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MRI and immunohistochemical analysis of toxic- induced (cuprizone) and congenital (B4galt1-null) demyelination time course

Mice with a disrupted B4galnt1 gene lack the enzyme UDP-GalNAc:GM3/GD3 Nacetylgalactosaminyltransferase or simply GM2/GD2 synthase. Subsequently, they lack complex brain gangliosides such as GD1a and GT1b, found on axons. Lacking GD1a and GT1b, myelin associated glycoprotein (MAG) on oligodendrocytes, cannot make proper connections with opposing complex gangliosides. With age, these mice exhibit axonal degeneration and demyelination resulting in motor and behavioral deficits. Cuprizone-induced demyelination is a well characterized model for central nervous system demyelination, due to specific cell death of oligodendrocytes. To compare these two mouse demyelination models, we performed protocol of 20 days of cuprizone feeding, brain magnetic resonance imaging (MRI) and immunohistochemistry at 3 and 6 months of age. We imaged ex vivo brains of 3 groups of mice: B4galnt1-null, wild- type (C57BL/6) and cuprizone-fed. Each group consisted of 3 subjects. Brains were perfused with 4% PFA, extracted and kept in the PBS, then underwent imaging. MRI data was acquired on 7T Bruker BioSpec using the spin-echo DTI sequence in 64 directions, and isotropic 150 μm voxels. Using anatomical markings, regions of interest were set in the rostral part of corpus callosum and analyzed according to their radial diffusivity (RD) maps. Immunohistochemistry was performed using typical myelination (MAG, MBP), and fiberphosphorylation markers (SMI 311, SMI 312) to compare groups and validate MRI results. Using Mann-Whitney and Kruskal-Wallis tests, results showed significant statistical differences across the groups at 3 and 6 months of age (both p=0.004). At 3 months of age, B4galnt1-null group had lower RD values from both cuprizone-fed (p=0.002) and wild-type group (p=0.026). At 6 months of age wildtype group had lower RD values comparing to the cuprizone-fed group(p=0.002), as well as B4galnt1-null group (p=0.038). In 3 months old group, highest RD values in the cuprizone-fed group can be explained solely due to demyelination. Moreover, lowest RD values in the B4galnt1-null group can be explained due to unwinding of myelin causing less diffusion of water perpendicularly to the axon. At 6 months of age, difference between cuprizone and wild-type group suggests higher demyelination in this group than at 3 months old one, which can suggest that cuprizone had more toxic effect on mice that were 6 months of age. RD being higher in B4galnt1-null group at 6 comparing to 3 months old animals, suggests apoptosis of oligodendrocytes with unwound myelin. Myelin markers confirmed MRI results and proved different demyelination pattern in two mice models. MAG expression was similar in B4galnt1-null group and wild-type at 3 months, but was significantly reduced at 6 months of age. Fiber markers (SMI 311, SMI 312) showed accompanied dephosphorylation of neurofilaments, suggesting axonal pathology.

MRI, immunohistochemical analysis, demyelination, cuprizone, B4galt1-null

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