Ultrastructural Axon–Myelin Unit Alterations in Multiple Sclerosis Correlate with Inflammation

Abstract

Objective: Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon-myelin unit in MS NAWM and determined how this correlates with low-grade inflammation. Methods: Human brain tissue obtained with short postmortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohis tochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon-myelin unit in MS NAWM (n = 8) and control white matter (WM) in the optic nerve. Results: In the MS NAWM, there were more activated and phagocytic microglia cells (HLA + P2RY12 À and Iba1 + CD68 + ) and more T cells (CD3 + ) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM, there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve. Interpretation: These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These subclinical alterations to the axon-myelin unit in MS NAWM may contribute to disease progression.

Micro‐diffusely abnormal white matter: An early multiple sclerosis lesion phase with intensified myelin blistering

“…36 Our myelin blister results further support the prelesional/early lesional nature of mDAWM. Myelin blisters are emerging as important MS pathology correlates, being spotted in several NAWM and peri-lesion areas of MS brains 5 and, more recently, in the optic nerve, 33 a region often associated with the earliest MS lesion formation. 42 Nonetheless, in line with the early involvement of myelin blisters in lesion formation, studies employing cuprizone mouse models of MS have reported the presence of similar morphological alterations in mice weeks prior to demyelination.…”

Seeking neuroprotection in multiple sclerosis: an ongoing challenge

“…Nodal and paranodal regions have been considered particularly vulnerable sites of immune-mediated injury in MS ( 15 ). Alterations in these regions can even be observed in normal-appearing white matter ( 16 , 17 ) and are implicated in contributing to disease progression in MS ( 17 ). Ultrastructure changes include uncompacting of myelin, unmasking of potassium channels, and increased axonal energy demand.…”

Profiling of microglia nodules in multiple sclerosis reveals propensity for lesion formation

“…The WM tissue of stroke brain donors did not contain infarcts. Cryo-protected frozen optic nerve samples of MS (n=4) and control donors (n=4) were previously obtained 29 .…”

“…For IHC, (NA)WM tissue of MS (n=9) and stroke (n=8) brain donors was cut from frozen (20 µm) or paraffin-embedded (8 µm) tissue blocks not containing any lesions or reactive sites. For SMI312 and PLP, normal-appearing optic nerve tissue of MS donors was fixed overnight in 4% paraformaldehyde, protected in 30% sucrose for 24 hours, frozen, and cut at 20 µm 29 . For paraffin-embedded tissue, antigen retrieval was performed as indicated in Table 3 for 10 min in a microwave at 700 W. Sections were incubated overnight at 4°C with primary antibodies (Table 3) diluted in incubation buffer (for paraffin tissue: 0.5% gelatin and 0.5% Triton X-100 in TBS; for frozen tissue: 1% bovine serum albumin and 0.5% Triton X-100 in phosphate-buffered saline, pH7.6).…”