Myelin Quantification in White Matter Pathology of Progressive Multiple Sclerosis Post-Mortem Brain Samples: A New Approach for Quantifying Remyelination

Abstract

Multiple sclerosis (MS) is a demyelinating and neurodegenerative disease of the central nervous system (CNS). Repair through remyelination can be extensive, but quantification of remyelination remains challenging. To date, no method for standardized digital quantification of remyelination of MS lesions exists. This methodological study aims to present and validate a novel standardized method for myelin quantification in progressive MS brains to study myelin content more precisely. Fifty-five MS lesions in 32 tissue blocks from 14 progressive MS cases and five tissue blocks from 5 non-neurological controls were sampled. MS lesions were selected by macroscopic investigation of WM by standard histopathological methods. Tissue sections were stained for myelin with luxol fast blue (LFB) and histological assessment of de- or remyelination was performed by light microscopy. The myelin quantity was estimated with a novel myelin quantification method (MQM) in ImageJ. Three independent raters applied the MQM and the inter-rater reliability was calculated. We extended the method to diffusely appearing white matter (DAWM) and encephalitis to test potential wider applicability of the method. Inter-rater agreement was excellent (ICC = 0.96) and there was a high reliability with a lower- and upper limit of agreement up to −5.93% to 18.43% variation in myelin quantity. This study builds on the established concepts of histopathological semi-quantitative assessment of myelin and adds a novel, reliable and accurate quantitative measurement tool for the assessment of myelination in human post-mortem samples.

Validating a minipig model of reversible cerebral demyelination using human diagnostic modalities and electron microscopy

“…We confirmed demyelination by diminished Luxol fast blue staining ( Fig. 4 d–g) and myelin inclusions in PAS-stained macrophages 46 ( Fig. 4 h).…”

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

“…Staining protocols using luxol fast blue (LFB), 5 , 20 antibodies against proteolipid protein (PLP) and major histocompatibility complex type‐II (MHC‐II) were used to assess the tissue presence of NAWM, mDAWM, WM lesions and remyelinating/remyelinated shadow lesions. For LFB staining, sections were incubated (58°C, overnight) in 0.1% LFB solution (Gurr, Electron Microscopy Sciences, Hatfield, PA, USA), further washed‐out in 96% ethanol and milli‐Q water (2–3 sec, and 3 sec, respectively), differentiated (0.05% lithium carbonate solution, 5 sec, Merck Millipore, Germany and 70% ethanol, 5–7 sec), dehydrated (Milli‐Q water followed by 96%–100% ethanol and xylene; 3–5 min, 2 × 5 min, 3 × 5 min, respectively), and coverslipped using Entellan (Sigma Aldrich, US).…”

“… 15 Moreover, to reduce the artifact risk we considered as mDAWM only those areas whose surface was higher than 0.1 mm 2 and excluding regions whose myelin density change could be otherwise defined by variation in fiber orientation. Myelin density quantification was performed adapting a previously used protocol, 20 which involved ROI selection and manual segmentation to retain the maximum amount of pixel associated to myelin signal. To confirm the quality of the mDAWM selection, random digitally acquired ROIs of the tissue were automatically processed using the Image‐J algorithm percentile (Fig.…”

APART-QSM: an improved sub-voxel quantitative susceptibility mapping for susceptibility source separation using an iterative data fitting method

“…The 3D GRE scan parameters were: FOV=71.0×84.0×51.2 mm 3 ; matrix size = 710×840×512; spatial resolution = 0.1×0.1×0.1 mm 3 ; TR = 75 ms; TE1/spacing/TE3 = 5.7/8.5/24.7 ms; FA = 35°; BW = 81967 Hz; scan time = 6.7 h. The 3D MSE sequence was conducted with the parameters: FOV = 71.0×82.0 mm 2 ; matrix size = 710×820; slice number = 512; spatial resolution = 0.1×0.1×0.12 mm 3 ; TR = 140 ms; TE = 15.2, 30.4 ms; BW = 66667 Hz; scan time = 12.6 h. The DTI data were acquired by a 3D spin-echo pulse sequence: FOV = 70.7×84.0×53.2 mm 3 ; matrix size = 202×240×152; spatial resolution = 0.35×0.35×0.35 mm 3 ; TR = 100 ms; TE = 25.2 ms; One non-diffusion weighted image and one b-value of 3000 s/mm 2 with 30 diffusion directions; BW = 45455 Hz; scan time = 19.9 h. The myelin staining procedure followed the steps described in (Huitema et al, 2021), which was first dehydrated and quickly frozen by dry ice, and cut into sections 80-μm thick at the coronal position. Then the sections were stained with Luxol Fast Blue (LFB, Beijing Solarbio Science & Technology Co., Ltd) solution.…”