Abstract
PurposeWe have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane.MethodsThy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption.ResultsThe pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence.ConclusionsInterconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection.
Highlights
The cornea is the most densely innervated structure in the human body and the trigeminal ganglion provides sensory innervation to the cornea
A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection; (ii) segmental transection; and (iii) annular transection
Several studies have reported that routine surgical procedures in ophthalmic practice such as corneal transplantation, photorefractive keratectomy (PRK), radial keratotomy and laser-assisted in situ keratomileusis (LASIK) cause disruption and dysfunction of corneal nerves [10]
Summary
The cornea is the most densely innervated structure in the human body and the trigeminal ganglion provides sensory innervation to the cornea. Corneal nerves influence stimuli (touch, temperature and pain) perception and blink reflex, tear formation and maintenance of hydration as well as wound healing and avoidance of injury [1,2,3,4,5,6,7]. Ocular diseases such as Neurotrophic Keratitis and Dry Eye Disease cause considerable morbidity which is attributed to corneal nerve dysfunction [8,9]. There are numerous studies on nerve loss, healing and regeneration after injury to the corneal nerves due to surgery, there is relatively scant knowledge on corneal nerve loss and regeneration in non-surgery scenarios of neurotrophic corneas wherein there is an absence of a surgical plane
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