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D. E. Berezovsky, A. R. Jani, E. S. Kim, B. E. McCarey, H. F. Edelhauser; Determination of Transscleral Diffusion of Fluorescein and Fluoresceinated Dextrans Into the Rabbit Eye Using Noninvasive Ocular Fluorometry. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5970.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the transscleral penetration of sodium fluorescein and FITC-labeled dextrans into the rabbit vitreous using noninvasive ocular fluorometry
Sodium fluorescein (NaF) and FITC-labeled dextran (FITC-D, MW 40kDa and 70kDa, Sigma Scientific) were diluted in Balanced Salt Solution to 25mg/mL. NZW rabbits (n=6) were euthanized in accordance with ARVO animal use guidelines. Four rabbits received a sub-Tenon injection of 200µL NaF solution in one eye, using a 30G needle in the superior temporal quadrant. Two rabbits received 200µL injections of 40kDa or 70kDa FITC-D in both eyes. Ocular fluorometry (Ocumetrics Fluorotron Master) was performed on all eyes immediately after injection, and at 10 min, 30 min, and hourly through six hrs. FITC-D eyes were also evaluated at 24 hrs. Fluorometer readings were taken along a line through the center of the pupil and lens, ending at the posterior retina and choroid. Dissection was then performed using a quick-freezing technique to directly visualize the intraocular distribution of flouorescein.
Fluorescence remained at baseline levels in all noninjected eyes. NaF injected eyes exhibited fluorescence above baseline in both anterior and posterior segments, reaching levels greater than 600ng/mL at the mid-vitreous at 6 hrs. Dissection of these eyes showed extensive lateral spread of NaF along the sclera, and large amounts of fluorescein within the lens and vitreous. A concentration gradient was visible within these structures, with the highest concentration near the injection site. All eyes injected with FITC-D remained at baseline fluorescence levels at 24 hours. However, when fluorometer readings were redirected toward the injection site, fluorescence above 100ng/mL was observed at 6hr. Frozen dissection of FITC-D eyes showed a small localized deposit of FITC-D on the sclera at the site of injection, and examination of the vitreous under cobalt blue light revealed fluorescein within the vitreous, adjacent to the site of injection and away from the center of the vitreous body.
Noninvasive ocular fluorometry is well suited for determining the transscleral penetration of NaF into the posterior segment of the rabbit eye. Vitreous levels of larger FITC-labeled dextrans was not well visualized using standard technique, although there was evidence of FITC-D within the peripheral vitreous body when a modified technique was used. Dissection of these eyes also suggests that 40kDa and 70kDa fluoresceinated dextrans enter the vitreous body, remaining localized near the injection site during the first 24 hours after sub-Tenon injection.
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