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Objectives:Explain the etiology of pellucid marginal corneal degeneration.Describe the appropriate evaluation of pellucid marginal corneal degeneration.Outline the treatment modalities for patients with pellucid marginal corneal degeneration.Summarize the importance of collaboration and communication amongst the interprofessional team to enhance the care for patients with pellucid marginal corneal degeneration.Access free multiple choice questions on this topic.

Corneal keratocytes from six New Zealand White Rabbits were isolated and cultured until Passage 1. The proliferative effects of EBN on corneal keratocytes were determined by MTT assay in serum-containing medium (FDS) and serum-free medium (FD). Keratocytes phenotypical changes were morphologically assessed and gene expression of aldehyde dehydrogenase (ALDH), collagen type 1 and lumican were determined through RT-PCR.

The cornea comprises of three distinct layers: the epithelium, the stroma and the endothelium. Each layer provides specific properties which are crucial to the optimal functionality of the cornea in normal vision while acting as a protective barrier from external environment [1]. The corneal stroma makes up 90% of the corneal volume and is filled with keratocytes bound by extracellular matrix which forms the structural backbone of the cornea [2, 3]. Keratocytes are mesenchymal-derived cells of the corneal stroma responsible for the synthesis and maintenance of the extracellular matrix (ECM) components [4]. Normal, quiescent keratocytes residue between collagen lamellae of the corneal stroma as a sparse population of flattened-cells [5], connecting to one other through a network of extensive processes [6]. The keratocytes have low cell turnover with undetectable cell remodelling overtime [5, 7, 8]. These homeostatic characteristics are important for corneal transparency. During deep injuries in which the epithelial basement membrane is disrupted, these keratocytes will change their morphological characteristics into 'activated' phenotype which resembles the fibroblasts: fusiform-shaped with multiple nucleoli and lack of cytoplasmic granules [9]. The activation of fibroblasts from quiescent keratocytes is also observed in cell culture models which show similar phenotypical changes of keratocytes during wounding. This was done by adding serum into the culture medium and by further passaging these cells, which were initially maintained in a serum-free condition [3, 7].

The 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the effects of EHMG-coded EBN on corneal keratocytes' proliferative capacity and viability. The cells were seeded overnight in 96-well cell culture plate (Cellstar, Greiner Bio-one, Germany) with the seeding density of 5 × 103 cells cm-2. Several concentrations of EHMG-coded EBN using dilution factor of 2 were added in the following day using 2 different media, serum-containing media (FDS) and serum-free media (FD). The cells were incubated at 37°C in a humidified incubator 5% CO2 atmosphere for 72 hours until confluence.

Viability of corneal keratocytes cultured in serum-containing medium (FDS, 1A), serum-free medium (FD, 1B) supplemented with EBN ranging from 0.05% to 100% and comparison on cell viability between the two groups (1C). Significant differences were noted at 0.05%, 0.1%, 75% and 100% in FDS (marked with *). No significant difference was found in keratocytes cultured in FD. Cells cultured in FDS showed significantly higher viability compared to groups in FD. # denotes significant difference (p < 0.05) between groups. Values were tested using Student t-test and expressed as mean ± SEM, n = 6.

Microscopic examination of cell morphology on corneal keratocytes showed higher mitotic figures in serum-containing media (Figure 2A & 2B) as compared to serum-free media (Figure 2C & 2D). This was most apparent in cells cultured in FDS + 0.05% EBN medium (Figure 2B). The corneal keratocytes exhibited typical fibroblast-like feature with fusiform shape in all media, particularly seen in serum-containing media. Similar morphology was observed in corneal keratocytes cultured in serum-free medium (Figure 2C & 2D), but with fewer corneal keratocytes. More importantly, corneal keratocytes did not show any abnormal morphological changes when cultured in the EBN-containing media.

Phase contrast micrographs under magnification 50 × showing the morphology of corneal keratocytes cultured in different media; A) Serum-containing medium, FDS, B) FDS plus 0.05% EBN, C) Serum-free medium, FD, D) FD plus 0.05% EBN; at Day 1, passage 1. Supplementation of 0.05% EBN was able to promote higher density of cells.

The real time PCR data showed a higher expression level in the serum-containing groups (with or without supplementation of EBN) compared to serum-free groups (with or without supplementation of EBN). This was probably because of the presence of serum which contains multiple growth factors to promote cells' proliferation and total RNA expression. In this study, the cultured corneal keratocytes were supplemented with 0.05% EBN concentration, since it was the optimal concentration for cell proliferation derived from the MTT assay. In collagen type 1, the expression level was significantly lower in the serum-free groups (with or without EBN) compared to serum-containing groups (p < 0.05) (Figure 3A). For aldehyde dehydrogenase (ALDH), both cells cultured with 0.05% EBN with or without the addition of serum (FDS+EBN and FD+EBN) showed a higher expression level compared to cells cultured in serum-free medium (FD) and serum-containing medium (FDS) alone (Figure 3B) with the significant difference observed in the serum-free groups only (p = 0.01). The expression level of lumican was found to be significantly higher in the EBN-supplemented media in both groups (FD+EBN and FDS+EBN) compared to FD and FDS (p < 0.05) [Figure 3C].

Quantitative gene expressions of cultured rabbit corneal keratocytes. The expression values for (A) Collagen Type 1, (B) ALDH and (C) Lumican relative to the expression values of GAPDH as the internal control. Higher expression level was showed in serum-containing groups (FDS, FDS+EBN) compared to serum-free groups (FD, FD+EBN). * denotes significant difference (p < 0.05) in the same group. # denotes significant difference (p < 0.05) between groups. Values were tested using Student t-test and expressed as mean ± SEM, n = 6

We also observed the corneal keratocytes displayed morphological features of 'activated' fibroblasts when cultured in serum-containing media added with 0.05% EBN. Similar morphological features were observed in keratocytes cultured in serum-free media because of the effect of pasaging. It was important to ensure that EBN besides inducing proliferation of corneal cells was also capable to maintain their phenotypes and functionality by synthesizing and organizing stromal constituents crucial in maintaining corneal transparency. This was further confirmed by the higher functional gene expression of collagen type 1, ALDH and lumican on cultured corneal keratocytes in 0.05% EBN supplemented medium (Figure 4). Collagen Type I is the major structural collagen of the cornea [49]. On the other hand, ALDH catalyzed the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids, with some ALDH have been identified as corneal crystallins which contribute to the protective and refractive properties of the cornea [50]. The rabbit abundantly expresses ALDHs in its cornea [51]. Lumican is essential for normal cornea morphogenesis during embryonic development and maintenance of corneal topography in adults. Lumican may have additional biological functions, such as modulation of cell migration and epithelium-mesenchyme transition in wound healing and regulating collagen fibrillogenesis [51].

The utilization of scleral lenses as drug delivery devices has been demonstrated.30 Scleral lenses have been reported as drug delivery systems to deliver antibiotics,31,32 anti-vascular endothelial growth factor (anti-VEGF) agents,33-35 and stem cells with compounded products.36 The large fluid reservoir provides a protected environment in which the corneal surface is continuously bathed in preservative-free fluid. These lenses are inherently stable to provide continuous ocular penetration of a drug. Scleral lenses exhibit minimal tear exchange after lens settling; tear exchange has been reported at 0.2% per minute of wear.37 If a topical drug is applied over a scleral lens, there is minimal accumulation in the post-lens fluid reservoir. Alternatively, drugs applied in the reservoir prior to lens application should remain for the majority of the scleral lens wearing time. The replenishment of fluid under a scleral lens requires more than 8 hours of lens wear. A study evaluated post-lens tear dynamics at two different time points, during and after scleral lens wear with post-lens tear fluorescence.38 Approximately one-third of the subjects had no tear flow into the post-lens fluid reservoir after 5 hours of lens wear.38 Scleral lenses may function as an ideal drug delivery system to provide a therapeutic level of drug at the desired target tissue, with minimal variability and have a duration appropriate for the therapeutic indication.30

A number of studies have indicated keratoconic corneas show signs of increased activity by proteases,[19] a class of enzymes that break some of the collagen cross-linkages in the stroma, with a simultaneous reduced expression of protease inhibitors.[24] Other studies have suggested that reduced activity by the enzyme aldehyde dehydrogenase may be responsible for a build-up of free radicals and oxidising species in the cornea.[25] Whatever the pathogenetical process, the damage caused by activity within the cornea likely results in a reduction in its thickness and biomechanical strength. At an ultrastructural level the weakening of the corneal tissue is associated with a disruption of the regular arrangement of the collagen layers and collagen fibril orientation.[26] While keratoconus is considered a noninflammatory disorder, one study shows wearing rigid contact lenses by people leads to overexpression of proinflammatory cytokines, such as IL-6, TNF-alpha, ICAM-1, and VCAM-1 in the tear fluid.[27] 2b1af7f3a8