To quantify microglial cells in the inner retinal layers (nerve fiber, ganglion cell, and inner plexiform layers) we modified (to adapt it to the rat retina) a semiautomated computerized routine (Image Pro Plus software) previously developed by our group to automatically quantify different retinal cell populations in mouse retinas.
54 Briefly, first the retinal area was drawn and measured. Then, the optic nerve and the limits of each retinal quadrant were manually pointed, usually at the center of the quadrant. Next, the automatic subroutine selected three standard equidistant areas in line between the optic disc and the marked quadrant limit. Thus, the subroutine selected 12 standard circular samples (0.25 mm
2 each) from each photomontage, and these areas were thus situated 4 in each retinal quadrant and 4 in each retinal region (central, equatorial, and peripheral,
Fig. 2). In the superotemporal quadrant, we avoided photographing the region of retinal injections (see Results) by marking the quadrant periphery far from these. Microglial cells were manually dotted on each of the circular samples described above (
Fig. 2). Next, the number of cells dotted was automatically quantified and the densities (± standard deviation of the mean, SD) of microglial (dotted) cells (cells/mm
2) were calculated in each sample. Finally, because there were no differences in the densities of microglial cells between the central, equatorial, and peripheral retina (see Results), we calculated the total numbers and densities of microglial cells for each retina using the total retinal area.