Characterisation of the neuroinflammatory response in a rat model of atrophic AMD

Abstract

Age-related macular degeneration (AMD) affects millions of individuals worldwide, and is the leading cause of blindness in the industrialised world. AMD is a multifactorial disorder, involving complex interaction between environmental and genetic factors. While the advent of anti-vascular endothelial growth factor (VEGF) therapy has allowed for the effective treatment of neovascular AMD, no effective treatments are available to mitigate 'dry' forms of AMD, including geographic atrophy. A role for inflammatory processes in the progression AMD has been postulated over a period of many years, mainly through observations of leukocyte infiltrates within AMD-affected eyes in traditional histological examinations and by electron microscopy. Recently however, rigorous genome-wide screening for short nucleotide polymorphisms (SNPs) has identified a significant association of a number of complement system gene variants with AMD, which together have firmly placed inflammation - particularly the involvement of innate immune pathways - as a key factor in the pathogenesis of AMD. Despite this, a number of key aspects of the inflammatory process remain to be clarified, including the spatiotemporal cellular events leading to local complement activation, and inflammatory cell recruitment in the macular region. In the investigations presented in this thesis, I aimed to investigate the suitability of light-induced retinal degeneration in rats as a model for atrophic AMD, and to explore the spatiotemporal emergence of inflammatory events in the retina in relation to light damage, with an emphasis on the recruitment of macrophages/microglia and the complement system.

Light-mediated retinal degeneration was induced in albino Sprague Dawley (SD) rats by exposure to bright continuous light (BCL) at 1000 lux for incremental periods between 0 and 24hrs, followed by a post-exposure period under standard dim-light (5 lux) conditions ranging from 0 to 56 days. Features of light-damage induced changes to the retina were compared to the histopathology of AMD using histological analyses, immunohistochemistry for markers of retinal stress (glial fibrillary acidic protein (GFAP) and fibroblast growth factor 2 (FGF-2)), and macrophage (ED1) markers, in relation to quantitative assessment of photoreceptor apoptosis, using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Following exposure to BCL, the spatiotemporal profile of monocyte-chemoattractant gene chemokine ( C-C motif) ligand 2 (Cc12) in the retina was investigated using a combination of quantitative real time polymerase chain reaction ( qPCR), in situ hybridisation, and immunohistochemistry. Expression of Cc12 was assessed in relation to photoreceptor death using TUNEL, and the infiltration of monocytes, quantified though counts of ED1-immunoreacitive (IR) macrophages/monocytes.

The expression of complement system genes in the retina following BCL was determined by microarray analysis, in which gene expression in animals reared in dim light conditions was compared with those exposed to 24hrs BCL. Genes of interest were validated using qPCR analysis on samples collected at 24 hours exposure, and over a protracted timecourse in the post-exposure period. Because it was highly expressed in the post-exposure period, and plays a pivotal role in the complement cascade, the spatiotemporal expression of complement component 3 (C3) in retinal sections was assessed using in situ hybridisation coupled with immunohistochemistry.

Exposing albino rats to BCL induced a rapid and prolonged emergence of focal histopathology at the area centralis - a homologue of the fovea centralis in the superior retina. Features of degeneration included breakdown of the blood retinal barrier, photoreceptor death, the prevalence of surviving cones over rods, macrophage recruitment, and reactive gliosis in Muller cells. Additionally the expression of stress factors GFAP and FGF-2 and photoreceptor degeneration continued at the margin of the lesion up to 56 days post-exposure. Progressive expansion of the lesion in the post exposure period - a feature of atrophic AMD - was a prominent aspect of the degeneration. The emergence of these features was accompanied by a focal recruitment of macrophages from the choroid and retinal vasculature at the lesion area. Each of these features is also observed in an atrophic AMD lesion, indicating that this light-damage model is a suitable model for atrophic AMD.

To understand the mechanisms that recruit macrophages into the light-damaged region, the spatiotemporal expression of Cc12 was assessed. It was observed that Cc12 is synthesised by Muller cells following BCL exposure, in spatiotemporal coincidence with the subsequent emergence of photoreceptor death at the area centralis. Following the expression of Cc12, a localised recruitment of macrophages from both retinal and choroidal vascular supplies was observed at the area centralis, correlating with the spatial distribution of Cc12-positive Muller cells. Recruited macrophages were also observed to express Cc12.

Expression of complement-related genes following exposure to BCL was investigated by microarray analysis. A suite differentially expressed complement genes was identified following exposure to 24hrs BCL, including opsonin mediators from classical and lectin pathways (C1s, C2, C4, Ficolin B), complement receptors (C1qR1, C3aR1, C5r1, CR3, CR4) and regulators (CD46, CD55, SERPING1, C4bp, a2m). A number of these were found by qPCR to positively correlate with levels of photoreceptor apoptosis, both during and after exposure to BCL. Most significantly the study shows by in situ hybridization that C3 deposited in the ONL and outer segments in the damaged region originates from recruited microglia/macrophages.

These findings pinpoint macrophages/microglia as key factors mediating activation of the complement system in the degenerating retina, and whose focal recruitment to the area centralis may be facilitated in part by chemotactic signals originating from the neural retina. These studies point to the retina as the primary site for initiation of degenerative mechanisms, rather that the supporting layers, including the RPE and choroid. Therapeutic attenuation of microglial/macrophage recruitment may be a useful strategy to control detrimental propagation of complement in the retina, particularly in retinal degenerations such as AMD. Show more