The adult mouse corneas (22 mice) have a radius of approximately 1.5 mm. To calculate the subbasal epithelial nerve densities, we divided the mouse cornea into central and peripheral zones. The central zone was defined by a radius of 0.5 mm starting at the apex, and the peripheral zone with a radius of 0.5 mm beginning at the limbus. To avoid overlap, approximately 0.5 mm of space between the two zones was left uncounted.
To get a better contrast, the fluorescent images were changed to grayscale mode and placed against a white background using imaging software (Photoshop; Adobe Systems, Inc., Mountain View, CA, USA). The subbasal nerve fibers in each image were carefully drawn with 4-pixel lines following the course of each fiber by using the brush tool in the imaging software (Adobe Systems, Inc.). The nerve area and the total area of the image were obtained by using the histogram tool. The percentage of total nerve area was quantified for each image as described previously.25 (link)28 (link, link, link) To compare nerve densities in the central and peripheral areas, eight images for each zone were randomly chosen from each cornea (two images/quadrant). A total of 80 images for each zone from 10 corneas of 10 mice (5 mice/sex) were averaged. Nerve terminals in the superficial epithelia within the central and peripheral zones were calculated by directly counting the number of terminals in each image. Twenty-four images per zone from six corneas were analyzed. The terminal numbers in each image were counted directly. Since each image comprised an area of 0.335 mm2, the terminal numbers per square millimeter were calculated.
To examine the relative content of neuropeptides in the subbasal nerves, 12 corneas that had been stained with anti-βIII-tubulin were double-stained with CGRP or SP. For each neuropeptide, a total of 24 whole-mount images from the central zone (one image/quadrant) were taken, and then the same numbers of images were taken for βIII-tubulin. In the same visual field, the percentage of βIII-tubulin equaled that of the total nerve area, and the ratio of the peptide-positive nerve area against βIII-tubulin represented the relative content.
To calculate CGRP- and SP-positive neurons in the TG, 20 images were selected randomly from 10 mice (1 section/ganglion) and counted in a blind fashion. Differences in central and peripheral corneal nerve densities, terminal numbers, and the relative content of neuropeptides in the central cornea and TG were expressed as means ± SEM and t-test was performed; P < 0.05 was considered a statistically significant difference between two groups.
To get a better contrast, the fluorescent images were changed to grayscale mode and placed against a white background using imaging software (Photoshop; Adobe Systems, Inc., Mountain View, CA, USA). The subbasal nerve fibers in each image were carefully drawn with 4-pixel lines following the course of each fiber by using the brush tool in the imaging software (Adobe Systems, Inc.). The nerve area and the total area of the image were obtained by using the histogram tool. The percentage of total nerve area was quantified for each image as described previously.25 (link)
To examine the relative content of neuropeptides in the subbasal nerves, 12 corneas that had been stained with anti-βIII-tubulin were double-stained with CGRP or SP. For each neuropeptide, a total of 24 whole-mount images from the central zone (one image/quadrant) were taken, and then the same numbers of images were taken for βIII-tubulin. In the same visual field, the percentage of βIII-tubulin equaled that of the total nerve area, and the ratio of the peptide-positive nerve area against βIII-tubulin represented the relative content.
To calculate CGRP- and SP-positive neurons in the TG, 20 images were selected randomly from 10 mice (1 section/ganglion) and counted in a blind fashion. Differences in central and peripheral corneal nerve densities, terminal numbers, and the relative content of neuropeptides in the central cornea and TG were expressed as means ± SEM and t-test was performed; P < 0.05 was considered a statistically significant difference between two groups.
