A novel method for quantifying axon degeneration

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.

Developmental Axon Degeneration Requires TRPV1-Dependent Ca2+Influx

“…Images were stitched automatically with Zen 2 software from Zeiss to produce a master image of all explants on the entire six-well plate. From this master image, quarter-DRG fields were cropped using NIH ImageJ (FIJI build) to create an image set for quantification as recently described in detail by our group (Johnstone et al, 2018).…”

“…Axon degeneration was quantified by implementing the R script Axoquant2.0 recently described in detail and made freely available by our group (Johnstone et al, 2018). For plotting and statistical analysis, each point represents the mean value of measurements from the complement of DRG cultured from a single embryo.…”

“…We recently reported that sensory axons are rescued from developmental degeneration by Ca 2+ chelation in vitro (Johnstone et al, 2018). Intriguingly, nerve terminals that innervate the skin can be locally ablated in the clinic by activation of Ca 2+ influx mediated by the cation channel transient receptor potential vanilloid family member 1 (TRPV1); topical application of the TRPV1 agonist capsaicin is used to alleviate chronic pain and itch in humans (Jancso et al, 1985; Gibbons et al, 2010; Chiang et al, 2015; Şavk, 2016).…”

“…Intriguingly, nerve terminals that innervate the skin can be locally ablated in the clinic by activation of Ca 2+ influx mediated by the cation channel transient receptor potential vanilloid family member 1 (TRPV1); topical application of the TRPV1 agonist capsaicin is used to alleviate chronic pain and itch in humans (Jancso et al, 1985; Gibbons et al, 2010; Chiang et al, 2015; Şavk, 2016). Since activation of TRPV1 can trigger degeneration in sensory neurons (Jancso et al, 1985; Sann et al, 1995; Wang et al, 2001; Gibbons et al, 2010; Chiang et al, 2015), and because we found that Ca 2+ is required for developmental degeneration (Johnstone et al, 2018), here we have explored the possibility that TRPV1 is required for developmental degeneration of sensory axons.…”

Collapsin Response Mediator Protein 4 (CRMP4) Facilitates Wallerian Degeneration and Axon Regeneration following Sciatic Nerve Injury

“…The axons were imaged at a 5Â magnification using a Zeiss Axioscope 2 Microscope. The quantification of the area covered by the axons was done with Axoquant 2.0 in R Studio as previously described (Johnstone et al, 2018).…”