Retinal ganglion cells from normal, untreated, and BDNF-treated
eyes were compared using a computer-based imaging system. The region
selected for quantitative analysis occupied 1.72
mm
2, and was located 3 mm above and 1.5 mm
temporal to the area centralis
(Fig. 1) . This region was chosen because of the relatively constant size and
density of ganglion cells in this location of the cat
retina.
22 A stage digitizer (AccuStage, Shoreview, MN) was
used to properly orient
30 each retina on the microscope,
and to standardize the starting point and stage movements used for cell
sampling. From the starting point, 42 digital images (41,000μ
m
2/image) were obtained systematically using a
high resolution video camera (chilled CCD, model C5985; Hamamatsu) and
40× objective. The retinal images were collected as three
dorsa
l-ventral passes composed of 14 images each. Double
counting was avoided by separating each sample column horizontally by
500 μm, and vertically by using the previous image as a reference.
Cell size, density, and number were determined directly from the
digital images using image analysis software (Image Pro Plus, Media
Cybernetics). Neurons were classified as ganglion cells based on the
criteria of Stone.
24 25 In brief, they had to display a
distinct nucleus and nucleolus, and have a clear ring of cytoplasm
completely surrounding the nucleus. Although this conservative approach
most likely underestimated the number of small ganglion cells, it also
reduced the number of nonganglion cells included in the
measurements.
26 27 Based on these criteria, our estimates
of ganglion cell density, as well as the proportions of small, medium,
and large ganglion cells in the normal sample region were comparable to
those reported by Stone.
24