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462 TRANSGENIC CORNEAL NEUROFLUORESCENCE IN MICE: A NEW MODEL FOR IN VIVO INVESTIGATION OF NERVE STRUCTURE AND REGENERATION.
  1. C. Q. Yu1,
  2. M. I. Rosenblatt1
  1. 1Department of Ophthalmology, University of California, Davis School of Medicine, Sacramento, CA

Abstract

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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