Wallerian degeneration: a major component of early axonal pathology in multiple sclerosis

Abstract

Axonal loss is a major component of the pathology of multiple sclerosis (MS) and the morphological basis of permanent clinical disability. It occurs in demyelinating plaques but also in the so-called normal-appearing white matter (NAWM). However, the contribution of Wallerian degeneration to axonal pathology is not known. Here, we analyzed the extent of Wallerian degeneration and axonal pathology in periplaque white matter (PPWM) and lesions in early multiple sclerosis biopsy tissue from 63 MS patients. Wallerian degeneration was visualized using an antibody against the neuropeptide Y receptor Y1 (NPY-Y1R). The number of SMI-32-positive axons with non-phosphorylated neurofilaments was significantly higher in both PPWM and plaques compared to control white matter. APP-positive, acutely damaged axons were found in significantly higher numbers in plaques compared to PPWM. Strikingly, the number of NPY-Y1R-positive axons undergoing Wallerian degeneration was significantly higher in PPWM and plaques than in control WM. NPY-Y1R-positive axons in PPWM were strongly correlated to those in the lesions. Our results show that Wallerian degeneration is a major component of axonal pathology in the periplaque white matter in early MS. It may contribute to radiological changes observed in early MS and most likely plays a major role in the development of disability.

Figure 4

Wallerian degeneration in PPWM, lesions and control white matter: High numbers of axons undergoing Wallerian degeneration are found in PPWM (A) and lesions (B) of MS patients. As control, high numbers of degenerating axons are found in a patient with cerebral infarction (C) but not in patients who had surgery for epilepsy (D). Original magnification: ×400. Scale bar: 50 m. NPY‐Y1R staining, patient #202, inactive lesion. 210 × 297mm (200 × 200 DPI).

Figure 1

Anti‐NPY‐Y1R antibody detects Wallerian degeneration in human and experimental PNS and CNS tissue: (A,B) Mouse sciatic nerve 6 days after transection immunostained with anti‐NPY‐Y1R antibody shows immunolabeled elongated and ovoid structures (B, brown, arrows) not seen in non‐transected control nerve (A). Human sural nerve biopsy showing single NPY‐Y1R‐positive structures (C, red, arrows). Wallerian degeneration is present in this sural nerve biopsy confirmed by toluidin‐blue stained semi‐thin sections (D, arrow) and teased‐fiber preparation (E). (F–H) NPY‐Y1R immunoreactive profiles in wt (F, arrows) and WLDs mice with EAE at peak of disease (G arrow). Significantly more NPY‐Y1R immunoreactive profiles are detected in wt mice compared to WLDs mice at a similar stage of lesion formation (P < 0.05; H). Original magnifications: A,B, D–G: ×400; C: ×200. Scale bars: A,B, D–G: 50 m, C: 100 m. 209 × 297 mm (300 × 300 DPI).

Figure 2

Axonal damage in plaques, periplaque white matter and controls: (A) Bielschowsky's silver impregnation. Relative axonal density in plaques was significantly lower than in PPWM (P < 0.01) and in control WM (P < 0.01). (B) SMI‐31 staining. Relative axonal density in stainings for phosphorylated neurofilaments was significantly lower in lesions compared to PPWM (P < 0.01) and control WM (P = 0.03). (C) SMI‐32 staining. Higher relative axonal density in stainings for SMI‐32‐positive neurofilaments was shown in both PPWM and plaques compared to control WM (P < 0.01 and P = 0.01, respectively). (D) APP staining. The number of APP‐positive axons was significantly higher in plaques compared to PPWM (P < 0.01) and control WM (P < 0.01). (E) NPY‐Y1R staining. The number of NPY‐Y1R‐positive axons was significantly higher in PPWM (P < 0.01) and plaques (P = 0.01) than in control WM. P values are the results of Kruskal‐Wallis test with three groups. *P < 0.05. Relative axonal density was expressed as percentage of axons crossing the stereological grid points to total number of grid points.

Figure 3

Axonal density and axonal damage in periplaque white matter and lesions: Bielschowsky's silver impregnation shows a significantly higher axonal density in PPWM (A) compared to lesions (B). The same is shown by staining for phosphorylated neurofilaments, although numbers are in general lower (SMI‐31, C = PPWM, D = lesion). Injured axons with non‐phosphorylated neurofilaments, as demonstrated by staining with SMI‐32‐antibodies, are found in high numbers in PPWM (E) as well as in lesions (F). However, acutely damaged axons are found in higher numbers in lesions (H) compared to PPWM (G) (APP staining). Original magnification: ×400. Scale bar: 50 m. Patient #262, early active lesion. 210 × 297mm (600 × 600 DPI).