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A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis


In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits1,2. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view2,3,4,5,6, demyelination—a hallmark of multiple sclerosis—is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.

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Figure 1: In vivo imaging of FAD.
Figure 2: Early FAD stages show mitochondrial alterations but no demyelination.
Figure 3: Activated macrophage/microglia-derived reactive species induce FAD.
Figure 4: Axonal changes consistent with FAD are present in acute human multiple sclerosis lesions.

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We would like to thank B. Fiedler, G. Heitmann, M. Schedensack, A. Schmalz and S. Knecht for excellent technical assistance; D. Matzek for animal husbandry; A. Dagkalis for help with immunizations; M. Krumbholz for advice on statistical analysis; and R. Hohlfeld, H. Wekerle, J. Sanes, J. Lichtman, L. Godinho, D. Kerschensteiner, P. Williams, T. Dick, E. Meinl and K. Dornmair for discussions or critical reading of the manuscript. Work in M. Kerschensteiner's laboratory is financed through grants from the Deutsche Forschungsgemeinschaft (DFG; Emmy Noether Program and Sonderforschungsbereich 571) and the 'Verein Therapieforschung für MS-Kranke e.V'. M. Kerschensteiner and W.B. are supported by a grant from the German Federal Ministry of Education and Research (Competence Network Multiple Sclerosis). T.M. is supported by the Institute for Advanced Study, Technische Universität München, by the Alexander von Humboldt Foundation and by the Center for Integrated Protein Science (Munich). D.M. and W.B. are supported by grants from the DFG (Sonderforschungsbereich Transregio 43). D.M. is supported by the Swiss National Science Foundation (PP00P3 128372). D.B. is supported by the US National Institutes of Health. This project was further financed by grants to M. Kerschensteiner and T.M. from the Dana Foundation and the Hertie Foundation, and by a grant from the Christopher and Dana Reeve Foundation to T.M. and D.B.

Author information

Authors and Affiliations



M. Kerschensteiner, T.M., D.B., D.M. and I.N. conceived the experiments. I.N. and C.S. did the imaging experiments. I.N., C.S., T.M. and M. Kerschensteiner did image analysis. M.B. and D.B. did and evaluated serial electron microscopy. I.N. and F.M.B. did therapy experiments. D.M., M. Kreutzfeldt and W.B. did histopathological evaluations of EAE and multiple sclerosis tissue. I.N., M. Kerschensteiner and T.M. wrote the paper.

Corresponding authors

Correspondence to Thomas Misgeld or Martin Kerschensteiner.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Methods (PDF 894 kb)

Supplementary Video 1

In vivo multi-photon time-lapse that illustrates the degeneration of a transgenically labeled stage 1 axon (white) in an acute EAE lesion in a Thy1-CFP-S × Cx3cr1GFP/+ mouse. Axonal degeneration is initiated near a putative node of Ranvier in close proximity to activated macrophages/microglia (magenta). (MOV 409 kb)

Supplementary Video 2

In vivo overview multi-photon time-lapse of the healthy lumbar spinal cord of a Thy1-YFP-16Thy1-MitoCFP-P) mouse in which axons are labeled with YFP (gray). The video illustrates that no obvious morphological changes are induced by our imaging approach. 300 min, 11 frames. (MOV 1533 kb)

Supplementary Video 3

In vivo overview multi-photon time-lapse of the lumbar spinal cord of a Thy1-YFP-16Thy1-MitoCFP-P) mouse, in which axons are labeled with YFP (gray; in some axons, CFP-labeled mitochondria are visible due to spectral cross-talk) 2 d after the EAE onset. The video illustrates stage 1 to stage 2 transitions in three axons during the observation period. 300 min, 11 frames. (MOV 2253 kb)

Supplementary Video 4

In vivo multi-photon time-lapse of the lumbar spinal cord of a Thy1-YFP-16Thy1-MitoCFP-P) mouse in which the axons are labeled with YFP (gray) 3 d after the EAE onset. This video illustrates the recovery of a stage 1 axon during the observation period. 330 min, 11 frames. (MOV 263 kb)

Supplementary Video 5

Video sequence of a stage 1 EAE axon (shown in Fig. 2a–d) that illustrates the correlation between in vivo multi-photon imaging and ssTEM. (MOV 3002 kb)

Supplementary Video 6

In vivo multi-photon microscopy time-lapse of an activated macrophage/microglia (one cell was manually pseudo-colored in magenta based on GFP expression) in apposition to an axon (white) in an acute EAE lesion in a Cx3cr1GFP/+ × Thy1-CFP-S mouse. The video illustrates how immune cells tracts were generated from time-lapse sequences. Note transition of the apposed axon from stage 0 to stage 1 during the time-lapse. Asterisk in first frame marks additional macrophage/microglia. 192 min, 20 frames. (MOV 180 kb)

Supplementary Video 7

In vivo multi-photon time-lapse of a T cell (one cell was manually pseudo-colored in cyan based on GFP expression) in apposition to an axon (white) in an acute EAE lesion in a Thy1-CFP-S × Cd2-GFP mouse. The video illustrates how immune cells tracts were generated from time-lapse sequences. Green asterisk in first frame marks additional T cell, gray asterisk marks axon fragment. 40 min, 27 frames. (MOV 249 kb)

Supplementary Video 8

In vivo multi-photon time-lapse of axonal (white) and mitochondrial (cyan) changes induced after application of H2O2 (330 mM) to the spinal cord of a Thy1-YFP-16 × Thy1-MitoCFP-P mouse. Note that axonal mitochondria change before the transition of the axons from stage 0 to stage 1. (MOV 1088 kb)

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Nikić, I., Merkler, D., Sorbara, C. et al. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nat Med 17, 495–499 (2011).

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