Vascular Leakage in the Retina

Detection of vascular leakage was performed using Evans Blue as previously described (Ou et al., 2019). Briefly, anesthetized mice (n = 5/group) received intravenous injections of saline containing 2% Evans Blue, which circulated for 10 min. Mice were then euthanized, and eyes were enucleated and incubated in 4% paraformaldehyde for 2 h. After fixation, retinas were isolated and flat mounts were imaged on a Leica DMI 600B inverted microscope connected to a Retiga EXi camera (Q-imaging Vancouver, BC). The number of leaking vessels was manually quantified, while vascular leakage was quantified by calculating fluorescence intensity in 10 field areas using Metamorph imaging software (Molecular Devices Downington, PA). Representative images of retinal flat mount (upper quadrant) and 100X magnified areas that were quantified (lower quadrant) is displayed in Figure 6A.

Vascular leakage in the retina of diabetic mice. (A) Representative images of vascular leakage in retinas of non-diabetic, 20 μM XMD8-92 treated diabetic, and untreated diabetic mice; following intravenous injections of Evans Blue. Upper quadrant is a full scan of the retinal vasculature, while the lower quadrant displays magnified sections of a leaking vessel. (B) Quantifications of the number of leaking vessels in the retinas (n = 5/group) of non-diabetic (white), XMD8-92 treated diabetic (grey), and diabetic (black) mice. (C) Quantification of vascular leakage measured by fluorescence intensity units (FIU) within a 1.10 μm² area of each retina of all mice. The p-value was first equated by determining differences between the three sets of data using a one-way factorial ANOVA, wherein these statistical differences are symbolized by the horizontal lines. Significant differences were then validated and the p-value was calculated using an unpaired t-test with Tukey’s post-hoc analysis. All samples were collected 8 months after diabetic conditions were confirmed.