Abstract
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.
Highlights
Glaucoma is a severe optic neuropathy leading to blindness without treatment and affecting more than 70 million people worldwide
We focused on three areas of interest: (1) the ocular surface with cornea and conjunctiva as the first ocular defense, (2) the iridocorneal angle and (3) the optic nerve area, the two ocular structures involved in glaucoma
The first model was based on the instillation of the 0.01% benzalkonium chloride (BAK) solution, which is the concentration commonly used in eye drops, twice a day for 5 months and the other model was based on the instillation of one drop of 0.2% BAK solution once daily for 1 month
Summary
Glaucoma is a severe optic neuropathy leading to blindness without treatment and affecting more than 70 million people worldwide. At a concentration ranging from 0.004 to 0.2% in eye drops, this preservative is required by pharmacopeia guidelines to prevent the multidose eye drop containers from bacterial and fungi contamination [4,5] It has the advantage of inducing fewer allergic-type side effects and of being relatively well tolerated, it has been reported to induce ocular surface disorders combining irritation, inflammation and cell death processes, especially in long-term treatment [6]. While the deleterious effects of BAK could be negligible for a short-term treatment, they need to be considered for long-term or repeated treatment such as in chronic open-angle glaucoma In this case, patients are most often treated for the rest of their life, often with several BAKcontaining eye drops, since about 40% of patients require multiple therapies to control their IOP and prevent further optic nerve damage [7]. BAK-containing antiglaucoma eye drops have been reported to cause disruption of the blood– aqueous barrier inducing cystoid macular edema following cataract surgery [8]
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