Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Silk fibroin in ocular surface reconstruction : what is its potential as a biomaterial in ophthalmics?

Hardly a month goes by within the scientific literature without some new material “X” being reported as a suitable material on which to grow cell type “Y”, for the potential purpose of treating disease “Z”. Thus when fibroin, a protein found in silk, was first proposed as a biomaterial for cell growth [1] it joined a long list of other materials of both natural as well as synthetic origin. Nevertheless, in the second decade of the Asian Century it is perhaps befitting that a material of so much importance to the continent’s cultural and economic history, should become the focus of cutting-edge biomedical research. Sentiments aside, however, silk fibroin possesses quite a unique combination of properties which make it a promising candidate for repairing the eye and especially for treating damage to the cornea, the transparent window at the front of the eye.

GABAergic modification of myopic and hyperopic ocular tissue effects on scleral fibroblast growth

Purpose: Myopia is a common eye disorder affecting up to 90% of children in South East Asia and 30% of the population worldwide. Myopia of high severity is a leading cause of blindness around the world (4th to 5th most common). Changes and remodelling of the sclera i.e. increase cellular proliferation & increase protein synthesis within scleral cells (↑ scleral DNA) and thinning and lose of extracellular matrix of sclera (↓ scleral GAG synthesis) have been linked to myopic eye growth in animal models. Signals acting on the sclera are thought to originate in the retina, and are modulated by the retinal pigment epithelium (RPE) with limited evidence suggesting that the RPE can modify scleral cell growth in culture. However, the mechanism of retinal signal transmission and the role of posterior eye cup tissue, including the RPE, in mediating changes in scleral fibroblast growth during myopia development are unclear. Retinal transmitter systems are critically involved in pathways regulating eye growth, which ultimately lead to alterations in the sclera if eye size is to change. A dopaminergic agonist and muscarinic antagonists decrease the proliferation of scleral chondrocytes when co-cultured with chick’s retinal pigment epithelium (RPE). GABA receptors have recently been localised to chick sclera. We therefore hypothesised that posterior eye cup tissue from myopic eyes would stimulate and from hyperopic eyes would inhibit growth of scleral fibroblasts in vitro and that GABAergic agents could directly interact with scleral cells or indirectly modify the effects of myopic and hyperopic posterior eye cup tissue on scleral fibroblast growth. Method: Fibroblastic cells obtained from 8-day-old chick sclera were used to establish cell banks. Two major experiments were performed. Experiment 1: To determine if posterior eye cup tissues from myopic eye stimulates and hyperopic eye inhibits scleral cell proliferation, when co-cultured with scleral cells in vitro. This study comprised two linked experiments, i) monocular visual treatments of FDM (form-deprivation myopia), LIM (lens-induced myopia) and LIH (lens-induced hyperopia) with assessment of the effect of full punch eye cup tissue on DNA and GAG synthesis by cultured chick scleral fibroblasts, and ii) binocular visual treatments comprising LIM and LIH with assessment of the effect of individual layers of eye cup tissues (neural retina, RPE and choroid) on cultured chick scleral fibroblasts. Visual treatment was applied for 3 days. Experiment 2: To determine the direct interaction of GABA agents on scleral cell growth and to establish whether GABA agents modify the stimulatory/inhibitory effect of myopic and hyperopic posterior eye cup tissues on cultured scleral cell growth in vitro. Two linked experiments were performed. i) GABA agonists (muscimol and baclofen) and GABA antagonists (bicuculine (-), CGP46381 and TPMPA) were added to scleral cell culture medium to determine their direct effect on scleral cells. ii) GABAergic agents (agonists and antagonists) were administered to scleral fibroblasts co-cultured with posterior eye cup tissue (retina, RPE, retina/RPE, RPE/choroid). Ocular tissues were obtained from chick eyes wearing +15D (LIH) or -15D lenses (LIM) for 3 days. In both experiments, tissues were added to hanging cell culture insert (pore size 1.0ìm) placed over each well of 24 well plates while scleral cells were cultured in DMEM/F12, Glutamax (Gibco) plus 10% FBS and penicillin/streptomycin (50U/ml)) and fungizone (1.25ug/ml) (Gibco), at seeding density of 30,000 cells/well at the bottom of the well and allowed to grow for 3 days. Scleral cells proliferation rate throughout the study was evaluated by determining GAG and DNA content of scleral cells using Dimethylmethylene blue (DMMB) dye and Quant-iTTm Pico Green® dsDNA reagent respectively. Results and analysis: Based on DNA and GAG content, there was no significant difference in tissue effect of LIM and LIH eyes on scleral fibroblast growth (DNA: 8.4 ± 1.1μg versus 9.3 ± 2.3 μg, p=0.23; GAG: 10.13 ± 1.4 μg versus 12.67 ± 1.2 μg, F2,23=6.16, p=0.0005) when tissues were obtained from monocularly treated chick eyes (FDM or +15D lens or -15D lens over right eyes with left eyes untreated) and co-cultured as full punch. When chick eyes were treated binocularly with -15D lens (LIM) right eye and +15D lens (LIH) left eyes and tissue layers were separated, the retina from LIM eyes did not stimulate scleral cell proliferation compared to LIH eyes (DNA: 27.2 ± 6.7 μg versus 23.2 ± 1.5 μg, p=0.23; GAG: 28.1 ±3.7 μg versus 28.7 ± 4.2 μg, p=0.21). Similarly, the LIH and LIM choroid did not produce a differential effect based on DNA (LIM 46.9 ± 6.4 μg versus LIH 53.5 ± 4.7 μg, p=0.18), however the choroid from LIH eyes induced higher scleral GAG content than from LIM eyes (32.5 ± 6.7 μg versus 18.9 ± 1.2 μg, p=0.023). In contrast, the RPE from LIM eyes caused a significant increase in fibroblast proliferation whereas the RPE from LIH eyes was relatively inhibitory (72.4 ± 6.3 μg versus 27.9 ± 2.3 μg, F1, 6=69.99, p=0.0005). GAG data were opposite to DNA data e.g. the RPE from LIH eyes increased (33.7 ± 7.9 μg) while the RPE from LIM eyes decreased (28.2 ± 3.0 μg) scleral cell growth (F1, 6=13.99, p=0.010). Based on DNA content, GABA agents had a small direct effect on scleral cell growth; GABA agonists increased (21.4 ± 1.0% and 18.3 ± 1.0% with muscimol and baclofen, p=0.0021), whereas GABA antagonists decreased fibroblast proliferation (-23.7 ± 0.9% with bicuculine & CGP46381 and -28.1 ± 0.5% with TPMPA, p=0.0004). GABA agents also modified the effect of LIM and LIH tissues (p=0.0005).The increase in proliferation rate of scleral fibroblasts co-cultured with tissues (RPE, retina, RPE/retina and RPE/choroid) from LIM treated eyes was enhanced by GABA agonists (muscimol: 27.4 ± 1.2%, 35.8 ± 1.6%, 8.4 ± 0.3% and 11.9 ± 0.6%; baclofen: 27.0 ± 1.0%, 15.8 ± 1.5%, 16.8 ± 1.2% and 15.4 ± 0.4%, p=0.014) whereas GABA antagonists further reduced scleral fibroblasts growth (bicuculine: -52.5 ± 2.5%, -36.9 ± 1.4%, -37.5 ± 0.6% and -53.7 ± 0.9%; TPMPA: 57.3 ± 1.3%, -15.7 ± 1.2%, -33.5 ± 0.4% and -45.9 ± 1.5%; CGP46381: -51.9 ± 1.6%, -28.5 ± 1.5%, -25.4 ± 2.0% and -45.5 ± 1.9% respectively, p=0.0034). GAG data were opposite to DNA data throughout the experiment e.g. GABA agonists further inhibited while antagonists relatively enhanced scleral fibroblasts growth for both LIM and LIH tissue co-culture. The effect of GABA agents was relatively lower (p=0.0004) for tissue from LIH versus LIM eyes but was in a similar direction. There was a significant drug effect on all four tissue types e.g. RPE, retina, RPE/retina and RPE/choroid for both LIM and LIH tissue co-culture (F20,92=3.928, p=0.0005). However, the effect of GABA agents was greatest in co-culture with RPE tissue (F18,36=4.865, p=0.0005). Summary and Conclusion: 1) Retinal defocus signals are transferred to RPE and choroid which then exert their modifying effect on scleral GAG and DNA synthesis either through growth stimulating factors or directly interacting with scleral cells in process of scleral remodeling during LIM and LIH visual conditions. 2) GABAergic agents affect the proliferation of scleral fibroblasts both directly and when co-cultured with ocular tissues in vitro.

Effect of age on components of peripheral ocular aberrations

Purpose: To investigate the effect of age on the contributions of the anterior cornea and internal components to ocular aberrations in the peripheral visual field. Methods: Ocular aberrations were measured in 10 young emmetropes and 7 older emmetropes using a modified commercial Hartmann-Shack aberrometer across 42° x 32° of central visual field. Anterior corneal aberrations were estimated from anterior corneal topography using theoretical ray-tracing. Internal aberrations were calculated by subtracting anterior corneal aberrations from ocular aberrations. Results: Anterior corneal aberrations of young subjects were reasonably compensated by the internal aberrations, except for astigmatism for which the internal contribution was small out to the 21° field limit. The internal coma and spherical aberration of the older subjects were considerably smaller in magnitude than those of the young subjects such that the compensation for anterior corneal aberrations was poorer. This can be explained by age-related changes in the lens shape and refractive index distribution. Conclusion: oss of balance between anterior cornea and internal components of higher order aberrations with increasing age, found previously for on-axis vision, applies also to the peripheral visual field.

Diurnal variation of ocular biometrics under natural and defocused conditions

It is well known that a broad range of ocular anatomical and physiological parameters undergo significant diurnal variation. However, the natural diurnal variations that occur in the length of the human eye (axial length) and their underlying causes have been less well studied. Improvements in optical methods for the measurement of ocular biometrics now allow more precise and comprehensive measurements of axial length to be performed than has previously been possible. Research from animal models also suggests a link between diurnal axial length variations and longer term myopic eye growth, and that retinal image defocus can disrupt these diurnal rhythms in axial length. This research programme has examined the diurnal variations in axial length in young normal eyes, the contributing components and the influence of optical stimuli on these changes. In the first experiment, the normal pattern and consistency of the diurnal variations in axial length were examined at 10 different times (5 measurements each day, at ~ 3-hour intervals from ~ 9 am to ~ 9 pm) over 2 consecutive days on 30 young adult subjects (15 myopes, 15 emmetropes). Additionally, variations in a range of other ocular biometric measurements such as choroidal thickness, intraocular pressure, and other ocular biometrics were also explored as potential factors that may be associated with the observed variations in axial length. To investigate the potential influence of refractive error on diurnal axial length variations, the differences in the magnitude and pattern of diurnal variations in axial length between the myopic and emmetropic subjects were examined. Axial length underwent significant diurnal variation that was consistently observed over the 2 consecutive days of measurements, with the longest axial length typically occurring during the day, and the shortest at night. Significant diurnal variations were also observed in choroidal thickness, IOP and other ocular biometrics (such as central corneal thickness, anterior chamber depth and vitreous chamber depth) of the eye. Diurnal variations in vitreous chamber depth, IOP (positive associations) and choroidal thickness (negative association) were all significantly correlated with the diurnal changes in axial length. Choroidal thickness was found to fluctuate approximately in antiphase to the axial length changes, with the average timing of the longest axial length coinciding with the thinnest choroid and vice versa. There were no significant differences in the ocular diurnal variations associated with refractive error. Given that the diurnal changes in axial length could be associated with the changes in the eye’s optical quality, whether the optical quality of the eye also undergoes diurnal variation in the same cohort of young adult myopes and emmetropes over 2 consecutive days was also examined. Significant diurnal variations were observed only in the best sphere refraction (power vector M) and in the spherical aberration of the eye over two consecutive days of testing. The changes in the eyes lower and higher order ocular optics were not significantly associated with the diurnal variations in axial length and the other measured ocular biometric parameters. No significant differences were observed in the magnitude and timing of diurnal variations in lower-order and higher-order optics associated with refractive error. Since the small natural fluctuations in the eye’s optical quality did not appear to be sufficient to influence the natural diurnal fluctuations in ocular biometric parameters, in the next experiment, the influence of monocular myopic defocus (+1.50 DS) upon the normal diurnal variations in axial length and choroidal thickness of young adult emmetropic human subjects (n=13) imposed over a 12 hour period was examined. A series of axial length and choroidal thickness measurements (collected at ~3 hourly intervals, with the first measurement at ~9 am and the final measurement at ~9 pm) were obtained over three consecutive days. The natural diurnal rhythms (Day 1, no defocus), diurnal rhythms with monocular myopic defocus (Day 2, +1.50 DS spectacle lens over the right eye), and the recovery from any defocus induced changes (Day 3, no defocus) were examined. Significant diurnal variations over the course of the day were observed in both axial length and choroidal thickness on each of the three measurement days. The introduction of monocular myopic defocus led to significant reductions in the mean amplitude of diurnal change, and phase shifts in the peak timing of the diurnal rhythms in axial length and choroidal thickness. These defocus induced changes were found to be transient in nature and returned to normal the day following removal of the defocus. To further investigate the influence of optical stimuli on human diurnal rhythms, in the final experiment, the influence of monocular hyperopic defocus on the normal diurnal rhythms in axial length and choroidal thickness was examined in young adult emmetropic subjects (n=15). Similar to the previous experiment, the natural diurnal rhythms (Day 1, no defocus), diurnal rhythms with monocular hyperopic defocus (Day 2, -2.00 DS spectacle lens over the right eye), and the recovery from any defocus induced changes (Day 3, no defocus) were examined over three consecutive days. Both axial length and choroidal thickness underwent significant diurnal variations on each of the three days. The introduction of monocular hyperopic defocus resulted in a significant increase in the amplitude of diurnal change, but no change in the peak timing of diurnal rhythms in both parameters. The ocular changes associated with hyperopic defocus returned to normal, the day following removal of the defocus. This research has shown that axial length undergoes significant diurnal variation in young adult human eyes, and has shown that the natural diurnal variations in choroidal thickness and IOP are significantly associated, and may underlie these diurnal fluctuations in axial length. This work also demonstrated for the first time that exposing young human eyes to monocular myopic and hyperopic defocus leads to a significant disruption in the normal diurnal rhythms of axial length and choroidal thickness. These changes in axial length with defocus may reflect underlying mechanisms in the human eye that are involved in the regulation of longer term eye growth.

In the blink of an eye : the circadian effects on ocular and subjective indices of driver sleepiness

Driver sleepiness contributes substantially to fatal and severe crashes and the contribution it makes to less serious crashes is likely to as great or greater. Currently, drivers’ awareness of sleepiness (subjective sleepiness) remains a critical component for the mitigation of sleep-related crashes. Nonetheless, numerous calls have been made for technological monitors of drivers’ physiological sleepiness levels so drivers can be ‘alerted’ when approaching high levels of sleepiness. Several physiological indices of sleepiness show potential as a reliable metric to monitor drivers’ sleepiness levels, with eye blink indices being a promising candidate. However, extensive evaluations of eye blink measures are lacking including the effects that the endogenous circadian rhythm can have on eye blinks. To examine the utility of ocular measures, 26 participants completed a simulated driving task while physiological measures of blink rate and duration were recorded after partial sleep restriction. To examine the circadian effects participants were randomly assigned to complete either a morning or an afternoon session of the driving task. The results show subjective sleepiness levels increased over the duration of the task. The blink duration index was sensitive to increases in sleepiness during morning testing, but was not sensitive during afternoon testing. This finding suggests that the utility of blink indices as a reliable metric for sleepiness are still far from specific. The subjective measures had the largest effect size when compared to the blink measures. Therefore, awareness of sleepiness still remains a critical factor for driver sleepiness and the mitigation of sleep-related crashes.

The Norfolk Island Eye Study (NIES) : rationale, methodology and distribution of ocular biometry (Biometry of the Bounty)

Aim: To describe the recruitment, ophthalmic examination methods and distribution of ocular biometry of participants in the Norfolk Island Eye Study, who were individuals descended from the English Bounty mutineers and their Polynesian wives. Methods: All 1,275 permanent residents of Norfolk Island aged over 15 years were invited to participate, including 602 individuals involved in a 2001 cardiovascular disease study. Participants completed a detailed questionnaire and underwent a comprehensive eye assessment including stereo disc and retinal photography, ocular coherence topography and conjunctival autofluorescence assessment. Additionally, blood or saliva was taken for DNA testing. Results: 781 participants aged over 15 years were seen (54% female), comprising 61% of the permanent Island population. 343 people (43.9%) could trace their family history to the Pitcairn Islanders (Norfolk Island Pitcairn Pedigree). Mean anterior chamber depth was 3.32mm, mean axial length (AL) was 23.5mm, and mean central corneal thickness was 546 microns. There were no statistically significant differences in these characteristics between persons with and without Pitcairn Island ancestry. Mean intra-ocular pressure was lower in people with Pitcairn Island ancestry: 15.89mmHg compared to those without Pitcairn Island ancestry 16.49mmHg (P = .007). The mean keratometry value was lower in people with Pitcairn Island ancestry (43.22 vs. 43.52, P = .007). The corneas were flatter in people of Pitcairn ancestry but there was no corresponding difference in AL or refraction. Conclusion: Our study population is highly representative of the permanent population of Norfolk Island. Ocular biometry was similar to that of other white populations. Heritability estimates, linkage analysis and genome-wide studies will further elucidate the genetic determinants of chronic ocular diseases in this genetic isolate.

Prevalence of chronic ocular diseases in a genetic isolate : the Norfolk Island Eye Study (NIES)

Purpose: Over 40% of the permanent population of Norfolk Island possesses a unique genetic admixture dating to Pitcairn Island in the late 18 th century, with descendents having varying degrees of combined Polynesian and European ancestry. We conducted a population-based study to determine the prevalence and causes of blindness and low vision on Norfolk Island. Methods: All permanent residents of Norfolk Island aged ≥ 15 years were invited to participate. Participants completed a structured questionnaire/interview and underwent a comprehensive ophthalmic examination including slit-lamp biomicroscopy. Results: We recruited 781 people aged ≥ 15, equal to 62% of the permanent population, 44% of whom could trace their ancestry to Pitcairn Island. No one was bilaterally blind. Prevalence of unilateral blindness (visual acuity [VA] < 6/60) in those aged ≥ 40 was 1.5%. Blindness was more common in females (P=0.049) and less common in people with Pitcairn Island ancestry (P<0.001). The most common causes of unilateral blindness were age-related macular degeneration (AMD), amblyopia, and glaucoma. Five people had low vision (Best-Corrected VA < 6/18 in better eye), with 4 (80%) due to AMD. People with Pitcairn Island ancestry had a lower prevalence of AMD (P<0.001) but a similar prevalence of glaucoma to those without Pitcairn Island ancestry. Conclusions: The prevalence of blindness and visual impairment in this isolated Australian territory is low, especially amongst those with Pitcairn Island ancestry. AMD was the most common cause of unilateral blindness and low vision. The distribution of chronic ocular diseases on Norfolk Island is similar to mainland Australian estimates.

Risk factors for ocular surface squamous neoplasia recurrence after treatment with topical mitomycin C and interferon alpha-2b

Purpose To determine the rate of recurrence and associated risk factors following the use of mitomycin C (MMC) and/or interferon alpha-2b (IFN) for management of non-invasive ocular surface squamous neoplasia (OSSN). Design Retrospective non-comparative interventional case series. Methods Clinical practice setting of 135 patients treated consecutively with topical MMC (0.4 mg/mL) and/or IFN (1 million units/mL) for OSSN observed for clinical recurrence. Results Clinical recurrences were diagnosed in 19 of 135 (14.1%) eyes following topical treatment. The mean time to recurrence was 17.2 months (range 4 - 61) with 14 (73.7%) recurring within a two year period. There was no greater risk of recurrence identified for variables including lesion size, lesion location, gender, age, treatment type or duration. Post-hoc log-Rank pairwise comparisons revealed that lesions initially treated using surgery alone had significantly reduced time to recurrence (21.1 ± 5.6 months) compared to previous topical treatment with MMC (with or without surgery) (29.6 ± 4.7 months) (p = 0.04) and primary OSSN (23.2 ± 1.8 months) (p = 0.09). Conclusions Topical MMC and IFN are an effective treatment modality for a wide range of non-invasive OSSN. Topical therapy avoids the morbidity of excisional surgery with equivalent or reduced recurrence rates and should be considered as primary therapy.

Through-focus performance with multifocal contact lenses : effect of binocularity, pupil diameter and inherent ocular aberrations

Purpose to evaluate the effects of the wearer’s pupil size and spherical aberration on visual performance with centre-near, aspheric multifocal contact lenses (MFCLs). The advantage of binocular over monocular vision was also investigated. Methods Twelve young volunteers, with an average age of 27±5 years, participated in the study. LogMAR Visual Acuity (VA) was measured under cycloplegia for a range of defocus levels (from +3.0 to -3.0D, in 0.5D steps) with no correction and with three aspheric MFCLs (Air Optix Aqua Multifocal, Ciba Vision, Duluth, GA, US) with a centre-near design, providing correction for “Low”, “Med” and “High” near demands. Measurements were performed for all combinations of the following conditions: i) artificial pupils of 6mm and 3mm diameter, ii) binocular and monocular (dominant eye) vision. Depth-of-focus (DOF) was calculated from the VA vs. defocus curves. Ocular aberrations under cycloplegia were measured using iTrace. Results VA at -3.0D defocus (simulating near performance) was statistically higher for the 3mm than for the 6mm pupil (p=0.006), and for binocular rather than for monocular vision (p<0.001). Similarly, DOF was better for the 3mm pupil (p=0.002) and for binocular viewing conditions (p<0.001, ANOVA). Both VA at –3.0D defocus and DOF increased as the “addition” of the MFCL correction increased. Finally, with the centre-near MFCLs a linear correlation was found between VA at –3.0D defocus and the wearer’s ocular spherical aberration (R2=0.20 p<0.001 for 6mm data), with the eyes exhibiting the higher positive spherical aberration experiencing lower VAs. By contrast, no correlation was found between VA and spherical aberration at 0.00D defocus (distance vision). Conclusions Both near VA and depth-of-focus improve with these MFCLs, with the effects being more pronounced for small pupils and binocular than for monocular vision. Coupling of the wearer’s ocular spherical aberration with the aberration profiles provided by MFCLs affects their functionality.

2. Contact lens care and ocular surface homeostasis

Moderator Opening The early focus of contact lens wear and ocular health was on oxygen delivery. However, as we learn more about how the eye works, and investigate how the contact lens interacts with the cornea, the role of the tear film has risen in prominence. A healthy tear film is critical for normal ocular homeostasis, and abnormalities of the tear film are the primary cause of dry eye. In order to improve patient eye health and comfort during lens wear, we need to further elucidate the relationship among contact lenses, contact lens solutions, the tear film, and the corneal epithelium, and find ways to maintain homeostasis of the ocular surface. In this section, we review the latest data and opinions on this complex relationship between contact lenses and lens care solutions

Ocular surface health with contact lens wear

Eye care practitioners (ECPs) would tend to agree that wearing contact lenses increases the risk for infection, but millions of patients are still fitted with lenses every year because ECPs feel that the risk is manageable and that their patients' eye health can be protected. The Fusarium and Acanthamoeba keratitis outbreaks of years past were a wake-up call to manufacturers, ECPs, and regulatory agencies that risk cannot be managed without diligence, and that the complex relationship between contact lens materials, contact lens solutions, and compliance needs to be better understood in order to optimize the efficacy of contact lens care and improve care guidelines.

The TFOS International Workshop on Contact Lens Discomfort: report of the contact lens interactions with the ocular surface and adnexa subcommittee

The report of this subcommittee concerns the impact of contact lenses (CLs) on the ocular surface, with a particular emphasis on CL discomfort (CLD). We define the ocular surface, its regional anatomy, and the physiological responses of each region to CL wear.