth factor beta genes in vitro. Furthermore, proliferation of fibroblasts, a major cause of corneal haze after injury, reduced. IHC staining of treated cornea with alpha-smooth muscle actin and CD34 + antibodies showed efficient prevention of myofibroblasts differentiation and angiogenesis, respectively. In conclusion, ocular delivery of rapamycin using NLCs after corneal injury may be considered as a promising antifibrotic/angiogenic treatment approach to preserve patient eyesight.

Cyclodextrin Enhances Corneal Tolerability and Reduces Ocular Toxicity Caused by Diclofenac.

With advances in refractive surgery and demand for cataract removal and lens replacement, the ocular use of nonsteroidal anti-inflammatory drugs (NSAIDs) has increased. One of the most commonly used NSAIDs is diclofenac (Diclo). In this study, cyclodextrins (CDs), α-, ß-, γ-, and HP-ß-CDs, were investigated with in vitro irritation and in vivo ulceration models in rabbits to reduce Diclo toxicity. Diclo-, α-, ß-, γ-, and HP-ß-CD inclusion complexes were prepared and characterized and Diclo-CD complexes were evaluated for corneal permeation, red blood cell (RBCs) haemolysis, corneal opacity/permeability, and toxicity. Guest- (Diclo-) host (CD) solid inclusion complexes were formed only with ß-, γ-, and HP-ß-CDs. Amphipathic properties for Diclo were recorded and this surfactant-like functionality might contribute to the unwanted effects of Diclo on the surface of the eye. Contact angle and spreading coefficients were used to assess Diclo-CDs in solution. Reduction of ocular toxicity 3-fold to16-fold and comparable corneal permeability to free Diclo were recorded only with Diclo-γ-CD and Diclo-HP-ß-CD complexes. These two complexes showed faster healing rates without scar formation compared with exposure to the Diclo solution and to untreated groups. This study also highlighted that Diclo-γ-CD and Diclo-HP-ß-CD demonstrated fast healing without scar formation.

Los reptiles en la historia de la oftalmología española: excrementos de lagarto ocelado y leucoma corneal / Reptiles in the history of Spanish ophthalmology: Ocellated lizard excrements and corneal leucoma

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Prevalence of Trachoma in Car-Nicobar Island, India after Three Annual Rounds of Mass Drug Administration with Azithromycin.

BACKGROUND: A high proportion of active trachoma infection in children of Car-Nicobar Island was reported through the Trachoma Rapid Assessment survey conducted in year 2010 by the same researchers. Annual mass drug treatment with azithromycin was administered from years 2010-12 to all individuals residing in this island for reducing the burden of active trachoma infection. A cross-sectional prevalence survey was conducted in the year 2013 to assess the post-treatment burden of trachoma in this population. METHODS: In the 15 randomly selected compact segments from each village of the island, children aged 1-9 years were examined for evidence of active trachoma infection and participants aged ten years and above were examined for trachomatous trichiasis and corneal opacity. RESULTS: A total of 809 children (1-9 years) and 2735 adults were examined. Coverage with azithromycin for all the three rounds was more than 80%. The prevalence of active trachoma infection in children aged 1-9 years old was 6.8% (95% CI 5.1, 8.5) and Trachomatous Trichiasis (TT) was 3.9% (95% CI 3.2, 4.6). The risk factors associated with active trachoma infection were older age and unclean faces. The risk factors associated with TT were older age and lower literacy level. CONCLUSION: Trachoma has not been eliminated from Car-Nicobar Island in accordance to 'Global Elimination of Trachoma, 2020' guidelines. Sustained efforts and continuous surveillance admixed with adequate programmatic response is imperative for elimination of trachoma in the island.

Angiogenin ameliorates corneal opacity and neovascularization via regulating immune response in corneal fibroblasts.

BACKGROUND: Angiogenin (ANG), a component of tears, is involved in the innate immune system and is related with inflammatory disease. We investigated whether ANG has an immune modulatory function in human corneal fibroblasts (HCFs). METHODS: HCFs were cultured from excised corneal tissues. The gene or protein expression levels of interleukin (IL)-1beta (ß), IL-4, IL-6, IL-8, IL-10, complements, toll-like receptor (TLR)4, myeloid differentiation primary response gene (MYD)88, TANK-binding kinase (TBK)1, IkappaB kinase-epsilon (IKK-ε) and nuclear factor-kappaB (NF-κB) were analyzed with or without ANG treatment in tumor necrosis factor-alpha (TNF-α)- or lipopolysaccharide (LPS)-induced inflammatory HCFs by real-time polymerase chain reaction (PCR), Western blotting and immunocytochemistry. Inflammatory cytokine profiles with or without ANG were evaluated through immunodot blot analysis in inflammatory HCFs. Corneal neovascularization and opacity in a rat model of corneal alkali burn were evaluated after application of ANG eye drops. RESULTS: ANG decreased the mRNA levels of IL-1ß, IL-6, IL-8, TNF-α receptor (TNFR)1, 2, TLR4, MYD88, and complement components except for C1r and C1s and elevated the mRNA expression of IL-4 and IL-10. Increased signal intensity of IL-6, IL-8 and monocyte chemotactic protein (MCP)-1 and MCP-2 induced by TNF-α or LPS was weakened by ANG treatment. ANG reduced the protein levels of IKK-ε by either TNF-α and LPS, and decreased TBK1 production induced by TNF-α, but not induced by LPS. The expression of NF-κB in the nuclei was decreased after ANG treatment. ANG application lowered corneal neovascularization and opacity in rats compared to controls. CONCLUSION: These results demonstrate that ANG reduces the inflammatory response induced by TNF-α or LPS in HCFs through common suppression of IKK-ε-mediated activation of NF-κB. This may support the targeting of immune-mediated corneal inflammation by using ANG.

Evaluation of the Ocular Tolerance of Three Tacrolimus Topical Pharmaceutical Preparations by Bovine Corneal Opacity and Permeability Test.

PURPOSE: Tacrolimus ocular preparations are commonly employed in autoimmune or inflammatory ocular disorders. However, currently there are not yet approved ocular formulations. Tacrolimus ocular side effects have been reported in clinical use, so the evaluation of different pharmaceutical preparations is mandatory. In this study, the local corneal tolerance and safety profile of three common tacrolimus 0.03% pharmaceutical preparations were evaluated. MATERIAL AND METHODS: Corneal irritation and permeability of tacrolimus preparations were evaluated with the bovine corneal opacity and permeability (BCOP) test. Complementary corneal hematoxylin/eosin and immunohistochemistry staining for tight junctions and adherent junctions E-cadherin, VE-cadherin and zonula occludens-1 were examined and scored to evaluate and to confirm corneal disruption and irritation scores obtained with the BCOP method. RESULTS: Commercial brand ointment (Protopic®), topical compounded eye ointment (pharmacy elaboration) and tacrolimus suspension eye drops (elaborated from parenteral prograf®) were tested as potential ocular preparations to be used in clinics. Tacrolimus preparations hereby studied do not alter the opacity and permeability of the bovine cornea by more than three units, measured by the In Vitro Irritancy Score, neither affected the immunohistochemical parameters, composite score or transepithelial electrical resistance. CONCLUSIONS: Tacrolimus preparations studied can be safely applied as a topical ocular treatment.

Effect of onion extract on corneal haze suppression after air assisted lamellar keratectomy.

This study evaluated the effect of onion extract on corneal haze suppression after applying the air assisted lamellar keratectomy. The air assisted lamellar keratectomy was performed on 24 canine eyes. They were treated with an artificial tear (group C), prednisolone acetate (group P), onion extract (group O) and TGF-ß1 (group T) three times per day from 7 to 28 days after the surgery. Corneal haze occurred on the all eyes and was observed beginning 7 days after the surgery. The haze was significantly decreased in groups P and O from day 14 compared with the group C using the clinical (group P; P=0.021, group O; P=0.037) and objective evaluation method (group P; P=0.021, group O; P=0.039). In contrast, it was significantly increased in group T from day 14 compared with group C based on the clinical (P=0.002) and objective evaluation method (P<0.001). Subsequently, these eyes were enucleated after euthanasia, and immunohistochemistry with α-SMA antibodies was done. The total green intensity for α-SMA was significantly more expressed in group T and significantly less expressed in groups P and O than in group C. Onion extract could have potential as a therapeutic in preventing corneal haze development by suppressing the differentiation of fibroblasts into myofibroblasts.

Idiopathic stromal keratitis resembling central toxic keratopathy.

UNLABELLED: A 28-year-old previously emmetropic woman presented with a 2-day history of bilateral photophobia and blurred vision. She had no history of corneal surgery or contact lens use. Snellen uncorrected distance visual acuity (UDVA) was 6/12 bilaterally. Bilateral anterior stromal infiltration of the central cornea was seen, with no overlying epithelial defect and intact sensation. There was a marked hyperopic shift, central corneal thinning, and associated keratometric flattening. The patient was started on topical corticosteroids. Over 12 weeks, the UDVA returned to 6/5 with emmetropization of the refractive status and thickening of the central cornea. The clinical findings in our patient resemble those of central toxic keratopathy (CTK) in post-refractive-surgery cases, suggesting a similar mechanism. To our knowledge, this is the first report of a patient presenting with a CTK-like syndrome in the absence of previous keratorefractive surgery or contact lens use. The pathophysiology of this condition remains elusive and requires further investigation. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.