Figure 1.

Hypoxia increases Mo resistance to RPE-induced elimination. (A) schematic representation of the experiments: mice were laser injured and either exposed to 10% O₂ hypoxia from d0–d4 and evaluated for the presence of subretinal IBA1⁺MPs at d4 or exposed to 10% O₂ hypoxia from d4–d10 and evaluated for MPs and the extent of Collagen 4⁺CNV at d10. Mice raised in normal 20.9% ambient O₂ served as controls. (B) Representative images of Collagen 4 (Coll4; red) and IBA-1 (green) immuno- stained RPE/choroid flatmounts of 10 days post laser-injured normoxic and hypoxic (d4–d10) mice. (C,D) Quantification of the density of subretinal IBA-1+ MPs per impact: (B) directly within the RPE-denuded lesion and (C) counted at a distance of 0–500μm to Coll4⁺CNV on the apical side of the RPE surrounding the lesion at d4 and d10 of normoxic and hypoxia-exposed 2-month-old mice (room air white columns, 10% O₂ hypoxia d0-d4 orange columns; 10% O₂ hypoxia d4–d10 blue columns) (n = 9–10 eyes; ^$ p = 0.0503; * p < 0.0001 Mann–Whitney versus their normoxic controls). (E) Quantification of the Coll4⁺CNV surface at d10 of normoxia and hypoxia-exposed (d4–d10) laser-injured mice (n = 9–10 eyes; * p = 0.0060 Mann–Whitney versus normoxic control). (F) Representative pictures of PU-1 OTX-2 co-stained co-cultures of PU1⁺human monocytes (green) and OTX-2⁺RPE cells (red) under normoxic-(20% O₂; white columns) and hypoxic-(2% O₂; black columns) conditions and their automated quantifications after 24 h co-culture (n = 5 wells; * p = 0.0079 Mann–Whitney versus the normoxic condition), (G) quantifications of PU1⁺human monocytes after 24 h of hypoxic (black column) or normoxic (white column) pre-incubation of Mo followed by 24 h of Mo/RPE coculture (n = 5 wells; * p = 0.0079 Mann–Whitney versus the normoxic condition) (H) automated quantifications of PU1⁺human monocytes after 24 h of hypoxic (black column) or normoxic (white column) pre-incubation of RPE followed by 24 h of Mo/RPE coculture. The in vivo results presented in (B–E) summarize two independently carried out experiments, the in vitro experiments (F–H) were repeated a minimum of five times and gave similar results. All values are reported as mean  ±  SEM. IBA-1: ionized calcium adapter molecule 1; Coll4: Collagen 4; PU1: hematopoietic transcription factor; OTX-2: Orthodenticle Homeobox 2; scale bar A = 400 μm, E = 500 μm.