Relationship of acute axonal damage, Wallerian degeneration, and clinical disability in multiple sclerosis

We investigated paraffin-embedded archival brain tissue from 17 autopsied multiple sclerosis patients and 14 biopsied patients diagnosed with inflammatory demyelination consistent with multiple sclerosis. A total of 34 [16] tissue specimens, which included non-demyelinated white matter regions, were used for the study. All biopsy samples analyzed contained non-demyelinated periplaque white matter (PPWM) areas contiguous with early/late active (n = 6) or inactive (n = 9) lesions. For direct comparison, non-demyelinated white matter tissue adjacent to 5 active lesions, 7 chronic active, and 7 chronic inactive lesions was analyzed in multiple sclerosis autopsy tissue. All lesions fulfilled the criteria for the diagnosis of multiple sclerosis [22]. Lesional activity was determined using previously described criteria [23]. The biopsies were performed in different neurosurgical centers to exclude neoplastic or infectious diseases. Specimens were sent to the Department of Neuropathology in Göttingen, Germany, for a second opinion. The patients’ clinical characteristics are summarized in Table 1.

Axonal transport disturbances and Wallerian degeneration are typical and abundant after brain ischemia. To study the spatial relation of the two phenomena in a prototypic human disease, we analyzed archival paraffin-embedded brain biopsy tissue from four patients (three females, one male; median age = 54 years; range 49–63 years) with ischemic stroke lesions. Lesions were characterized by tissue necrosis as evidenced by massive axonal loss and dense macrophage infiltration. Axonal swellings in a typical ischemic pattern were abundant at the lesion borders.

Female 8–10-week-old Wld ^S (C57BL/6 OlaHsd) and C57BL/6 mice were obtained from the Harlan Laboratories, UK. The Wld ^S mouse strain is characterized by an 85-kb tandem triplication on chromosome four that occurred as a spontaneous mutation in the B6 strain in the 1940s, leading to the expression of an Ube4b/Nmnat chimeric protein. Mutant mice do not show a spontaneous phenotype. All mice had free access to water and chow and were included in the experiments after at least 5 days of acclimatization.

EAE was induced by subcutaneous injection of 200 μg myelin oligodendrocyte glycoprotein (MOG)-peptide_35–55 emulsified in complete Freund’s adjuvant (CFA) containing 1 mg/ml inactivated Mycobacterium tuberculosis. Control mice were injected with CFA alone. Three hundred nanogram pertussis toxin was injected i.p. at day 0 and day 2 after immunization. Clinical deficits were assessed daily by a blinded observer using the following scoring system: 0=no symptoms, 0.5=partial tail paresis, 1.0=complete tail paralysis, 1.5=slight hind limb paresis, 2.0=distinct hind limb paresis, 2.5=severe hind limb paresis, 3.0=complete hind limb paralysis, 3.5=slight forelimb paresis, 4.0=tetraparesis, 4.5=moribund, and 5.0=death. Mice were euthanized when reaching a score of 3.5.

At the end of the EAE experiments, animals were deeply anesthetized and perfused with phosphate buffered saline (PBS) (pH 7.4) followed by 4% paraformaldehyde (PFA) in PBS. The spinal cords (SC) were dissected, and at least eight transverse sections were embedded in paraffin. One to three micrometer-thick sections were stained with hematoxylin-eosin (HE), Luxol Fast Blue/periodic acid Schiff’s reagent (LFB/PAS), and Bielschowsky silver impregnation to determine inflammation, demyelination, and axonal loss. Immunohistochemistry (IHC) was performed using the primary antibodies listed in Table 2. For antigen retrieval, tissue slices were microwaved in 10 mM citrate buffer (pH 6.0) 3 × 5 min. Bound antibodies were visualized using an appropriate biotinylated secondary antibody and an avidin-peroxidase-DAB technique. Negative control sections were incubated without primary antibodies or with irrelevant primary antibodies of the respective isotypes. Slices were counterstained with hemalaun and cover-slipped. Double fluorescence labeling with two mouse monoclonal primary antibodies was carried out as described previously [24].

Four female C57BL/6 mice were used to study sciatic nerve axotomy. They were deeply anesthetized by intraperitoneal injection of ketamine hydrochloride (“Ketanest Inresa,” 50 mg/ml, Inresa, Freiburg, Germany) mixed with xylazine hydrochloride (“Rompun” 2%, Bayer, Leverkusen, Germany) in a ratio of 2:1 (0.4 mg Ketanest and 2 mg Rompun for each mouse). Skin and muscles above the right femur were opened by fine scissors, and the sciatic nerve was completely transected. Subsequently, muscle and skin were closed by suture (Ethicon). The mice were kept for 6 days under a 12-h dark-light cycle and given food and water ad libitum. The animals were perfused transcardially with PBS and 4% PFA, and the sciatic nerves dissected. The contralateral nerves and a sciatic nerve from an animal without axotomy served as controls. Sciatic nerves were post-fixed in 4% PFA overnight and embedded in paraffin. Microtome sections of 1–3 μm thickness were de-paraffinized, pre-treated by cooking in citrate buffer (pH 6) for 10 min, and subjected to IHC.

Tissue sections were analyzed using an Olympus BX51 fluorescence microscope equipped with a DP71 CCD camera (Olympus Optical Co, Ltd., Hamburg, Germany), a Zeiss Cell Observer microscope with an AxioCam ICc 3 CCD camera (Carl Zeiss MicroImaging, Ltd., Göttingen, Germany), or by confocal laser scanning microscopy with a Fluoview 1000 Olympus microscope. Transverse spinal cord (SC) sections at the cervical, thoracic, lumbar, and sacral levels were used for quantitative analysis. The extent of acute axonal damage of axons undergoing Wallerian degeneration and the density of healthy phosphorylated axons was calculated by counting APP⁺, NPY-Y1R⁺, or SMI31⁺ profiles in spinal cross sections within at least five white matter lesions per mouse at ×400 magnification using an ocular counting grid. Counts are given as immunopositive axonal profiles per square millimeter. Relative axonal densities within lesions were determined in sections stained with Bielschowsky’s silver impregnation by using an axonal counting grid with 25 cross-points [25]. The number of axons intersecting with the crossing points was determined as a fraction of the total number of cross-points at a magnification of ×1000, under oil immersion. The value obtained in control animals was set to 100%. The degree of axon reduction in the lesion is given as the percentage of axon density compared with the control animals. To determine the extent of demyelination, digital images of LFB/PAS-stained SC cross sections were recorded through an Olympus light microscope with a CCD DP71 camera at ×100 magnification. Using the computer program Cell^F (Soft Imaging Systems^®), demyelinated white matter areas were measured, and the percentage with respect to total white matter was calculated. The inflammatory index was defined as the mean number of perivascular infiltrates within the SC parenchyma of 8–10 spinal cross sections per animal. All the images were prepared in Adobe Photoshop CS4 Version 11.0.2.

Statistical analyses were carried out using Microsoft Office Excel 2007 and GraphPad Prism (GraphPad Software, La Jolla, USA). After normality testing, an unpaired t test was applied to determine potential differences in the mean clinical severity scores of WT and Wld ^S mice at day 20 and day 40 after disease onset. Histological data were analyzed by the non-parametric Mann-Whitney U test; animals in two groups (i.e., Wld ^S and WT) were compared on the basis of closely matched clinical scores. The influence of genotype and disease stage on inflammation and demyelination was determined using a two-way-ANOVA. Statistical significance was defined as p < 0.05. All quantitative morphological data are expressed as mean ± standard error of the mean (SEM) in the EAE studies.

Wallerian degeneration is thought to reflect a clinically relevant component of disability and disease progression in multiple sclerosis. However, the contribution of Wallerian degeneration to multiple sclerosis pathology is not yet known. Here, we studied the occurrence and extent of Wallerian degeneration in biopsy multiple sclerosis tissue from patients with short duration as well as in autopsied patients with chronic multiple sclerosis. Using NPY-Y1R immunohistochemistry, Wallerian degeneration was quantified in areas of PPWM brain tissue from 31 multiple sclerosis patients. We observed widespread occurrence of NPY-Y1R⁺ degenerating axonal fibers in non-demyelinated PPWM areas in multiple sclerosis patients (Fig. 1). Patients were divided into early and chronic disease groups according to disease duration, i.e., time from the first symptoms to biopsy/autopsy. The number of NPY-Y1R⁺ axons in multiple sclerosis PPWM was significantly higher in the early as compared to the chronic disease stage (early; 113.6 ± 7, chronic; 4.6 ± 0.8 profiles/mm²; ***p < 0.001). In early multiple sclerosis, the number of axons undergoing Wallerian degeneration was significantly higher in PPWM connected to active lesions as compared to early inactive lesions (PPWM-active; 129.1 ± 5, early inactive; 85.9 ± 5 profiles/mm²; **p < 0.01). Indeed, even in chronic disease stages, the number of axons undergoing Wallerian degeneration was significantly lower in PPWM areas of chronic inactive lesions (1.4 ± 0.6 profiles/mm²) compared to PPWM of active (6.8 ± 1.3 profiles/mm²; **p < 0.01), and chronic active lesions (5.1 ± 0.9 profiles/mm²; *p = 0.03). Our findings in brains of patients with multiple sclerosis indicate a high incidence of Wallerian degeneration in early stages of multiple sclerosis, which may be related to axonal transections in focal demyelinated lesions.

To assess the impact of the Wld ^S fusion gene on inflammatory disease severity, MOG_35–55 EAE was induced in Wld ^S mutant and WT C57BL/6 mice. No statistically significant difference in disease severity, i.e., mean disease score ± SD (standard deviation) was observed between Wld ^S and WT mice on specifically testing the acute (day 20, p = 0.94) and chronic (day 40, p = 0.85) disease phase (Fig. 2) [26]. Also, the mean maximal cumulative score 40 days after disease onset was comparable in WT (2.5 ± 0.32) and Wld ^S (2.31 ± 0.28) mice. A qualitative histopathological analysis revealed an increase in lesion sizes from day 20 to day 40 after disease onset in both experimental groups. Experiments were repeated at least three times, and consistent results were obtained.

Differences in lesion acuteness and extent of inflammation and demyelination might obscure differences in the extent of axonal vulnerability between the two mouse strains. We thus examined the number of perivascular and subpial inflammatory infiltrates per SC cross section (inflammatory index) and the percentage of demyelinated white matter in Wld ^S vs. WT mice with acute EAE (Fig. 3a–f). The mean inflammatory index was 4.8 ± 0.8 in Wld ^S and 4.9 ± 0.7 in WT mice (Fig. 3c); the mean demyelinated white matter area was 5.4 ± 1.3% in Wld ^S and 5.7 ± 0.7% in WT mice (Fig. 3f). Inflammatory index and demyelination after immunization with MOG_35–55 in Wld ^S and WT mice were thus virtually the same, making a direct comparison of axonal pathology between the two mouse strains possible [27]. To ensure that lesions with similar inflammatory demyelinating activity were examined for axonal damage and loss, IHC for the early macrophage activation antigen S100A9 was performed, and all animals were found to have active lesions with recent monocyte invasion [28]. Also, morphometry on H&E and LFB/PAS stained sections of Wld ^S and WT mice with chronic EAE revealed no significant differences with regard to inflammation (mean inflammatory index Wld ^S ; 1.3 ± 0.5 and WT; 1.4 ± 0.3 inflammatory infiltrates per spinal cord cross section) or demyelination (mean demyelinated white matter Wld ^S ; 3.3 ± 1.2% and WT; 2.7 ± 0.9%) between the strains (Fig. 3g-l). However, a significant decrease in the extent of inflammation and a trend towards reduced demyelination from day 20 to day 40 after disease onset was observed for both experimental groups (two-way ANOVA; inflammatory index, F (1, 25), p = 0.0002); demyelination, F (1, 25), p = 0.0519).

The clinical deficit in an acute inflammatory demyelinating disease may not reflect the extent of structural axonal damage due to the effects of cytokines and edema on ion homeostasis and axonal conduction. In fact, elevated cytokine levels in the CNS could affect neuroaxonal function and may at least be partly responsible for the clinical manifestations during acute exacerbation of multiple sclerosis and EAE [29, 30]. Therefore, to identify the extent of acute axonal damage in Wld ^S mutant and WT mice, we applied antibodies to APP (Fig. 4a–d) to visualize axons with impaired axonal transport. In the acute as well as chronic disease stage, the densities of APP⁺ axonal profiles in inflammatory demyelinated lesions did not differ between Wld ^S and WT mice [Wld ^S ; 370 ± 58, WT; 424 ± 37 APP⁺ profiles/mm² (acute, p = 0.46); Wld ^S ; 350 ± 47, WT; 387 ± 64 APP⁺ profiles/mm² (chronic, p = 0.79)], thus indicating that axons are damaged to a similar degree in both mouse strains, irrespective of the genotype and disease phase (Fig. 4e).

As characterized previously [31, 32], the NPY-Y1R antiserum recognized elongated and ovoid axonal structures in Wld ^S and WT EAE mice (Fig. 4f–g). In acute disease, a mean of 67 ± 13 NPY-Y1R⁺ axonal profiles/mm² in Wld ^S mutant mice and 1153 ± 241 NPY-Y1R⁺ axonal profiles/mm² in WT mice were detected (Fig. 4f–g and j), indicating markedly reduced Wallerian degeneration in Wld ^S mice in the acute phase of the disease (*p = 0.02). In contrast, no significant difference in Wallerian degeneration was observed in the chronic disease phase; 535 ± 154 NPY-Y1R⁺ axonal profiles per square millimeter were detected in Wld ^S mice and 633 ± 164 per mm² in Wld ^S mice (p = 0.69) (Fig. 4h–j). Intriguingly, a significant increase in the densities of NPY-Y1R⁺ axonal profiles was observed in chronic Wld ^S EAE mice as compared to the acute stage (*p = 0.03). This implies that axon degenerative mechanisms in Wld ^S mice are averted primarily during the acute stage of an autoimmune inflammatory demyelinating attack.

To examine whether the Wallerian degeneration-induced expression of NPY-Y1R is associated with degenerating axonal structures, immunofluorescence double labelings were performed in mouse EAE lesions (Fig. 5). We observed only rarely a co-localization of NPY-Y1R immunoreactivity with accumulated APP in axons (Fig. 5a–c). Also, NPY-Y1R expression did not co-localize with phosphorylated neurofilaments (NFs) of healthy axons detected by the SMI31 antibody (Fig. 5d–f), dephosphorylated NFs of damaged axons detected by the SMI32 antibody (Fig. 5g–i), and the low-molecular-weight NF-68 (Additional file 1). Hypophosphorylated NFs found in normal axons recognized by the SMI35 antibody were only occasionally co-localized with NPY-Y1R⁺ axons (Additional file 1). However, substantial co-localization of NPY-Y1R was observed with the high-molecular-weight NF-200, detected by the N52 antibody (Fig. 5j–l, Table 2). Besides axonal degeneration, Wallerian degeneration is characterized by disintegration of myelin with the formation of the pathognomonic myelin ovoids [33]. Hence, to assess whether the antigen detected by the anti-NPY-Y1R antiserum is associated with myelin ovoids, we performed fluorescent double IHC with antibodies against myelin basic protein (MBP), myelin proteolipid protein (PLP), MOG, myelin-associated glycoprotein (MAG), and 2′,3′-Cyclic-nucleotide 3′-phosphodiesterase (CNPase) in spinal EAE lesions from WT mice (Fig. 5m–o, Additional file 2). NPY-Y1R did not co-localize with any of the myelin proteins. In addition, to test whether this spatial co-localization pattern is also observed in areas of Wallerian degeneration after stroke, human ischemic brain lesions were double-labeled for NPY-Y1R and axonal markers (data not shown). In line with our findings in mouse EAE, co-localization of NPY-Y1R and NF-200 was observed, but not of NPY-Y1R and APP or SMI32.

Wallerian degeneration after mouse sciatic nerve axotomy features a rapid degeneration of axons during the first 6 days after injury [34, 35]. Double immunofluorescence on mouse sciatic nerve 6 days after transection showed significantly more elongated and ovoid NPY-Y1R⁺ axonal structures enwrapped by MBP⁺ myelin sheaths than degenerated axons lacking any myelin (Additional file 3). This further supports that NPY-Y1R expression is present on axonal structures, but not myelin, in both the CNS and PNS, thus identifying comparatively early stages of Wallerian degeneration.

We hypothesized that the lower numbers of axons undergoing Wallerian degeneration in the early disease stage might lead to less net axonal loss in established EAE lesions of Wld ^S mice. Healthy SMI31⁺ axons as well as relative axonal densities (Bielschowsky silver impregnation) were determined in EAE lesions (Fig. 6). In the acute stage, the number of healthy SMI31⁺ axons was significantly higher in Wld ^S compared to WT lesions (Wld ^S ; 2658 ± 335, WT; 1156 ± 265 SMI31⁺ profiles/mm²; *p = 0.02), whereas the counts were very similar in the chronic stage (Wld ^S ; 193 ± 21, WT; 173 ± 14 SMI31⁺ profiles/mm²; p = 0.39) (Fig. 6a and c). Furthermore, the densities of healthy axons in both genotypes were significantly higher in the acute compared to the chronic stage (Wld ^S ; *p = 0.03; WT; *p = 0.01). Importantly, axons after an inflammatory attack may become dephosphorylated but do not necessarily vanish. Therefore, we next quantified axons in defined lesion areas after Bielschowsky silver impregnation (Fig. 6b and d). Relative axonal densities were significantly higher in Wld ^S mice than WT mice in the acute stage (Wld ^S ; 91.52 ± 2.3%, WT; 76.34 ± 2.3%; *p = 0.04) but virtually identical in the chronic stage (Wld ^S ; 56.8 ± 8.4%, WT; 55.1 ± 11.6%; p = 0.93). Moreover, comparable with the SMI31⁺ counts, relative axonal densities of silver impregnated axons in Wld ^S mice were significantly lower in the chronic than in the acute stage (*p = 0.03). This clearly indicates that the Wld ^S mutation did not protect against long-term inflammatory axonal loss. Hence, consistent with other CNS lesion paradigms tested [36‐38], our results suggest that also in inflammatory demyelination, Wallerian degeneration is merely slowed down or delayed in Wld ^S mice.

De- and regenerative processes in axons may be closely related. We thus tested whether markers putatively associated with axonal regeneration are co-expressed with NPY-Y1R. Growth-associated protein 43 (GAP43) and synaptophysin (Syn) are synaptic proteins that are associated with axonal sprouting and synaptogenesis in various neurodegenerative diseases and may accumulate in regenerating neurons and axons. Therefore, expression of GAP43 and Syn in damaged axons could reflect a regenerative attempt [24]. However, immunofluorescence double labelings on WT EAE spinal lesions did not reveal any co-localizations of NPY-Y1R with GAP43 (Additional file 4 A-C) or Syn (Additional file 4 D-F).