(a) overview image of control (vehicle-treated OIR) and Everolimus-treated OIR retinas (from n = 2 control and n = 2 drug-treated retinas). Avascular areas were visibly larger in Everolimus-treated retinas (marked with white line and adjacent to the optic nerve, yellow arrow) as previously reported. Scale bar, 300 μm. (b) tuft volume comparison n = 2 control OIR and n = 2 Everolimus-treated OIR, (c) quantification of tuft volume vs nuclei count, with and without drug treatment. n = 5 tufts from one drug-treated retina compared to comparable (medium) tuft volume data in Figure 5b. (d) Overview of Everolimus-treated OIR retina with many small tufts demonstrating highly active filopodia. Vessels were visualised with CD31, Scale bar, 20 μm. (e–f) Overhead view of three active adjacent tufts with similar morphology to d, from the top (e) and side (f). Vessels were visualised with CD31, nuclei were visualised with ERG. Scale bar, 20 μm. (g) Surface render (Imaris) of rapamycin-treated OIR tuft, which shows a swirl structure, as well as a visible hole through the centre of the tuft (white arrow). (h) Top and (i) side view of a Everolimus-treated tuft with a ring-like, layered arrangement of cells, and splayed filopodia representative of others observed in these retinas. Tufts were visualised using CD31 (green), while nuclei were visualised using ERG (blue). (j) Schematic of Everolimus treated tuft in c: viewing from the top, it is possible to see a hole or invagination (black circle) to the tuft and/or nuclei circled around it, while from the side, there are two layers of nuclei within the tuft, similar to the layered swirl of a control tuft from an LSFM imaged OIR retina in Figure 6d and microCT imaged OIR retina in Figure 6—figure supplement 2c,d.