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Shandiz Tehrani, Lauren Davis, William O Cepurna, Elaine C Johnson, John C Morrison; In Vivo Small Molecule Delivery to the Optic Nerve in a Rat Model. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Small molecule delivery to the optic nerve head (ONH) would allow probing of cellular pathways involved in glaucomatous optic neuropathy and provide a tool for screening therapeutics in animal models. This study was designed to show quantitative delivery of small molecules to the optic nerve in vivo.
Brown Norway rats (n = 4) were anesthetized with intraperitoneal ketamine injections, followed by soft tissue dissection to expose the superior junction of the optic nerve and globe. The superior optic nerve sheath (ONS) was dissected to expose the superior optic nerve at its junction with the globe (Fig. 1), followed by placement of a 1.5 x 5mm gelatin foam pledget (Fig. 2) soaked in a solution of 100µM cytochalasin D (a small molecule actin inhibitor that may alter astrocyte morphology within the ONH). A second approach without ONS dissection was followed by injection of 100µL of 100µM cytochalasin D in the sub-ONS space via a 31g needle. Controls (n = 2) included placement of cytochalasin D pledgets without ONS dissection or injection along the superior junction of the optic nerve and globe. Animals were sacrificed after 4 hours, followed by isolation of the anterior 2mm of the optic nerve (including the ONH). Tissue samples were sonicated in phosphate-buffered saline, analyzed for total protein content by absorbance spectroscopy, followed by cytochalasin D quantification by liquid chromatography and mass spectroscopy. Standard errors of the mean were calculated for each group.
Cytochalasin D content within the optic nerve (ng) was normalized to total protein content within the sample (mg). In the ONS dissection and sub-ONS injection groups, 12.60 ± 0.34 ng/mg and 5.94 ± 1.81 ng/mg cytochalasin D was detected within the optic nerve tissue, respectively. This is equivalent to a final concentration of 5.6 µM and 2.5 µM cytochalasin D within the optic nerve tissue of the ONS dissection and sub-ONS injection groups, respectively (assuming equal distribution within a cylinder with a radius of 150µm and a length of 2mm). This range of cytochalasin D concentration is active in inducing actin depolymerization in cell culture models. Minimal cytochalasin D was detected in control groups.
Local, quantitative, in vivo small molecule delivery to the optic nerve in a rat model is feasible. This approach may be useful in testing various modulators of ONH cellular activity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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