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 (c(FG)), from the emission spectrum. c(FG) is proportional to the real FG concentration as it corrects for variations of retinal protein concentration in the lysate. After SC injection, c(FG) monotonously increases with time (p = 0.002). Optic nerve axonal damage caused a significant decrease of c(FG) (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.

Figure 1. Spectrometry of FG and retinal lysate.

A) Spectra of pure FG (interrupted line; 1: 5000 dilution) and retinal lysate (black line). The diluent for both was RIPA lysis buffer (grey line). B) Spectra of retinal lysate containing different amounts of FG (white dotted lines). Pure retinal lysate without FG (black dots) and RIPA lysis buffer (grey dots) are shown for comparison. With increasing FG concentration the slope of the curve between 520 and 610 nm increases.

Figure 2. Raw and normalised spectra of retinal lysate from untreated animals with FG added in vitro.

A and B) Dilution series of retinal lysate containing a constant proportion of FG. 100% is the undiluted sample. 80% and 50% is the content of lysate in the diluted samples. The dilution was done with RIPA lysis buffer. A) Raw emission spectra. The dotted vertical lines mark the 520 to 610 nm range that was normalised in B. B) Spectra after normalisation to the E₅₂₀ value. The curves of the undiluted and the diluted samples are now congruent, indicating that the normalisation compensates for the effect of different sample dilutions. C and D) Retinal lysate with increasing concentrations of FG. C) Raw emission spectra. The dotted vertical lines mark the 520 to 610 nm range that was normalised in D. D) Spectra after normalisation to the E₅₂₀ value. The end points of the normalised curves are encircled. They increase with higher FG concentration.

Figure 3. FG accumulation in the retina by retrograde transport over time.

Normalised emission curves (A) and relative FG content in the lysate (c_FG; B) in control retinal lysate and at three different timepoints after FG injection. The data points and bars are the mean and SEM for each group. The group size is given in panel A. The difference between control and days five and seven are statistically significant (p<0.01 and 0.001, respectively).

Figure 4. FG accumulation in the retina after different types of optic nerve injury.

A-F) Normalised emission curves (A, C, E) and c_FG (B, D, F) five days after optic nerve injury. Data points and error bars are mean and SEM. A, B) Optic nerve crush, n = 3, p = 0.029. C, D) Laser induced ocular hypertension, n = 5, p = 0.025. E, F) Colchicine injection to the superior colliculus, n = 5 for treatment and control group, p = 0.006). G) Western blot for phospho-Erk1/2 from control retinal lysate after superior colliculus injection of PBS (lane “C”) and from retinal lysate used for FG spectrometry (lane “F”). Both lanes show a similar band pattern indicating that despite the FG labelling and spectrometric measurements the retinal lysate can be further used for western blots.