Investigation of pathomechanisms and treatment safety of age-related and juvenile macular degeneration

In humans, high-resolution vision relies on a specialised anatomical structure of the retina, the so called macula. While only 5.5 mm in diameter, the macula is indispensable for visual tasks depending on visual fixation of an object, such as reading and facial recognition. Loss of the macula therefore heavily impairs affected individuals, even though peripheral vision is not inhibited and spatial orientation is still possible. Macular degeneration usually arises either as multifactorial age-dependent disease (age-related macular degeneration, AMD) or as rare hereditary disease (the most common one being Stargardt disease, STGD1). AMD can be sub-classified in a “wet” (exudative) form that is characterised by neovascularisation with collateral oedema and bleeding, and a “dry” (non-exudative) form defined by local degeneration of photoreceptors, retinal pigment epithelium cells and the supporting capillary bed. Inherited Stargardt disease has a similar clinical presentation to dry AMD, even though the underlying disease mechanisms differ. To date, no cure for AMD or Stargardt disease is available. However, since wet AMD is accompanied with a surplus of growth factors, e.g. vascular endothelial growth factor (VEGF), the repeated intravitreal application of anti-VEGF therapeutics allows for a reversal of acute visual distortions due to oedema and a reduction of disease progression. This thesis deals with the pathologic processes and pharmacological therapy of macular degeneration and therefore investigates three aspects: 1) pathomechanisms in AMD and Stargardt disease, investigated by light- and electron microscopy on human donor samples and three Stargardt mouse models, respectively 2) resolving the subcellular origin of near-infrared autofluorescence (NIR-AF) used in diagnostics and monitoring of retinal diseases, such as AMD and STGD1 3) adverse events on the ultrastructural level after intravitreal use of antiVEGF compounds for treatment of wet AMD A light and electron microscopic investigation of human ocular tissue with and without AMD revealed that a loss of choriocapillaris, the nourishing capillary bed underneath the retina, precedes retinal degeneration in both the wet and the dry form of AMD. This study answered the open question of which retinal tissue is the first to degenerate in AMD. Stargardt disease progression was investigated in three commonly used Stargardt mouse models and revealed distinctive pathologic changes only present in an albino, but not in two pigmented mouse models, highlighting the potential beneficial impact of ocular melanin in disease modulation. NIR-AF was shown to not be an intrinsic property of melanin, as previously thought, but rather a consequence of melanin damage due to photic and/or oxidative stress. Furthermore, NIR-AF was found to not only stem from melanosomes, but also partly from lipofuscin, a byproduct of the visual cycle that accumulates with age and/or disease in the retinal pigment epithelium. These findings are relevant for both elucidating the pathomechanisms in melanin- and lipofuscinassociated retinal diseases and the refinement of NIR-AF based diagnostics. And lastly, previous ultrastructural investigations of adverse events of the immunoglobulin G (IgG)-based anti-VEGF compound bevacizumab suggested that the fragment crystallisable (Fc) unit is involved in the development of said adverse events. This was confirmed by studies analysing ultrastructural changes after intravitreal application of anti-VEGF compounds with and without Fc fragment in monkey eyes and intravitreal application of isolated Fc fragments in rat eyes. These works emphasize the role of the Fc fragment in adverse events of IgG-based pharmacological compounds used in ophthalmology. To conclude, the present thesis investigates key events in the pathomechanisms of AMD (order of tissue loss) and STGD1 (indicated protective role of melanin), identifies the subcellular origin of the NIR-AF signal used in ophthalmologic diagnostics and disease monitoring and confirms the involvement of the Fc fragment in adverse events observed after intravitreal application of anti-VEGF compounds

Ultrastructural alterations in the retinal pigment epithelium and photoreceptors of a Stargardt patient and three Stargardt mouse models: indication for the central role of RPE melanin in oxidative stress

Background Stargardt disease (SD) is characterized by the accumulation of the age-pigment lipofuscin in the retinal pigment epithelium (RPE) and subsequent neuroretinal degeneration. The disease leads to vision loss early in life. Here, we investigate age-dependent ultrastructural changes in three SD mouse models: albino Abca4-/- and pigmented Abca4-/- and Abca4-/-.Rdh8-/- mice. Since we found indications for oxidative stress primarily in albino SD mice, we tested RPE melanin for its antioxidative capabilities. Methods SD mouse eyes were investigated by light, fluorescence and electron microscopy and were compared to the respective albino and pigmented wild type mice and to a human donor SD eye. To confirm the role of RPE melanin in scavenging oxidative stress, melanin from S. officinalis as a standard and porcine RPE were tested for their capability to quench superoxide anions. Results Histological alterations indicative of oxidative stress and/or lysosomal dysfunction were present in albino Abca4-/- and Abca4-/-.Rdh8-/- mice. Retinal damage, such as inner segment rupture and pyknotic or free photoreceptor nuclei in the subretinal space and RPE vacuolization were exclusively found in albino Abca4-/- mice. Shortened and disorganized photoreceptor outer segments and dead RPE cells were found in albino Abca4-/- and Abca4-/-.Rdh8-/- mice, with earlier onset in albino Abca4-/- mice. Undegraded phagosomes and lipofuscin accumulation were present in the RPE of all three SD strains, but numbers were highest in Abca4-/-.Rdh8-/- mice. Lipofuscin morphology differed between SD strains: (melano-)lipofuscin granules in pigmented Abca4-/- mice had a homogenous electron density and sharp demarcations, while lipofuscin in albino Abca4-/- mice had a flocculent electron density and often lacked a surrounding membrane, indicating loss of lysosomal integrity. Young Abca4-/-.Rdh8-/- mice showed (melano-)lipofuscin granules with homogenous electron density, while in aged animals granules with flocculent electron density predominated. Both strains of pigmented SD mice had melanolipofuscin clusters as found in the human SD eye. Like melanin from S. officinalis, porcine RPE melanin can also quench superoxide anions. Discussion The presented pathologies in albino Abca4-/- and Abca4-/-.Rdh8-/- mice suggest oxidative stress and/or lysosomal dysfunction within the RPE. Since albino Abca4-/- mice have the earliest onset and severest damage and as absence of melanin and also melanin turnover with age are known to diminish RPEs anti-oxidative properties, we assume that RPE melanin plays a role in SD related damages. A lack of pathology in pigmented Abca4-/- mice due to lower stress levels as compared to the Abca4-/-.Rdh8-/- mice underlines this hypothesis. It is also supported by the finding that RPE melanin can quench superoxide anions. We therefore suppose that RPE melanin is important in retinal health and we discuss its role as an oxidative stress scavenger

Fundus autofluorescence, spectral-domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease

Fundus autofluorescence, spectral‐domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease

npSCOPE: A New Multimodal Instrument for In Situ Correlative Analysis of Nanoparticles

Over the last few decades, nanoparticles have become a key element in a number of scientific and technological fields, spanning from materials science to life sciences. The characterization of nanoparticles or samples containing nanoparticles, in terms of morphology, chemical composition, and other parameters, typically involves investigations with various analytical tools, requiring complex workflows and extending the duration of such studies to several days or even weeks. Her; we report on the development of a new unique in situ correlative instrument, allowing us to answer questions about the shape, size, size distribution, and chemical composition of the nanoparticles using a single probe. Combining various microscopic and analytical capabilities in one single instrument allows a considerable increase in flexibility and a reduction in the duration of such complex investigations. The new instrument is based on focused ion beam microscopy technology using a gas field ion source as a key enabler and combining it with specifically developed secondary ion mass spectrometry and scanning transmission ion microscopy technology. We will present the underlying concept, the instrument and its main components, and proof-of-concept studies performed on this novel instrument. For this purpose, different pure titanium dioxide nanoparticle samples were investigated. Furthermore, the distribution and localization of the nanoparticles in biological model systems were studied. Our results demonstrate the performance and usefulness of the instrument for nanoparticle investigations, paving the way for a number of future applications, in particular, nanotoxicological research.ISSN:1520-6882ISSN:0003-270