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Kamila Mizerska, Valentina Dallacasagrande, Harumitsu Hirata, Carl F. Marfurt, Mark Rosenblatt; Mechanisms of corneal intraepithelial nerve damage after exposure to hyperosmolar solutions in rat.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):799.
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© ARVO (1962-2015); The Authors (2016-present)
Our recent work has shown that exposure of the rat cornea to hyperosmolar solutions induces morphological alterations in corneal intraepithelial nerves. The mechanisms for these changes remain unknown. The purpose of this study was to investigate, using a novel nerve fragmentation quantification technique, possible neural and epithelial mechanisms underlying these changes.
Immunohistochemistry: The eyes of each rat were exposed to hyperosmolar solutions (HOS; 600 and 1000 mOsm NaCl) or artificial tears (ATs), respectively, for 30 min to 3 hours. Another group of animals was treated with 200 µM Nifedipine or 2% Lidocaine in 1000 mOsm NaCl. The morphology of the corneal epithelial innervation was evaluated by staining corneal whole mounts with an antibody against beta-III tubulin. Damage (fragmentation) of corneal intraepithelial nerves acquired from z-stack images of central corneal areas was assessed quantitatively and expressed as nerve fragments/sq mm. Corneal cross sections were stained for ZO-1 as a marker for epithelial tight junctions. Electrophysiology: Single corneal neurons were recorded extracellularly from rat trigeminal ganglia. The neuronal responses to corneal drying (an important stimulus for tearing) were noted before, and after, applying 200 µM Nifedipine or 2% Lidocaine in 1000 mOsm NaCl or ATs.
Immunohistochemistry: The morphometric analyses indicated that corneal nerve damage appeared even after short (30 min) exposure to 600 mOsm NaCl (p<0.0001). Corneal nerve morphology was not changed after adding a Ca++ channel blocker to the 1000 mOsm NaCl solution. Corneal cross sections stained for ZO-1 appeared to reveal diminished immunostaining intensity in 1000 mOsm NaCl-treated animals compared to controls. Electrophysiology: The responses of corneal neurons to hyperosmolar solutions (1000 mOsm) were abolished by 200 µM Nifedipine or 2% Lidocaine.
The novel morphometric approach used in this study enables automated and accessible evaluation of corneal nerve damage caused by short term exposure to hyperosmolar solutions. These results demonstrate for the first time that corneal nerve damage due to hyperosmotic tears might be a result of a combination of different mechanisms (neurotoxicity and decreased tight junction protein expression). These findings may contribute to a better understanding of signs and symptoms observed in dry eye patients.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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