Digitized image analysis reveals diffuse abnormalities in normal‐appearing white matter during acute experimental autoimmune encephalomyelitis

Abstract

Demyelination of the central nervous system is a hallmark of multiple sclerosis and its widely used animal model, experimental autoimmune encephalomyelitis (EAE). Recent studies using magnetic resonance imaging and spectroscopy on multiple sclerosis patients have revealed abnormalities of central nervous system normal-appearing white matter suggesting that micro-demyelination and/or extensive membrane turnover accompanies and perhaps precedes the appearance of manifest inflammatory lesions. In the present study, we induced EAE in SWXJ mice and analyzed digitized images of immunocytochemically stained spinal cord for detection of myelin proteolipid protein (PLP). We found that digitized image analysis is a highly sensitive, objective methodology for measuring the extent of myelin loss during EAE. Our data show that two-thirds of the measured reduction of myelin PLP occurring in EAE spinal cord could be attributed to a loss of myelin in normal-appearing white matter. The marked decrease in detection of PLP was accompanied by a corresponding decrease in PLP mRNA in the central nervous system. Our results indicate that during acute EAE, diffuse myelin abnormalities extend far beyond visibly detectable inflammatory foci and are characterized by a global decrease in the expression of myelin genes and their encoded proteins.

Differential DM20 mRNA expression distinguishes two distinct patterns of spontaneous recovery from murine autoimmune encephalomyelitis

“…Dorsal columns were chosen for analysis because they represent a well‐defined uniformly myelinated area that can be easily identified and outlined throughout the full length of the spinal cord. Digitized images of spinal cord sections were captured, normalized, and analyzed as previously described (Kawczak et al, 1998) to determine the level of PLP immunostaining in dorsal columns. For the purpose of comparison, the levels of PLP immunostaining measured in dorsal columns from normal mice were considered to represent maximum staining intensity and were assigned a value of 100% immunostaining.…”

“…Immunocytochemistry and quantitative digitized image analysis were performed as previously described (Kawczak et al, 1998). At the same time brains were isolated for RNA analysis, spinal cords were removed to 10% phosphate‐buffered formalin.…”

Mechanisms Underlying the Process of Demyelination in Multiple Sclerosis

“…Some of such factors can intensify, e.g., proliferation and, at the same time, inhibit differentiation and maturation of oligodendrocytes. This is why a detailed elucidation of the extra-and intracellular factors influencing the above processes is a very important and promising direction of the corresponding experimental studies [77].…”

Pre-Emptive Targeting of the Epitope Spreading Cascade with Genetically Modified Regulatory T Cells During Autoimmune Demyelinating Disease

“…Spinal cords were fixed in 10% phosphate-buffered formalin and cut into multiple pieces, and paraffin-embedded tissue sections were cut (30 m each) for immunostaining as described previously (21). Sections were pretreated with 0.04% OsO 4 and 1% H 2 O 2 in 10% Triton X-100 (Electron Microscopy Sciences, Fort Washington, PA) and blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA) and 5% nonfat dehydrated milk for 60 min.…”

“…Demyelination was quantified by analysis of digitized images of PLP-immunostained spinal cord sections as previously described (21). Briefly, digital images of PLP-immunostained spinal cord sections were captured at 640 ϫ 480 pixel resolution and ϫ11.67 magnification using the AlphaImager 2000 System (Alpha Innotech, San Leandro, CA).…”