Rat Optic Nerve Vascular Imaging

Rodent ON vascular filling was performed following Nicholson et al (2012) [16 (link)]. Quantitative ON vascular analysis was performed using tissue from terminally anesthetized rats. After euthanasia, animals were placed on a warming pad (~38°C) and transcardially perfused sequentially with the following heated (~38°C) solutions: 120 ml heparinized saline (50 units/ml) with 2 μg/ml atropine sulfate (Sigma Chemicals) and 100 μM adenosine (HAAS solution), 50 ml fluorescein-conjugated bovine serum albumen (FITC-BSA) with 2% dissolved gelatin (300 bloom, Sigma Chemicals) in HAAS solution, 20 ml FITC-BSA with 4% dissolved gelatin in HAAS solution. Tissues were fixed in 4% neutral-buffered paraformaldehyde (PFA), washed in phosphate buffered saline (PBS), embedded in 10% gelatin (#G1890, Sigma), and re-fixed for an additional 24 h in PFA. Tissues were cryosectioned to 40 μm and imaged by confocal microscopy using tiled z-stacks imaged on a Zeiss LSM510 Duo (Carl Zeiss Microscopy, Gottingen) fitted with a 40x 1.4 NA oil objective. Vascular data was quantified using a filament model constructed using Imaris software (Bitplane Software) (See Nicholson et al. (2012) for method details [16 (link)]).