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Francoise Brignole-Baudouin, Nicolas Desbenoit, Gregory Hamm, Alain Brunelle, Hong Liang, Maxence Wisztorski, Jonathan Stauber, Jean-Pierre Both, Olivier Laprevote, Christophe Baudouin; Distribution of Benzalkonium Chloride in Rabbit Eyes using Mass Spectrometry Imaging MALDI and Tof-SIMS. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4983.
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Antiglaucoma eye drops preserved with benzalkonium chloride (BAK) have been reported to cause ocular surface disorders with tear film alteration, eye irritation and to promote dry eye. BAK was also suspected to induce cystoid macular edema following cataract surgery in BAK-receiving eyes. In fact, little is known about BAC distribution in the eye. Our aim was to use new mass spectrometry imaging (MSI) techniques to describe BAK distribution in the eye in an in vivo model and investigate the physiopathological consequences at a molecular level.
The eyes of New Zealand rabbits were instilled with different BAK solutions at 0.01%, 2 drops BID for 1 month or 5 months or 0.2% at 1 drop/day for 1 month. After sacrifice, eyes were quickly enucleated and embedded in tragacanth gum and frozen at -80°C. Serial cryosections were deposited on glass slides for Hematoxylin-eosin, on silicon wafers for ToF-SIMS imaging, and ITO or stainless steel plates for MALDI-ToF/ToF imaging. Ion images were acquired in positive ion mode.
Two ions were detected in BAK-instilled rabbit eye samples corresponding to a mixture of benzododecinium C12 (C21H38N+, m/z 304.32) and myristalkonium C14 (C23H42N+, m/z 332.36). The characteristic fragments of these compounds were identified by MALDI-ToF/ToF at m/z 212.42 and 240.50 corresponding to BAK C12 and C14 respectively, as well as tropylium ion, common to both species, at m/z 91.21. The ToF-SIMS ion image of m/z 332.33 ion showed a distribution in the outer periphery of eyeball, in the cornea and conjunctiva as well as in the limbus and near the iridocorneal angle and the trabecular meshwork. An increase of BAK accumulation was observed in eyes treated for 5 months. Ion images and immunohistology were put side by side to correlate inflammatory areas. MALDI-ToF/ToF imaging confirmed these data and also showed the presence of BAK in the retina and near the optic nerve. All these localizations were confirmed with the BAK 0.2% model.
These two MSI techniques highlight here the BAK distribution in the eye from the cornea to the optic nerve, which could affect sensitive areas involved in the glaucoma pathophysiology. These complementary techniques offer new powerful tools in the field of ophthalmotoxicology to investigate the distribution of various compounds like this amphiphilic eye drop excipient with known deleterious effects.
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