A Novel Mouse Model for Neurotrophic Keratopathy: Trigeminal Nerve Stereotactic Electrolysis through the Brain

The cornea is the most innervated tissue in the entire human body (400 times more than skin).1 It receives dense sensory innervation from the trigeminal ganglion and, in some species, modest autonomic innervation. The trigeminal nerve divides into three main branches, one of which, the ophthalmic branch, provides corneal innervation. The organization of corneal nerves, from the depth to the surface of the cornea, is made up of penetrating stromal nerve bundles, subepithelial plexus, sub-basal nerve plexus, and intraepithelial nerve terminals.2 More than a dozen different neurotransmitters and neuropeptides, whose functions are still incompletely understood, are secreted by corneal nerves.3 Corneal nerves are lost because of many pathologic conditions, such as ocular infection, topical anesthetic abuse, surgery, diabetes, stroke, and dry eye syndrome,1,4–6 which affect millions of people annually. When corneal nerves are severely reduced, neurotrophic keratopathy (NK), a poorly treatable disease, develops. This includes the development of persistent epithelial defects and potentially stromal ulceration that may progress to perforation. However, the exact mechanisms and interactions between trigeminal nerves and corneal cells, including stem cells, epithelial cells, and keratocytes, remain unclear. It is known that nerve-secreted peptides influence corneal cell proliferation in vitro,7 and corneal epithelial cell mitosis has been shown to be altered in denervated rats.8,9 Apoptosis and cell proliferation play key roles in ocular wound healing.10 Reduced cell proliferation and thinning have been described in the denervated skin11; however, it is not known whether enhanced apoptosis or simply reduced cell proliferation is associated with NK. In addition, apoptosis and proliferation have not been studied simultaneously in this condition. This has significant clinical implications because it is a well-known fact that NK patients have delayed healing and often experience persistent epithelial defects12 and that even minor injuries can seriously threaten ocular integrity. To address these shortcomings in our current understanding of these important pathophysiologic mechanisms, we have developed, for the first time, a mouse model by removing corneal nerves reproducibly using electrolysis of the ophthalmic branch of the trigeminal nerve with a stereotactic approach, trigeminal stereotactic electrolysis (TSE). This procedure aims to destroy only the fibers providing corneal innervation, leaving the rest of the trigeminus undamaged. Other denervation models have been proposed in monkeys,13 rabbits,14–16 and rats.17,18 NK induction with a hot metal probe through the roof of the mouth has been described in mice; however, no report of death or success rate has been provided, and no stereotactic technique was used.19 When the same procedure was performed on rats, the authors reported a success rate of 60%, and animals could be kept alive for only 3 to 6 days, which precludes long-term follow-up.9 Our method represents an advancement of one of the most effective denervation procedures.20 In addition, this technique applies to the mouse, which is the most widespread experimental mammal used, and is available with the full array of research tools. The procedure was followed by the development of progressive degeneration of the corneal tissue, confirmed clinically by biomicroscopy, functionally by examination of the blink reflex, and histologically by the loss of corneal nerves and the degeneration of corneal tissues. Finally, we show histologic evidence of a simultaneous increase in corneal cell apoptosis and a reduction in basal epithelial cell proliferation.