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Hugo Adrian Barba, Melanie Spedale, Jason Zhang, Asadolah Movahedan, Urooba Nadeem, Nini Deng, Eugene Chang, Betty Theriault, Dimitra Skondra; Germ-Free Laser-Induced Murine Model Shows The Effect Of The Microbial Organ On Choroidal Neovascularization.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):959 – A0428.
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© ARVO (1962-2015); The Authors (2016-present)
Emerging data link the gut microbiome with neovascular age related macular degeneration (nAMD) and choroidal neovascularization (CNV), its hallmark lesion. Our team has previously described how the microbial organ modulates the retinal expression of genes/pathways associated with nAMD such as vascular endothelial growth factor (VEGF), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha and hypoxia-inducible factor (HIF) 1 pathway. To further confirm the effects of the gut microbiome on nAMD, we compared the laser-induced CNV formation between germ-free (GF) mice and specific pathogen free mice (SPF).
Male GF C57Bl/6 mice were bred/kept in sterile isolators in the UChicago Gnotobiotic Facility. Age/gender-matched SPF C57Bl/6 mice were used as control. Around 16 weeks of age, mice underwent laser photocoagulation (power 120mW/duration 0.1 sec/50um spot size, Argon 532 nm, Iridex OcuLight GL) using our specialized sterile protocol for GF mice and conventional protocol for SPF mice. Choroidal flatmounts were immunostained with isolectin IB4 for CNV and IBA-1 for activated microglia. Images were taken using a Leica-SP5 confocal microscope. CNV and IBA-1 signal were analyzed with Image-J software and data were analyzed with R Studio. Sterility of GF mice was checked weekly with cultures and PCR.
PCR and cultures were negative before and after the laser sessions confirming GF sterility. GF group showed decreased CNV size by 35% (12,940.05um2±5,627um2, n=9 vs 19,873.39um2±13,336um2, n= 20, P= 0.0345 ) but had similar CNV formation rate compared to SPF group ( 92% vs 94% respectively). Number of IBA-1 positive activated microglia within 100um outside the lesion was decreased in the GF mice (5.36 and ± 4 vs 9.03 ± 5, P = 0.03). However, IBA-1 signal within CNV lesion was similar between the two groups (55.66 ± 2.79 vs 55.86 ± 6.55, P > 0.05).
These results, together with the RNA-seq data previously obtained, suggest that the microbial organ may have a considerable influence on the development of nAMD lesions. The GF laser-induced CNV model allowed us to eliminate other factors such as antibiotics showing the direct effects of the overall microbiome over the CNV formation. Further studies are needed to determine which microbes, either protective or pathogenic, have the highest impact on the development of nAMD and the mechanisms involved.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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