Evaluation of biophysical alterations in the epithelial and endothelial layer of patients with Bullous Keratopathy.

The cornea is a fundamental ocular tissue for the sense of sight. Thanks to it, the refraction of two-thirds of light manages to participate in the visual process and protect against mechanical damage. Because it is transparent, avascular, and innervated, the cornea comprises five main layers: Epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Each layer plays a key role in the functionality and maintenance of ocular tissue, providing unique ultrastructural and biomechanical properties. Bullous Keratopathy (BK) is an endothelial dysfunction that leads to corneal edema, loss of visual acuity, epithelial blisters, and severe pain, among other symptoms. The corneal layers are subject to changes in their biophysical properties promoted by Keratopathy. In this context, the Atomic Force Microscopy (AFM) technique in air was used to investigate the anterior epithelial surface and the posterior endothelial surface, healthy and with BK, using a triangular silicone tip with a nominal spring constant of 0.4 N/m. Six human corneas (n = 6) samples were used for each analyzed group. Roughness data, calculated by third-order polynomial adjustment, adhesion, and Young's modulus, were obtained to serve as a comparison and identification of morphological and biomechanical changes possibly associated with the pathology, such as craters and in the epithelial layer and exposure of a fibrotic layer due to loss of the endothelial cell wall. Endothelial cell membrane area and volume data were calculated, obtaining a relevant comparison between the control and patient. Such results may provide new data on the physical properties of the ocular tissue to understand the physiology of the cornea when it has pathology.

Pathogenic Role of Endoplasmic Reticulum Stress in Diabetic Corneal Endothelial Dysfunction.

PURPOSE: Progressive corneal edema and endothelial cell loss represent the major corneal complications observed in diabetic patients after intraocular surgery. However, the underlying pathogenesis and potential treatment remain incompletely understood. METHODS: We used streptozotocin-induced type 1 diabetic mice and db/db type 2 diabetic mice as diabetic animal models. These mice were treated with the endoplasmic reticulum (ER) stress agonist thapsigargin; 60-mmHg intraocular pressure (IOP) with the ER stress antagonist 4-phenylbutyric acid (4-PBA); mitochondria-targeted antioxidant SkQ1; or reactive oxygen species scavenger N-acetyl-l-cysteine (NAC). Corneal thickness and endothelial cell density were measured before and after treatment. Human corneal endothelial cells were treated with high glucose with or without 4-PBA. The expression of corneal endothelial- and ER stress-related genes was detected by western blot and immunofluorescence staining. Mitochondrial bioenergetics were measured with an Agilent Seahorse XFp Analyzer. RESULTS: In diabetic mice, the appearance of ER stress preceded morphological changes in the corneal endothelium. The persistent ER stress directly caused corneal edema and endothelial cell loss in normal mice. Pharmacological inhibition of ER stress was sufficient to mitigate corneal edema and endothelial cell loss in both diabetic mice after high IOP treatment. Mechanistically, inhibiting ER stress ameliorated the hyperglycemia-induced mitochondrial bioenergetic deficits and improved the barrier and pump functional recovery of the corneal endothelium. When compared with NAC, 4-PBA and SkQ1 exhibited better improvement of corneal edema and endothelial cell loss in diabetic mice. CONCLUSIONS: Hyperglycemia-induced ER stress contributes to the dysfunction of diabetic corneal endothelium, and inhibiting ER stress may offer therapeutic potential by improving mitochondrial bioenergetics.

RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice.

Slc4a11 KO mice show significant corneal edema, altered endothelial morphology, and mitochondrial ROS at an early age without a decrease in endothelial cell density. We examined the differential gene expression profile between wild type (WT) and KO with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function that can explain the corneal edema. Freshly dissected Corneal Endothelium-Descemet's Membrane (CEDM) and cultured Mouse Corneal Endothelial Cells (MCEC) were obtained from WT and Slc4a11 KO mice. RNA sequencing Ingenuity Pathway Analysis (IPA) predicted activation, inhibition or differential regulation of several pathways. QPCR and Western analysis validated downregulation of Glycolytic enzymes, Mitochondrial complex components and Ion transporters. Functional testing revealed decreases in endothelial lactate production, Extracellular Acidification Rate (ECAR), glutaminolysis, and Oxygen Consumption Rate (OCR) of KO CEDM in the presence of Glutamine (Gln) that was not compensated by fatty acid oxidation. Stromal lactate was significantly elevated in KO along with decreased expression of MCT1 and MCT4 lactate transporters in endothelial cells. ATP levels were 2x higher in KO CEDM, concomitant with a 3-fold decrease in Na-K-ATPase activity and reduced basolateral membrane localization. Genes for cholesterol biosynthesis, glutathione metabolism and tight and adherens junctions were elevated. Alteration of tight junction structure and cortical cytoskeleton is evident in KO corneal endothelium with a significant increase in trans-endothelial fluorescein permeability. We conclude that Slc4a11 KO induces a coordinated decrease in glycolysis, glutaminolysis, lactate transport and Na-K-ATPase activity. These changes together with an altered barrier function cause an accumulation of stromal lactate in Slc4a11 KO mice leading to chronic corneal edema.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity. Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined. Results: At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO. Conclusions: The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Topical administration of the kappa opioid receptor agonist nalfurafine suppresses corneal neovascularization and inflammation.

Corneal neovascularization (CNV) causes higher-order aberrations, corneal edema, ocular inflammation, and corneal transplant rejection, thereby decreasing visual acuity. In this study, we investigated the effects of topical administration of the kappa opioid receptor agonist nalfurafine (TRK-820) on CNV. To induce CNV, intrastromal corneal sutures were placed on the corneal stroma of BALB/c mice for 2 weeks. Nalfurafine (0.1 µg/2 µL/eye) was topically administered to the cornea once or twice daily after CNV induction. The CNV score, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in neovascularized corneas were evaluated using slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. The mRNA expression of the kappa opioid receptor gene Oprk1 was significantly upregulated following CNV induction. Topical administration of nalfurafine twice daily significantly suppressed CNV and lymphangiogenesis, as well as reduced the mRNA levels of angiogenic and pro-inflammatory factors in the neovascularized corneas. Moreover, nalfurafine administration twice daily reduced the numbers of infiltrating leukocytes, neutrophils, macrophages, and interferon-γ-producing CD4+ T cells in the neovascularized corneas. In this study, we demonstrated that topical administration of nalfurafine suppressed local CNV in a mouse model along with the activation of KOR, suggesting that nalfurafine may prevent and control CNV in humans.

Effects of scleral-lens oxygen transmissibility on corneal thickness: A pilot study.

PURPOSE: To investigate the effect of various oxygen transmissibilities (Dk/t) of scleral lenses and corneal thickness recovery time from overnight eye closure with patching on corneal edema during 5 h lens wear. METHODS: Scleral lenses (hofocon A, 15.6 mm diameter) were worn bilaterally with three different Dks (100, 140, and 160 Barrer). Central and peripheral corneal thickness (CCT and PCT) were measured using optical coherence tomography. Four subjects were randomly selected for one additional visit and asked to patch one eye before night sleeping. The patch was not removed until lens insertion to avoid corneal deswelling. Then CCT of both eyes was measured. RESULTS: Ten neophytes with healthy eyes participated in the study. Mean [95% CI] Dk/t of the study lenses was 32.0 [29.2, 34.7] hBarrer/cm. Mean [95% CI] CCT immediately upon lens insertion and after 5 h of lens wear were 532.4 [520.3, 544.5] µm and 538.7 [526.5, 551.0] µm, respectively. Mean [95% CI] percentage change (%Δ) in CCT was 1.2% [0.9%, 1.5%], 1.2% [0.9%, 1.4%], and 0.8% [0.6%, 1.1%] for CCT, nasal PCT, and temporal PCT, respectively. There was an inverse relationship between temporal Dk/t and %ΔPCT (p < 0.05) while Dk/t was not found significantly associated with either CCT or nasal PCT. The patched eyes maintained a relatively stable CCT and showed progressive deswelling, starting and ending with 2.8% and 0.6%, respectively. In contrast, the unpatched eyes swelled, starting with nearly 0% and ending with 0.7% with a maximum swelling of 1.8%. CONCLUSION: There was limited amount of corneal edema induced by short-term scleral lens wear with lens Dk/t ranging from 21 to 47 hBarrer/cm and lenses with lower lens Dk/t did not induce significantly higher corneal swelling. Scleral lens insertion soon after overnight eye closure with patching did not introduce additional swelling for young and healthy eyes.

The Use of Conjunctival Staining to Measure Ocular Surface Inflammation in Patients With Dry Eye.

PURPOSE: To investigate the expression levels of inflammatory cytokines in the conjunctival epithelium and correlations with clinical parameters in dry eye disease (DED). METHODS: This study evaluated 28 patients with Sjögren syndrome (SS) DED, 28 patients with non-SS DED, and 10 controls. The messenger ribonucleic acid (mRNA) expression of inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, interferon (IFN)-γ, IL-17, and matrix metalloproteinase 9 (MMP9) from conjunctival epithelium was investigated by real-time polymerase chain reaction. Protein expression was confirmed by immunofluorescence staining. Correlations were evaluated between the mRNA expression of inflammatory cytokines and clinical DED parameters such as ocular surface disease index score, Schirmer I value, tear film breakup time, and corneal and conjunctival staining scores. RESULTS: Patients with non-SS DED expressed significantly more IFN-γ, IL-6, and MMP9 genes in the conjunctival epithelium than the controls (P < 0.05), and all cytokine gene expression was significantly higher in patients with SS DED than in the controls (P < 0.01). Tumor necrosis factor-α, IL-1ß, IL-6, and IL-17 gene expression was higher in patients with SS DED than in the non-SS DED group (P < 0.05). Immunofluorescence staining of conjunctival epithelium demonstrated that positive cells with IL-6 or MMP9 were significantly higher in non-SS DED than in controls (P < 0.01) and much higher in SS DED than in non-SS DED (P < 0.05). Conjunctival staining scores significantly correlated with the expression of IFN-γ, IL-6, IL-17, and MMP9 in both DED groups (P < 0.05 in non-SS DED and P < 0.01 in SS-DED). Interestingly, correlation coefficients of all cytokines were much higher in SS DED compared to non-SS DED. Corneal staining scores showed positive correlations with IFN-γ, IL-17, and MMP9 (P < 0.05), and correlation coefficients were lower than those of conjunctival staining scores. CONCLUSIONS: Conjunctival staining scores may be useful to measure ocular surface inflammation in SS and non-SS DED.

Effects of amantadine on corneal endothelium.

PURPOSE: An adverse effect of amantadine, a drug used for Parkinson's disease, is corneal edema. While corneal endothelial cell loss is noted with amantadine toxicity, the reversibility of corneal edema suggests that amantadine affects active mechanisms regulating corneal hydration. Although mainly known as a NMDA receptor antagonist, amantadine is also a K+-channel blocker. The purpose of this study was to investigate potential mechanisms of amantadine's toxic effects on corneal endothelium. METHODS: Bovine corneas were used for short-circuit current measurements of corneal endothelial active ion transport to compare the effects of amantadine with an NMDA receptor agonist (NMDA) and antagonist (D-APV), and the K+-channel blockers BaCl2 and clotrimazole. Cell death and changes in cell morphology were observed using annexin V stain, alizarin red S staining of the intercellular junctions, ZO-1 immunolocalization, and phalloidin stain of the actin cytoskeleton. RESULTS: Amantadine caused a transient decrease in the short-circuit current that mimicked the effect of clotrimazole. BaCl2, and the NMDA receptor agonist and antagonist had no effect on the short-circuit current. Tissue incubation with amantadine caused an increase in cell area (measured by ZO-1 localization) and cell height (measured by phalloidin stain) but did not increase apoptotic cell death (annexin V stain). CONCLUSIONS: The similarity of amantadine and clotrimazole effects on the short-circuit current and the effects on cell volume suggest that amantadine's actions on corneal endothelium are mediated via K+ channels. The observed absence of cell death and transient effect on short-circuit current support the reported reversibility of amantadine-induced corneal edema.

Cytokine Levels in the Aqueous Humor Are Associated With Corneal Thickness in Eyes With Bullous Keratopathy.

PURPOSE: We sought to investigate the association between the severity of bullous keratopathy and proinflammatory cytokine levels in the aqueous humor (AqH). DESIGN: Cross-sectional study. METHODS: This study included a total of 95 eyes: 62 with bullous keratopathy and 33 that underwent cataract surgery. Central corneal thickness (CCT) and central corneal volume within 4 and 6 mm (CCV4mm and CCV6mm, respectively) were determined using anterior segment optical coherence tomography. A total of 95 AqH samples were collected at the beginning of surgery. The levels of cytokines (interleukins [ILs]-1α, -1ß, -4, -6, -8, -10, -12p70, -13, -17A, interferon [IFN]-α, IFN-γ, monocyte chemotactic protein [MCP]-1, E-selectin, P-selectin, and soluble intercellular adhesion molecule-1 [sICAM-1]) in the AqH were measured using multiplex beads immunoassay. We evaluated the correlation among AqH cytokine levels, CCT, CCV4mm, and CCV6mm in eyes with bullous keratopathy. RESULTS: The levels of protein, ILs-4, -6, -8, -10, -12p70, and -17A, MCP-1, IFN-γ, E-selectin, P-selectin, and sICAM-1 were significantly higher in eyes with bullous keratopathy compared with those of the normal control subjects (all P < .0025). CCT was significantly correlated with the levels of IL-13 (r = 0.551, P = .0009) and sICAM-1 (r = 0.448, P = .0005). CCV4mm was significantly correlated with the levels of IL-13 (r = 0.514, P = .0022) and sICAM-1 (r = 0.404, P = .0019). CCV6mm was significantly correlated with the level of sICAM-1 (r = 0.459, P = .0003). CONCLUSION: The severity of corneal edema in eyes with bullous keratopathy was associated with the levels of specific cytokines in the AqH.

Central Corneal Edema with Scleral-Lens Wear.

PURPOSE: To evaluate the safety of scleral-lens designs, we model and clinically assess central corneal edema induced by scleral-lens wear for healthy subjects. MATERIALS AND METHODS: Central corneal swelling during scleral-lens wear is measured using optical coherence tomography (OCT). Transport resistances are modeled for oxygen diffusion through the scleral lens and post-lens tear-film (PoLTF), and into the cornea. Oxygen deficiency in the cornea activates anaerobic metabolic reactions that induce corneal edema. Oxygen permeability, carbon-dioxide permeability, settled-lens PoLTF thickness, and scleral-lens thickness are varied in the calculations to mimic different lens fits. RESULTS: Transport modeling predicts that for open eyes, increasing PoLTF thickness from 50 to 400 µm increases central corneal swelling by approximately 1-1.5% when oxygen transmissibility (Dk/L) is greater than 10 hBarrer/cm (i.e., hectoBarrer/cm). Although swelling is larger for oxygen Dk/L < 10 hBarrer/cm, PoLTF thickness has minimal impact in this range. For open eye, oxygen transmissibility of the lens plays a significant role in corneal edema, but is negligible when oxygen Dk/L is > 40 hBarrer/cm. For closed eye, central corneal swelling is greater than 5% for an oxygen Dk/L range of 0-100 hBarrer/cm with typical lens-fitting parameters. For carbon-dioxide transmissibilities increasing from 50 to 250 hBarrer/cm and with a fixed oxygen Dk/L of 25 hBarrer/cm, calculated swelling diminishes by an additional 0.5%. Comparison of model calculations to clinical-swelling data is within the error range of the clinical measurements. CONCLUSIONS: Oxygen/metabolite transport calculations for open-eye scleral-lens wear show that typical PoLTF thicknesses fitted by clinicians (i.e., PoLTF thicknesses < 400 µm) with modern scleral lenses (i.e., oxygen Dk/L > 25 hBarrer/cm) produce corneal swelling of less than 2% in agreement with experiment. Therefore, scleral lenses prescribed today evoke less than physiological hypoxic swelling (i.e., less than 4%) for healthy corneas during open-eye. Closed-eye wear, however, appears clinically unsafe.

Comparison of femtosecond laser-assisted corneal intrastromal xenotransplantation and the allotransplantation in rhesus monkeys.

BACKGROUND: In our previous study, we showed that both allogeneic and autogeneic small-incision femtosecond laser-assisted corneal intrastromal transplantation are safe and effective surgeries. However, the results of small-incision femtosecond laser-assisted intrastromal xenotransplantation have not yet been explored. Additionally, we suggest that glycerol-dehydrated corneal lamellae might provide a possible alternative for this xenogenic implantation approach. METHODS: Corneal inlay lamellae were produced from rabbits and humans using femtosecond laser-assisted surgeries and were dehydrated in glycerol for 1 week at 4 °C. These xenogeneic glycerol-dehydrated grafts and fresh allogeneic monkey lamellae were then implanted into rhesus monkeys using small-incision femtosecond laser assistance. Postoperatively, clinical examinations, AS-OCT measurements and tear inflammatory mediator assays were performed. RESULTS: There were no significant changes in the transparency of the corneal lamellae after glycerol dehydration. Following implantation, no evidence of tissue rejection or severe inflammatory responses was observed in the monkeys, and the host corneas remained transparent throughout a 6-month observation period. The grafts were clearly visible via AS-OCT. Corneal thickness increased 1 week postoperatively but subsequently declined and remained unchanged 1 month after surgery. Significant changes were observed in all tear inflammatory mediators in the 'Rabbit to Monkey' group. The trends in changes of tear inflammatory mediators in the 'Human to Monkey' group were similar to those in the 'Rabbit to Monkey' group. At 1 month post-surgery, the levels of most tear inflammatory mediators had decreased, with the exception of IL-1ß, TGF-ß1 and IFN-γ in the allotransplantation group. CONCLUSION: Small-incision femtosecond laser-assisted intrastromal transplantation minimized invasiveness and improved surgical efficiency. In addition, the host cornea maintained a high level of biocompatibility. Glycerol-dehydrated corneal lamellae might be potentially useful as an alternative inlay xenogeneic material. In this study, we also describe a new treatment that can be used in keratoconus, corneal ectasia, presbyopia, hyperpresbyopia and other diseases.

Ocular surface inflammation impairs structure and function of meibomian gland.

Dysfunction of the meibomian glands alters secreted meibum quantitatively and qualitatively that can lead to damage to the ocular surface epithelium. In response to an unstable tear film cause by meibomian gland dysfunction, ocular surface epithelium is damaged and expresses inflammatory cytokines leading to secondary ocular inflammation. In turn, inflammatory disorders of the palpebral conjunctiva and lid margin may affect the structure and function of meibomian gland. The disorders include allergic conjunctivitis, long-term usage of contact lenses, dermatological diseases that affect conjunctival homeostasis, Stevens-Johnson's syndrome or chemical burning of the ocular surface and lid margin.

Interpreting the corneal response to oxygen: Is there a basis for re-evaluating data from gas-goggle studies?

When anoxia (0% oxygen) is created within a gas-tight goggle, ocular physiological responses, including corneal swelling, limbal hyperaemia and pH change, are known to vary, depending on the presence or absence of a low, oxygen transmissibility contact lens. A new theory is proposed to account for this discrepancy based on the concept of lid derived oxygen, whereby oxygen originating from the vascular plexus of the palpebral conjunctiva supplements that available to the ocular surface in an open, normally blinking eye, even when the surrounding gaseous atmosphere is anoxic. The effect of a lid derived contribution to corneal oxygenation was assessed by using existing experimental data to model open-eye, corneal swelling behavior as a function of atmospheric oxygen content, both with and without the presence of a contact lens. These models predict that under atmospheric anoxia, contact lens wear results in 13.2% corneal swelling compared with only 5.4% when the lens was absent. Lid derived oxygen acts to provide the ocular surface in the non-contact lens wearing, normally blinking, open-eye with up to 4.7% equivalent oxygen concentration, even within the anoxic environment of a nitrogen filled goggle. Correcting for lid derived oxygen eliminates previously observed discrepancies in corneal swelling behavior and harmonizes the models for the contact lens wearing and gas-goggle cases. On this basis it is proposed that true anoxia at the ocular surface cannot be achieved by atmospheric manipulation (i.e. a gas-goggle) alone but requires an additional presence, e.g. a low, oxygen transmissibility contact lens, to prevent access to oxygen from the eyelids. Data from previously conducted experiments in which the gas-goggle paradigm was used, may have been founded on underestimates of the real oxygen concentration acting on the ocular surface at the time and if so, will require re-interpretation. Future work in this area should consider if a correction for lid derived oxygen is necessary.

Longitudinal Changes to Tight Junction Expression and Endothelial Cell Integrity in a Mouse Model of Sterile Corneal Inflammation.

PURPOSE: We previously reported that applying toll-like receptor (TLR) ligands to an injured cornea induces corneal edema at 24 hours, which subsides by 1 week. We tested the hypotheses that endothelial expression of the tight-junction protein, zonula occludens-1 (ZO-1), would be altered during experimental sterile corneal inflammation and that endothelial cell density (ECD) would remain unaffected. METHODS: Anesthetized C57BL/6J mice received central 1-mm corneal abrasions followed by topical application of saline or cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN, TLR-9 agonist). At 24 hours, 1 week and 4 weeks post treatment, spectral-domain optical coherence tomography images were captured. Eyes were enucleated and processed for zonula occludens-1 (ZO-1) immunofluorescent staining. Corneal flatmounts were analyzed for endothelial ZO-1 expression, cell density, polymegethism, and polymorphism. Corneal stromal inflammatory cell infiltration was evaluated at 4 weeks by immunostaining for CD45. RESULTS: Central corneal thickness (CCT) was increased in CpG-ODN treated eyes at 24 hours, had normalized by 1 week, but was again thickened by 4 weeks. In eyes with CpG-ODN, endothelial cell ZO-1 expression was reduced at 24 hours but returned to normal levels by 1 week. Endothelial cell density was not altered at 24 hours or 1 week. By 4 weeks, only CpG-ODN eyes showed relatively reduced ECD, as well as large numbers of CD45+ cells in the stroma. Changes to ECD correlated with CCT (r = -0.53, P < 0.01). Compared with naïve controls, more saline- and CpG-ODN-treated eyes exhibited polymegethism. CONCLUSIONS: This study provides novel insights into the interplay between endothelial cell integrity, corneal edema, and chronic stromal leukocyte activation during sterile corneal inflammation in mice.

Potential Application of Biological Products for the Treatment of Ocular Surface Inflammation.

Various biological products have been introduced for the treatment of autoimmune diseases. The injection of tocilizumab [anti-interleukin (IL)-6R antibody] and a tumor necrosis factor receptor fusion protein (TNFR-Fc) has been approved for the treatment of rheumatoid arthritis. We investigated the effect of the anti-IL-6R antibody and TNFR-Fc on corneal inflammation. Topical instillation of the anti-IL-6R antibody (MR16-1, 2 µg/µL; anti-IL-6R group) or TNFR1-Fc (100 µg/mL; TNFR1 group) was performed after corneal wounds were induced in BALB/c mice by alkali burns. The injured eye was analyzed on day 14 or 28 after injury, and topical instillation was performed until day 14 or day 28. Corneal stromal sections were made using a laser capture microdissection system, and total RNA from the specimens was subjected to quantitative polymerase chain reaction array analysis. Topical instillation of phosphate-buffered saline (PBS) served as a control. The vascularized area was significantly reduced in the anti-IL-6R (day 14) and TNFR1 groups (day 28) compared with that in the PBS group. In the anti-IL-6R group, the expression levels of matrix metalloproteinase-13, monocyte chemotactic protein-1, and C-C motif ligand-22 were downregulated compared with those in the PBS group. In the TNFR1 group, expression of mitogen-activated protein kinase 8 was downregulated. These results indicate the possible application of biological products for topical instillation for the treatment of corneal inflammation.

An Immunohistochemical Study of Inflammatory Cell Changes and Matrix Remodeling With and Without Acute Hydrops in Keratoconus.

PURPOSE: To determine the inflammatory cell and matrix changes in advanced keratoconus, including acute hydrops, using immunohistochemical analysis. METHODS: The corneal tissue from eight subjects with keratoconus undergoing corneal transplantation (three keratoconic buttons, five buttons post acute hydrops­one of them with extensive neovascularization following hydrops) was compared with tissue from two normal corneoscleral rims (n = 10). The corneas were sectioned and analyzed with specific markers for macrophages, lymphocytes, dendritic cells, and scar associated matrix molecules laminin, fibronectin, tenascin-C, and type III collagen. RESULTS: Populations of cells using markers for macrophages, leucocytes and antigen presenting cells were found to be associated with the epithelium and stroma of keratoconic tissue. Populations of these cells appeared decreased in hydrops-associated keratoconus except for a large increase in leucocytes in the stroma and endothelium associated with neovascularization. Extracellular matrix deposition was found to be uniquely demonstrated in localized areas of the stroma, corresponding to the site of hydrops involvement. CONCLUSIONS: Immunohistochemical analysis revealed a chronic, inflammatory process with recruitment of immunoinflammatory cells and deposition of scar tissue in keratoconus. The inflammatory markers were somewhat attenuated in hydrops-associated keratoconus corneas and thus inflammation was not considered to be a major factor in the development of acute corneal hydrops.

Inhibition of Lymphangiogenesis and Hemangiogenesis in Corneal Inflammation by Subconjunctival Prox1 siRNA Injection in Rats.

PURPOSE: Prospero homeobox 1 (Prox1) siRNA is a small interfering RNA that is designed to specifically bind Prox1 mRNA. We determined whether Prox1 siRNA inhibits lymphangiogenesis and hemangiogenesis after acute corneal inflammation. METHODS: Three Prox1 siRNAs were synthesized and investigated for their effects on Prox1 mRNA expression and tube formation in human dermal lymphatic endothelial cells (HDLECs) in vitro. The in vivo effects of Prox1 siRNA were assessed in alkali burn-induced inflammatory corneal neovascularization in rats. Prox1 siRNA was administered via subconjunctival injection. Corneal flat mounts were stained for lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 to show lymphatic vessels. Lymphangiogenesis and hemangiogenesis were analyzed morphometrically using Image J software. Corneal inflammatory cell infiltration was evaluated by immunostaining for F4/80 and CD45. Protein levels of LYVE-1, podoplanin, VEGF receptor 2 (VEGFR2), and VEGFR3 were analyzed by Western blotting. RESULTS: Prox1 siRNA treatment decreased Prox1 mRNA expression and tube formation in cultured HDLECs. Subconjunctival injection of Prox1 siRNA significantly inhibited alkali burn-induced lymphangiogenesis and hemangiogenesis in the cornea compared with those of scrambled siRNA (negative control). This inhibition was comparable to that induced by bevacizumab (positive control). Prospero homeobox 1 knockdown by Prox1 siRNA also inhibited macrophage and leukocyte infiltration into the cornea. Prox1 siRNA downregulated the expression of all four proteins. CONCLUSIONS: Prox1 siRNA is a strong inhibitor of inflammatory corneal lymphangiogenesis and hemangiogenesis in vivo. Prox1 siRNA may be useful in preventing immune rejection after penetrating keratoplasty by suppressing lymphangiogenesis.

Oxygen therapy for corneal edema after cataract surgery.

PURPOSE: To evaluate the effects of oxygen therapy on corneal edema after cataract surgery. SETTING: Imam Khomeini Hospital, Ahvaz, Iran. DESIGN: Randomized controlled trial. METHODS: Patients with severe corneal edema were randomized into 3 groups. Group 1 (control) received conventional therapy including topical sodium chloride, timolol, and betamethasone. Group 2 received the same therapy in addition to systemic normobaric oxygen at a flow rate of 10 L/min for 1 hour twice daily for 3 weeks. Group 3 received conventional therapy and transcorneal oxygen at a flow rate of 5 L/min for 1 hour twice daily for 3 weeks. Preoperative pachymetry and specular microscopy were performed. Pachymetry was performed 1, 3, 5, 7, 10, and 14 days postoperatively. At 1, 3 and 12 months, pachymetry and specular microscopy were performed. RESULTS: The study enrolled 45 patients. Preoperatively, there was no significant difference between the groups. Pachymetry was more than 1000 µm 1 day postoperatively in all patients. The preoperative pachymetry was restored in 14 days in Group 3 only. After 1 year, the endothelial cell count (ECC) was 1603 cells/mm(2), 1693 cells/mm(2), and 1911 cells/mm(2) with a loss of 37%, 32%, and 25% in Group 1, Group 2, and Group 3, respectively (P = .034, Groups 1 and 3). Group 3 had a higher ECC than the control group at 3 months (P = .037) and 1 year (P = .025). One patient in Group 1 and 1 patient in Group 2 developed bullous keratopathy. CONCLUSION: Transcorneal oxygen decreased corneal edema more rapidly than conventional and systemic oxygen therapies and preserved more endothelial cells than conventional therapy. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Glaucoma tardío por partículas de cristalino en síndrome de Marfan / Late onset lens particle glaucoma in Marfan syndrome

CASO CLÍNICO: Glaucoma agudo por partículas de cristalino, en una paciente afecta de síndrome de Marfan que presentaba un cristalino luxado en vítreo de más de 20 años de evolución. DISCUSIÓN: El síndrome de Marfan es un trastorno hereditario, autosómico dominante, del tejido conectivo causado por mutaciones del gen de la fibrilina. La ectopia lentis es la alteración ocular predominante y el criterio mayor de diagnóstico, siendo frecuente el desarrollo de glaucoma en los pacientes afectos de síndrome de Marfan. El caso que se expone es particular, dado que presenta una luxación completa, espontánea y bilateral del cristalino, que además desemboca en un glaucoma secundario de ángulo abierto por liberación de partículas del mismo desde la cámara vítrea

CASE REPORT: A case is presented of an acute onset lens particle glaucoma originating from a crystalline lens spontaneously dislocated into the vitreous for more than 20 years in a patient diagnosed with Marfan syndrome. DISCUSSION: Marfan syndrome is a connective tissue disorder with autosomal dominant inheritance caused by fibrillin gene mutation. Ectopia lentis is the predominant ocular abnormality and a major diagnostic criterion. An association between Marfan syndrome and glaucoma has also been demonstrated. The reported case is unusual in that a complete spontaneous lens dislocation to vitreous was present and progressed to secondary lens particle open angle glaucoma

Endoftalmitis postoperatoria por Candida parapsilosis / Post-operative endophthalmitis due to Candida parapsilosis

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