Tear analysis and lens-tear interactions:part II. Ocular lipids-nature and fate of meibomian gland phospholipids

Purpose: Published data indicate that the polar lipid content of human meibomian gland secretions (MGS) could be anything between 0.5% and 13% of the total lipid. The tear film phospholipid composition has not been studied in great detail and it has been understood that the relative proportions of lipids in MGS would be maintained in the tear film. The purpose of this work was to determine the concentration of phospholipids in the human tear film. Methods: Liquid chromatography mass spectrometry (LCMS) and thin layer chromatography (TLC) were used to determine the concentration of phospholipid in the tear film. Additionally, an Amplex Red phosphatidylcholine-specific phospholipase C (PLC) assay kit was used for determination of the activity of PLC in the tear film. Results: Phospholipids were not detected in any of the tested human tear samples with the low limit of detection being 1.3 µg/mL for TLC and 4 µg/mL for liquid chromatography mass spectrometry. TLC indicated that diacylglycerol (DAG) may be present in the tear film. PLC was in the tear film with an activity determined at approximately 15 mU/mL, equivalent to the removal of head groups from phosphatidylcholine at a rate of approximately 15 µM/min. Conclusions: This work shows that phospholipid was not detected in any of the tested human tear samples (above the lower limits of detection as described) and suggests the presence of DAG in the tear film. DAG is known to be at low concentrations in MGS. These observations indicate that PLC may play a role in modulating the tear film phospholipid concentration.

Clinical evaluation of the Oculus Keratograph

AIM: To determine the validity and reliability of the measurement of corneal curvature and non-invasive tear break-up time (NITBUT) measures using the Oculus Keratograph. METHOD: One hundred eyes of 100 patients had their corneal curvature assessed with the Keratograph and the Nidek ARKT TonorefII. NITBUT was then measured objectively with the Keratograph with Tear Film Scan software and subjectively with the Keeler Tearscope. The Keratograph measurements of corneal curvature and NITBUT were repeated to test reliability. The ocular sensitivity disease index questionnaire was completed to quantify ocular comfort. RESULTS: The Keratograph consistently measured significantly flatter corneal curvatures than the ARKT (MSE difference: +1.83±0.44D), but was repeatable (p>0.05). Keratograph NITBUT measurements were significantly lower than observation using the Tearscope (by 12.35±7.45s; pp < 0.001) and decreased on subsequent measurement (by -1.64 ± 6.03s; p < 0.01). The Keratograph measures the first time the tears break up anywhere on the cornea with 63% of subjects having NI-TBUT's <5s and a further 22% having readings between 5 and 10s. The Tearscope results were found to correlate better with the patients symptoms (r = -0.32) compared to the Keratograph (r = -0.19). Conclusions: The Keratograph requires a calibration off-set to be comparable to other keratometry devices. Its current software detects very early tear film changes, recording significantly lower NITBUT values than conventional subjective assessment. Adjustments to instrumentation software have the potential to enhance the value of Keratograph objective measures in clinical practice.

Ophthalmic engineering:the development of novel instrumentation to further research in the field

The principle theme of this thesis is the advancement and expansion of ophthalmic research via the collaboration between professional Engineers and professional Optometrists. The aim has been to develop new and novel approaches and solutions to contemporary problems in the field. The work is sub divided into three areas of investigation; 1) High technology systems, 2) Modification of current systems to increase functionality, and 3) Development of smaller more portable and cost effective systems. High Technology Systems: A novel high speed Optical Coherence Tomography (OCT) system with integrated simultaneous high speed photography was developed achieving better operational speed than is currently available commercially. The mechanical design of the system featured a novel 8 axis alignment system. A full set of capture, analysis, and post processing software was developed providing custom analysis systems for ophthalmic OCT imaging, expanding the current capabilities of the technology. A large clinical trial was undertaken to test the dynamics of contact lens edge interaction with the cornea in-vivo. The interaction between lens edge design, lens base curvature, post insertion times and edge positions was investigated. A novel method for correction of optical distortion when assessing lens indentation was also demonstrated. Modification of Current Systems: A commercial autorefractor, the WAM-5500, was modified with the addition of extra hardware and a custom software and firmware solution to produce a system that was capable of measuring dynamic accommodative response to various stimuli in real time. A novel software package to control the data capture process was developed allowing real time monitoring of data by the practitioner, adding considerable functionality of the instrument further to the standard system. The device was used to assess the accommodative response differences between subjects who had worn UV blocking contact lens for 5 years, verses a control group that had not worn UV blocking lenses. While the standard static measurement of accommodation showed no differences between the two groups, it was determined that the UV blocking group did show better accommodative rise and fall times (faster), thus demonstrating the benefits of the modification of this commercially available instrumentation. Portable and Cost effective Systems: A new instrument was developed to expand the capability of the now defunct Keeler Tearscope. A device was developed that provided a similar capability in allowing observation of the reflected mires from the tear film surface, but with the added advantage of being able to record the observations. The device was tested comparatively with the tearscope and other tear film break-up techniques, demonstrating its potential. In Conclusion: This work has successfully demonstrated the advantages of interdisciplinary research between engineering and ophthalmic research has provided new and novel instrumented solutions as well as having added to the sum of scientific understanding in the ophthalmic field.

Factors affecting corneoscleral topography

PURPOSE: To evaluate factors affecting corneoscleral profile (CSP) using Anterior Segment Optical Coherence Tomography (AS-OCT) in combination with conventional videokeratoscopy. METHODS: OCT data were collected from 204 subjects of mean age 34.9 years (SD: ±15.2 yrs, range 18 to 65) using the Zeiss Visante AS-OCT and Medmont M300 corneal topographer. Measurements of corneal diameter (CD), corneal sagittal height (CS), iris diameter (ID), corneoscleral junction angle (CSJ) and scleral radius (SR) were extracted from multiple OCT images. Horizontal visible iris diameter (HVID) and vertical palpebral aperture (PA) were measured using a slit lamp graticule. Subject body height was also measured. Associations were then sought between CSP variables and age, height, ethnicity, sex and refractive error data collected. Results: Significant correlations were found between age and ocular topography variables of HVID, PA, CSJ, SR and ID (P<0.0001), while height correlated with HVID, CD and ID, and power vector terms only with vertical plane keratometry, CD and CS. Significant differences were noted between ethnicities with respect to CD (P=0.0046), horizontal and vertical CS (P=0.0068 and P=0.0095), and also horizontal ID (P=0.0010), while the same variables, with the exception of vertical CS, also varied with sex; horizontal CD (P=0.0018), horizontal CS (P=0.0018) and ID (P=0.0012). Age accounted for up to 36% of the variance in CSP variables. Conclusion: Age is the main factor influencing corneoscleral topography; consequently, this should be taken into consideration in contact lens design, in the optimization of surgical procedures involving the cornea and sclera and in IOL selection.

Decellularization of bovine corneas for tissue engineering applications

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas’ biocompatibility.

The predictive ability of clinical tests for contact lens induced dry eye

Approximately half of current contact lens wearers suffer from dryness and discomfort, particularly towards the end of the day. Contact lens practitioners have a number of dry eye tests available to help them to predict which of their patients may be at risk of contact lens drop out and advise them accordingly. This thesis set out to rationalize them to see if any are of more diagnostic significance than others. This doctorate has found: (1) The Keratograph, a device which permits an automated, examiner independent technique for measuring non invasive tear break up time (NITBUT) measured NITBUT consistently shorter than measurements recorded with the Tearscope. When measuring central corneal curvature the spherical equivalent power of the cornea was measured as being significantly flatter than with a validated automated keratometer. (2) Non-invasive and invasive tear break-up times significantly correlated to each other, but not the other tear metrics. Symptomology, assessed using the OSDI questionnaire, correlated more with those tests indicating possible damage to the ocular surface (including LWE, LIPCOF and conjunctival staining) than with tests of either tear volume or stability. Cluster analysis showed some statistically significant groups of patients with different sign and symptom profiles. The largest cluster demonstrated poor tear quality with both non-invasive and invasive tests, low tear volume and more symptoms. (3) Care should be taken in fitting patients new to contact lenses if they have a NITBUT less than 10s or an OSDI comfort rating greater than 4.2 as they are more likely to drop-out within the first 6 months. Cluster analysis was not found to be beneficial in predicting which patients will succeed with lenses and which will not. A combination of the OSDI questionnaire and a NITBUT measurement was most useful both in diagnosing dry eye and in predicting contact lens drop out.

Evaluating transglutaminase crosslinked collagen gel systems for hard and soft tissue repair

Tissue Transglutaminase (TG2) and FXIIIa, members of the transglutaminase (TG) family, catalyses a transamidating reaction and form covalent bond between or within proteins. In bone development, both enzymes expressions correlate with the initial of the mineralisation process by osteoblasts and chondrocytes. Exogenous TG2 also promotes maturation of chondrocytes and mineralisation in pre-osteoblasts. To understand the role of endogenous TG2 in osteoblast mineralisation, the TG2 expression was examined during the human osteoblast (HOB) mineralisation. The expression of the endogenous TG2 increased during the mineralisation, yet, its expression was not essential for mineral deposition due to the compensation effect by other members in the TG family. The extracellular transamidating activity of HOBs was found increased during mineralisation and a shift from FXIIIa dominant- to TG2-dominant crosslinking activity was suggested after differentiation. However, the transamidating activity of both TG2 and FXIIIa were not critical for cell mineralisation. On the other hand, Exogenous TG2 was found to enhance wild type HOB and TG2 knockdown HOB mineral deposition. The transamidating activity of TG2 was not required but most likely a close conformation was essential for this enhancement. Results also demonstrated that exogenous TG2 may activate the ß-catenin pathway through LRP5 receptor thus contribute in cell mineralisation. This enhancement could be abolished by addition of ß-catenin inhibitors. Finally, using of TG2 crosslinked collagen gel for bone and cornea repair was evaluated. Crosslinked collagen gel showed promising results in improving HOB mineralisation, human corneal fibroblast (hCF) proliferation and migration. These effects might be resulted from the trapped TG2 within the collagen matrix and the alteration of matrix topography by TG2.

Anterior segment anomalies and effects on visual quality

The tear film, cornea and lens dictate the refractive power of the eye and the retinal image quality is principally defined by diffraction, whole eye wavefront error, scatter, and chromatic aberration. Diffraction and wave aberration are fundamentally pupil diameter dependent; however scatter can be induced by refractive surgery and in the normal ageing eye becomes an increasingly important factor defining retinal image quality. The component of visual quality most affected by the tear film, refractive surgery and multifocal contact and intraocular lenses is the wave aberration of the eye. This body of work demonstrates the effects of each of these anomalies on the visual quality of the eye. When assessing normal or borderline self-diagnosed dry eye subjects using aberrometry, combining lubricating eye drops and spray does not offer any benefit over individual products. However, subjects perceive a difference in comfort for all interventions after one hour. Total higher order aberrations increase after laser assisted sub-epithelial keratectomy performed using a solid-state laser on myopes, but this causes no significant decrease in contrast sensitivity or increase in glare disability. Mean sensitivity and reliability indices for perimetry were comparable to pre-surgery results. Multifocal contact lenses and intraocular lenses are designed to maximise vision when the patient is binocular, so any evaluation of the eyes individually is confounded by reduced individual visual acuity and visual quality. Different designs of aspheric multifocal contact lenses do not provide the same level of visual quality. Multifocal contact lenses adversely affect mean deviation values for perimetry and this should be considered when screening individuals with multifocal contact or intraocular lenses. Photographic image quality obtained through a multifocal contact or intraocular lens appears to be unchanged. Future work should evaluate the effect of these anomalies in combination; with the aim of providing the best visual quality possible and supplying normative data for screening purposes.

Astigmatism and vision:should all astigmatism always be corrected?

As technology and medical devices improve, there is much interest in when and how astigmatism should be corrected with refractive surgery. Astigmatism can be corrected by most forms of refractive surgery, such as using excimer lasers algorithms to ablate the cornea to compensate for the magnitude of refractive error in different meridians. Correction of astigmatism at the time of cataract surgery is well developed and can be achieved through incision placement, relaxing incisions and toric intraocular lens (IOL) implantation. This was less of an issue in the past when there was a lower expectation to be spectacle independent after cataract surgery, in which case the residual refractive error, including astigmatism, could be compensated for with spectacle lenses. The issue of whether presurgical astigmatism should be corrected can be considered separately depending on whether a patient has residual accommodation, and the type of refractive surgery under consideration. We have previously reported on the visual impact of full correction of astigmatism, rather than just correcting the mean spherical equivalent. Correction of astigmatism as low as 1.00 dioptres significantly improves objective and subjective measures of functional vision in prepresbyopes at distance and near.

Ultraviolet damage to the eye revisited:eye-sun protection factor (E-SPF®), a new ultraviolet protection label for eyewear

Ultraviolet (UV) radiation potentially damages the skin, the immune system, and structures of the eye. A useful UV sun protection for the skin has been established. Since a remarkable body of evidence shows an association between UV radiation and damage to structures of the eye, eye protection is important, but a reliable and practical tool to assess and compare the UV-protective properties of lenses has been lacking. Among the general lay public, misconceptions on eye-sun protection have been identified. For example, sun protection is mainly ascribed to sunglasses, but less so to clear lenses. Skin malignancies in the periorbital region are frequent, but usual topical skin protection does not include the lids. Recent research utilized exact dosimetry and demonstrated relevant differences in UV burden to the eye and skin at a given ambient irradiation. Chronic UV effects on the cornea and lens are cumulative, so effective UV protection of the eyes is important for all age groups and should be used systematically. Protection of children's eyes is especially important, because UV transmittance is higher at a very young age, allowing higher levels of UV radiation to reach the crystalline lens and even the retina. Sunglasses as well as clear lenses (plano and prescription) effectively reduce transmittance of UV radiation. However, an important share of the UV burden to the eye is explained by back reflection of radiation from lenses to the eye. UV radiation incident from an angle of 135°-150° behind a lens wearer is reflected from the back side of lenses. The usual antireflective coatings considerably increase reflection of UV radiation. To provide reliable labeling of the protective potential of lenses, an eye-sun protection factor (E-SPF®) has been developed. It integrates UV transmission as well as UV reflectance of lenses. The E-SPF® compares well with established skin-sun protection factors and provides clear messages to eye health care providers and to lay consumers. © 2014 Behar-Cohen et al, This work is published by Dove Medical Press Ltd.

Fiberised set-up for eye length measurement

We describe a configuration consisting of two fiberised Michelson interferometers and show that, with an optical balanced technique and suitable electronics, the signal corresponding to coherent interference in the first interferometer can be sufficiently attenuated in the channel processing the signal from the second interferometer. In this way it is possible to display simultaneous reflectograms of two different regions in the eye (e.g. cornea and retina) and infer the eye length from these measurements.

The eye and vision:an overview

The eye is the major organ of vision and highly specialized for photoreception. It focusses light from an object onto the light-sensitive retina. Changes in specialized neurons in the retina result in nerve action potentials which are relayed to the brain via the optic nerve. Visual processing by the brain results in ‘visual perception’, the construction of a sensory image which is consciously appreciated as vision. All other structures of the eye are subsidiary to this function, either by facilitating focusing of light rays or by supporting the tissues of the eye. This chapter is an introduction to the various parts of the eye including the eyelids and associated structures, conjunctiva, cornea, sclera, iris, lens, vitreous body, retina, optic disc and nerve, and orbit. This chapter describes the functions of these various structures and their importance in achieving a visual image.

Use of imaging technology to better understand soft contact lens fit dynamics

The principal theme of this thesis is the identification of additional factors affecting, and consequently to better allow, the prediction of soft contact lens fit. Various models have been put forward in an attempt to predict the parameters that influence soft contact lens fit dynamics; however, the factors that influence variation in soft lens fit are still not fully understood. The investigations in this body of work involved the use of a variety of different imaging techniques to both quantify the anterior ocular topography and assess lens fit. The use of Anterior-Segment Optical Coherence Tomography (AS-OCT) allowed for a more complete characterisation of the cornea and corneoscleral profile (CSP) than either conventional keratometry or videokeratoscopy alone, and for the collection of normative data relating to the CSP for a substantial sample size. The scleral face was identified as being rotationally asymmetric, the mean corneoscleral junction (CSJ) angle being sharpest nasally and becoming progressively flatter at the temporal, inferior and superior limbal junctions. Additionally, 77% of all CSJ angles were within ±50 of 1800, demonstrating an almost tangential extension of the cornea to form the paralimbal sclera. Use of AS-OCT allowed for a more robust determination of corneal diameter than that of white-to-white (WTW) measurement, which is highly variable and dependent on changes in peripheral corneal transparency. Significant differences in ocular topography were found between different ethnicities and sexes, most notably for corneal diameter and corneal sagittal height variables. Lens tightness was found to be significantly correlated with the difference between horizontal CSJ angles (r =+0.40, P =0.0086). Modelling of the CSP data gained allowed for prediction of up to 24% of the variance in contact lens fit; however, it was likely that stronger associations and an increase in the modelled prediction of variance in fit may have occurred had an objective method of lens fit assessment have been made. A subsequent investigation to determine the validity and repeatability of objective contact lens fit assessment using digital video capture showed no significant benefit over subjective evaluation. The technique, however, was employed in the ensuing investigation to show significant changes in lens fit between 8 hours (the longest duration of wear previously examined) and 16 hours, demonstrating that wearing time is an additional factor driving lens fit dynamics. The modelling of data from enhanced videokeratoscopy composite maps alone allowed for up to 77% of the variance in soft contact lens fit, and up to almost 90% to be predicted when used in conjunction with OCT. The investigations provided further insight into the ocular topography and factors affecting soft contact lens fit.

An overview of eye-related photophobias

This chapter provides an overview of the various eye-related causes of photophobia and the likely mechanisms responsible. Photophobia is the experience of discomfort affecting the eyes as a result of exposure to light. It has a variety of causes, including the result of eye or brain disease, or it can be a side effect of various drugs or laser surgery. Photophobia can also be a symptom of a more serious disorder such as meningitis and therefore, requires appropriate investigation, diagnosis, and treatment. Trauma or disease affecting several structures of the eye are a common cause of photophobia and can be associated with: (1) the ocular adnexia, such as blepharitis and blepharospasm, (2) the cornea, including abrasion, ulcerative keratitis, and corneal dystrophy, (3) problems in eye development, such as aniridia, buphthalmos, coloboma, and aphakia, (4) various eye inflammations, including uveitis, and (5) retinal disorders, such as achromatopsia, retinal detachment, and retinal dystrophy. There may be two main explanations for photophobia associated with these conditions: (1) direct stimulation of the trigeminal nerve due to damage, disease, or excessive light entering the eye and (2) overstimulation of the retina including a specific population of light-sensitive ganglion cells.

Biomaterial interactions with compromised tissue surfaces

This thesis is concerned with the nature of biomaterial interactions with compromised host tissue sites. Both ocular and dermal tissues can be wounded, following injury, disease or surgery, and consequently require the use of a biomaterial. Clear analogies exist between the cornea/tear film/contact lens and the dermal wound bed/wound fluid/skin adhesive wound dressing. The work described in this thesis builds upon established biochemistry to examine specific aspects of the interaction of biomaterials with compromised ocular and dermal tissue sites, with a particular focus on the role of vitronectin. Vitronectin is a prominent cell adhesion glycoprotein present in both tear fluid and wound fluid, and has a role in the regulation and upregulation of plasmin. The interaction of contact lenses with the cornea was assessed by a novel on-lens cell-based vitronectin assay technique. Vitronectin mapping showed that vitronectin-mediated cell adhesion to contact lens surfaces was due to the contact lens-corneal mechanical interaction rather than deposition out of the tear film. This deposition is associated predominantly with the peripheral region of the posterior contact lens surface. The locus of vitronectin deposition on the contact lens surface, which is affected by material modulus, is potentially an important factor in the generation of plasmin in the posterior tear film. Use of the vitronectin mapping technique on ex vivo bandage contact lenses revealed greater vitronectin-mediated cell adhesion to the contact lens surfaces in comparison to lenses worn in the healthy eye. The results suggest that vitronectin is more readily deposited from the impaired corneal tissue bed than the intact healthy tissue bed. Significantly, subjects with a deficient tear film were found to deposit high vitronectin-mediated cell adhesion levels to the BCL surface, thus highlighting the influence of the contact lens-tissue interaction upon deposition. Biomimetic principles imply that adhesive materials for wound applications, including hydrogels and hydrocolloids, should closely match the surface energy parameters of skin. The surface properties of hydrocolloid adhesives were found to be easily modified by contact with siliconised plastic release liners. In contrast, paper release liners did not significantly affect the adhesive surface properties. In order to characterise such materials in the actual wound environment, which is an extremely challenging task, preliminary considerations for the design of an artificial wound fluid model from an animal serum base were addressed.