Exploitation of an ex vivo disease model to characterise early events in retinal degeneration

Aims

This project aims to replace the use of some animal studies by developing and exploiting an ex vivo culture model of age-related macular degeneration which reproduces key features of early disease.

Background

Age-related macular degeneration (AMD) is a degenerative disease that poses significant risks to those over 50 years and affects two-thirds of individuals in their 80s, resulting in the irreversible loss of central vision.  A critical process in visual loss is the gradual impairment of the retinal pigment epithelium (RPE), a monolayer of cells in the retina which maintains photoreceptors on its apical surface and basally preserves the blood-retinal barrier. Animal models of AMD including rabbits, rodents and pigs, do not reproduce all the features of the disease and in particular, rodents lack a macula, the main structure damaged in the human eye.

The recent discovery that amyloid beta is involved in key stages of retinal pathology offers the opportunity to develop new approaches to treat early stages of the disease. The use of an ex vivo model to study the role of the amyloid beta protein and inflammation in early RPE impairment could potentially reduce animal use by up to 50%.

Research details and methods

This project will use confluent monolayers of mouse RPE cells grown on a porous support which mimics the Bruch’s membrane, the structure which separates the RPE from the fenestrated choroidal capillaries. This ex vivo culture model has previously been shown to have characteristics observed in early AMD such as drusen formation and inflammation. A variety of methods including live fluorescence and utrastructural imaging as well as molecular biology and biochemical approaches will be used to investigate amyloid beta driven changes in the RPE. The model will also be used to study amyloid beta-mediated inflammatory events. 

Age-related Macular Degeneration (AMD) is a degenerative disease of a critical region of the retina known as the macula. AMD affects 50 million people globally and poses increasing risks from midlife onwards, resulting in the irreversible loss of central vision. A key process in AMD is the gradual impairment of the Retinal Pigment Epithelium (RPE), a monolayer of cells in the retina which maintains photoreceptors as well as the blood-retinal barrier. Early AMD is asymptomatic, but typified by the presence of sub-RPE deposits known as drusen, associated with local inflammation. Loss of central vision in late AMD is correlated with RPE/photoreceptor atrophy and proliferation of new vessels through the blood-retinal barrier. Although some treatment is available, the majority of patients eventually lose their central vision. AMD is a complex disease, where underlying processes are incompletely understood, particularly during early stages. To address this, numerous experimental models of AMD have been created in rodents, rabbits, pigs and primates. However, no single animal model has reproduced the full disease spectrum, while the widely utilised rodent models suffer from the absence of a macula, the key structure damaged in the human eye. Here we aim to replace, reduce and refine the number of animals used in early AMD studies by exploiting an ex vivo culture model of AMD which reproduces salient features of early disease including drusen formation and inflammation. This model will be used to elucidate the role of Abeta and inflammation in early RPE dysfunction. We are supported by KalVista Pharmaceuticals, an industry leader in the design/development of anti-inflammatory inhibitors, providing translational capacity to any findings from this work.

Ratnayaka, J A & Lotery, A J (2020) Challenges in studying geographic atrophy (GA) age-related macular degeneration: the potential of a new mouse model with GA-like features Neural Regen Res 15:863-4 doi: 10.4103/1673-5374.268972

Keeling E et al. (2019). Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE). Molecular Nutrition & Food Research e1800951. doi :10.1002/mnfr.201800951

Keeling E et al. (2018). Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases. Cells 7(2):16. doi: 10.3390/cells7020016

Lynn SA et al. (2018). A convenient protocol for establishing a human cell culture model of the outer retina. F1000Research 7:1107. doi: 10.12688/f1000research.15409.1

Lynn SA et al. (2017). Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE. Tissue & Cell 49(4):447-460. doi: 10.1016/j.tice.2017.06.003

Lynn SA et al. (2017). The complexities underlying age-related macular degeneration: could amyloid beta play an important role? Neural regeneration research 12(4):538-548. doi: 10.4103/1673-5374.205083

Ratnayaka JA and Lynn SA (2016). Alzheimer’s-Related Amyloid Beta Peptide Aggregates in the Ageing Retina: Implications for Sight Loss and Dementia, Update on Dementia, Davide Vito Moretti, IntechOpen doi: 10.5772/64790

Taylor-Walker G et al. (2016). The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2). Experimental eye research 153(110-121). doi: 10.1016/j.exer.2016.10.013

Ratnayaka JA et al. (2015). Dementia of the Eye: the role of Amyloid beta in Retinal Degeneration. Eye 29(8):1013–1026. doi: 10.1038/eye.2015.100

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PhD Studentship

Status:

Closed

Principal investigator

Dr J. Arjuna Ratnayaka

Institution

University of Southampton

Co-Investigator

Professor Andrew Lotery

Grant reference number

NC/L001152/1

Award date:

Nov 2014 - Oct 2017

Grant amount

£90,000