Influence of substrate on corneal epithelial cell viability within ocular surface models

Corneal tissue engineering has improved dramatically over recent years. It is now possible to apply these technological advancements to the development of superior in vitro ocular surface models to reduce animal testing. We aim to show the effect different substrates can have on the viability of expanded corneal epithelial cells and that those which more accurately mimic the stromal surface provide the most protection against toxic assault. Compressed collagen gel as a substrate for the expansion of a human epithelial cell line was compared against two well-known substrates for modeling the ocular surface (polycarbonate membrane and conventional collagen gel). Cells were expanded over 10 days at which point cell stratification, cell number and expression of junctional proteins were assessed by electron microscopy, immunohistochemistry and RT-PCR. The effect of increasing concentrations of sodium lauryl sulphate on epithelial cell viability was quantified by MTT assay. Results showed improvement in terms of stratification, cell number and tight junction expression in human epithelial cells expanded upon either the polycarbonate membrane or compressed collagen gel when compared to a the use of a conventional collagen gel. However, cell viability was significantly higher in cells expanded upon the compressed collagen gel. We conclude that the more naturalistic composition and mechanical properties of compressed collagen gels produces a more robust corneal model.

The mechanical properties of amniotic membrane influence its effect as a biomaterial for ocular surface repair

The human amniotic membrane (AM) is a tissue of fetal origin and has proven to be clinically useful as a biomaterial in the management of various ocular surface disorders including corneal stem cell transplantation. However, its success rate displays a degree of clinical unpredictability. We suggest that the measured variability inAMstiffness offers an explanation for the poor clinical reproducibility when it is used as a substrate for stem cell expansion and transplantation. Corneal epithelial stem cells were expanded upon AM samples possessing different mechanical stiffness. To investigate further the importance of biological substrate stiffness on cell phenotype we replaced AM with type I collagen gels of known stiffness. Substrate stiffness was measured using shear rheometry and surface topography was characterized using scanning electron microscopy and atomic force microscopy. The differentiation status of epithelial cells was examined using RT-PCR, immunohistochemistry and Western blotting. The level of corneal stem cell differentiation was increased in cells expanded upon AM with a high dynamic elastic shear modulus and cell expansion on type I collagen gels confirmed that the level of corneal epithelial stem cell differentiation was related to the substrate’s mechanical properties. In this paper we provide evidence to show that the preparatory method of AM for clinical use can affect its mechanical properties and that these measured differences can influence the level of differentiation within expanded corneal epithelial stem cells.

Effects of the Ottonia martiana Miq. (Piperaceae) extract on dog's ocular surface

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Role of Dyslipidemia on Ocular Surface, Lacrimal and Meibomian Gland Structure and Function

Purpose: Dyslipidemia is characterized by high lipid blood levels that are risk factors for cardiovascular diseases, which are leading causes of death. However, it is unclear whether dyslipidemia is a cause of the dry eye syndrome (DES). Therefore we determined in transgenic mice models of dyslipidemia, whether there is an association with DES development. Methods: Dyslipidemic models included male and female adult mice overexpressing apolipoprotein CIII (Apo CIII), LDL receptor knockout (LDLR-KO) and ApoE knockout (ApoE-KO). They were compared with age-and gender-matched C57BL/6 mice. Ocular health was evaluated based on corneal slit lamp assessment, phenol red thread test (PRT) and impression cytology. Blood lipid profiles and histology of meibomian and lacrimal glands were also evaluated. Effects of high-fat diet and aging were observed in LDLR-KO and ApoCIII strains, respectively. Results: Body weight and lacrimal gland weight were significantly higher in male mice compared to females of the same strain (P < 0.05). Body weight was significantly lower in LDLRKO mice receiving high lipid diet compared to their controls (P = 0.0043). ApoE-KO were hypercholesterolemic and ApoCIII hypertriglyceridemic while LDLR-KO showed increases in both parameters. The PRT test was lower in male LDLR-KO mice with high-fat diet than control mice with standard diet (P = 0.0273). Aging did not affect lacrimal structural or functional parameters of ApoCIII strain. Conclusions: DES development is not solely dependent on dyslipidemia in relevant mice models promoting this condition. On the other hand, lacrimal gland structure and function are differentially impacted by lipid profile changes in male and female mice. This dissociation suggests that other factors beside dyslipidemia impact on tear film dysfunction and DES development.

Proteomic analysis of ocular surface components by use of HPLC based mass spectrometric strategies

In der vorliegenden Arbeit wurde eine Top Down (TD) und zwei Bottom Up (BU) MALDI/ESI Massenspektrometrie/HPLC-Methoden entwickelt mit dem Ziel Augenoberfächenkomponenten, d.h. Tränenfilm und Konjunktivalzellen zu analysieren. Dabei wurde ein detaillierter Einblick in die Entwicklungsschritte gegeben und die Ansätze auf Eignung und methodische Grenzen untersucht. Während der TD Ansatz vorwiegend Eignung zur Analyse von rohen, weitgehend unbearbeiteten Zellproben fand, konnten mittels des BU Ansatzes bearbeitete konjunktivale Zellen, aber auch Tränenfilm mit hoher Sensitivität und Genauigkeit proteomisch analysiert werden. Dabei konnten mittels LC MALDI BU-Methode mehr als 200 Tränenproteine und mittels der LC ESI Methode mehr als 1000 Tränen- sowie konjunktivale Zellproteine gelistet werden. Dabei unterschieden sich ESI- and MALDI- Methoden deutlich bezüglich der Quantität und Qualität der Ergebnisse, weshalb differente proteomische Anwendungsgebiete der beiden Methoden vorgeschlagen wurden. Weiterhin konnten mittels der entwickelten LC MALDI/ESI BU Plattform, basierend auf den Vorteilen gegenüber dem TD Ansatz, therapeutische Einflüsse auf die Augenoberfläche mit Fokus auf die topische Anwendung von Taurin sowie Taflotan® sine, untersucht werden. Für Taurin konnten entzündungshemmende Effekte, belegt durch dynamische Veränderungen des Tränenfilms, dokumentiert werden. Außerdem konnten vorteilhafte, konzentrationsabhängige Wirkweisen auch in Studien an konjunktival Zellen gezeigt werden. Für die Anwendung von konservierungsmittelfreien Taflotan® sine, konnte mittels LC ESI BU Analyse eine Regenerierung der Augenoberfläche in Patienten mit Primärem Offenwinkel Glaukom (POWG), welche unter einem “Trockenem Auge“ litten nach einem therapeutischen Wechsel von Xalatan® basierend auf dynamischen Tränenproteomveränderungen gezeigt werden. Die Ergebnisse konnten mittels Microarray (MA) Analysen bestätigt werden. Sowohl in den Taurin Studien, als auch in der Taflotan® sine Studie, konnten charakteristische Proteine der Augenoberfläche dokumentiert werden, welche eine objektive Bewertung des Gesundheitszustandes der Augenoberfläche ermöglichen. Eine Kombination von Taflotan® sine und Taurin wurde als mögliche Strategie zur Therapie des Trockenen Auges bei POWG Patienten vorgeschlagen und diskutiert.

Influence of corneal asymmetry on the phakometric measurement of ocular surface alignment

Phakometric measurements of corneal and crystalline lens surface alignment are influenced by corneal asymmetry in which the corneal apex does not coincide with the limbal centre. The purpose of this study was to determine the horizontal separation (e) between these corneal landmarks. Measurements were made in 60 normal eyes (30 subjects) using the Orbscan Ilz corneal analysis workstation. Our results show that both corneal landmarks typically coincide, so that e = 0, but that inter-subject variations of about ±1 mm can be expected (so that the corneal apex may fall nasal or temporal to the visual axis). This suggests that no correction for corneal asymmetry is required when estimating average amounts of ocular alignment from samples of eyes but that the measurement of e is strongly recommended for measurements in individual eyes. © 2004 The College of Optometrists.

Phakometric measurement of ocular surface radii of curvature, axial separations and alignment in relaxed and accommodated human eyes

Measurements (autokeratometry, A-scan ultrasonography and video ophthalmophakometry) of ocular surface radii, axial separations and alignment were made in the horizontal meridian of nine emmetropes (aged 20-38 years) with relaxed (cycloplegia) and active accommodation (mean ± 95% confidence interval: 3.7 ± 1.1 D). The anterior chamber depth (-1.5 ± 0.3 D) and both crystalline lens surfaces (front 3.1 ± 0.8 D; rear 2.1 ± 0.6 D) contributed to dioptric vergence changes that accompany accommodation. Accommodation did not alter ocular surface alignment. Ocular misalignment in relaxed eyes is mainly because of eye rotation (5.7 ± 1.6° temporally) with small amounts of lens tilt (0.2 ± 0.8° temporally) and decentration (0.1 ± 0.1 mm nasally) but these results must be viewed with caution as we did not account for corneal asymmetry. Comparison of calculated and empirically derived coefficients (upon which ocular surface alignment calculations depend) revealed that negligible inherent errors arose from neglect of ocular surface asphericity, lens gradient refractive index properties, surface astigmatism, effects of pupil size and centration, assumed eye rotation axis position and use of linear equations for analysing Purkinje image shifts. © 2004 The College of Optometrists.

Phakometric measurement of ocular surface radius of curvature and alignment: evaluation of method with physical model eyes

Ophthalmophakometric measurements of ocular surface radius of curvature and alignment were evaluated on physical model eyes encompassing a wide range of human ocular dimensions. The results indicated that defocus errors arising from imperfections in the ophthalmophakometer camera telecentricity and light source collimation were smaller than experimental errors. Reasonable estimates emerged for anterior lens surface radius of curvature (accuracy: 0.02–0.10 mm; precision 0.05–0.09 mm), posterior lens surface radius of curvature (accuracy: 0.10–0.55 mm; precision 0.06–0.20 mm), eye rotation (accuracy: 0.00–0.32°; precision 0.06–0.25°), lens tilt (accuracy: 0.00–0.33°; precision 0.05–0.98°) and lens decentration (accuracy: 0.00–0.07 mm; precision 0.00–0.07 mm).

The relation between physical properties of the anterior eye and ocular surface temperature

PURPOSE. To examine the relation between ocular surface temperature (OST) assessed by dynamic thermal imaging and physical parameters of the anterior eye in normal subjects. METHODS. Dynamic ocular thermography (ThermoTracer 7102MX) was used to record body temperature and continuous ocular surface temperature for 8 s after a blink in the right eyes of 25 subjects. Corneal thickness, corneal curvature, and anterior chamber depth (ACD) were assessed using Orbscan II; noninvasive tear break-up time (NIBUT) was assessed using the tearscope; slit lamp photography was used to record tear meniscus height (TMH) and objective bulbar redness. RESULTS. Initial OST after a blink was significantly correlated only with body temperature (r = 0.80, p < 0.0005), NIBUT (r = -0.68, p < 0.005) and corneal curvature (r = -0.40, p = 0.05). A regression model containing all the variables accounted for 70% (p = 0.002) of the variance in OST, of which NIBUT (29%, p = 0.004), and body temperature (18%, p = 0.005) contributed significantly. CONCLUSIONS. The results support previous theoretical models that OST radiation is principally related to the tear film; and demonstrate that it is less related to other characteristics such as corneal thickness, corneal curvature, and anterior chamber depth. © 2007 American Academy of Optometry.

Ocular surface temperature:a review

Purpose. To review the evolution in ocular temperature measurement during the last century and examine the advantages and applications of the latest noncontact techniques. The characteristics and source of ocular surface temperature are also discussed. Methods. The literature was reviewed with regard to progress in human thermometry techniques, the parallel development in ocular temperature measurement, the current use of infrared imaging, and the applications of ocular thermography. Results. It is widely acknowledged that the ability to measure ocular temperature accurately will increase the understanding of ocular physiology. There is a characteristic thermal profile across the anterior eye, in which the central area appears coolest. Ocular surface temperature is affected by many factors, including inflammation. In thermometry of the human eye, contact techniques have largely been superseded by infrared imaging, providing a noninvasive and potentially more accurate method of temperature measurement. Ocular thermography requires high resolution and frame rate: features found in the latest generation of cameras. Applications have included dry eye, contact lens wear, corneal sensitivity, and refractive surgery. Conclusions. Interest in the temperature of the eye spans almost 130 years. It has been an area of research largely driven by prevailing technology. Current instrumentation offers the potential to measure ocular surface temperature with more accuracy, resolution, and speed than previously possible. The use of dynamic ocular thermography offers great opportunities for monitoring the temperature of the anterior eye. © 2005 Contact Lens Association of Ophthalmologists, Inc.

The effect of contact lens wear on dynamic ocular surface temperature

Aim: To determine the dynamic emitted temperature changes of the anterior eye during and immediately after wearing different materials and modalities of soft contact lenses. Method: A dynamic, non-contact infrared camera (Thermo-Tracer TH7102MX, NEC San-ei) was used to record the ocular surface temperature (OST) in 48 subjects (mean age 21.7 ± 1.9 years) wearing: lotrafilcon-A contact lenses on a daily wear (LDW; n = 8) or continuous wear (LCW; n = 8) basis; balafilcon-A contact lenses on a daily wear (BDW; n = 8) or continuous wear (BCW; n = 8) basis; etafilcon-A contact lenses on a daily disposable regimen (EDW; n = 8); and no lenses (controls; n = 8). OST was measured continuously five times, for 8 s after a blink, following a minimum of 2 h wear and immediately following lens removal. Absolute temperature, changes in temperature post-blink and the dynamics of temperature changes were calculated. Results: OST immediately following contact lens wear was significantly greater compared to non-lens wearers (37.1 ± 1.7 °C versus 35.0 ± 1.1 °C; p < 0.005), predominantly in the LCW group (38.6 ± 1.0 °C; p < 0.0001). Lens surface temperature was highly correlated (r = 0.97) to, but lower than OST (by -0.62 ± 0.3 °C). There was no difference with modality of wear (DW 37.5 ± 1.6 °C versus CW 37.8 ± 1.9 °C; p = 0.63), but significant differences were found between etafilcon A and silicone hydrogel lens materials (35.3 ± 1.1 °C versus 37.5 ± 1.5 °C; p < 0.0005). Ocular surface cooling following a blink was not significantly affected by contact lens wear with (p = 0.07) or without (p = 0.47) lenses in situ. Conclusions: Ocular surface temperature is greater with hydrogel and greater still with silicone hydrogel contact lenses in situ, regardless of modality of wear. The effect is likely to be due to the thermal transmission properties of a contact lens. © 2004 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

The ageing ocular surface:challenges for biomaterials design and function

Changing demographics and in particular an increasingly ageing population, in combination with improved longevity, will have a major impact on changing the face of human diseases and likewise the demand for appropriate biomaterials. The ocular surface is a multifaceted system that combines to create a unique mucosal surface, which includes the cornea, conjunctiva, sclera and lids of the eye. Physical parameters such as the eyelids and eyelashes, combined with the numerous secretory glands that produce the complex tear film, act together to protect and maintain the cornea. Unfortunately an ageing tear film and lacrimal functional unit can lead to impairment of this magnificently orchestrated structure. No single mechanism or modification is responsible but, whatever the cause, the consequence is a reduction in tear stability. An uncompromised tear film is fundamental to a healthy ocular surface. In the face of progressively changing demographics and consequent requirements for medical intervention and medical device developments, it is important to understand what effects the ageing process has on these anterior ocular structures.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.

Novel micelle carriers for cyclosporin A topical ocular delivery: in vivo cornea penetration, ocular distribution and efficacy studies.

Cornea transplantation is one of the most performed graft procedures worldwide with an impressive success rate of 90%. However, for "high-risk" patients with particular ocular diseases in addition to the required surgery, the success rate is drastically reduced to 50%. In these cases, cyclosporin A (CsA) is frequently used to prevent the cornea rejection by a systemic treatment with possible systemic side effects for the patients. To overcome these problems, it is a challenge to prepare well-tolerated topical CsA formulations. Normally high amounts of oils or surfactants are needed for the solubilization of the very hydrophobic CsA. Furthermore, it is in general difficult to obtain ocular therapeutic drug levels with topical instillations due to the corneal barriers that efficiently protect the intraocular structures from foreign substances thus also from drugs. The aim of this study was to investigate in vivo the effects of a novel CsA topical aqueous formulation. This formulation was based on nanosized polymeric micelles as drug carriers. An established rat model for the prevention of cornea graft rejection after a keratoplasty procedure was used. After instillation of the novel formulation with fluorescent labeled micelles, confocal analysis of flat-mounted corneas clearly showed that the nanosized carriers were able to penetrate into all corneal layers. The efficacy of a 0.5% CsA micelle formulation was tested and compared to a physiological saline solution and to a systemic administration of CsA. In our studies, the topical CsA treatment was carried out for 14 days, and the three parameters (a) cornea transparency, (b) edema, and (c) neovascularization were evaluated by clinical observation and scoring. Compared to the control group, the treated group showed a significant higher cornea transparency and significant lower edema after 7 and 13 days of the surgery. At the end point of the study, the neovascularization was reduced by 50% in the CsA-micelle treated animals. The success rate of cornea graft transplantation was 73% in treated animals against 25% for the control group. This result was as good as observed for a systemic CsA treatment in the same animal model. This new formulation has the same efficacy like a systemic treatment but without the serious CsA systemic side effects. Ocular drug levels of transplanted and healthy rat eyes were dosed by UPLC/MS and showed a high CsA value in the cornea (11710 ± 7530 ng(CsA)/g(tissue) and 6470 ± 1730 ng(CsA)/g(tissue), respectively). In conclusion, the applied formulation has the capacity to overcome the ocular surface barriers, the micelles formed a drug reservoir in the cornea from, where a sustained release of CsA can take place. This novel formulation for topical application of CsA is clearly an effective and well-tolerated alternative to the systemic treatment for the prevention of corneal graft rejection.

Transpalpebral proton beam radiotherapy of choroidal melanoma.

BACKGROUND: Collateral damage to upper eyelid margin during proton beam radiotherapy (PBR) for choroidal melanoma may cause squamous metaplasia of the tarsal conjunctiva with keratinisation, corneal irritation, discomfort and, rarely, corneal perforation. We evaluated transpalpebral PBR as a means of avoiding collateral damage to the upper eyelid margin without increasing the risk of failure of local tumour control. METHODS: Retrospective study of consecutive patients who underwent PBR for choroidal melanoma between 1992 and 2007 at the Royal Liverpool University Hospital and the Douglas Cyclotron at Clatterbridge Cancer Centre, UK. RESULTS: Sixty-three patients were included in this study. Mean basal tumour diameter and tumour thickness were 11.8 mm and 3.6 mm, respectively. PBR mean beam range and modulation were 26.5 mm and 16.9 mm respectively. The eyelid margin was included in the radiation field in 15 (24%) eyes. The median follow-up was 2.5 years. Local tumour recurrence developed in 2 (3.2%) patients. In these two cases that developed tumour recurrence the transpalpebral treatment did not involve the eyelid margin. Six (9.5%) patients died of metastatic disease. No eyelid or ocular surface problems developed in any of the 48 patients who were treated without eyelid rim involvement, while 7 of the 15 patients with unavoidable irradiation of the eyelid rim developed some degree of madarosis. These seven patients all received more than 26.55 proton Gy to the eyelid margin. Symptoms, such as grittiness occurred in 12% of 48 patients without eyelid margin irradiation as compared with 53% of 15 patients whose lid margin was irradiated. CONCLUSIONS: Transpalpebral PBR of choroidal melanoma avoids eyelid and ocular surface complications without increasing failure of local tumour control.