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S. B. Robinson, H. Kim, W. He, K. G. Csaky; Quantitative Evaluation of Lymphatic Elimination of Subconjunctivally Administered Sodium Fluorescein (NaF) in Rats. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3187. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Transscleral drug delivery is an attractive route of treating retinal disease, because it is less invasive than intravitreal injection. However, transscleral administration has not yet proven a clinically effective means of vitreous drug delivery because of limited delivery efficiency. Information regarding the role of conjunctival lymphatics on the elimination of subconjunctivally administered drugs is particularly limited. This study proposed a method to quantitatively assess the conjunctival lymphatic elimination of a fluorescent drug surrogate.
Long-Evans rats were injected with 20ul of 50mg/ml Patent Blue V subconjunctivally to visualize the lymph vessels and "sentinel" nodes draining the eye. 5ul of 100mg/ml (500ug) NaF was injected into the subconjunctival space and the submandibular sentinel node(s) positive for blue dye isolated at 30, 90, and 120 minutes post-NaF injection. The nodes were homogenized with a sonicator in 150ml of water, incubated at 4°C overnight, centrifuged at 10,000 rpm for 10 min., and 50ml of supernatant from each sample added to a luminescence plate well read at 485nm excitation/525nm emission. A standard curve was generated using homogenated blue dye positive nodes from rats that had blue dye injected subconjunctivally. NaF concentration was determined by comparison with the standard curve, and total NaF in the lymph node(s) calculated from this concentration data.
The standard calibration curve of NaF in the lymph node showed linear correlation (R2 = 0.999) in a concentration range from 50 ug/mL to 0.5 ng/mL. The total amount of NaF in the lymph node(s) at 30, 90, and 120 minutes after a 500ug NaF injection was 254.7ng, 168.3ng, and 118.1ng, representing .051%, .034%, and .024% of the total 500ug injection, respectively.
This quantitative assay associated lymph node fluorescence with the orbital lymphatic elimination of a fluorescing compound. It facilitates a quantitative assessment of NaF in the lymph node(s) draining the eye to evaluate the contribution of conjunctival lymphatic elimination. NaF, a small and hydrophilic drug surrogate, was eliminated by conjunctival lymphatic vessels, demonstrating that conjunctival lymphatics compromise the efficiency of transscleral drug delivery to the vitreous. However, the amount of NaF in the lymph node decreased with time after 30 minutes instead of accumulating, suggesting NaF elimination from the sentinel node is more rapid than uptake into the node during this time.
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