To study corneal lymphangiogenesis after corneal transplantation, corneal allogenic transplantation models were established in rats. 8 female Wister rats were used as donors, and 16 Sprague Dawley (SD) rats were used as recipients and 2 SD served as controls. Corneal lymphangiogenesis and hemangiogenesis was examined by electron microscopy 1 and 2 weeks after corneal penetrating transplantation, and the expression of lymphatic vessel endothelial receptor (LYVE-1) was examined 1, 3, 7, 14 days after the transplantation respectively. In addition, 19 allograft failed human corneas were examined by 5'-nase-alkaline phosphatase (5'-NA-ALP) double-enzyme-histochemistry staining to detect corneal lymphangiogenesis and hemangiogenesis. By immunohistochemistry for LYVE-1, it was found that blown lymphatics were localized in the stroma 3 days after the corneal transplantation. With electron microscopy, new lymphatic vessels and blood vessels were found 1 and 2 weeks after the corneal transplantation. By 5'-NA-ALP enzyme-histochemistry, corneal hemangiogenesis was found in all allograft failed human corneas and 5 of 19 (26.3 %) cases had developed corneal lymphangiogenesis. It is concluded that corneal lymphangiogenesis is present after corneal transplantation, which may play an important role in allograft rejection.
Cornea/*blood supply ; Cornea/chemistry ; Cornea/ultrastructure ; Corneal Neovascularization/etiology ; Corneal Neovascularization/metabolism ; Corneal Transplantation/adverse effects ; Corneal Transplantation/*methods ; Immunohistochemistry ; Lymphangiogenesis ; Microscopy, Electron ; Rats, Sprague-Dawley ; Rats, Wistar ; Vesicular Transport Proteins/biosynthesis

A 62-year-old female visited our clinic with progressively decreased vision in both eyes beginning 12 years prior. Idiopathic corneal opacity in all layers of the cornea was found in both eyes. One year later, we performed penetrating keratoplasty on the undiagnosed right eye. During post-surgical follow-up, corneal edema and stromal opacity recurred, and penetrating keratoplasty was performed two more times. The patient's total serum protein level, which had previously been normal, was elevated prior to the final surgery. She was diagnosed with monoclonal gammopathy of undetermined significance. We made a final diagnosis of monoclonal gammopathy-associated crystalline keratopathy after corneal biopsy. Monoclonal gammopathy-associated crystalline keratopathy is difficult to diagnose and may lead to severe visual loss. A systemic work-up, including serologic tests like serum protein or cholesterol levels, is needed in patients with unexplainable corneal opacity.
Corneal Edema/etiology/*metabolism/physiopathology/surgery ; Corneal Neovascularization/etiology/*metabolism/physiopathology/surgery ; Corneal Opacity/etiology/*metabolism/physiopathology/surgery ; Crystallins/*metabolism ; Female ; Humans ; Keratoplasty, Penetrating ; Microscopy, Electron ; Middle Aged ; Monoclonal Gammopathy of Undetermined Significance/*complications/pathology ; Reoperation ; Visual Acuity

OBJECTIVETo investigate the effect of integrin α9β1 on corneal angiogenesis and the expression of vascular endothelial growth factor A (VEGF-A) in rats after corneal suture.

METHODSThirty-nine SD rats were randomly divided into normal control group, corneal suture model group, α9β1 group, and α9β1 suppression group, and in the latter 3 groups, the rats received topical administration of levofloxacin, levofloxacin + α9β1, and levofloxacin+ anti-α9β1 monoclonal antibody (Y9A2) twice daily after corneal suture, respectively. At 3, 5, 7, 14 days after the surgery, 3 rats were randomly selected from each group to observe corneal neovascularization (CNV) under slit lamp. RT-PCR and Western blotting were used to detect the expressions of VEGF-A.

RESULTSCNV was not observed in normal cornea. Neovascularization occurred in the corneal limbus in the 3 corneal suture groups, and the leak neovascularization count (CNVC), which occurred on the 7th postoperative day, was significantly increased after α9β1 treatment compared with that in the model group (4.57∓0.31 vs 3.21∓0.19, P<0.05) but markedly decreased after α9β1 suppression (2.03∓0.26, P<0.05). VEGF-A was expressed at a low level in normal cornea, and increased significantly in the model group, reaching the peak level on 7th day (P<0.05); corneal VEGF-A expression was further enhanced by α9β1 treatment (P<0.05) while significantly lowered by α9β1 suppression (P<0.05).

CONCLUSIONIntegrin α9β1 can promote CNV by up-regulating VEGF-A expression and α9β1 suppression produces the opposite effect after corneal suture in rats.

Animals ; Cornea ; drug effects ; metabolism ; Corneal Neovascularization ; metabolism ; Female ; Integrins ; antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Sutures ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization.

METHODSThe rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells.

RESULTSThe cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection.

CONCLUSIONarAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.

Cell Line ; Corneal Neovascularization ; therapy ; Dependovirus ; genetics ; Endothelial Cells ; cytology ; metabolism ; Genetic Therapy ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the effects of ursolic acid on corneal graft rejection in a rat model of othotopic corneal allograft transplantation.

METHODSForty-eight recipient Wistar rats were divided into normal control group with saline treatment (group A), autograft group with saline treatment (group B), SD rat allograft group with saline treatment (group C), and SD rat allograft group with intraperitoneal ursolic acid (UA) treatment group (group D). The rats received saline or UC (20 mg·kg(-1)·d(-1)) treatment for 12 days following othotopic graft transplantation. The grafts were evaluated using the Larkin corneal rejection rating system, and the graft survival was assessed with Kaplan-Meier analysis. On day 14, the grafts were harvested for histological examination, Western blotting, and assessment of expressions of interlukin-2 (IL-2), interferon-γ (IFN-γ), nuclear transcription factor-κB (NF-κB) p65, vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1).

RESULTSThe allograft survival was significantly longer in group D than in group C (29.12±9.58 vs 9.67±2.16 days, P<0.05). UC treatment obviously reduced the expression levels of IL-2, IFN-γ, NF-κBp65, ICAM-1 and VEGF and increased inhibitory kappa B alpha (IκB-α) expression in the grafts, where no obvious inflammatory cell infiltration or corneal neovascularization was found.

CONCLUSIONAs a NF-κB inhibitor, ursolic acid can prevent corneal neovascularization and corneal allograft rejection to promote graft survival in rats following orthotopic corneal allograft transplantation.

Animals ; Cornea ; metabolism ; Corneal Neovascularization ; prevention & control ; Corneal Transplantation ; Graft Rejection ; prevention & control ; Graft Survival ; drug effects ; I-kappa B Proteins ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Interferon-gamma ; metabolism ; Kaplan-Meier Estimate ; NF-KappaB Inhibitor alpha ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Transcription Factor RelA ; metabolism ; Transplantation, Homologous ; Triterpenes ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism

PURPOSE: To determine the efficacy of topical application and subconjunctival injection of bevacizumab in the treatment of corneal neovascularization. METHODS: Corneal neovascularization was induced with a silk suture of the corneal stroma in 12 rabbits (24 eyes). One week after suturing, four rabbits were treated with topical bevacizumab at 5 mg/mL (group A) and another four rabbits were treated with topical bevacizumab 10 mg/mL (group B) in the right eyes twice a day for two weeks. A subconjunctival injection of bevacizumab 1.25 mg/mL was done in the right eyes of four rabbits (group C). All of the left eyes (12 eyes) were used as controls. The area of corneal neovascularization was measured after one and two weeks, and the concentration of vascular endothelial growth factor (VEGF) in corneal tissue was measured after two weeks. RESULTS: The neovascularized area was smaller in all treated groups than in the control group (p<0.001). Upon analysis of the neovascularized area, there was no significant difference between groups A and B. However, the mean neovascularized area of group B was significantly smaller than that of group C after two weeks of treatment (p=0.043). The histologic examination revealed fewer new corneal vessels in all treated groups than the control group. The concentration of VEGF was significantly lower in all treated groups compared to the control group (p<0.01), but no difference was shown between treated groups. CONCLUSIONS: Topical and subconjunctival bevacizumab application may be useful in the treatment of corneal neovascularization and further study is necessary.
Angiogenesis Inhibitors/*administration & dosage ; Animals ; Antibodies, Monoclonal/*administration & dosage ; Cornea/metabolism/*pathology ; Corneal Neovascularization/*drug therapy/metabolism/pathology ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Drug Administration Schedule ; Female ; Follow-Up Studies ; Male ; Ophthalmic Solutions ; Rabbits ; Treatment Outcome ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

PURPOSE: To evaluate factors that can influence the prevalence of amblyopia in children with anisometropia. METHODS: We retrospectively reviewed the records of 63 children 2 to 13 years of age who had anisometropic amblyopia with a difference in the refractive errors between the eyes of at least two diopters (D). The type of anisometropia (myopia, hyperopia, and astigmatism), degree of anisometropia (<2-3 D, <3-4 D, or >4 D), best corrected visual acuity (BCVA) of the amblyopic eye at the time of initial examination, BCVA differences between sound and amblyopic eyes, whether or not occlusion therapy was performed, compliance with occlusion therapy, and the patient's age when eyeglasses were first worn were investigated. RESULTS: There was an increase in the risk of amblyopia with increased magnitude of anisometropia (p=0.021). The prevalence of amblyopia was higher in the BCVA <20/40 group and in patients with BCVA differences >4 lines between sound and amblyopic eyes (p=0.008 and p=0.045, respectively). There was no statistical relationship between the prevalence of amblyopia and the type of anisometropia or the age when eyeglasses were first worn. Poor compliance with occlusion therapy was less likely to achieve successful outcome (p=0.015). CONCLUSIONS: Eyes with poor initial visual acuities of <20/40, a high magnitude of anisometropia, and a >4 line difference in the BCVA between sound and amblyopic eyes at the initial visit may require active treatment.
Angiogenesis Inhibitors/*administration & dosage ; Animals ; Antibodies, Monoclonal/*administration & dosage ; Cornea/metabolism/*pathology ; Corneal Neovascularization/*drug therapy/metabolism/pathology ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Drug Administration Schedule ; Female ; Follow-Up Studies ; Male ; Ophthalmic Solutions ; Rabbits ; Treatment Outcome ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.
Vascular Endothelial Growth Factor A/biosynthesis ; Sirolimus/pharmacokinetics/*pharmacology ; Rabbits ; Humans ; Fibroblast Growth Factor 2/pharmacokinetics/pharmacology ; Female ; Endothelial Cells/cytology/drug effects/metabolism ; Drug Implants ; Dose-Response Relationship, Drug ; Corneal Neovascularization/*drug therapy/pathology ; Cells, Cultured ; Cell Proliferation/drug effects ; Cell Movement/drug effects ; Animals ; Angiogenesis Inhibitors/pharmacokinetics/*pharmacology

OBJECTIVETo evaluate the efficacy of epigallocatechin gallate (EGCG) in treatment of corneal alkali burn injury in mice.

METHODSCorneal alkali burn injury was induced by sodium hydroxide method in C57BL/6J mice. The mice with cornea burns were treated intraperitoneally with EGCG solution or phosphate buffer solution (PBS) respectively. The healing of corneal epithelium, the formation of corneal neovascularization (CNV) and the inflammation reaction were assessed by slit -lamp microscopy and histological examination. Expression of vascular endothelial growth factor (VEGF) mRNA and protein in cornea was evaluated by real -time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Myeloperoxidase (MPO) assay was used to quantitatively evaluate the polymorphonuclear neutrophils (PMNs) infiltration in the corneas.

RESULTSThe healing rate of corneal epithelium in EGCG group was significantly higher than that of PBS group at d1, d3 and d7 after treatment (d1: 41.0%±13.0% vs 23.8%±7.6%; d3: 76.6%±7.5% vs 61.2%±6.8%; d7: 87.8%±8.5% vs 74.0%±9.1%; all P <0.05). The CNV scores and the number of CNV in the corneal sections of EGCG group were significantly lower than those of PBS group at d3, d7 and d14 after treatment (CNV score: d3: 1.1±0.5 vs 6.6±1.0; d7: 1.3±0. 3 vs 8.1±1.0; d14: 0.9±0.2 vs 9.2±1.1; CNV number: d3: 1.68±0.61 vs 2.92±0.95; d7: 4.80±1.36 vs 7.92±1.28; d14: 3.64±0.71 vs 5.88±0.76; all P<0.05) . The expression of VEGF protein at d3 (0.19±0.05 vs 0.45±0.08) and d7 (0.42±0.07 vs 0.84±0.09), the expression of VEGF mRNA at d1, d3 and d7 in EGCG group were significantly lower than those in PBS group (all P <0.05). Compared to PBS group, the inflammatory index at d3 (3.2±0.4 vs 3.7±0.5) and d7 (2.3±0.5 vs 4.0±0.0), the number of PMNs in the corneal sections and the MPO values at d3, d7 and d14 in EGCG group were significantly decreased (PMNs: d3: 34.5±15.7 vs 90.0±28.8; d7: 17.1±11.4 vs 54.9±25.9; d14: 12. 8±4.6 vs 39.0±17.9; all P <0.05).

CONCLUSIONIn the murine corneal alkali burn model, intraperitoneal injection of EGCG solution can promote the healing of corneal epithelium, inhibit the formation of CNV and reduce the inflammatory cell infiltration in the corneas.

Alkalies ; Animals ; Burns, Chemical ; drug therapy ; Catechin ; analogs & derivatives ; therapeutic use ; Cornea ; drug effects ; pathology ; Corneal Neovascularization ; prevention & control ; Disease Models, Animal ; Eye Burns ; drug therapy ; Inflammation ; drug therapy ; immunology ; Mice ; Mice, Inbred C57BL ; Neutrophils ; cytology ; RNA, Messenger ; Vascular Endothelial Growth Factor A ; metabolism