PMC3971924_01
Stereomicroscope images of the right cornea with diagnosed EBMD. A) Dark-field illumination at a low magnification showed that numerous irregular geographic shaped, faint gray-white patches were present in the central area of the inferior quadrants of the cornea. B) Highly oblique illumination of the large framed area in A showed that the pathologic lesions were present as finger-prints (arrows), dots (arrows with circles at the end) and ridge-like patterns (dashed arrows). C) Dark-field of the central area (dashed frame in A) in higher magnification and D) oblique illumination showed the details of four finger-print lesions.
nihms-565270-f0001.jpg
PMC3971924_01_nihms-565270-f0001.jpg
F1
In the EBMD eye, dark-field illumination (Figure 1A) showed numerous irregular geographically-shaped, faint gray-white patches that appeared as finger-print dots and ridge-like lesions. Figure 1B shows the frame area from Figure 1A using oblique illumination. Figure 1D; this was taken from the dashed frame area of Figure 1A. Figure 1C is the dark-field image of Figure 1D.
A) Whole mount view of epithelial nerve distribution in the normal left cornea. Images were acquired in a time-lapse mode with a Nikon Eclipse TE200 and with a 5X lens in compliance with the natural shape of the cornea. The epithelial nerve bundles ran in a radial pattern from the periphery to merge in an area within the inferior-nasal quadrant beyond the corneal apex. Highlighted images showed the detailed architecture of the epithelial nerve distribution in the peripheral (B), central (C), and vortex areas (D). E) High magnification image recorded from the central area as marked in Figure 2C shows the nerve terminals at the superficial epithelia. F) Whole mount view of corneal epithelial nerve distribution in the central area of the inferior quadrant of the right cornea. The total area is about 45 mm2, consisting of 38 images recorded in a time-lapse mode with a 10X lens. Arrows indicate the pathological loci. G) and H) are highlighted images showing the detailed architecture of the epithelial nerve distribution in the central and vortex area as marked in F). I) Topographic images, recorded at the plane of superficial epithelia (i), wing cells (ii), basal cells (iii) and the basement membrane (iv), show detailed epithelial innervation in a finger-print lesion (arrow) at the central cornea.
nihms-565270-f0002.jpg
PMC3971924_01_nihms-565270-f0002.jpg
F2
Figure 2A shows the whole mount view of the epithelial nerve architecture of the normal cornea. Highlighted images (Figure 2B-D) show the details of the epithelial nerve architecture in the periphery, center and vortex areas. Free nerve endings budding from the network innervated the epithelial cells (Figure 2E). Figure 2F shows the whole mount view of the epithelial nerve distribution within the pathological area in the EBMD cornea. Highlighted images show the epithelial nerve distribution in pathological loci at the central (Figure 2G) and vortex (Figure 2H) areas; arrows show the pathological lesions. Topographic images in Figure 2I showed the detailed epithelial nerve architecture at different depths from superficial (I) to the subbasal layer of the epithelia (IV) in a finger-shaped lesion located in the central area.
Effect of EBMD on corneal subbasal nerve density and nerve terminals. (A) Subbasal nerve density in the central area of the inferior quadrant was calculated as percentage of total area in each image. A total of 36 images were taken at a magnification of 10 x lens from each eye. These images were changed to grayscale mode and placed against a white background to get better contrast using Photoshop imaging software. The nerve fibers of each image were carefully drawn with four-pixel lines following the course of each fiber. Percentage of nerve area was quantified for each image using the image analysis program. Data are expressed as average +- SD. (B) Representative images of EBMD and normal subbasal nerves. (C) Number of nerve terminals. 20 images for each eye from the central corneas were used. Nerve terminals in superficial epithelia in each image were counted. Because each image took up an area of 0.335 mm2, the terminal numbers per mm2 were calculated. Data are expressed as average +- SD. (D) Representative images of nerve terminals in both eyes.
nihms-565270-f0003.jpg
PMC3971924_01_nihms-565270-f0003.jpg
F3
As shown in Figure 3A and B, a big difference in subbasal nerve density was found between the EMBD and the normal eye. There was an obvious decrease in nerve terminals in the EMBD eye (Figure 3C, D).