Abstract
Purpose :
Current approaches to glaucoma diagnosis are reliant on the detectable loss of vision by perimetry and loss of retinal ganglion cells (RGCs) by optical coherence tomography (OCT). Even before these dramatic changes, histopathology and electron microscopy (EM) studies show significant morphological changes within RGCs in experimental models of glaucoma such as altered of the axonal cytoskeleton, mitochondria, and dendrites. Studies with fluorophore-labeled cholera toxin subunit B (CTB) confirm that these early-stage axonal cytoskeletal changes are associated with predictable deficits of axonal transport, a critical function within RGCs. However, a direct link between the ultrastructural breakdown and the transport deficits has not yet been established at the sub-cellular level by EM. We hypothesize that CTB-conjugated gold nanoparticles can be used to study axonal transport across multiple imaging modalities and scales.
Methods :
We have designed a biocompatible hybrid lipid-coated coated gold nanoparticles (HMNPs) with lipids that were conjugated with Oregon Green dyes and CTB proteins on the surface using click chemistry. Conjugation of the CTB to the hybrid lipid coating was confirmed using thin-layer chromatography, NMR, and fluorescence. CTB-conjugated HMNPs were injected into 5 adult male brown Norway rats and imaged using a confocal scanning laser ophthalmoscope (fl-CLSO). At the experimental endpoint, eyes were perfused, enucleated, and stored at 4 °C in PBS buffer. A 2 mm trephine was used to biopsy a specimen of the retina from the inferotemporal edge of each optic disc before processing for light microscopy and EM.
Results :
Our in vivo studies show that the CTB-conjugated HMNPs are visualized towards the optic nerve within 12 h post-injection by fl-CSLO. EM studies of the same retinal tissue show that the nanoparticles are localized in the RGC cells and their axons. The presence of the CTB-conjugated HMNPs was confirmed by EM.
Conclusions :
Our results confirm that CTB-conjugated HMNPs can be targeted towards RGC cells with minimal inflammation when injected intravitreally into the rat eye. Furthermore, these materials can be used to visually track the axonal transport of the nanotracer in the living eye across multiple scales, fl-CLSO, light microscopy, and EM. This nanotracer will our understanding of the pathophysiological sequence of axonal cytoskeletal breakdown and transport failure.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.