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. 2018 Jul 18;13(7):e0199570.
doi: 10.1371/journal.pone.0199570. eCollection 2018.

A novel method for quantifying axon degeneration

Aaron D Johnstone  1   2 , Robin M Hallett  3 , Andrés de Léon  1   2 , Bruno Carturan  2 , Julien Gibon  2 , Philip A Barker  2
Affiliations

A novel method for quantifying axon degeneration

Aaron D Johnstone et al. PLoS One. .

Abstract

Axons normally degenerate during development of the mammalian nervous system, but dysregulation of the same genetically-encoded destructive cellular machinery can destroy crucial structures during adult neurodegenerative diseases. Nerve growth factor (NGF) withdrawal from dorsal root ganglia (DRG) axons is a well-established in vitro experimental model for biochemical and cell biological studies of developmental degeneration. Definitive methods for measuring axon degeneration have been lacking and here we report a novel method of axon degeneration quantification from bulk cultures of DRG that enables objective and automated measurement of axonal density over the entire field of radial axon outgrowth from the ganglion. As proof of principal, this new method, written as an R script called Axoquant 2.0, was used to examine the role of extracellular Ca2+ in the execution of cytoskeletal disassembly during degeneration of NGF-deprived DRG axons. This method can be easily applied to examine degenerative or neuroprotective effects of gene manipulations and pharmacological interventions.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Axoquant 2.0 quantifies neurite degeneration from quarter-field images of DRG explants.
DRG explants are dissected from E13.5 mouse embryos and seeded on 6-well plates (two wells shown in A; NGF (top) & anti-NGF 24h (bottom), scale bar = 1 mm for 5x images and 10 μm for 40x images). To quantify the degree of degeneration following a phase of NGF withdrawal and to assess the effect of pharmacological or genetic manipulations on its progression, the entire culture is imaged (after fixation and tubulin cytoskeletal immunostaining) by automatic tile-scanning on a motorized microscope stage, and quarter-fields are cropped and saved according to embryo and treatment (A; fields containing axons from only a single DRG are chosen, indicated by dotted box and green checkmark). The user directs Axoquant 2.0 to the experimental parent folder, where subfolders organized by treatment and embryo are crawled and quantified automatically (B).
Fig 2
Fig 2. Axoquant 2.0 workflow.
The R script automatically crawls folders and opens each quarter-field image (A) and if necessary, auto-orients the explant centre to the origin (B). Images are converted to binary masks with adaptive thresholding (C), and the area of the substrate occupied by axons is measured in bins radiating from the explant centre (stylized in D). Axoquant 2.0 automatically saves a comma separated file (*.csv) to the experiment parent folder for import into graphing and statistical analysis software. Example axon density curves are shown as embryo means (E) and treatment means with standard error (F).
Fig 3
Fig 3. Axon degeneration following NGF withdrawal is rescued by Ca2+ chelator EGTA.
(A) Explant ganglia were cultured in NGF and then either maintained in NGF (left, scale bar = 500 μm), deprived of NGF with a function-blocking anti-NGF antibody (middle), or deprived of NGF but in the presence of 6 mM EGTA (right). After 24 hours, cultures were fixed and immunostained with anti-ß-III tubulin primary antibody. (B) Axon density as a function of distance from the explant center was quantified by Axoquant 2.0 and (C) binned in 500 μm increments for statistical testing. NGF deprivation induced a significant loss of axons compared to NGF-supplied axons (p<0.05 within the bin at 500–1000 μm, and ps<0.0001 within the 1000–1500, 1500–2000, and 2000–2500 μm bins). Axons incubated with EGTA during the phase of NGF deprivation were significantly more dense than axons deprived of NGF without EGTA (ps<0.0001 within 1000–1500, 1500–2000 and 2000–2500 μm bins) indicating rescue of axons by Ca2+ chelation. n = 9 embryos per condition; plotted are mean and SEM. *p<0.05, ****p<0.0001, obtained by Dunnett’s post hoc comparison with anti-NGF control following two factor ANOVA, F (6, 72) = 37.87, p<0.0001.
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