Microglial nodules in early multiple sclerosis white matter are associated with degenerating axons

Abstract

Microglial nodules in the normal-appearing white matter have been suggested as the earliest stage(s) of multiple sclerosis (MS) lesion formation. Such nodules are characterized by an absence of leukocyte infiltration, astrogliosis or demyelination, and may develop into active demyelinating MS lesions. Although the etiology of MS is still not known, inflammation and autoimmunity are considered to be the central components of this disease. Previous studies provide evidence that Wallerian degeneration, occurring as a consequence of structural damage in MS lesions, might be responsible for observed pathological abnormalities in connected normal-appearing white matter. As innate immune cells, microglia/macrophages are the first to react to even minor pathological changes in the CNS. Biopsy tissue from 27 MS patients and autopsy and biopsy tissue from 22 normal and pathological controls were analyzed to determine the incidence of microglial nodules. We assessed MS periplaque white matter tissue from early disease stages to determine whether microglial nodules are associated with altered axons. With immunohistochemical methods, the spatial relation of the two phenomena was visualized using HLA-DR antibody for MHC II expression by activated microglia/macrophages and by applying antibodies against damaged axons, i.e., SMI32 (non-phosphorylated neurofilaments) and amyloid precursor protein as well as neuropeptide Y receptor Y1, which marks axons undergoing Wallerian degeneration. Our data demonstrate that the occurrence of microglial nodules is not specific to MS and is associated with degenerating as well as damaged axons in early MS. In addition, we show that early MS microglial nodules exhibit both pro- and antiinflammatory phenotypes.

Fig. 1

Microglial nodules are found in the normally myelinated periplaque white matter in MS biopsy tissue. Staining for LFB/PAS (a) and immunohistochemistry for PLP (b), MBP (c), MOG (d) and MAG (e) on sequential tissue sections showed normal myelin (MS no. 7). Activated microglia/macrophages and circumscribed nodules (arrows) that express HLA-DR are shown in (f). Insets in (b, c, d, e, f) show higher magnification of the respective tissue immunostaining. Scale bars = (a–f) 250 μm

Fig. 2

Microglial nodules are associated with underlying axonal pathology in early multiple sclerosis. LFB/PAS stain and HLA-DR immunohistochemistry were used to identify microglial nodules in PPWM (a, b). Sequential tissue sections (a, b, c, d) were matched using a blood vessel (asterisk) (MS no. 5). The region of interest exhibits intact myelin (a). Microglial nodule representing cluster of activated HLA-DR expressing microglia/macrophages localized in normally myelinated PPWM tissue (b). Sequential section identified injured/damaged axons associated with microglial nodules (c, d). APP⁺, acutely damaged axons are detected in close association with the microglial nodule (c). SMI32⁺, axonal ovoids occur in the same region (d and inset). Inset in (d) shows higher magnification of the marked region. Scale bars = (a) 100 μm; (b–d) 50 μm

Fig. 3

Axons undergoing Wallerian degeneration in close spatial association with activated microglia/macrophages in MS PPWM. NPY-Y1R⁺ axons (green) undergoing Wallerian degeneration apposed to HLA-DR⁺ microglia/macrophages (red) with an activated morphology (arrows, a–b) (MS no. 14). NPY-Y1R⁺ axons were frequently surrounded by activated microglia/macrophages throughout the PPWM (c–d) (MS no. 1). Activated microglia/macrophage cells were visualized clustering along the length of the NPY-Y1R⁺ axonal segment (arrow, e–f) (MS no. 4). Stainings are merged with DAPI, which stains the nuclei (a–f, blue). Scale bars = (a–b) 25 μm; (c–d) 10 μm; (e–f) 20 μm

Fig. 4

Degenerating axons undergoing Wallerian degeneration associated with microglial nodule in MS. Fluorescent immunohistochemical staining for HLA-DR (red) counterstained with DAPI (blue) shows a microglial nodule consisting of several microglial/macrophage cells (MS no. 11). The association of NPY-Y1R⁺ fragments (green) with the microglial nodule suggests Wallerian degeneration induced formation of microglial nodule. Scale bar = 30 μm

Fig. 5

Intracellular NPY-Y1R⁺ axonal fragments in a HLA-DR⁺ nodule-forming microglial/macrophage cell. Nuclei of nodule-forming microglia/macrophage cells (blue, a), NPY-Y1R⁺ particles (green, b) present within the microglial/macrophage cytoplasm (red, c). The merge is shown in (d). The presence of intracellular NPY-Y1R⁺ fragments (arrow) in nodule-forming HLA-DR⁺ cell indicates engulfment of axonal debris by activated microglia/macrophages. Scale bar = 15 μm

Fig. 6

Identification of microglial nodules in OND and MS. Microglia/macrophage activation (HLA-DR⁺) throughout the perilesional white matter in the autopsy tissue from an infarct case (a). b Represents a magnification of the region containing nodules in (a). Microglial nodule in the white matter biopsy tissue from a patient with cerebral infarction (c). White matter region surrounding contusional lesion in an autopsy case (d). Disperse microglial/macrophage activation in biopsied epilepsy white matter (e, inset). Nodules present adjacent to the biopsied MS lesion in MS no. 3 (f). Immunohistochemistry for HLA-DR: (a–f). Arrows: nodules. Scale bars = (a, e) 250 μm; (b, d, f) 100 μm; (c) 25 μm

Fig. 7

Microglial nodules in MS display both classically (M1) and alternatively (M2) activated macrophage markers. CD40 was expressed in cells associated with microglial nodules present in PPWM (a) (MS no. 13). iNOS was present in phagocytes in demyelinating MS lesions and very frequently found to be expressed in the clusters forming a small foci of microglial/macrophage cells (arrows) in PPWM (b, c) (MS no. 6). c Represents a higher magnification of the PPWM region marked with black box in (b). M2 marker mannose receptor was present perivascularly (inset of d) and was only rarely expressed on parenchymal cells (arrowheads) in PPWM (d) (MS no. 13). CD163 was detected in activated microglia/macrophages and microglial nodules (arrow) as well as in perivascular spaces (arrowhead) (d) (MS no. 8). Scale bars = (a, d, e) 100 μm; (b) 200 μm; (c) 50 μm