Enfoques en la fijación de objetivos

A plethora of animal models have been used to investigate the pathogenesis of retinal degeneration and AMD, as well as potential treatment options (as reviewed in [117]). The previously described changes resulting from ageing in humans have been demonstrated in rodent models [50, 62, 118-123].

Animal models have been developed to study both wet and dry AMD [124]. Laser- induced damage models have been the leading technique used in the study of wet AMD due to its quick and reproducible administration [125]. A larger series of animal models have been characterised to mimic facets of dry AMD, reflecting its multifaceted nature. This involves targeting pathways involved in complement, chemokines, cytokines and oxidative stress [125, 126]. These pathways have been targeted using a mixture of genetic modification and experimentally administered techniques.

Genetic modifications in mice have been at the forefront of techniques used to study AMD. The target genetic modifications have been informed by leading studies such as GWAS, which offered valuable insight into the genes that may be implicated in AMD. Although a single mice strain that mimics all facets of AMD has yet to be identified, knock- out mice strains for key genes including C3 [127], Cfh [128, 129], Ccl2 [130], Cx3c [131],

Sod-1 [132, 133], and APOE [134-136] have provided an avenue to further enhance our current understanding of AMD.

In contrast to the use of genetic modifications, experimentally administered models have become commonplace in the study of dry AMD. One such example includes the use of intravenous injection of sodium iodate (NaIO3) to chemically-induce retinal degeneration in

the rodent retina [137]. This technique has been employed to mimic RPE and photoreceptor cell death observed in AMD [137].

A further example is the use of photo-oxidative damage (PD) to induce retinal degeneration in rodents. This is a well-established model and has been widely used since the 1960s [138]. Although rodents do not have a macula or fovea, they have a functionally analogous region, the area centralis. Located in the central region of the retina, the area centralis has the highest concentration of photoreceptor cells [139]. The existing literature has been built upon through the establishment and characterisation of PD models in both rats and mice [140, 141]. These models mimic key facets of AMD including photoreceptor degeneration at a focal point in the superior retina, expansion of this lesion over time, upregulation of oxidative stress and inflammatory stress markers implicated in the pathogenesis of AMD, recruitment of retinal microglia and macrophages, and reduced retinal function [140, 141]. This thesis includes the use of both of these rodent models. The focus of Chapter 3 and monitoring retinal damage meant that the use of rats over a long, protracted time course was the preferred model. The focus of later chapters in this thesis relate to potential therapeutics and therefore lends itself to use of the PD model in mice, that while still mimicking the facets of AMD, could also be efficiently achieved over a shorter time course. In addition to animal models used to study retinal degeneration in vivo, the techniques used to undertake the analysis are of great importance.

1.6.2 Techniques: optical coherence tomography (OCT)

Traditional histological techniques have been the leading form of analysis used to enhance our understanding of retinal degeneration. However, the development of medical technologies has been pertinent to advancing the approach to diagnosis and treatment of disease. One key example is optical coherence tomography (OCT). OCT has revolutionised

cross-sectional images of the retina in vivo, in real time [143]. The use of OCT in guiding personalised retinal disease treatment has reduced US Medicare costs by $9 billion US dollars [144-148], highlighting the magnitude of its impact and its effectiveness.

The basic principle underpinning OCT is that coherent long-wavelength light waves are propagated to retinal tissue and the reflection of these light waves is measured in a form that adapts to an image [142, 143, 149, 150]. First used in 1991 [151], the OCT is functionally analogous to an ultrasound, except it uses light instead of sound. OCT works by emitting light that travels via an optical beam directed at retinal tissue [143]. The image is captured by measuring the backscattered light waves from internal structures [143]. Specifically, OCT depends on time-delay information contained in light waves, which has been reflected from different depths inside the sample and returned to the detector in order to form an image [142]. The OCT functions at wavelengths of 800 nm to 1300 nm, with detection of shorter wavelengths indicating shallow penetration of the light, and the longer wavelengths indicating deeper penetration [142].

The leading advantage of this technique is that it is non-invasive, while still enabling the observation of minute structural changes to the retina. It also has the ability to trace advancement of disease in individual animals and provide paired observations, offering an alternative to the use of multiple animals per time point for a single study [150, 152].

OCT is a powerful tool for the visualisation of morphological changes observed in retinal disease (as reviewed in [153, 154]). OCT has been used in the ophthalmic clinical setting in the study of retinal diseases including AMD [155, 156], diabetic and cystoid macular edema [156-162], retinitis pigmentosa [163], glaucoma [164-170], and vitreoretinal conditions [171]. The advancement of OCT angiography has also enabled an effective method of visualising changes to retinal vasculature resulting from disease [172, 173]. It has been

well recognised in clinical practice and the literature as having great potential to assist in the therapeutic targeting of retinal degenerative diseases [174-176].