Date Published: June 11, 2018
Publisher: Public Library of Science
Author(s): André Vicente, Berit Byström, Mona Lindström, Ulf Stenevi, Fátima Pedrosa Domellöf, Alexander V. Ljubimov.
To study structural changes in naïve and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK).
Two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor α11 integrin and laminin α3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, α-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naïve ARK, KLAL ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous.
Given the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run.
Aniridia is a rare disorder caused by mutations in the PAX6 gene, essential for the development of the eye. The age-specific prevalence in a Swedish cohort of cases < 20 years of age is 1:47000 . The developmental disturbances associated with aniridia affect several different structures of the eye, including the cornea, anterior chamber, iris, lens, retina and optic nerve  and one of the most visually significant consequences is aniridia related keratopathy (ARK). The present study comprised two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time (cases A and B), one corneal button from an ARK patient that had undergone a KLAL (case C), two corneal buttons from ARK patients who had previously undergone centered (case D) or decentered (case E) transplantation and were now retransplanted and two adult healthy donor control corneas; one (74-year-old male, who had previously undergone cataract surgery), obtained immediately after evisceration due to an ocular tumor, and another (82-year-old male, who had not undergone any ocular surgery) obtained from a donor who had chosen, when alive, to donate his corneas for research post-mortem. This control cornea was collected postmortem within the time frame of the European guidelines for tissue use for human transplantation, guaranteeing the viability of the tissue samples. Control corneas were not age-matched with the patients. However, it is well known that corneas normally maintain their transparency and structure throughout life, irrespective of age and that age does not affect the viability of corneal buttons for transplantation [27, 28]. The diagnosis of aniridia was based on typical clinical characteristics [5, 29] and genetic analysis had also been performed in cases D and E and identified a heterozygous mutation in the PAX6 gene. All five aniridia corneas had a rather similar pathological appearance in hematoxylin-eosin sections (Fig 2A–2E). In both naïve ARK, KLAL ARK and transplanted ARK corneas, the epithelium was irregular with large variation in cell size and form and with varying thickness throughout consecutive sections, in contrast to the epithelium with regular thickness and morphology present in control corneas (Fig 2). The epithelial basement membrane of all aniridia corneas was fragmented and appeared to be absent in long segments in which the Bowman’s layer could not be identified (Fig 2). A thick subepithelial pannus was present in the anterior stroma (Fig 2A–2E, arrows). In the two naïve ARK corneas, the pannus comprised less than half of the central corneal thickness in case A and approximately half of the thickness in case B (Fig 2). In the KLAL ARK (case C) and in the centered transplanted ARK (case D) corneas, the subepithelial pannus accounted for approximately half of the corneal thickness. In the decentered transplanted ARK cornea (case E) it comprised less than half of the corneal thickness (Fig 2). Importantly, the subepithelial pannus was also present in parts of the periphery of corneal buttons of both naïve ARK, KLAL ARK and transplanted ARK corneas. The endothelium and Descemet´s membrane appeared normal in all ARK corneas. This is the first study to investigate both naïve corneas of aniridia patients with severe ARK, an ARK cornea from a patient that had previously undergone a keratolimbal autograft transplant but without success in restoring a normal ocular surface; and transplanted corneal buttons that were initially from healthy donors but had been transplanted to aniridia patients that subsequently developed severe ARK and required re-transplantation. Decentered keratolimbal transplantation is considered to be the best first-line surgical treatment for advanced ARK [5, 11], as centered penetrating keratoplasty alone has a failure rate of 64–100% . KLAL, in particular the limbal allograft transplantation with the modified protocol described by Holland and co-workers  is also considered a valid option for patients with corneal pathologies that primarily involve limbal stem cells, such as ARK [15, 16]. Nevertheless, both decentered keratolimbal transplantation and KLAL are also associated with limited success in aniridia, with Boston keratoprosthesis being the last surgical treatment option available. Our samples from failed decentered corneal transplantations and KLAL constitute, therefore, an opportunity to further evaluate the pathophysiology associated with the recurrence of ARK. Source: http://doi.org/10.1371/journal.pone.0198822