Retinal Glial and Choroidal Vascular Pathology in Donors Clinically Diagnosed With Stargardt Disease

Abstract

The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD). METHODS. Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the disease-causing mutations. Eyes were enucleated and fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. After imaging, retinas were dissected and immunostained for glial fibrillary acidic protein, vimentin, and peanut agglutin. Following RPE removal, the choroid was immunostained with Ulex europaeus agglutinin lectin. For each choroid, the area of affected vasculature, percent vascular area, and choriocapillaris luminal diameters were measured. The retina from one donor was hemisected and cryopreserved or embedded in JB-4 for cross-section analysis. RESULTS. Genetic testing confirmed the STGD diagnosis in donor 1, whereas a mutation in peripherin 2 was identified in donor 3. Genetic testing was not successful on donor 2. Therefore, only donor 1 can definitively be classified as having STGD. All donors had areas of RPE atrophy within the macular region, which correlated with underlying choriocapillaris loss. In addition, Müller cells formed pre-and subretinal membranes. Subretinal gliotic membranes correlated almost identically with RPE and choriocapillaris loss. CONCLUSIONS. Despite bearing different genetic mutations, all donors demonstrated choriocapillaris loss and Müller cell membranes correlating with RPE loss. Müller cell remodeling was most extensive in the donor with the peripherin mutation, whereas choriocapillaris loss was greatest in the confirmed STGD donor. This study emphasizes the importance of genetic testing when diagnosing macular disease.

Histopathological assessments reveal retinal vascular changes, inflammation, and gliosis in patients with lethal COVID-19

“…The retina was dissected and washed overnight in TBS. The RPE/choroid was incubated in the disodium salt of ethylenediaminetetraacetic acid for 1.5 h, and RPE was removed using a pipette before washing with TBS overnight, and wholemount staining was performed as described with the exception that the tissues were incubated with the antibodies and the UEA lectin for 4 days [17]. Retinas were stained with chicken anti-glial fibrillary acidic protein (GFAP; 1:500; Millipore, Burlington, MA, USA), mouse anti-SARS-CoV S Protein (1:100; NR-614, BEI Resources, NIAID, Manassas, VA, USA), rabbit anti-Iba-1 (1:250; Wako Chemicals USA, Inc., Richmond, VA), and Ulex europaeus agglutinin-FITC (UEA lectin1:100; GeneTex, Irvine, CA, USA).…”

Histopathological assessments reveal retinal vascular changes, inflammation and gliosis in patients with lethal COVID-19

“…The retina was dissected and washed overnight in TBS. The RPE/choroid was incubated in the disodium salt of ethylenediaminetetraacetic acid for 1.5 hours and RPE was removed using a pipette before washing with TBS overnight and wholemount staining was performed as described with the exception that the tissues were incubated with the antibodies and the UEA lectin for 4 days 16 . Retinas were stained with chicken anti-glial fibrillary acidic protein (GFAP; 1:500; Millipore, Burlington, MA, USA), mouse anti-SARS-CoV S Protein (1:100; NR-614, BEI Resources, NIAID, Manassas, VA, USA), rabbit anti-Iba-1 (1:250; Wako Chemicals USA, Inc., Richmond, VA) and Ulex europaeus agglutinin-FITC (UEA lectin1:100; Genetex, Irvine, CA, USA).…”

Evidence of complement dysregulation in outer retina of Stargardt disease donor eyes

“…All three globes displayed different levels of central atrophy of the retina by fundus macroscopy: STGD #1 showed a classic STGD phenotype with severe RPE loss and hyperpigmentation in the perimacular region [ 34 ]; STGD #2 exhibited a small parafoveal atrophic area while STGD #3 displayed a large region of macular atrophy [ 35 ]. Genetic information could not be collected for STGD #2 due to the poor DNA quality while STGD #3 was found to carry a heterozygous mutation (c.629C > G, p.Pro210Arg) in the PRPH2 gene.…”

“…Here, we showed that electron-dense lipofuscin and melanolipofuscin granules, both key pathological hallmarks for STGD, were much more abundant in the STGD donor eyes versus control eyes. Additional morphological features of the same STGD donor eyes were described in a recent study by Edwards et al [ 35 ]. Analyzing the flatmounts of both retinas and choroids from these STGD donor eyes, they evidenced severe attenuation of the choriocapillaris in areas with RPE loss.…”

“…Analyzing the flatmounts of both retinas and choroids from these STGD donor eyes, they evidenced severe attenuation of the choriocapillaris in areas with RPE loss. Furthermore, they found that the area of RPE atrophy was accompanied by an extension of Müller cell processes into the subretinal compartment unlike the area with an intact monolayer which displayed choriocapillaris with normal density and lumenal diameter [ 35 ]. Previously, Taubitz et al reported severely degenerated photoreceptors resulting in geographic atrophy in the macula of the STGD #3 retina, a typical clinical appearance for Stargardt patients [ 37 ].…”