Fig. 6

Diabetes increases optic nerve vascularization, while repeated glucose challenge enlarges PVS in the optic nerve. (A) Representative images of lectin-stained vasculature in optic nerve cross sections from groups of four-month diabetic and control mice (top) and one-month glucose or saline injected mice (bottom). Scale bar: 50 μm. (B) Percentage of lectin-positive stained area in optic nerve cross sections for anterior (first 100 μm behind orbit) (left) and posterior (4–5 mm behind orbit) (right) optic nerve sections (n = 3–4 mice, 1 data point = average of 2–4 analyzed cross sections). Right hand graphs represent the summary for anterior and posterior sections. (C) Blood vessel length (µm) measured in optic nerve cross sections at different segments (control/diabetic: n(anterior) = 16–18, n(posterior) = 26–27; saline/glucose: n(anterior) = 31–37, n(posterior) = 30). (D-E) Measurement of perivascular size in optic nerve cross sections of four-month diabetic and control mice (D) and for one-month glucose of saline-injected healthy mice (E), along with representative images (scale bar: 5 μm). Perivascular space size was calculated by subtracting lectin signal diameter (blue line ROIs) from the tracer signal diameter (yellow line ROIs) (left graphs), and width ratio calculated by diving tracer signal diameter with lectin signal diameter, thus normalizing for different vessel sizes (right graphs); (n(control/diabetic) = 34–39, n(saline/glucose) = 35–42; 3–4 different animals). (C-E) Color gradings indicate single mice. B) Unpaired two-tailed t-test with Welch’s correction; (C-E) Linear mixed-effects model. ****P ≤ 0.0001, **P ≤ 0.01, ns = P > 0.05. All graphs show mean ± SD