Intraocular methotrexate for epithelial downgrowth: long-term outcomes in a multicentre case series.

BACKGROUND/AIMS: Sheet-like type of epithelial downgrowth (EDG) is not easily amenable to surgical excision. We describe long-term outcomes in patients with EDG treated with intraocular methotrexate (MTX). METHODS: This is a retrospective, multicentric case series including 10 eyes (nine patients) treated with intraocular MTX for sheet-like EDG. Relevant ocular history, previous EDG treatments, MTX injection regimen, long-term outcomes and complications are reported. RESULTS: All cases were associated with intraocular surgery. Most patients were treated with 400 µm/0.1 mL MTX injections with a starting frequency of two times per week or weekly injections. Mean and SD number of injections per eye was 16±13 injections and duration of follow-up was 54±36 months (range: 7-120 months). Eradication of EDG was achieved in seven eyes of which one required a second MTX treatment course to achieve eradication, while clinical resolution with recurrence was observed in two. One treatment failure occurred despite eight weekly injections which slowed but did not halt EDG progression; the patient later requested that treatments be stopped given difficulty to come to follow-ups. Surface epitheliopathy developed in eight patients and was used to titrate MTX treatment. Six patients also developed endothelial failure. CONCLUSION: We report the largest case series of diffuse, sheet-like EDG treated with intraocular MTX with follow-ups up to 10 years. Intraocular MTX may be used effectively to achieve eradication of EDG in cases where surgery is not amenable. However, further recommendations to guide treatment remain warranted.

Anterior segment optical coherence tomography angiography in the assessment of ocular surface lesions.

PURPOSE: Describe the utility of anterior segment optical coherence tomography angiography (AS-OCTA) to assess ocular surface lesions. METHODS: Retrospective, case-control study of 10 eyes of 9 patients with malignant lesions and 23 eyes of 22 patients with benign lesions. Lesions included 13 epithelial, 10 pigmented and 10 lymphoid lesions. Graders performed an average of 3 depth and diameter measurements of peri-lesional vessels entering each lesion on AS-OCTA. Statistical models to assess differences between groups accounted for bilateral eye inclusion and lesion thickness (on AS-OCT). Receiver operating characteristic (ROC) curve and area under the curve (AUC) were performed for each parameter. RESULTS: In the benign and malignant groups, age was 49.5 ± 22.4 and 64.3 ± 10.6 years (p = 0.145) with 45% males and 55% males (p = 0.458), in their respective groups. AS-OCTA showed greater peri-lesional vessel depth and diameter in malignant lesions (315.2 ± 73.0 µm, p < 0.001 and 76.4 ± 18.2 µm, p < 0.001; respectively) compared to benign lesions (199.4 ± 34.1 µm and 44.0 ± 9.4 µm, respectively). Malignant lesions showed deep and dilated peri-lesional vessels, which may represent feeder vessels. Vessel depth showed AUC = 0.980, 90.9% sensitivity and 100.0% specificity with a 236.5 µm cutoff. Vessel diameter showed AUC = 0.960, 100.0% sensitivity and 88.9% specificity with a 53.9 µm cutoff. CONCLUSION: AS-OCTA shows greater peri-lesional vessel depth and diameter of malignant lesions compared to benign lesions. This imaging modality provides novel and non-invasive functional vascular parameters that can potentially aid the assessment of ocular surface lesions.

Effect of human tears on acanthamoeba-induced cytopathic effect.

OBJECTIVE: To determine whether tears of healthy individuals provide protection against Acanthamoeba-induced cytopathic effect (CPE) in vitro. METHODS: Acanthamoebae were added to confluent cultures of corneal epithelium in 24-well plates, and co-cultures were incubated overnight in a serum-free medium containing varying amounts of tears or immunoglobulin A (IgA)-depleted tears. At the end of the incubation period, the cells were stained with Giemsa, and the extent of target cell damage (ie, CPE) was quantified. RESULTS: Acanthamoebae produced extensive CPE. The presence of even a low concentration of tears (10 microL of undiluted tears per milliliter of media) almost completely inhibited Acanthamoeba-induced CPE. The CPE was inhibited by pretreatment of the parasites with tears. In contrast, the pretreatment of host cells with tears was not protective. This finding suggests that the target of the inhibitory factor is the parasite. IgA-depleted tears also inhibited Acanthamoeba-induced CPE, albeit with a lower potency than total tears. CONCLUSION: In addition to known IgA-dependent protective factors, human tears contain factors that inhibit Acanthamoeba-induced CPE independently of IgA. Clinical Relevance Identification and characterization of factors that protect against Acanthamoeba-induced CPE should help in the development of novel, rationally designed strategies to manage and protect against keratitis.

Galectin-7 as a potential mediator of corneal epithelial cell migration.

OBJECTIVE: To assess the role of a carbohydrate-binding protein, galectin-7, in reepithelialization of corneal wounds. METHODS: Transepithelial excimer laser ablations were performed on mouse corneas, and the wounds were allowed to partially heal in vivo for 18 to 22 hours. At the end of the healing period, expression levels of galectin-7 messenger RNA and protein were analyzed using semiquantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemical localization studies. To determine the effect of exogenous galectin-7 on reepithelialization of corneal wounds, corneas with 2-mm alkali burn wounds were allowed to partially heal in vitro for 20 to 24 hours in serum-free media in the presence or absence of recombinant galectin-7. At the end of the healing period, the wound areas were photographed and quantified. RESULTS: Expression of galectin-7 messenger RNA and protein was markedly up-regulated in the corneal epithelium after injury. Exogenous galectin-7 stimulated reepithelialization of corneal wounds. The stimulatory effect of galectin-7 on corneal epithelial wound closure was specifically inhibited by a competing sugar, beta-lactose, but not by an irrelevant disaccharide, sucrose. CONCLUSIONS: Galectin-7 has the potential to mediate corneal epithelial cell migration and reepithelialization of wounds. CLINICAL RELEVANCE: These findings have broad implications for developing novel, galectin-based, therapeutic strategies for treatment of nonhealing corneal epithelial defects.

Galectins-3 and -7, but not galectin-1, play a role in re-epithelialization of wounds.

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study, using cornea as a model, we demonstrate for the first time the importance of carbohydrate-binding proteins galectins-3 and -7 in re-epithelialization of wounds. In two different models of corneal wound healing, re-epithelialization of wounds was significantly slower in galectin-3-deficient (gal3(-/-)) mice compared with wild-type (gal3(+/+)) mice. In contrast, there was no difference in corneal epithelial wound closure rates between galectin-1-deficient and wild-type mice. Quantitation of the bromodeoxyuridine-labeled cells in gal3(+/+) and gal3(-/-) corneas revealed that corneal epithelial cell proliferation rate is not perturbed in gal3(-/-) corneas. Exogenous galectin-3 accelerated re-epithelialization of wounds in gal3(+/+) mice but, surprisingly, not in the gal3(-/-) mice. Gene expression analysis using cDNA microarrays revealed that healing corneas of gal3(-/-) mice contain markedly reduced levels of galectin-7 compared with those of gal3(+/+) mice. More importantly, unlike galectin-3, galectin-7 accelerated re-epithelialization of wounds in both gal3(-/-) and gal3(+/+) mice. In corresponding experiments, recombinant galectin-1 did not stimulate the corneal epithelial wound closure rate. The extent of acceleration of re-epithelialization of wounds with both galectin-3 and galectin-7 was greater than that observed in most of the published studies using growth factors. These findings have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Detection of differentially expressed genes in healing mouse corneas, using cDNA microarrays.

PURPOSE: To identify differentially expressed genes in healing mouse corneas by using cDNA microarrays. METHODS: Transepithelial excimer laser ablations were performed on mouse corneas, and the wounds were allowed to heal partially in vivo for 18 to 22 hours. Total RNA was isolated from both normal and healing corneas and was used for synthesis of cDNA probes. 33P-labeled exponential cDNA probes were hybridized to mouse cDNA nylon arrays. RESULTS: Of the 1176 genes on the nylon arrays, the expression of 37 was upregulated and that of 27 was downregulated more than fivefold in the healing corneas compared with the normal, uninjured corneas. Interleukin (IL)-1beta, laminin-5, and thrombospondin-1, which have been shown to be upregulated in healing corneas, were all found to be induced in the corneas in response to excimer laser treatment. Many genes were identified for the first time to be differentially regulated during corneal wound healing. Among the upregulated genes were intercellular adhesion molecule (ICAM)-1, macrophage inflammatory proteins, suppressors of cytokine signaling proteins (SOCS), IL-10 receptor, and galectin-7. Among the downregulated genes were connexin-31, a gap junction protein; ZO1 and occludin, tight junction proteins; and Smad2, a key component in the TGFbeta signaling pathway. Microarray data were validated on a limited number of genes by semiquantitative RT-PCR and Western blot analyses. CONCLUSIONS: Gene array technology was used to identify for the first time many genes that are differentially regulated during corneal wound healing. These differentially expressed genes have not previously been investigated in the context of wound healing and represent novel factors for further study of the mechanism of wound healing.

Astigmatism and LASIK.

Although laser in situ keratomileusis (LASIK) enjoys a high success rate, postoperative residual or induced astigmatism may limit uncorrected visual acuity and cause starbursts and glare at night. Irregular astigmatism can also cause loss of best-corrected visual acuity, monocular diplopia, and ghosting of images. Astigmatism may be measured by keratometry and refraction, while corneal topographic techniques help to define irregular astigmatism, in particular. Further information may be obtained regarding induced higher-order aberrations with aberrometry. Because astigmatism has both direction and magnitude, its analysis is more complex than that of the spherical component of the treatment. There are multiple approaches to the analysis of surgically induced astigmatism, including vector analysis, conversion to a Cartesian coordinate system, matrix formalism, and linear optics. Both excimer laser and incisional techniques may be used to correct astigmatism after LASIK, but the treatment of irregular astigmatism requires selective zonal ablation techniques or customized corneal ablations, using topographic or wavefront derived data.