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An Animal Model Of Corneal Neurotisation Using The Thy1-GFP+ Rat To Further Investigate How Reinnervation Influences Corneal Health
Joseph Catapano, MD, Jennifer J. Zhang, MD, PhD, Kira Antonyshyn, BSc, Gregory H. Borschel, MD, FACS.
University of Toronto, Toronto, ON, Canada.

PURPOSE: Patients with corneal anaesthesia develop neurotrophic keratopathy, causing corneal scarring and vision loss. Corneal neurotisation restores sensation but it remains unknown whether donor nerves reinnervating the cornea contain neuromediators that are required for corneal epithelial maintenance and repair. Further investigation of the effect of neurotisation on corneal health requires an animal model in which tissue can be harvested for analysis. Here we describe a rat model of corneal neurotisation.
METHODS:Thy1-GFP+ rats, which express green fluorescent protein in axons, were used to monitor corneal denervation and reinnervation. Parameters for the stereotactic electrocautery of the ophthalmic branch (V1) of cranial nerve V, which innervates the cornea, was determined using serial imaging of whole mount corneas at 1, 2 and 4 weeks to determine optimal parameters for corneal denervation. Blink reflexes were assessed to confirm corneal denervation. After establishing a method of corneal denervation, corneal neurotisation was accomplished using common peroneal (CP) and sural nerve grafts coapted to the contralateral infraorbital nerve. Four corneas were harvested 4 weeks after neurotisation and denervation to determine corneal nerve density and four corneas were retrograde labelled with 4% Fluorogold to determine whether reinnervating axons derived from the contralateral or ipsilateral trigeminal ganglion (TG). Neurotised corneas were compared with uninjured (normal) and denervated (injured) controls. CP and sural grafts were harvested for histomorphometry.
RESULTS: Optimal corneal denervation was achieved by ablating V1 at the stereotactic coordinates (+ 1.5 mm, + 2.0 mm, 10 mm) with 3 W for 60s. Stereotactic electrocautery of V1 was well tolerated, however injury to the TG resulted in unacceptable morbidity. Corneal neurotisation using CP and sural nerve grafts was successful resulting in a significant increase in corneal axon density (Figure 1). Denervated corneas demonstrated minimal reinnervation after 4 weeks (2301 μm/mm2 ± 1347) and reinnervation was restricted to the peripheral stroma. Neurotised corneas exhibited significantly greater corneal nerve density (62872 μm/mm2 ± 12400; p < 0.0001), which extended to the central cornea and subbasal layer and was comparable to uninjured (normal) controls (46165 μm/mm2 ± 3965). Histomorphometry demonstrated significant growth of myelinated axons across the grafts. Retrograde-labelling of uninjured cornea controls labeled 478 ± 16 neurons in the ipsilateral TG innervating the cornea with no labeled neurons in the contralateral TG. In contrast, labelling of neurotised corneas demonstrated no labeled neurons in the ipsilateral TG (0 ± 0) with a significant number of labelled sensory neurons in the contralateral TG (353 ± 215), suggesting axons reinnervating the cornea after neurotisation derived from the donor grafts and contralateral face.
CONCLUSION: The described animal model of corneal neurotisation is valuable to further investigate how reinnervation of the cornea using foreign donor nerves influences corneal epithelial health, including epithelial healing and protein expression.
Figure 1. Axons in the uninjured cornea (A) are absent four weeks after injury (B). Significant axon reinnervation is present after neurotisation (C).


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