Photochemically Induced Crosslinking of Tarsal Collagen as a Treatment for Eyelid Laxity: Assessing Potentiality in Animal Tissue.

PURPOSE: An experimental study to demonstrate in animal eyelids that the controlled exposure of excised tarsal plate to ultraviolet-A radiation can induce a rigidification effect due to photochemical crosslinking of the constitutive collagen. METHODS: Excised strips of sheep tarsus were irradiated with ultraviolet-A rays (wavelength 365 nm) at low and high irradiances, in the presence of riboflavin as a photosensitizer, using radiation sources available for corneal collagen crosslinking procedure. The tensile strength and Young's modulus (stiffness) of irradiated and control samples were measured in a mechanical tester and analyzed statistically. Histologic examination of the specimens was carried out to evaluate the effect of radiation on the meibomian glands and collagen organization. RESULTS: Mechanical evaluation showed that irradiation induced both stiffening and strengthening of the tarsal plate specimens, and this effect was enhanced at the higher levels of irradiance. The changes in mechanical properties can be attributed to a process of photochemically induced crosslinking of tarsal collagen. Histology revealed no changes in the meibomian glands or in the fibrous collagen system of the tarsus. CONCLUSIONS: These findings indicate that irradiation of tarsal collagen leading to tissue stiffening could be a safe procedure for treating lax eyelid conditions in human patients.

Nature and incidence of severe limbal stem cell deficiency in Australia and New Zealand.

BACKGROUND: This study aimed to determine the nature and incidence of severe limbal stem cell deficiency (LSCD) in Australia and New Zealand. DESIGN: A 1-year pilot surveillance study with a 1-year follow-up period was conducted in association with the Australian and New Zealand Ophthalmic Surveillance Unit. PARTICIPANTS: The study included patients reported by practising ophthalmologists on the Surveillance Unit's database. METHODS: Ophthalmologists were provided with a definition of severe limbal stem cell deficiency, contacted on a monthly basis by the Unit and asked to report newly diagnosed cases. MAIN OUTCOME MEASURES: Severe LSCD was defined as at least 6 clock hours of whorl-like epitheliopathy, an opaque epithelium arising from the limbus, late fluorescein staining of the involved epithelium and superficial corneal neovascularization or conjunctivalization. RESULTS: On average, 286 report cards were sent by the Surveillance Unit to practising ophthalmologists each month (total 3429 over 12 months) and the Unit received an average of 176 responses per month (total 2111; 62% response rate). During the 1-year study period from April 2013 to March 2014, 14 positive cases were reported to the Unit. A range of underlying aetiologies were implicated, with contact lens over-wear and cicatrizing conjunctivitis being the most common (n = 3). CONCLUSIONS: This surveillance study is the first worldwide to document the incidence of limbal stem cell deficiency; however, because of study design limitations, it is likely to have been under-reported. It provides novel data on the demographics, clinical conditions and management of patients with limbal stem cell deficiency as reported by treating ophthalmologists.

Characterization of Goblet Cells in a Pterygium Biopsy Using Laser Scanning Confocal Microscopy and Immunohistochemistry.

PURPOSE: To confirm that structures presumed to be GCs observed using laser scanning confocal microscopy (LSCM) are actually GCs. METHODS: A single tissue sample was obtained from a pterygium that was freshly excised from a 33-year-old male. After viewing what were believed to be GCs in the tissue sample using LSCM, the same sample was observed using laboratory confocal microscopy and immunohistochemistry. GCs were identified by a combination of classic morphologic appearance and the use of immunofluorescence to antibodies for mucin 5AC and cytokeratin-7. The LSCM and immunohistochemistry results were compared. RESULTS: Using LSCM, GCs were observed between 7 and 41 µm deep, at the level of the superficial basal cells of the tissue sample. GCs were estimated to have a diameter of 35-40 µm near the surface and 20-30 µm in the deeper layers. A small dark dot was visible in some GCs, indicating cell nuclei and/or the opened apical portion of cells representing the site of mucin release. GCs were more reflective and larger than the surrounding cells. Positively stained GCs in immunofluorescence showed a similar distribution pattern to those observed with LSCM. The tissue sample stained intensely for GC-specific mucin type 5AC. CONCLUSIONS: The pattern of discrete, large reflective cells observed using LSCM are likely to be GCs.

Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit.

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury is difficult to control using chemical-based methods. Moreover, the anterior chamber can be easily perforated while surgically excising the corneal limbus. In the interest of creating a safer and more defined LSCD model, we have therefore evaluated a mechanical debridement technique based upon use of the AlgerBrush II rotating burr. An initial comparison of debridement techniques was conducted in situ using 24 eyes in freshly acquired New Zealand White rabbit cadavers. Techniques for comparison (4 eyes each) included: (1) non-wounded control, (2) surgical limbectomy followed by treatment with 100% (v/v) n-heptanol to remove the corneal epithelium (1-2 min), (3) treatment of both limbus and cornea with n-heptanol alone, (4) treatment of both limbus and cornea with 20% (v/v) ethanol (2-3 min), (5) a 2.5-mm rounded burr applied to both the limbus and cornea, and (6) a 1-mm pointed burr applied to the limbus, followed by the 2.5-mm rounded burr applied to the cornea. All corneas were excised and processed for histology immediately following debridement. A panel of four assessors subsequently scored the degree of epithelial debridement within the cornea and limbus using masked slides. The 2.5-mm burr most consistently removed the corneal and limbal epithelia. Islands of limbal epithelial cells were occasionally retained following surgical limbectomy/heptanol treatment, or use of the 1-mm burr. Limbal epithelial cells were consistently retained following treatment with either ethanol or n-heptanol alone, with ethanol being the least effective treatment overall. The 2.5-mm burr method was subsequently evaluated in the right eye of 3 live rabbits by weekly clinical assessments (photography and slit lamp examination) for up to 5 weeks, followed by histological analyses (hematoxylin & eosin stain, periodic acid-Schiff stain and immunohistochemistry for keratin 3 and 13). All 3 eyes that had been completely debrided using the 2.5-mm burr displayed symptoms of ocular surface failure as defined by retention of a prominent epithelial defect (∼40% of corneal surface at 5 weeks), corneal neovascularization (2-3 quadrants), reduced corneal transparency and conjunctivalization of the corneal surface (demonstrated by the presence of goblet cells and/or staining for keratin 13). In conclusion, our findings indicate that the AlgerBrush II rotating burr is an effective method for the establishment of ocular surface failure in New Zealand White rabbits. In particular, we recommend use of the 2.5-mm rotating burr for improved efficiency of epithelial debridement and safety compared to surgical limbectomy.

Concise reviews: can mesenchymal stromal cells differentiate into corneal cells? A systematic review of published data.

The majority of stem cell therapies for corneal repair are based upon the use of progenitor cells isolated from corneal tissue, but a growing body of literature suggests a role for mesenchymal stromal cells (MSC) isolated from noncorneal tissues. While the mechanism of MSC action seems likely to involve their immuno-modulatory properties, claims have emerged of MSC transdifferentiation into corneal cells. Substantial differences in methodology and experimental outcomes, however, have prompted us to perform a systematic review of the published data. Key questions used in our analysis included: the choice of markers used to assess corneal cell phenotype, the techniques used to detect these markers, adequate reporting of controls, and tracking of MSC when studied in vivo. Our search of the literature revealed 28 papers published since 2006, with half appearing since 2012. MSC cultures established from bone marrow and adipose tissue have been best studied (22 papers). Critically, only 11 studies used appropriate markers of corneal cell phenotype, along with necessary controls. Ten out of these eleven papers, however, contained positive evidence of corneal cell marker expression by MSC. The clearest evidence is observed with respect to expression of markers for corneal stromal cells by MSC. In comparison, the evidence for MSC conversion into either corneal epithelial cells or corneal endothelial cells is often inconsistent or inconclusive. Our analysis clarifies this emerging body of literature and provides guidance for future studies of MSC differentiation within the cornea as well as other tissues.

Tamoxifen therapy conveys increased risk of developing a macular hole.

PURPOSE: To determine etiological factors in the development of, as well as anatomic success rate and visual outcome of a large consecutive series of macular hole surgeries. METHODS: Retrospective analysis of 300 consecutive cases of macular hole surgery by a single surgeon (RDB) between 1999 and 2003. Patients' medical and surgical histories were recorded and analysed for factors involved in aetiology and visual outcome. RESULTS: There were 8 (4.12%) women, on tamoxifen in the study, two of these women had bilateral macular holes. When this study prevalence of tamoxifen therapy (4.12%) was compared to the estimated percentage of women in the same age group in the Australian population on tamoxifen (0.82%), a statistically significant difference (p value 0.0001) was found. Analysis of the number of bilateral holes in the tamoxifen group compared to the non-tamoxifen group was suggestive of an increased incidence of bilateral holes but not to a significantly significant degree. CONCLUSION: Whilst no published reports link tamoxifen and macular holes, this may be due to the low incidence of the condition. Our study demonstrates a strong link between tamoxifen use and macular holes. Patients being commenced on tamoxifen should be advised of possible ocular complications and receive prompt ophthalmic review if symptoms develop.