Abstract
Small molecule delivery to the optic nerve would allow for exploration of molecular and cellular pathways involved in normal physiology and optic neuropathies such as glaucoma, and provide a tool for screening therapeutics in animal models. We report a novel surgical method for small molecule drug delivery to the optic nerve head (ONH) in a rodent model. In proof-of-principle experiments, we delivered cytochalasin D (Cyt D; a filamentous actin inhibitor) to the junction of the superior optic nerve and globe in rats to target the actin-rich astrocytic cytoskeleton of the ONH. Cyt D delivery was quantified by liquid chromatography and mass spectrometry of isolated optic nerve tissue. One day after Cyt D delivery, anterior ONH filamentous actin bundle content was significantly reduced as assessed by fluorescent-tagged phalloidin labeling, relative to sham delivery. Anterior ONH nuclear counts and axon-specific beta-3 tubulin levels, as well as peripapillary retinal ganglion cell layer nuclear counts were not significantly altered after Cyt D delivery relative to sham delivery. Lastly, the surgical delivery technique caused minimal observable axon degeneration up to 10 days post-surgery. This small molecule delivery technique provides a new approach to studying optic neuropathies in in vivo rodent models.
Highlights
Optic neuropathies such as glaucoma[1] result from injury to optic nerve axons at the level of the optic nerve head (ONH)[2,3,4,5,6,7]
The superior ONH is identified by the white box. (D,E) ONH after sham delivery and 1 mM cytochalasin D (Cyt D) delivery to the junction of the superior optic nerve and globe. (F) ONH F-actin content assessed by fluorescence intensity measurement of fluorescentlabeled phalloidin after sham (n = 4) and 1 mM Cyt D delivery (n = 6). *p < 0.05
The amount of Cyt D detected by liquid chromatography and mass spectrometry (LC/MS) was normalized to the total anterior optic nerve tissue protein content analyzed, and was found to depend on the concentration of Cyt D in the pledget that was surgically delivered (Fig. 1A; 12.6 ± 0.3 and 41.9 ± 6.2 ng/mg of Cyt D detected by LC/MS after 0.1 and 1 mM Cyt D in the pledget, respectively)
Summary
Optic neuropathies such as glaucoma[1] result from injury to optic nerve axons at the level of the optic nerve head (ONH)[2,3,4,5,6,7]. Small molecule delivery to the ONH would allow for probing of potential cellular and molecular pathways involved in axon injury in previously established animal models of glaucoma[3,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50]. We describe a surgical method for small molecule delivery to the optic nerve and ONH in a rat model, and quantitatively confirm delivery of small molecules to the optic nerve tissue using liquid chromatography and mass spectrometry (LC/MS). Astrocytes and their actin-based stellate morphology[25,26,57], and using an actin inhibitor allowed for direct assessment of the effect of small molecule delivery on the ONH
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