Fibroin-based materials support co-cultivation of limbal epithelial and stromal cells

The silk protein fibroin (Bombyx mori) provides a potential substrate for use in ocular tissue reconstruction. We have previously demonstrated that transparent membranes produced from fibroin support cultivation of human limbal epithelial (HLE) cells (Tissue Eng A. 14(2008)1203-11). We extend this body of work to studies of human limbal stromal cell (HLS) growth on fibroin in the presence and absence of serum. Also, we investigate the ability to produce a bi-layered composite scaffold of fibroin with an upper HLE layer and lower HLS layer.

Peripapillary choroidal thickness in childhood

Changes in the thickness of the invivo peripapillary choroid have been documented in a range of ocular conditions in adults; however, choroidal thickness in the peripapillary region of children has not been examined in detail. This study therefore aimed to investigate the thickness of the peripapillary choroid and the overlying retinal nerve fibre layer (RNFL) in a population of normal children with a range of refractive errors. Ninety-three children (37 myopes and 56 non-myopes) aged between 11 and 16 years, had measurements of peripapillary choroidal and RNFL thickness derived from enhanced depth imaging optical coherence tomography images (EDI-OCT, Heidelberg Spectralis). The average thickness was determined in a series of five 0.25 mm width concentric annuli (each divided into 8 equal sized 45° sectors) centred on the optic nerve head boundary, accounting for individual ocular magnification factors and the disc-fovea angle. Significant variations in peripapillary choroidal thickness were found to occur with both annulus location (p<0.001) and sector position (p<0.001) in this population of children. The innermost annulus (closest to the edge of the optic disc) exhibited the thinnest choroid (mean 77 ± 16 μm) and the outermost annulus, the thickest choroid (191 ± 52 μm). The choroid was thinnest inferior to the optic nerve head (139 ± 38 μm) and was thickest in the superior temporal sector (157 ± 40 μm). Significant differences in the distribution of choroidal thickness were also associated with myopia, with myopic children having significantly thinner choroids in the inner and outer annuli of the nasal and temporal sectors respectively (p<0.001). RNFL thickness also varied significantly with annulus location and sector (p<0.001), and showed differences in thickness distribution associated with refractive error. This study establishes the normal variations in the thickness of the peripapillary choroid with radial distance and azimuthal angle from the optic nerve head boundary. A significant thinning of the peripapillary choroid associated with myopia in childhood was also observed in both nasal and temporal regions. The changes in peripapillary RNFL and choroidal thickness associated with refractive error are consistent with a redistribution of these tissues occurring with myopic axial elongation in childhood.

The effect of BCMO1 gene variants on macular pigment optical density in young healthy Caucasians

Background Serum lutein (L) and zeaxanthin (Z) positively correlate with macular pigment optical density (MPOD), hence the latter is a valuable indirect tool for measuring L and Z content in the macula. L and Z have been attributed antioxidant capacity and protection from certain retinal diseases but their uptake within the eye is thought to depend on genetic, age and environmental factors. In particular gene variants within beta-carotene monooxygenase (BCMO1) are thought to modulate MPOD in the macula. Objectives: To determine the effect of BCMO1 single nucleotide polymorphisms (SNPs) rs11645428, rs6420424 and rs6464851 on macular pigment optical density (MPOD) in a cohort of young healthy participants of Caucasian origin with normal ocular health. Design In this cohort study, MPOD was assessed in 46 healthy participants (22 male and 24 female) with a mean age of 24 ± 4.0 years (range 19-33). The three SNPs, rs11645428, rs6420424, rs6564851 that have established associations with MPOD were determined using MassEXTEND (hME) Sequenom assay. One-way analysis of variance (ANOVA) was performed on groups segregated into homozygous and heterozygous BCMO1 genotypes. Correlations between body mass index (BMI), iris colour, gender, central retinal thickness (CRT), diet and MPOD were investigated. Results MPOD did not significantly vary with BCMO1 rs11645428 (F2,41 = 0.700, p = 0.503), rs6420424 (F2,41 = 0.210, p = 0.801) nor rs6464851 homozygous or heterozygous genotypes (F2,41 = 0,13, p = 0.88), in this young healthy cohort. The combination of these three SNPs into triple genotypes based on plasma conversion efficiency did not affect MPOD (F2,41 = 0.07, p = 0.9). There was a significant negative correlation with MPOD and central retinal thickness (r = - 0.39, p = 0.01) but no significant correlation between BMI, iris colour, gender and MPOD. Conclusion Our results indicate that macular pigment deposition within the central retina is not dependent on BCMO1 gene variants in young healthy people. We propose that MPOD is saturated in younger persons and/or other gene variant combinations determine its deposition.

Treatment of silk fibroin with poly(ethylene glycol) for the enhancement of corneal epithelial cell growth

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

In response: Changes in ultraviolet transmittance of hydrogel and silicone hydrogel contact lenses induced by wear

We thank Dr Shedden and Dr Pall for their insightful comments and the opportunity to clarify a number of points from our work.1 The “protection factor” (PF) expressed as the inverse of the transmittance of contact lens (CL) material (1/Tλ), where T is the percentage transmittance of ultraviolet radiation (UVR) in a given waveband (UVC, UVB or UVA) of the UV spectrum for contact lenses is the standard method for reporting PF values and as such there should not be any controversy. We have calculated the PF for each wavelength across the entire UV spectrum (UVC, UVB, UVA) as presented in figure 3 of our previous publication.1 In that article, we were simply stating the observation when transmission in the UVC spectra band is considered especially because appreciable amounts of potentially carcinogenic short UV wavelengths was shown to be present in sunlight in our region three decades ago2 and these short wavelength photons are reported to be more biologically damaging to ocular tissues.3 In addition, the depletion of the Ozone layer is still continuing. Nevertheless, we understand the concern of the authors that the results of the PF might be confusing to those who are not familiar with the science of UVR and as such we have made some revisions to the findings of the calculated PF...

Persistent anterior chamber silicone oil and myopia

A 60-year-old male experienced a marked unilateral myopic shift of 20 D following attempted removal of intravitreal heavy silicone oil (HSO) used in the treatment of inferior proliferative vitreous retinopathy following retinal detachment. Examination revealed HSO adherent to the corneal endothelium forming a convex interface with the aqueous, obscuring the entire pupil, which required surgical intervention to restore visual acuity. This case highlights the potential ocular complications associated with silicone oil migration into the anterior chamber, which include corneal endothelial decompensation and a significant increase in myopia.

The short-term accommodation response to aniso-accommodative stimuli in isometropia

Purpose There have been only a limited number of studies examining the accommodative response that occurs when the two eyes are provided with disparate accommodative stimuli, and the results from these studies to date have been equivocal. In this study, we therefore aimed to examine the capacity of the visual system to aniso-accommodate by objectively measuring the interocular difference in the accommodation response between fellow dominant and non-dominant eyes under controlled monocular and binocular viewing conditions during short-term exposure to aniso-accommodative stimuli. Methods The accommodative response of each eye of sixteen young isometropic adults (mean age 22 ± 2 years) with normal binocular vision was measured using an open-field autorefractor during a range of testing conditions; monocularly (accommodative demands ranging from 1.32 to 4.55 D) and binocularly while altering the accommodation demand for each eye (aniso-accommodative stimuli ranging from 0.24 to 2.05 D). Results Under monocular viewing conditions, the dominant and non-dominant eyes displayed a highly symmetric accommodative response; mean interocular difference in spherical equivalent 0.01 ± 0.06 D (relative) and 0.22 ± 0.06 D (absolute) (p>0.05). During binocular viewing, the dominant eye displayed a greater accommodative response (0.11 ± 0.34 D relative and 0.24 ± 0.26 D absolute) irrespective of whether the demand of the dominant or non-dominant eye was altered (p = 0.01). Astigmatic power vectors J0 and J45 did not vary between eyes or with increasing accommodation demands under monocular or binocular viewing conditions (p>0.05). Conclusion The dominant and non-dominant eyes of young isometropic individuals display a similar consensual lag of accommodation under both monocular and binocular viewing conditions, with the dominant eye showing a small but significantly greater (by 0.12 to 0.25 D) accommodative response. Evidence of short-term aniso-accommodation in response to asymmetric accommodation demands was not observed.

A Bruch’s membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a similar thickness as Bruch’s membrane (3 μm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell). Cultures established on either material developed a cobblestoned morphology with partial pigmentation within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned medium collected from above and below both membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrate that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model.

Comparison of visual status of Iranian military and commercial drivers

Background There is no legal requirement for Iranian military truck drivers to undergo regular visual checkups as compared to commercial truck drivers. Objectives This study aimed to evaluate the impact of drivers’ visual checkups by comparing the visual function of Iranian military and commercial truck drivers. Patients and Methods In this comparative cross-sectional study, two hundred military and 200 commercial truck drivers were recruited and their Visual Acuity (VA), Visual Field (VF), color vision and Contrast Sensitivity (CS) were assessed and compared using the Snellen chart, confrontation screening method, D15 test and Pelli-Robson letter chart, respectively. A questionnaire regarding driving exposure and history of motor-vehicle crashes (MVCs) was also filled by drivers. Results were analyzed using an independent samples t-test, one-way ANOVA (assessing difference in number of MVCs across different age groups), chi-square test and Pearson correlation at statistical significance level of P < 0.05. Results Mean age was 41.6 ± 9.2 for the military truck drivers and 43.4 ± 10.9 for commercial truck drivers (P > 0.05). No significant difference between military and commercial drivers was found in terms of driving experience, number of MVCs, binocular VA, frequency of color vision defects and CS scores. In contrast, the last ocular examination was significantly earlier in military drivers than commercial drivers (P < 0.001). In addition, 4% of military drivers did not meet the national standards to drive as opposed to 2% of commercial drivers. There was a significant but weak correlation between binocular VA and age (r = 0.175, P < 0.001). However, CS showed a significantly moderate correlation with age (r = -0.488, P < 0.001). Conclusions The absence of legal requirement for regular eye examination in military drivers caused the incompetent drivers to be missed in contrast to commercial drivers. The need for scientific revision of VA standard for Iranian drivers is also discussed. The CS measurement in visual checkups of older drivers deserves to be investigated more thoroughly.

Refractive indices used by the Haag-Streit Lenstar to calculate axial biometric dimensions

PURPOSE To estimate refractive indices used by the Lenstar biometer to translate measured optical path lengths into geometrical path lengths within the eye. METHODS Axial lengths of model eyes were determined using the IOLMaster and Lenstar biometers; comparing those lengths gave an overall eye refractive index estimate for the Lenstar. Using the Lenstar Graphical User Interface, we noticed that boundaries between media could be manipulated and opposite changes in optical path lengths on either side of the boundary could be introduced. Those ratios were combined with the overall eye refractive index to estimate separate refractive indices. Furthermore, Haag-Streit provided us with a template to obtain 'air thicknesses' to compare with geometrical distances. RESULTS The axial length estimates obtained using the IOLMaster and the Lenstar agreed to within 0.01 mm. Estimates of group refractive indices used in the Lenstar were 1.340, 1.341, 1.415, and 1.354 for cornea, aqueous, lens, and overall eye, respectively. Those refractive indices did not match those of schematic eyes, but were close in the cases of aqueous and lens. Linear equations relating air thicknesses to geometrical thicknesses were consistent with our findings. CONCLUSION The Lenstar uses different refractive indices for different ocular media. Some of the refractive indices, such as that for the cornea, are not physiological; therefore, it is likely that the calibrations in the instrument correspond to instrument-specific corrections and are not the real optical path lengths.

Evaluation of the relationship between Chlamydia pecorum sequence types and disease using a species-specific multi-locus sequence typing scheme (MLST)

Chlamydia pecorum is globally associated with several ovine diseases including keratoconjunctivitis and polyarthritis. The exact relationship between the variety of C. pecorum strains reported and the diseases described in sheep remains unclear, challenging efforts to accurately diagnose and manage infected flocks. In the present study, we applied C. pecorum multi-locus sequence typing (MLST) to C. pecorum positive samples collected from sympatric flocks of Australian sheep presenting with conjunctivitis, conjunctivitis with polyarthritis, or polyarthritis only and with no clinical disease (NCD) in order to elucidate the exact relationships between the infecting strains and the range of diseases. Using Bayesian phylogenetic and cluster analyses on 62 C. pecorum positive ocular, vaginal and rectal swab samples from sheep presenting with a range of diseases and in a comparison to C. pecorum sequence types (STs) from other hosts, one ST (ST 23) was recognised as a globally distributed strain associated with ovine and bovine diseases such as polyarthritis and encephalomyelitis. A second ST (ST 69) presently only described in Australian animals, was detected in association with ovine as well as koala chlamydial infections. The majority of vaginal and rectal C. pecorum STs from animals with NCD and/or anatomical sites with no clinical signs of disease in diseased animals, clustered together in a separate group, by both analyses. Furthermore, 8/13 detected STs were novel. This study provides a platform for strain selection for further research into the pathogenic potential of C. pecorum in animals and highlights targets for potential strain-specific diagnostic test development.

Regional changes in choroidal thickness associated with accommodation

Purpose. To characterize the changes occurring in choroidal thickness (ChT) across the posterior pole during accommodation using enhanced-depth imaging optical coherence tomography (OCT). Methods. Forty participants (mean age 21 ± 2 years) had measures of ChT and ocular biometry taken during accommodation to 0, 3, and 6 diopter (D) stimuli, with the Spectralis OCT and Lenstar biometer. A Badal optometer and cold mirror system was mounted on both instruments, allowing measurement collection while subjects viewed an external fixation target at varying accommodative demands. Results. The choroid exhibited significant thinning during accommodation to the 6 D stimulus in both subfoveal (mean change, −5 ± 7 μm) and parafoveal regions (P < 0.001). The magnitude of these changes varied by parafoveal meridian, with the largest changes seen in the temporal (−9 ± 12 μm) and inferotemporal (−8 ± 8 μm) meridians (P < 0.001). Axial length increased with accommodation (mean change, +5 ± 11 μm at 3 D, +14 ± 13 μm at 6 D), and these changes were weakly negatively associated with the choroidal changes (r2 = 0.114, P < 0.05). Conclusions. A small, but significant thinning of the choroid was observed at the 6 D accommodation demand, which was greatest in the temporal and inferotemporal parafoveal choroid, and increased with increasing eccentricity from the fovea. The regional variation in the parafoveal thinning corresponds to the distribution of the nonvascular smooth muscle within the uvea, which may implicate these cells as the potential mechanism by which the choroid thins during accommodation.

Genome-wide association study identifies susceptibility loci for open angle glaucoma at TMCO1 and CDKN2B-AS1

We report a genome-wide association study for open-angle glaucoma (OAG) blindness using a discovery cohort of 590 individuals with severe visual field loss (cases) and 3,956 controls. We identified associated loci at TMCO1 (rs4656461[G] odds ratio (OR) = 1.68, P = 6.1 × 10-10) and CDKN2B-AS1 (rs4977756[A] OR = 1.50, P = 4.7 × 10-9). We replicated these associations in an independent cohort of cases with advanced OAG (rs4656461 P = 0.010; rs4977756 P = 0.042) and two additional cohorts of less severe OAG (rs4656461 combined discovery and replication P = 6.00 × 10-14, OR = 1.51, 95% CI 1.35-1.68; rs4977756 combined P = 1.35 × 10-14, OR = 1.39, 95% CI 1.28-1.51). We show retinal expression of genes at both loci in human ocular tissues. We also show that CDKN2A and CDKN2B are upregulated in the retina of a rat model of glaucoma. © 2011 Nature America, Inc. All rights reserved.

Effect of age and refractive error on the melanopsin mediated Post-Illumination Pupil Response (PIPR)

Melanopsin containing intrinsically photosensitive Retinal Ganglion cells (ipRGCs) mediate the pupil light reflex (PLR) during light onset and at light offset (the post-illumination pupil response, PIPR). Recent evidence shows that the PLR and PIPR can provide non-invasive, objective markers of age-related retinal and optic nerve disease, however there is no consensus on the effects of healthy ageing or refractive error on the ipRGC mediated pupil function. Here we isolated melanopsin contributions to the pupil control pathway in 59 human participants with no ocular pathology across a range of ages and refractive errors. We show that there is no effect of age or refractive error on ipRGC inputs to the human pupil control pathway. The stability of the ipRGC mediated pupil response across the human lifespan provides a functional correlate of their robustness observed during ageing in rodent models.

Standardised antibacterial Manuka honey in the management of persistent post-operative corneal oedema: A case series

Background Corneal oedema is a common post-operative problem that delays or prevents visual recovery from ocular surgery. Honey is a supersaturated solution of sugars with an acidic pH, high osmolarity and low water content. These characteristics inhibit the growth of micro-organisms, reduce oedema and promote epithelialisation. This clinical case series describes the use of a regulatory approved Leptospermum species honey ophthalmic product, in the management of post-operative corneal oedema and bullous keratopathy. Methods A retrospective review of 18 consecutive cases (30 eyes) with corneal oedema persisting beyond one month after single or multiple ocular surgical procedures (phacoemulsification cataract surgery and additional procedures) treated with Optimel Antibacterial Manuka Eye Drops twice to three times daily as an adjunctive therapy to conventional topical management with corticosteroid, aqueous suppressants, hypertonic sodium chloride five per cent, eyelid hygiene and artificial tears. Visual acuity and central corneal thickness were measured before and at the conclusion of Optimel treatment. Results A temporary reduction in corneal epithelial oedema lasting up to several hours was observed after the initial Optimel instillation and was associated with a reduction in central corneal thickness, resolution of epithelial microcysts, collapse of epithelial bullae, improved corneal clarity, improved visualisation of the intraocular structures and improved visual acuity. Additionally, with chronic use, reduction in punctate epitheliopathy, reduction in central corneal thickness and improvement in visual acuity were achieved. Temporary stinging after Optimel instillation was experienced. No adverse infectious or inflammatory events occurred during treatment with Optimel. Conclusions Optimel was a safe and effective adjunctive therapeutic strategy in the management of persistent post-operative corneal oedema and warrants further investigation in clinical trials.