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. 2020 Nov 18;18(1):170.
doi: 10.1186/s12915-020-00869-2.

In vivo imaging of injured cortical axons reveals a rapid onset form of Wallerian degeneration

Alison Jane Canty  1 , Johanna Sara Jackson  2 , Lieven Huang  3 , Antonio Trabalza  3 , Cher Bass  3 , Graham Little  3 , Maria Tortora  3 , Shabana Khan  3 , Vincenzo De Paola  4   5
Affiliations

In vivo imaging of injured cortical axons reveals a rapid onset form of Wallerian degeneration

Alison Jane Canty et al. BMC Biol. .

Abstract

Background: Despite the widespread occurrence of axon and synaptic loss in the injured and diseased nervous system, the cellular and molecular mechanisms of these key degenerative processes remain incompletely understood. Wallerian degeneration (WD) is a tightly regulated form of axon loss after injury, which has been intensively studied in large myelinated fibre tracts of the spinal cord, optic nerve and peripheral nervous system (PNS). Fewer studies, however, have focused on WD in the complex neuronal circuits of the mammalian brain, and these were mainly based on conventional endpoint histological methods. Post-mortem analysis, however, cannot capture the exact sequence of events nor can it evaluate the influence of elaborated arborisation and synaptic architecture on the degeneration process, due to the non-synchronous and variable nature of WD across individual axons.

Results: To gain a comprehensive picture of the spatiotemporal dynamics and synaptic mechanisms of WD in the nervous system, we identify the factors that regulate WD within the mouse cerebral cortex. We combined single-axon-resolution multiphoton imaging with laser microsurgery through a cranial window and a fluorescent membrane reporter. Longitudinal imaging of > 150 individually injured excitatory cortical axons revealed a threshold length below which injured axons consistently underwent a rapid-onset form of WD (roWD). roWD started on average 20 times earlier and was executed 3 times slower than WD described in other regions of the nervous system. Cortical axon WD and roWD were dependent on synaptic density, but independent of axon complexity. Finally, pharmacological and genetic manipulations showed that a nicotinamide adenine dinucleotide (NAD+)-dependent pathway could delay cortical roWD independent of transcription in the damaged neurons, demonstrating further conservation of the molecular mechanisms controlling WD in different areas of the mammalian nervous system.

Conclusions: Our data illustrate how in vivo time-lapse imaging can provide new insights into the spatiotemporal dynamics and synaptic mechanisms of axon loss and assess therapeutic interventions in the injured mammalian brain.

Keywords: Acute Axonal Degeneration; Axon dieback; Axon fragmentation; Brain; Cortex; Cortical axons; In vivo imaging; Laser microsurgery; NAD+; Synapses; Wallerian degeneration.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Single-axon-resolution imaging of axon degeneration in the living brain. a Timeline of imaging and injury to study cortical axon degeneration in vivo. b A region of axon ready for lesioning (left); a binary mask (4 μm) is placed over the axon (green circle) and a focused laser beam used to induce axotomy, verified by imaging immediately after lesion (right). Proximal is up and distal is down. c Representative drawings of axon segments before lesioning used throughout the study. Black filled circles indicate terminal axon endings. d A representative axon before lesioning, total length 1289 μm. e Representative drawings of the disconnected portion of the axon in the dotted box in d illustrating axonal degeneration over selected time points. Time in hours post-lesion and percentage of axon remaining are indicated. f Representative example of quantification of the degeneration onset (23 h) and g calculation of fragmentation rate for the axon in d, e (1.45 μm/min). Scale bar = 5 μm (b), 150 μm (c), 200 μm (d) and 300 μm (e)
Fig. 2.
Fig. 2.
A rapid onset form of Wallerian degeneration in the adult cortex. a Representative images of a long axon ending undergoing Wallerian degeneration (WD) with time post-lesion and percentage of axon lost indicated. b Representative images of a shorter axon ending undergoing rapid onset WD (roWD) with time post-lesion and percentage of axon lost indicated. c Scatter plot indicating the presence of two axonal populations with different onset time of fragmentation. d Surviving length of each in vivo injured axon (n = 38) over the time points indicated illustrating a population undergoing roWD (blue lines). e Histogram demonstrating significant early onset time of fragmentation of axons < 600 μm in length (roWD, blue) compared to longer axons (WD, grey). Mean ± SEM. Mann Whitney U test. ****p < 0.0001. f, g No correlation between roWD onset time and length (f, n = 21 axons < 600 μm from 13 mice, p > 0.05) or for longer axons undergoing WD (g, n = 17 axons > 600 μm from 14 mice, p > 0.05). Scale bar: 135 μm (a), 70 μm (b). For further examples of fragmenting axons as in panels a-b, see Additional file 1
Fig. 3
Fig. 3
Probing axon degeneration kinetics in vivo. a Schematic illustrating single and double lesion experiments from different regions of the axon. b Average fragmentation onset time for double lesioned distal axons (black circles, n = 13 axons, 10 mice), double lesioned proximal axons (open circles, n = 8 axons, 7 mice) and single lesioned axons (blue circles, n = 18 axons, 9 mice). Grey lines link data points from double lesioned distal and proximal segments along the same axon (n = 5 axons, 3 mice). Wilcoxon test, *** p < 0.001. c Slower fragmentation rate of shorter axons undergoing roWD (blue points) compared to longer axons undergoing WD (grey points) after a single axonal lesion, **** p < 0.0001. d No correlation between roWD fragmentation rate and axon length (blue points, n = 21 axons, 13 mice, p > 0.05) or for longer axon WD fragmentation rate and axon length (e, grey points, n = 14 axons, 14 mice, p > 0.05)
Fig. 4
Fig. 4
Synaptic boutons delay the onset of degeneration in cortical axons. a, b Representative images of axons with relatively low (a) and high (b) densities of terminaux boutons (TBs). c, d There is a significant correlation between bouton density and the onset of fragmentation for segments undergoing WD (c, p < 0.05, n = 13 axons, 13 mice) and for shorter axons undergoing roWD (d, p < 0.01, n = 20 axons, 12 mice). e, f There is no correlation between bouton density and fragmentation rate for segments undergoing WD (e, p > 0.05) or roWD (f, p > 0.05). Scale bar 40 μm (a, b)
Fig. 5
Fig. 5
AAD is negligible in injured cortical axons in vivo. a No evidence of AAD within 60 min of injury when the length of severed cortical axon distal segments is monitored with a membrane marker (Thy1-L15). Note length stability in all but one axon (red line), indicating absence of fragmentation over the first 60 min post-lesion (n = 16 axons, 7 mice). b Example of the retraction of the severed axon away from the lesion site when observed with a cytosolic GFP marker (GFP-M line). Note the absence of fragmentation over the first 60 min post-lesion. Arrowheads indicate intact axon. Axon ending is on the right edge of the frame. Scale bar = 10 μm. c Quantification of the distance from the lesion site to the distal axon stump in cytosolic GFP labelled axons (Thy1-GFPM line) at the indicated post-lesion times (n = 10 axons, 7 mice)
Fig. 6
Fig. 6
A local NAD+-dependent pathway controls cortical axon WD. a, b Representative examples of a lesioned WldS–Thy1-L15 and WT axons of comparable distal length. c WldS axons have a significantly later fragmentation onset time than WT axons, WldS, n = 24 axons, 11 mice; WT n = 21 axons, 13 mice, ** p < 0.01. d The fragmentation rates are not significantly different in WT and WldS axons although the data suggested a trend towards faster fragmentation in the WldS axons. e Representative images of a lesioned axon in cortical slice culture treated with NAD+. f Fragmentation onset time in lesioned axons in cortical slice cultures is significantly earlier in axons < 600 μm (roWD, blue, n = 9 axons, 5 slices, 5 mice) than axons > 600 μm in length (WD, grey, n = 8 axons, 6 slices, 2 mice), *** p < 0.001. g–i Degeneration of in vitro lesioned axons with no treatment (control, g), with addition of NAD+ prelesion (h) and postlesion (i). Blue dotted line shows the 24 h time point. j Axons are protected by NAD+ when added both before or after the lesion. Mean ± SEM. Mann Whitney U test. Scale bar 70 μm (a, b), 100 μm (e)
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