Quantification of retrograde axonal transport in the rat optic nerve by Fluorogold spectrometry

Abstract: Purpose
Disturbed axonal transport is an important pathogenic factor in many neurodegenerative diseases, such as glaucoma, an eye disease characterised by progressive atrophy of the optic nerve. Quantification of retrograde axonal transport in the optic nerve usually requires labour intensive histochemical techniques or expensive equipment for in vivo imaging. Here, we report on a robust alternative method using Fluorogold (FG) as tracer, which is spectrometrically quantified in retinal tissue lysate.

Methods
To determine parameters reflecting the relative FG content of a sample FG was dissolved in retinal lysates at different concentrations and spectra were obtained. For validation in vivo FG was injected uni- or bilaterally into the superior colliculus (SC) of Sprague Dawley rats. The retinal lysate was analysed after 3, 5 and 7 days to determine the time course of FG accumulation in the retina (n=15). In subsequent experiments axona transport was impaired by optic nerve crush (n=3), laser-induced ocular hypertension (n=5) or colchicine treatment to the SC (n=10).

Results
Spectrometry at 370 nm excitation revealed two emission peaks at 430 and 610 nm. We devised a formula to calculate the relative FG content (cFG), from the emission spectrum. cFG is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, cFG monotonously increases with time (p=0.002). Optic nerve axonal damage caused a significant decrease of cFG (crush p=0.029; hypertension p=0.025; colchicine p=0.006). Lysates are amenable to subsequent protein analysis.

Conclusions
Spectrometrical FG detection in retinal lysates allows for quantitative assessment of retrograde axonal transport using standard laboratory equipment. It is faster than histochemical techniques and may also complement morphological in vivo analyses