(T1530-03-17) Imaging Analysis of Corneal Fluorescein Permeation Kinetics

Purpose: The application site of aqueous and vitreous humor continues to challenge the development of ophthalmic drug formulations. The rapid clearance through tear flow and strong biological and physical barrier properties of the cornea require the development of optimized formulations. Understanding pharmacokinetics through the cornea and its epithelium using simple ex vivo methods is of great importance. The aim of the study is to present an ex vivo model of semi-quantification of fluorescent active pharmaceutical ingredients (API)s and/or fluorescently labeled formulations using the model substance fluorescein sodium (FS). The studies were performed on sheep cornea, as a readily available substitute for human cornea in a Franz-cell-based vertical diffusion setup which offers a well-established and stable method to investigate permeation in and through biological tissue. Viability of sheep cornea was confirmed. The visualization of the kinetic processes in the cornea was achieved via confocal laser scanning microscopy (CLSM).

Methods: The corneas of daily fresh provided sheep heads were carefully dissected from the eyeball, stored in artificial aqueous humour (AAH) composed of NaCl, 130 mM; KCl, 5 mM; CaCl₂, 1 mM; MgCl₂, 1 mM; D-glucose, 2.5 mM; NaHCO₃, 10 mM; HEPES, 10 mM; NaN₃ 3 mM (pH 7.3); and used within 2 hours [1]. Sheep cornea was assayed for viability in AAH by measuring tetrazolium reductase activity, reflecting the number of viable cells present [2]. For imaging of permeation kinetics corneas were clamped in a Franz cell diffusion apparatus and equilibrated to a surface temperature of 35±1 °C. AAH functioned as acceptor phase whereas a solution of FS (0.8 mg/mL) provided as donor. After 10, 30 and 60 minutes, respectively epithelium was washed with AAH and cornea was immediately frozen. Fluorescence images were acquired in cross-sections of 50 µm using CLSM (Nikon, Japan). Z-Stacks were analysed semi quantitatively using ImageJ software. FS signal in the acceptor phase was measured fluorometrically (PerkinElmer, US).

Results: Tissue viability proved to be stable within 24 hours. No significant (p < 0,05) change in viability could be found between non-incubated sheep cornea which functioned as positive control, and cornea that was incubated with AAH. Blank cornea revealed no fluorescence signal under the selected settings. However, fluorescence signal in incubated corneas increased with time. FS penetrated upper cell layers after 10 minutes, whereas it diffused to the basal lamina (BL) of the corneal epithelium and beyond within 60 minutes (Fig. 1). FS showed the highest intensity in the cornea after 60 minutes (Fig. 2). The results also suggested a second maximum for 60 minutes incubation time at a distance of 100-125 µm, which corresponds to the depth of the BL as presented in figure 2D. FS fluorescence signal in the acceptor medium was earliest detectable after 60 min.

Conclusion: Sheep cornea proved to be of adequate stability and viability for the ex vivo model presented. The depth profile of FS in corneal tissue demonstrated that visualization via fluorescence with accompanying quantification via gray-value provides insights into kinetic processes in the cornea. This is applicable for fluorescent labelable formulations and their comparison. Since especially the first 60 minutes are decisive considering the short duration time of e.g. eyedrops, an insight into the cornea is of value.

References: [1] Linetsky M, Chemoganskiy VG, HuF, Ortwerth BJ. Effects of UVA light on the avtivity of several aged human lens enzymes. Invest Opthalmol Vis Sci 2003; 44(1):264-74.
[2] Siefen T, Lokhnauth J, Liang A, Larsen CC, Lamprecht A. An ex-vivo model for transsynovial drug permeation of intraarticular injectables in naive and arthritic synovium. Journal of controlled release: official journal of the Controlled Release Society 332, S. 581-591.


Fig. 1 Confocal Laser Scanning Microscopy. Cross sections of blank sheep cornea as transmitted image scan (A) and at excitation with 488 nm laser after incubation with fluorescein sodium: 10 (B), 30 (C) and 60 minutes (D). BL – Basal lamina. Scalebar is 250 µm


Fig. 2 Fluorescence signal in cross sections of sheep cornea after 10, 30 and 60 minutes of incubation with fluorescein sodium as gray-value versus corneal depth