PMC

Genetic profiles of GA-like lesions in lasered mice correlate with genes upregulated in GA patients. (a) mRNA expression in retinas of mice at 4 weeks post laser measured by SYBR Green qPCR. Values are shown as an average increase in gene expression in lasered eyes compared to the non-lasered fellow eye of each animal. Pooled data for 11 animals (8 mice aged 5 months, and 3 mice aged 11–13 months). Multiple paired one-tailed t-test followed by Holm-Sidak multiple comparison correction. Significance denoted by *p < 0.05 and **p < 0.01. (b) A genetic profile of lasered retinas created by grouping upregulated mRNAs into distinct but potentially overlapping disease processes; (1) complement activation, (2) glial/macrophage activation and (3) inflammatory pathway.

A diagrammatic figure summarising early GA-like pathology in lasered mouse retinas. Based on colour fundus photography, OCT, light and electron microscopy, confocal-immunofluorescence, ERG as well as genetic profiling, we present a summary of histopathological features in a representative lasered mouse retina at 12 weeks post treatment. Distinct zones of GA-like histopathology can be identified in a manner akin to those described by Sarks and colleagues in donor GA tissues. Structural changes are associated with functional retinal defects as well as activation of glial/macrophage, complement and inflammatory pathways reported in GA patients. With rapid disease onset, a high degree of experimental reproducibility as well as other important disease features, this model is useful for studying early stages of GA.

GA-like pathology corresponds to functional deficits in the mouse retina. Electroretinography (ERG) was used in longitudinal studies to measure the function of different retinal cell types. Average ERG traces are shown for lasered and non-lasered groups at (a) 1 week before laser, (b) 1 week after laser, (c) 2 weeks after laser, (d) 4 weeks after laser, and (e) at 8 weeks after laser. ERG values for lasered eyes were normalised to the fellow/control eye in each animal. (f) Comparison of A-wave amplitudes between lasered and non-lasered eyes. (g) Comparison of B-wave amplitudes, and (h) B-wave implicit times between lasered and non-lasered eyes. Arrows indicate point of the light stimulus. An average of 3 ERG measurements were obtained per eye at each time point (n = 8, five month old mice). Analysis by two-way ANOVA (repeated measures by both factors, factor 1: control vs. lasered eye, factor 2: time) followed with Holm-Sidak post-hoc testing. Graphs presented as mean ± SEM, significance denoted by *p < 0.05, **p < 0.01 and ***p < 0.0001.

The anatomy of geographic atrophy (GA) and development of GA-like pathology in lasered mice. (a) Schematic diagram of the outer retina showing arrangement of different cell layers. Outer nuclear layer (ONL), Photoreceptor IS (inner segments) and OS (outer segments), Retinal pigment epithelium (RPE), Bruch’s membrane (BrM) and Choriocapillaris. An arrow indicates the path of light. (b) A colour fundus photograph (CFP) of a healthy human retina with the macula encircled in yellow, (c) compared to a diseased retina from a GA patient. Notice presence of a central/macular lesion with defined borders. (d) Black and white optical coherence tomography scan of a GA lesion taken from a Heidelberg OCT, and (e) a cross-section (from central green line in d) showing respective layers of the outer retina affected by disease. (f) Development of GA-like pathology in lasered mice. Representative CFP from mice before and after laser and in subsequent weeks. Notice how multiple lasered spots in the central murine retina coalesce after 1 week to create a focused atrophic region. Longitudinal CFP imaging of a five month old mouse shows this GA-like lesion to persist for up to 8 weeks after laser treatment. The timeline of developing GA-like pathology alongside experimental end-points are also shown.

Longitudinal optical coherence tomography (OCT) of lasered mouse retinas show development of GA-like pathology. Representative OCT scan (a) 1 week before and (b) 1 week after laser treatment. Notice obliteration of the ONL and photoreceptor IS/OS. The clear demarcation of the RPE-BrM layer is also lost, although further details of pathology are difficult to distinguish. The OPL tapers into lesions and is no longer discernible as a distinct layer above the lasered spot. (c–e) Over the following weeks, lesions continue to persist, indicated by thickened areas of hyper-reflective pathology with progressive involvement of the INL and IPL, which appear to merge into lasered areas. Use of OCT scans thus provided insights into the involvement of distinct retinal layers over a 2 month period following laser treatment. Retinal ganglion cells (RGC), Inner plexiform layer (IPL), Inner nuclear layer (INL), Outer plexiform layer (OPL), Outer nuclear layer (ONL), IS/OS (inner and outer segments) of photoreceptors and Retinal pigment epithelium (RPE). OCT scans however do not allow the RPE and Bruch’s membrane (BrM) to be distinguished as separate layers. (f) OCT data was used to quantify the relative thickness of retinal lesions between weeks 1, 2, 4 and 8 following laser ablation. A statistically significant decrease in retinal thickness at lesion sites was observed between weeks 1, 2 and 4, after which the thickness of the retina became stable between weeks 4 and 8. Average of three measurements per lesion (n = 7, five month old mice). SEM with significance denoted by *p < 0.05 and ***p < 0.0001. [g] Speculative diagram based on OCT data showing arrangement of retina and associated tissues within lasered spots.

Structural changes and chronic inflammation within lesions and adjacent tissues in lasered mice correspond to early GA-like pathology. (a) Representative semi-thin tissue sections from an eleven month old mouse at 3 months post-treatment and stained with Toluidine blue show a characteristic GA-like lesion where photoreceptors are absent in lasered areas. (b) Magnified insert from (a) (yellow box) where the INL is observed lying in close apposition to the RPE. The inner retina had collapsed into the area that had been occupied by the degenerated photoreceptor layer. Lesion margins could be observed as well-defined wedge-shapes, with photoreceptor IS/OS and ONL as well as the OPL taking on a more normal appearance further away from the lesion. The RPE and BrM remains intact alongside a choroid that also appears to be unaffected. Scale bars in A and B corresponds to 100 μm. (c) Representative electron micrograph showing pathology in lesion margins. Areas where RPE cells had become atrophic can be observed (white arrows) alongside photoreceptor OS horizontally positioned next to the underlying BrM. However, the RPE monolayer was intact in most micrographs. Importantly, the BrM itself appears to remain unaffected. Photoreceptor OS followed by IS and the ONL gradually tapers closer to the lesion. (d) Micrograph from within the lesion showing the INL next to the RPE in the absence of a photoreceptor layer. The RPE monolayer shows neighbouring hypopigmented as well as hyperpigmented cells. Microvilli on the apical RPE surface appear to be disorganised and shorter in length (yellow arrows), whilst the BrM shows varying thicknesses (white arrows). Scale bars in c and d correspond to 1000 nm. (e) Representative confocal-immunofluorescence comparing GFAP (red) and FcγRI (green) staining in non-lasered tissues vs. (f) adjacent lasered areas at 4 weeks post treatment. Notice GFAP staining in processes extending into what appears to be remaining photoreceptors within lesions after 4 weeks, which had disappeared by 12 weeks when visualised by light and electron microscopy. GFAP expression was also observed in the INL and IPL layers, extending beyond the lesion into surrounding tissues. No GFAP expression was observed in non-lasered tissues except for constitutive staining near the vitreous interface. Upregulated FcγRI expression was observed clustered within lesions and in marginal tissues (yellow arrows). Only minimal FcγRI expression was observed in non-lasered tissues. Nuclei were labelled with DAPI and appear blue. Scale bars in e and f correspond to 100 μm. Retinal ganglion cells (RGC), Inner plexiform layer (IPL), Inner nuclear layer (INL), Outer plexiform layer (OPL), Outer nuclear layer (ONL), IS/OS (inner and outer segments) of photoreceptors and Retinal pigment epithelium (RPE).