Permeability measurements

Retinal explants were mounted for visualisation and further experimentation on an upright Zeiss Axiophot fluorescence microscope. A radial vessel of a superfused retinal explant was cannulated at ∼150 μm from the optic nerve using a microinjection needle (tip diameter 1–5 µm, sharpened to a bevel of <30°) and the entire retinal vasculature injected with sulforhodamine B (1 mg/ml in Krebs solution). Illumination was switched to fluorescence, and the vessels were visualised under a TRITC filter using an Olympus 40× water immersion objective. For permeability measurements, a microvessel was chosen at least 200 µm away from the cannulated radial vessel. Fluorescent content of the vessel was recorded continuously by time-lapse imaging (1 frame/2 s) on a Hamamatsu CCD camera for at least 2 min. A baseline was recorded for ∼30 s, before VEGF-A or BK (in Krebs solution) was added on the top of the retina. Time-lapse series were analysed using ImageJ. Time-dependent fluorescence intensity data of the chosen vessel was derived from a square region of interest (∼18×18 pixels; Fig. S1A,B). Fluorescence in the immediate vicinity of the microvessel was measured and subtracted from the vessel fluorescence measurements. Pixel intensity measurements were charted against time, and permeability values were computed by fitting data to the exponential equation C_t=C₀×e^−kt, where k=4P/d and d is the diameter of the vessel (Hudson et al., 2014). The difference in permeability between pre-treatment and post-treatment resulted in the absolute permeability change associated with the treatment regimen.