A New Safety Concern for Glaucoma Treatment Demonstrated by Mass Spectrometry Imaging of Benzalkonium Chloride Distribution in the Eye, an Experimental Study in Rabbits

• Published in: PloS one Vol. 7; no. 11; p. e50180
• Main Authors: Brignole-Baudouin, Françoise, Desbenoit, Nicolas, Hamm, Gregory, Liang, Hong, Both, Jean-Pierre, Brunelle, Alain, Fournier, Isabelle, Guerineau, Vincent, Legouffe, Raphael, Stauber, Jonathan, Touboul, David, Wisztorski, Maxence, Salzet, Michel, Laprevote, Olivier, Baudouin, Christophe
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27.11.2012; Public Library of Science (PLoS)
• Subjects: Animals; Anti-Infective Agents, Local - adverse effects; Apoptosis; Benzalkonium chloride; Benzalkonium Compounds - adverse effects; Biology; CD45 antigen; Cell activation; Cellular stress response; Chemical Sciences; Chlorides; Data analysis; Data processing; Desorption; Eye; Eye (anatomy); Eye - drug effects; Eye - pathology; Glaucoma; Glaucoma - drug therapy; Glaucoma - metabolism; Immunohistochemistry - methods; Infiltration; Inflammation; Information management; Intermediate filament proteins; Ionization; Ions; Lasers; Leukocyte Common Antigens - biosynthesis; Lipids; Mass spectrometers; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Medicine; Neuroglia - cytology; Ophthalmic agents; Ophthalmic Solutions - adverse effects; Ophthalmology; Optic nerve; Optic Nerve - drug effects; Organic chemistry; Preservatives; Rabbits; Retina; Retina - drug effects; Safety; Sample preparation; Scientific imaging; Spectrometers; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy; Stainless steel; Steel plates; Surface active agents; Time Factors; Tissues; Toxicity testing; Tragacanth; Vimentin; Vimentin - biosynthesis; France
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0050180

We investigated in a rabbit model, the eye distribution of topically instilled benzalkonium_(BAK) chloride a commonly used preservative in eye drops using mass spectrometry imaging. Three groups of three New Zealand rabbits each were used: a control one without instillation, one receiving 0.01%BAK twice a day for 5 months and one with 0.2%BAK one drop a day for 1 month. After sacrifice, eyes were embedded and frozen in tragacanth gum. Serial cryosections were alternately deposited on glass slides for histological (hematoxylin-eosin staining) and immunohistological controls (CD45, RLA-DR and vimentin for inflammatory cell infiltration as well as vimentin for Müller glial cell activation) and ITO or stainless steel plates for MSI experiments using Matrix-assisted laser desorption ionization time-of-flight. The MSI results were confirmed by a round-robin study on several adjacent sections conducted in two different laboratories using different sample preparation methods, mass spectrometers and data analysis softwares. BAK was shown to penetrate healthy eyes even after a short duration and was not only detected on the ocular surface structures, but also in deeper tissues, especially in sensitive areas involved in glaucoma pathophysiology, such as the trabecular meshwork and the optic nerve areas, as confirmed by images with histological stainings. CD45-, RLA-DR- and vimentin-positive cells increased in treated eyes. Vimentin was found only in the inner layer of retina in normal eyes and increased in all retinal layers in treated eyes, confirming an activation response to a cell stress. This ocular toxicological study confirms the presence of BAK preservative in ocular surface structures as well as in deeper structures involved in glaucoma disease. The inflammatory cell infiltration and Müller glial cell activation confirmed the deleterious effect of BAK. Although these results were obtained in animals, they highlight the importance of the safety-first principle for the treatment of glaucoma patients.