Purpose This study quantifies the level of neuron-specific fluorescence in the cornea of transgenic mice expressing yellow fluorescent protein (YFP) driven by the thy1 promoter and examines the viability of using thy1-YFP mice as a model for studying nerve regeneration in vivo.
Methods The structure of corneal innervation in mice homozygous for the thy1-YFP transgene visible with reporter gene fluorescence was compared with that visible with traditional immunofluorescence techniques. The percentage of corneal nerves with YFP fluorescence in whole-mounted corneas and trigeminal neuron cultures was determined. Regeneration of fluorescent corneal neuronal processes after wounding was monitored in vivo.
Results In the mouse cornea, neuron-specific immunostaining determined that nerve bundles traveled in the stroma after entering at the limbus and then sent branches anteriorly to form a sub-basal plexus beneath the corneal epithelium. Fine nerves from this plexus traveled superficially to the ocular surface. Neuron-specific expression of YFP allowed visualization of nearly all large nerve bundles of the stroma, but many finer nerves of the sub-basal plexus and surface were not visible. In the sub-basal neuron plexus, 46% of the total neuronal processes exhibited YFP neurofluorescence. In vitro, 22% of cultured trigeminal neurons exhibited YFP neurofluorescence. After corneal nerve sectioning, nerve processes distal to the site of injury degenerated, whereas those proximal to the site regenerated in a pattern different from original nerve architecture.
Conclusions Thy1-YFP mice display corneal neurofluorescence and provide a novel model for monitoring the patterning, injury, and growth of corneal nerves in vivo.
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