A quantitative study on the effects of corneal crosslinking on corneas in an ex vivo model

Project code:  MHS066

Department

Ophthalmology

Location

Auckland

Supervisor

Dr Jie Zhang

Keratoconus is a progressive, non-inflammatory thinning disorder of the cornea that compromises the stromal collagen matrix resulting in protrusion and alteration of corneal shape resulting in decreased vision, or in severe cases even blindness. Data from the New Zealand National Eye Bank over the past two decades have consistently reported keratoconus as the leading indication for corneal transplantation, accounting for between 40-45% of corneal transplants performed annually.

Corneal collagen crosslinking (CXL) is a technique used to strengthen corneal tissue utilising riboflavin as a photosensitizer and Ultraviolet-A (UVA). As a side effect, this procedure results in keratocyte loss in the corneal stroma, and in very thin corneas, irreversible loss of corneal endothelial cells which can be devastating. Currently there is no objective measurement of the depth and extent of CXL in vivo and ex vivo. CXL efficacy in patients is evaluated by corneal topography and stabilization of disease progression. The aim of this project is to investigate whether formation of reactive oxygen species (ROS) can be used as a quantitative measurement of the extent of CXL, and to compare ROS levels to the depth of CXL in ex vivo corneas. 

Methods

1. ROS quantification.

CXL induces crosslinks in collagen fibrils via the formation of ROS.  The amount of ROS released after CXL will be assessed using a Fluorometric Intracellular ROS detection Kit. Ex vivo porcine corneas will be obtained from a local butcher, sterilized using iodine-povidine and PBS, debrided of their epithelium, and treated with CXL (treatment group) or no CXL (control group), and compared to intact porcine corneas without epithelium debridement. Directly following the treatments corneas will be snap-frozen in OCT and sectioned. The extent and location of ROS formation after CXL will be assessed using the Fluorometric Intracellular ROS detection Kit (Sigma MAK142). Fresh frozen 16 µm thick sections of nasal, central and temporal cornea will then be imaged using fluorescence microscopy, 3 images from each section, to compare the production of ROS in the treatment and control groups. The total amount of ROS labelling in each image will be quantified using the ‘Analyze particles’ function in Image J software. Results will be analysed using one-way ANOVA followed by post-hoc tests. The appropriate post-hoc test will be chosen after determining homogeneity of variances.

2. CXL depth quantification.

Keratocytes undergo apoptosis due to debridement and UV damage within one day of CXL in humans. The magnitude and depth of CXL will be assessed histologically by evaluating the depth of keratocyte apoptosis at four hours post treatment. Half of the corneas will be fixed in 4% paraformaldehyde for 2 hours followed by cryoprotection in 20% and 30% sucrose. Each cornea will be embedded in OCT compound and rapidly frozen in liquid nitrogen. Sagittal 16 μm cryosections will be processed for immuno-labelling with rabbit anti-Caspase 3 antibody and TUNEL labelling. For each cornea, three locations at nasal, central, and temporal cornea will be imaged by confocal microscopy, therefore 9 images per cornea will be analysed. The depth of apoptosis and the total number of apoptotic cells in each image will be quantified using ImageJ software. Results will be analysed using one-way ANOVA followed by post-hoc tests. The appropriate post-hoc test will be chosen after determining homogeneity of variances.  

Skills

Tissue processing, immunohistochemistry, confocal microscopy, Reactive oxygen species detection.

Investigation of corneal epithelium matrix production in healthy and keratoconic corneas

Project code:  MHS068

Department

Ophthalmology

Location

Auckland

Supervisor

Carol Greene

Keratoconus, an ectatic corneal dystrophy, affects approximately 1 in 2000 individuals worldwide. The progressive thinning of the corneal stroma typically occurs over decades and results in a conical shaped cornea that then impairs vision due to irregular astigmatism and myopia. Keratoconus can show the following pathologic findings, including, fragmentation of Bowman’s layer, thinning of stroma and overlying epithelium, folds or breaks in Descemet’s membrane, and variable amounts of diffuse corneal scarring. The goal of this project is to  compare extracellular matrix deposition (ECM) by corneal epithelium cells from healthy and keratoconic corneas. Findings will provide insight into whether or not there is aberrant matrix production associated with keratoconus and will help in the development of novel strategies for regenerating the corneal layers.

Skills

Cell and tissue culture, immunohistochemistry, confocal microscopy

Quantification of vascular lesions in optical coherence tomography (OCT) and fundus images in a choroidal neovascularisation (CNV) mouse model

Project code:  MHS119

Department

Ophthalmology

Location

Auckland

Supervisor

Dr Ilva Rupenthal

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are major causes of blindness in the elderly and the working population, respectively. Both diseases are associated with extensive vascular disruption leading to the formation of new but leaky blood vessels (also known as neovascularisation). This results in fluid accumulation within sensitive areas of the retina, resulting in visual impairment. In order to achieve such leaky blood vessels in a mouse model, laser burns can be applied to the back of the eye to induce choroidal neovascularisation (CNV). The resulting damage as well as the reduction in injury after treatment with a novel peptide drug can be assessed using optical coherence tomography (OCT) and fundus imaging. The overall aim of the project is to develop the laser-induced CNV mouse model and compare changes in vascular lesion size of untreated and treated mice. In order to achieve this, the role of the summer student will be:

  1. To examine vascular lesions in OCT and fundus images obtained from a CNV mouse model
  2. To develop methods by which the observed vascular lesions can be quantified 

Skills

  • Characterising OCT and fundus images
    • Identification of vascular lesions
    • Haemorrhage scoring
  • Image processing and analysis
    • ImageJ software
    • Micron IV software
  • Statistical analysis
    • Microsoft Excel software
    • GraphPad Prism software

In vivo tolerability and safety of semi-fluorinated alkanes for treatment of dry eye syndrome

Project code:  MHS128

Department

Ophthalmology

Location

Auckland

Supervisor

Dr Ilva Rupenthal

Dry eye syndrome (DES) is one of the most prevalent ocular surface disorders, which is generally characterized by increased evaporation or decreased production of tear fluid, resulting in damage to the interpalpebral ocular surface and moderate to severe discomfort. Standard DES therapy usually involves a combination of symptomatic (artificial tears) and pathogenic (antibiotics) approaches. Semi-fluorinated alkanes (SFAs), a novel class of amphiphilic liquids, have recently been suggested for the management of DES. In vitro studies have shown that SFAs have excellent wetting and spreading properties and could have a lubricating effect on the ocular surface providing symptomatic relief. Furthermore, SFAs have also been found to be efficient drug delivery carriers for several hydrophobic drugs, such as Cyclosporine A, making it possible to treat DES by a dual therapeutic approach. However, there is currently limited information about the effect of SFAs on ocular surface properties and tear fluid dynamics. The aim of this this study is to evaluate the tear fluid dynamics after topical instillation of SFAs and eliminate any potential ocular surface irritation in vivo.

Skills

  • Animal handling for in vivo studies
  • Inferometry to assess tear film integrity and lipid layer thickness
  • Evaporimetry to evaluate the rate of tear film evaporation
  • Tear film osmolarity measurements
  • Phenol red test to measure tear volume
  • Sodium fluorescein staining and clinical scoring

Rocket science and ‘super’ vision

Project code:  MHS145

Department

Ophthalmology

Location

Grafton and Greenlane Clinical Centre

Supervisor

Dr Stuti Misra

Although being initially developed for aeronautics, wavefront analysis evaluates imperfections in optical systems and has been applied to characterise the optics of the eye. Currently, several wavefront sensing devices are available for the assessment of higher order aberrations in the human eye. This information can be used for selection of the most appropriate intraocular lens to maximise the visual outcome following cataract surgery, laser vision surgery and even customised contact lens fitting. The primary aim of the project is to assess the repeatability and comparability of ocular higher order aberrations and corneal aberrations obtained by the Zywave, OPDIII, iTrace and Galilei 2, clinical aberrometers. It is hypothesised that the four imaging devices are comparable and can be used inter-changeably. Furthermore, all of these devices are based on different wavefront measuring principles, including Hartmann-Shack, ray tracing and skiascopy so the accuracy and repeatability of each modality may vary. The study will evaluate reliability of each type of aberrometer.

Research team:

Dr Stuti Misra

Dr James McKelvie

Professor Charles McGhee

Skills

It is envisaged that the successful student will learn: basic methodologies of wavefront sensing devices, acquisition of wavefront data using clinical aberrometers, application of statistics and data analysis, and how to structure and prepare the first draft of a scientific paper.

It is envisaged that this project will result in sufficient quality to be published in a scientific journal.