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Stavros Sgouris, Christian van Oterendorp, Jens F. Jordan, Wolf A. Lagrèze; Quantification of Optic Nerve Retrograde Axonal Transport by Fluorogold Spectrometry. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2695.
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© ARVO (1962-2015); The Authors (2016-present)
Retrograde axonal transport is an essential function in neurons. Failure of the transport system has been associated with neurodegenerative diseases such as glaucoma. Fluorogold (FG) is known to be actively transported in the optic nerve axons. To quantify retrograde axonal transport in retinal ganglion cells (RGCs) a method to spectrometrically quantify FG accumulating in the retina after stereotactict injection into the superior colliculus of rats was developed.
Excitation and emission maxima of FG in retina lysate as well as autofluorescence of retina lysate were determined by spectrometry (Tecan infinite M200). The changes in spectral emission with different FG concentrations were analysed in vitro by adding increasing amounts of FG to retina lysate (n=4). To measure retrograde axonal transport in vivo 0.1ng FG were injected into the superior colliculus of 15 sprague dawley rats. The animals were killed after 3, 5 or 7 days, the retina was lysed and analysed by spectrometry.
Spectrometry revealed two peaks at 440 and 610nm with a trough at 520nm. The peak at 440nm interfered with retinal autoflourescence which peaks at 420 nm. As the 520nm value was still influenced by retinal autofluorescence we chose the ratio of 610/520nm emission (peak/trough) at 370nm excitation as a measure of FG content in lysate that provides certain correction for variations in retinal protein concentration in the fixed volume of lysate buffer. Using the 610/520 values obtained by adding given amounts of FG in vitro to retina lysate, a standard curve following a simple polynomial function was fitted (R squared 0.99). In the in vivo experiments we observed an increase of the retina lysate 610/520 ratio with increasing time after FG injection into the superior colliculus. The 610/520 ratio was significantly different between control versus day 5 (p=0.003) and day 7 (p=0.002) as well as day 3 versus day 5 timepoints (p=0.04, one way ANOVA).
Fluorogold injected into the superior colliculus using injection volumes and concentrations similar to what is used for RGC back-labelling can be detected spectrometrically in retina lysate. The 610/520nm ratio correlates well with the amount of FG in retina lysate samples. Our quantification method detects significant FG signals 5 days after superior colliculus injection and allows measurement of the increase in FG over time. After spectrometry the lysate may be further used for analyses, such as western blot. Thus, the spectrometric quantification of FG seems a useful tool to study retrograde axonal transport in RGCs in vivo.
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