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Mass spectrometry imaging (MALDI and TOF-SIMS) of benzalkonium chloride (BAK) distribution in rabbit

F. Brignole-Baudouin, (1,2,3,4,9), N. Desbenoit, (1,2,3,5), G. Hamm, (8), H. Liang, (1,2,3,4), R. Nanache, (1), J. Both, (6), A. Brunelle, (5), I. Fournier, (7), V. Guérineau, (5), R. Legouffe, (8), J. Stauber, (8), D. Touboul, (5), M. Wisztorski, (7), O. Laprévote, (9) and C. Baudouin, (1,2,3,4,10,11)

1 ImaBiotech, MS Imaging Department, Lille, France;
2 UPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris, F-75012, France;
3 CNRS UMR_7210, Paris, F-75012, France;
4 Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, F-75012, France.
5 Centre de recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, Avenue
de la Terrasse, 91198 Gif-sur-Yvette, France;
6 Laboratoire d’intégration des Systèmes et des Technologies, CEA-LIST, Plateau de Saclay, 91191 Gif-sur-Yvette, France;
7 Laboratoire de Spectrométrie de Masse Biologique, Fondamentale et Appliquée, EA 4550, Université¸ Lille Nord de France Université¸ Lille 1, 59655 Villeneuve d’Ascq, France;
8 Imabiotech Campus Cité¸ Scientifique, 59655 Villeneuve d’Ascq, France
9 Chimie Toxicologie Analytique et Cellulaire, EA 4463, Faculté¸ des Sciences Pharmaceutiques et Biologiques, Université¸ Paris Descartes, 4 Avenue de l’Observatoire, 75006 Paris, France;
10 Université¸ Versailles Saint-Quentin-en-Yvelines, Versailles, F-78000, France;
11 Assistance Publique – Hopitaux de Paris Hopital Ambroise Paré¸, Service d’Ophtalmologie, Boulogne-Billancourt, F-92100, France

Abstract
Purpose
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.

Methods
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’. 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 recorded in positive ion mode.

Results
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.

Conclusions
These two MSI techniques highlight here the BAK distribution in the eye from the cornea to the optic nerve that could affect sensitive areas involved in the glaucoma pathophysiology. These complementary techniques offer new powerful tools in the field of ophthalmo toxicology to investigate the distribution of various compounds like this amphiphilic eye drop excipient with known deleterious effects.

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