Statistical diffusion tensor histology reveals regional dysmyelination effects in the shiverer mouse mutant
Introduction
Shiverer is a mouse mutation characterized by an almost complete absence of compact myelin in the central nervous system (CNS). The mutation produces a shivering phenotype in affected mice (Biddle et al., 1973, Bird et al., 1978, Dupouey et al., 1979, Privat et al., 1979) and a shivering gait develops within a few weeks from birth with a reduced life span of between 50 and 100 days. In contrast, the peripheral nervous system appears normal. Myelin basic protein (MBP) is absent in the CNS due to a deletion of the major part of the MBP gene (Roach et al., 1985, Readhead et al., 1987, Kimura et al., 1989). Shiverer has relevance to both dysmyelinating and demyelinating disease in humans since it decouples dysmyelination from inflammatory processes seen in multiple sclerosis and experimental allergic encephalomyelitis (Ahrens et al., 1998).
The apparent diffusion coefficient (ADC) of water as measured by MRI is likely a mixture of an extracellular hindered component and a restricted intracellular component (Assaf et al., 2004). The diffusion tensor is a popular model of the anisotropy of the ADC observed by MRI in biological tissues (Basser and Pierpaoli, 1998, Basser et al., 1994). The eigenvalues of the tensor, λi, can be interpreted as the apparent diffusivity in the axial (λ1) and radial (λ2 and λ3) directions of a fiber tract. The trace of the tensor, Tr(D), equal to the sum of the eigenvalues, is a convenient representation of the orientation-independent mean diffusivity. Diffusion anisotropy is typically represented by an anisotropy index, such as the fractional anisotropy (FA), derived from the eigenvalues (Skare et al., 2000). We refer the reader to literature reviews of DTI for a more complete picture of the model (Le Bihan et al., 2001, Basser and Jones, 2002).
Early work by Beaulieu and Allen demonstrated the relative lack of influence of myelin on diffusion anisotropy in myelinated and unmyelinated garfish nerves (Beaulieu and Allen, 1994, Beaulieu et al., 1998). DTI studies of mouse and human white matter development confirm that myelination is not a prerequisite for diffusion anisotropy in white matter, but may still influence the magnitude of tensor indices (Mori et al., 2001, Neil et al., 2002, Zhang et al., 2003). Diffusion tensor microscopy of fixed spinal cords has shown significant increases in both axial and radial diffusivity in myelin-deficient rats (Gulani et al., 2001). However, increased radial diffusivity without significantly increased axial diffusivity has also been observed using in vivo DTI of shiverer mouse white matter tracts (Song et al., 2002).
In this study, we demonstrate how high-resolution MR diffusion tensor microscopy of fixed brains reveals a much broader range of detectable dysmyelination effects in the shiverer CNS than might be predicted from study of the major white matter tracts alone. Acquisition of high-resolution volumetric DTI data allows coregistration and statistical comparison of group diffusion measurements on a voxel-by-voxel-basis. This approach eliminates prior assumptions regarding regions of significant change, resulting in an unbiased picture of dysmyelination and its effect on water diffusion within CNS tissue.
Section snippets
Animal protocol
The brains of congenic male homozygous shiverer mutants (C3Fe.SWV Mbpshi/Mbpshi, Jackson Laboratories, mean age at fixation = 6.0 ± 0.2 weeks, n = 6) and control males with the same background as the shiverers (C3HeB/FeJ, Jackson Laboratories, mean age at fixation = 6.9 ± 0.2 weeks, n = 6) were studied using diffusion tensor imaging. Mice were anesthetized deeply using 2.5% Avertin (0.017 ml/g body weight). The mouse was then fixed by transcardiac perfusion using 30 ml of room temperature
Results and discussion
Diffusion-weighted images demonstrated consistently high signal-to-noise ratios and low artifact levels resulting from equilibration in contrast agent and simple spin echo data acquisition, respectively (Fig. 1). The median diffusion-weighted white matter SNR was found to be 28 in controls and 34 in shiverers. For comparison, the median white matter SNR without diffusion weighting was 34 in controls and 40 in shiverers. Analysis of the voxel-wise differences in the coregistered tensor invariant
Conclusions
High-resolution contrast enhanced DTI allows whole-brain searches for statistically significant differences between the fixed brains of shiverer mice and wild-type controls. This approach eliminates the need for prior assumptions regarding anatomic regions of interest. Statistical parametric mapping revealed a general increase in both axial and radial diffusivity in the white matter of fixed shiverer brains. Highly significant increases in diffusivity were also detected in the thalamus,
Acknowledgments
The authors wish to thank Tim Hiltner for mouse perfusions and brain preparation, Natasha Kovacevic, Josette Chen and Mark Henkelmann for invaluable discussions regarding image coregistration and tissue fixation. This work was funded in part by the Human Brain Project (EB00232) with contributions from the National Institute of Biomedical Imaging and Bioengineering and the National Institute of Mental Health, the NCRR (RR13625) and NIHM (MH61223).
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