Gillian M. Clover

Indications for corneal transplantation in New Zealand: 1991-1999.

Purpose. To identify the indications for keratoplasty in patients supplied with donor tissue through the New Zealand National Eye Bank. Methods. Analysis of penetrating and lamellar keratoplasty data collected by the New Zealand National Eye Bank, Auckland, from 1991 to 1999. Results. In this 9-year period, donor material was supplied for 1370 corneal grafts; 1308 for penetrating keratoplasty, 26 for lamellar keratoplasty, and 36 for unspecified grafts. This accounts for a minimum of 85% of the penetrating keratoplasties performed in New Zealand from 1991 to 1999. The leading indications for penetrating keratoplasty were keratoconus (45.6%), pseudophakic or aphakic corneal edema (17.9%), regraft (8.7%), viral keratitis (7.3%), and trauma (5.5%). The average age of patients was 47.5 years (SD = 22.6) and age distribution was bimodal, with peaks in the 3rd and 8th decades. Keratoconus, regraft, and trauma were significantly more common as indicators for penetrating keratoplasty in male patients than female patients; however, pseudophakic or aphakic corneal edema was more common in female patients. Conclusion. The majority of transplantation surgery in New Zealand is performed using corneal tissue from the New Zealand National Eye Bank. In this representative study, keratoconus is the leading indicator for penetrating keratoplasty in New Zealand, accounting for a higher proportion than in any other published literature. The other indications, age distribution and gender differences correlate with previous reports. These findings suggest that keratoconus leading to transplantation may have increased prevalence in New Zealand.

Involvement of corneal nerves in the progression of keratoconus

Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowmans layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowmans layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte-epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to alpha-tubulin (nerves), alpha3beta1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowmans layer, and as the disease progressed and Bowmans layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.

Clinical manifestations of a unique X‐linked retinal disorder in a large New Zealand family with a novel mutation in CACNA1F, the gene responsible for CSNB2

Purpose: To describe the phenotype in a New Zealand family with an unusual severe X‐linked retinal disorder with a novel I745T mutation in CACNA1F, the gene responsible for incomplete congenital stationary night blindness (CSNB2).

The New Zealand National Eye Bank study 1991-2003: a review of the source and management of corneal tissue.

Purpose: To evaluate donor demographics and source, donor tissue processing and storage, biologic contamination, and the utilization and distribution of corneal tissue procured by the New Zealand National Eye Bank. Methods: As part of a prospective longitudinal study, the electronic records of the NZNEB for the 13-year period 1991-2003 were analyzed for each year with respect to donor demographics, donor source and cause of death, death-to-preservation interval, storage methods, endothelial assessment, biologic contamination, corneal tissue utilization, and distribution. Results: During the study period, 3221 corneas were retrieved from 1628 donors (69.8% male, 30.2% female), with the mean age of donors 59.4 years (SD 18.3 years) and range 4 to 95 years. No significant correlation was identified between donor age group (using 10-year intervals) and the proportion of corneas suitable for transplantation. Donors were procured from the Coroners service (67.6%), public hospitals, (23.5%) and multiorgan donors (7.1%). The most common causes of donor death were cardiovascular disease, trauma, and cerebrovascular disease. Average storage duration increased from 3.5 to 11.8 days when organ culture replaced hypothermic storage in 1992. Biologic contamination occurred in 5% of all donor corneas. The most common bacterial and fungal isolates were coagulase-negative staphylococci and Candida spp, respectively. A significant decrease in contamination rate over the years of the study was identified. Overall, 79.4% of corneal tissue procured was used for corneal transplantation (75.8% for penetrating keratoplasty, 2.1% for lamellar keratoplasty, and 1.5% for unspecified transplants), and 21.6% was discarded. Most common reasons for discarding tissue were biologic contamination, abnormal serology, and failed endothelial assessment. Conclusion: Analysis of the NZNEB database provides valuable information in relation to eye banking and corneal transplantation in New Zealand. Significant trends were identified in donor demographics, donor procurement source, improved donor tissue processing and storage, decreased biologic contamination, and increased utilization of corneal tissue.

Confocal imaging of the human keratocyte network using the vital dye 5-chloromethylfluorescein diacetate.

Background: The human corneal stroma consists of intercalated layers of collagen and keratocytes. These cells are known to maintain the stroma and aid in repair but it is likely they have other crucial roles throughout the cornea. The complexity of their anatomy is revealed in this study by ex vivo in situ images of the human keratocyte covering a range of ages.

Confocal imaging of the keratocyte network in porcine cornea using the fixable vital dye 5-chloromethylfluorescein diacetate.

This study reports on the combined use of an aldehyde fixable, cell viability fluoroprobe, 5-chloromethylfluorescein diacetate (CMFDA), confocal laser scanning microscopy and digital image reconstruction, to produce high resolution images of corneal keratocyte preparations in situ. The central region of freshly enucleated porcine corneae were removed and stained overnight at 4 degrees C with CMFDA. The tissue was washed, fixed, and frozen for cryosectioning in either a horizontal or antero-posterior orientation. Sections from anterior, central and posterior stroma were examined with a confocal microscope, and the digital images rendered as three-dimensional stereo reconstructions. Fluorescent CMFDA which completely permeated the cell bodies and extremes of the finest ramifying cell processes of all keratocytes provided exceptional high resolution images of the three morphologically distinct cell subpopulations at different levels of the stroma, and enabled improved characterisation of each cell type. Anteriorly was a thin, dense, non-lamellar network of keratocytes subjacent Bowmans membrane. In the central stroma, keratocytes were arranged in layers, the cell bodies had a flattened pyramidal or stellate shape, and the fine cell processes formed extensive distal ramifications. Immediately anterior to Descemets membrane a small subpopulation of keratocytes with large cell bodies and short branched processes was identified. Extensive and diverse cell-to-cell contacts were orientated in all stromal planes, including ramping cell bridges between keratocyte lamellae in the central stroma. The use of the cell viability dye CMFDA is feasible and valuable for enhancing the visibility of entire keratocyte population in the intact cornea. Diverse multi-directional cell processes and intercellular contacts throughout the keratocyte network suggest a strong capacity for direct communication and cohesion in the maintenance and repair of the stromal matrix. Keratocytes closely related to the epithelium and endothelium have unique morphologies which may relate to specialised functions of these interface cells.