Most corneal nerve fibers are derived from the ophthalmic branch of the trigeminal nerve. Nerve bundles enter the peripheral mid-stroma in a radial pattern. These nerves lose their perineurium and myelin sheath within 1 mm of limbus, aiding the maintenance of corneal transparency, and are subsequently surrounded by only Schwann cell sheaths.
3 The nerves course anteriorly, giving rise to multiple branches innervating the anterior and mid-stroma.
1 Branches form the subepithelial nerve plexus that lies at the interface between Bowman’s layer and the anterior stroma. This plexus is sparse and patchy in distribution, largely limited to the mid-peripheral cornea, and may not be present in the central cornea.
7 Fifty percent of these nerves exhibit varicosities or beads.
1 Nerve bundles penetrate Bowman’s membrane throughout the central and peripheral cornea
4 5 at approximately 400 sites.
8 Bundles then divide and run parallel to the corneal surface between Bowman’s layer and the basal epithelium, forming the sub-basal nerve plexus.
4 5 These nerve bundles consist of straight and beaded fibers, with the beaded fibers located in the periphery of the bundle. The beads have been identified as axonal efferent and sensory terminals
6 8 and have been shown to consist of accumulations of mitochondria and glycogen.
2 Only the beaded fibers subsequently form branches that enter the corneal epithelium, where they terminate.
3 4 8
Because early studies of corneal nerves were unable to clearly identify the corneal position, the orientation of the sub-basal nerves was uncertain.
4 5 The introduction of the clinical in vivo confocal microscope enabled imaging of the living human sub-basal nerve plexus.
9 On the basis of data from serial section light and electron microscopy studies, in vivo confocal microscopy, and immunohistochemistry, it has been postulated that nerve bundles are preferentially orientated in a superior-inferior direction at the corneal apex and in a nasal-temporal direction in surrounding areas, with no defined focus.
3
The two-dimensional reconstructions we report reveal the architecture of the sub-basal nerve plexus over the central 5 to 6 mm of the cornea. Sub-basal corneal nerve architecture in vivo has not previously been elucidated to this extent because most in vivo confocal microscopy studies of the sub-basal nerves have restricted examination to the corneal apex. This is mainly because of the relative ease of obtaining good-quality tangential images in this region. The observation that the sub-basal nerves form a complex series of anastomoses is consistent with the pattern seen in the human dermal nerve plexus, though the latter shows a less regular arrangement.
10
Human histologic studies have shown that epithelial nerve branches are orientated perpendicularly to the corneal surface.
5 This suggests that if there is centripetal epithelial slide, the epithelial cells and epithelial nerves must be moving in the same direction and at the same velocity. Using in vivo confocal microscopy, Auran et al.
7 provided evidence that corneal epithelial nerves participate in centripetal migration. Sub-basal nerves were demonstrated to shift centripetally toward an area just inferior to the corneal apex, with velocities ranging from 1.7 to 32.0 μm a day. Shapes and lengths of nerve segments varied slightly as axons slid centripetally. Studies have also shown that, unlike the sub-basal plexus, the stromal nerves, subepithelial plexus, and nerve perforation points through Bowman’s layer remain stationary over extended periods of time and may be used as landmarks for study.
7 11
The radiating pattern of nerve fibers converging on a whorl-like complex reported here echoes the pattern seen in the epithelium in corneal verticillata and toxic keratopathies.
12 This suggests that epithelial cells and nerves may migrate centripetally in tandem. The patterns identified in the latter two conditions are typically clockwise.
13 Dua et al.
13 postulated that the combined effect of the electric and magnetic fields on centripetally migrating epithelial cells would result in a clockwise whorled pattern. Other theories regarding the driving force for centripetal movement of epithelial cells include: (1) preferential desquamation of the central corneal epithelium, drawing peripheral cells toward the central cornea; (2) population pressure from limbus and peripheral cornea because of proliferation and migration of cells; (3) gradient of chemical signals emanating from limbal capillary vessels; and (4) stimulation by epithelial sympathetic nerves.
14
A possible explanation for the location of the whorl in the inferocentral cornea is that cell and nerve migration may be affected by shearing forces exerted by the eyelids on blinking, and the focus of the whorl is at the site of the end of upper and lower lid excursion.
The mean central nerve density reported here (21,668 μm/mm
2) is significantly higher than values previously reported in studies using white light in vivo confocal microscopy ([11,110 μm/mm
2]
1 [8404 μm/mm
2]
15 ), probably because of differences in image quality and contrast. Images obtained using slit-scanning in vivo confocal microscopy are brightest along a central vertical strip and become darker laterally. Thus, nerve fibers at the edges of the image may not be clearly visible. Images obtained using the RCM are relatively uniform in contrast and brightness throughout the image. Additionally, the light source for this modality is coherent and brighter. This appears to enable visualization of the thinner nerve fiber bundles, which are not otherwise visible with slit-scanning in vivo confocal microscopy (authors’ unpublished data, May 2005). The recent observation that there appears to be no correlation between age and sub-basal nerve density
15 suggests that differences in the ages of the subjects in this study, compared with other in vivo confocal studies of nerve density, are unlikely to explain the observed differences in sub-basal nerve density.
The central corneal location was determined by use of a charge-coupled device (CCD) camera attachment enabling live imaging of the cornea from the temporal side during examination
(Fig. 1b) . Although this technique is useful for localizing the approximate area of the cornea under examination, it is not sufficiently accurate to enable determination of the exact point location of the corneal apex.
The use of a fixation grid is a novel method of facilitating in vivo confocal examination of different corneal locations. Theoretically, a precise relationship between points on the grid and locations on the cornea could be developed; however, factors such as disconjugate eye movements, microsaccades, involuntary patient movements, and difficulty aligning the center of the grid with the corneal apex make such topographic measurements inaccurate at present.
The multiple bright particles observed in two of the subjects resemble those recently reported by Zhivov et al.,
16 who postulated that these represent Langerhans cells. In the latter study, these cells were present in the central cornea and in the inferior periphery of 85.7% of healthy subjects.
We report the first study to elucidate the distribution of sub-basal nerves in the living human cornea. Although this pilot study is limited by the small number of subjects and although the power of the study was low (50% on post hoc power analysis), it provides interesting new data regarding the architecture of the corneal sub-basal nerve plexus. Knowledge of the orientation of these nerves in various regions of the cornea may be helpful in aiding localization during in vivo confocal microscopy, when only small areas of the cornea are examined. Future studies may be directed at analyzing nerve architecture over time or in disease states known to affect corneal nerve structure and corneal sensation.