Diffusion tensor imaging detects and differentiates axon and myelin degeneration in mouse optic nerve after retinal ischemia

Abstract

Both axon and myelin degeneration have significant impact on the long-term disability of patients with white matter disorder. However, the clinical manifestations of the neurological dysfunction caused by white matter disorders are not sufficient to determine the origin of neurological deficits. A noninvasive biological marker capable of detecting and differentiating axon and myelin degeneration would be a significant addition to currently available tools. Directional diffusivities derived from diffusion tensor imaging (DTI) have been previously proposed by this group as potential biological markers to detect and differentiate axon and myelin degeneration. To further test the hypothesis that axial (λ_‖) and radial (λ_⊥) diffusivities reflect axon and myelin pathologies, respectively, the optic nerve was examined serially using DTI in a mouse model of retinal ischemia. A significant decrease of λ_‖, the putative DTI axonal marker, was observed 3 days after ischemia without concurrently detectable changes in λ_⊥, the putative myelin marker. This result is consistent with histological findings of significant axonal degeneration with no detectable demyelination at 3 days after ischemia. The elevation of λ_⊥ observed 5 days after ischemia is consistent with histological findings of myelin degeneration at this time. These results support the hypothesis that λ_‖ and λ_⊥ hold promise as specific markers of axonal and myelin injury, respectively, and, further, that the coexistence of axonal and myelin degeneration does not confound this utility.

Introduction

The underlying mechanisms leading to neurological dysfunction in white matter disorders may consist of myelin dysfunction/demyelination, axonal dysfunction/injury, or both Li et al., 1999, McGavern et al., 1999, Perry and Anthony, 1999, Trapp et al., 1999, van der Valk and De Groot, 2000, Bjartmar et al., 2001, De Stefano et al., 2001, Phillips, 2001. However, the pathological processes underlying neurological dysfunction in white matter diseases remain unclear. Magnetic resonance imaging (MRI) is a well-established method for detecting various central nervous system (CNS) injuries Arfanakis et al., 2002, De Stefano et al., 2002, Parkinson et al., 2002. Unfortunately, conventional MRI techniques, such as T1- and T2-related measurements, are not capable of differentiating axonal vs myelin injury in CNS white matter disorders (Song et al., 2002).

Diffusion tensor magnetic resonance imaging (DTI) has been widely applied in CNS for detailed developmental and pathological analyses Filippi et al., 2001, Ito et al., 2001, Maldjian and Grossman, 2001, Nusbaum et al., 2001. After generation of the diffusion tensor matrix from a series of diffusion-weighted images, the three eigenvalues, also referred to as diffusivities (λ₁, λ₂, and λ₃), are calculated by matrix diagonalization Pierpaoli and Basser, 1996, Basser and Pierpaoli, 1998. These are scalar indices that describe water diffusion in a local, i.e., voxel specific, frame of reference that coincides with the geometry of white matter tracts.

Parameters derived from the three eigenvalues include the trace of the diffusion tensor, Tr(D) = λ₁ + λ₂ + λ₃ = 3 × ADC (apparent diffusion coefficient), the relative anisotropy (RA, the anisotropy index, defined as the standard deviation of the three eigenvalues, normalized by the ADC), and the fractional anisotropy (FA, anisotropy index normalized to the magnitude of diffusion tensor). These secondary parameters are reference frame independent and prove to be sensitive to pathology Horsfield et al., 1998, Werring et al., 1999, Filippi et al., 2001. As secondary parameters that allow a simplified expression of water diffusion, neither RA (or FA) nor Tr(D) is suited for a pathology-specific analysis. The current study proposes a more analytical approach to obtain specific information defining the underlying pathology in white matter injury.

The eigenvalues (λ₁, λ₂, and λ₃) derived by diffusion tensor matrix diagonalization can be separated into components parallel (λ₁) and perpendicular (λ₂ and λ₃) to the axonal tract Basser, 1995, Basser et al., 2000, Xue et al., 1999. The axial diffusivity, λ_‖ ≡ λ₁ > λ₂, λ₃, is defined as the magnitude of water diffusion parallel to the tract within the voxel of interest. Similarly, the radial diffusivity, λ_⊥ ≡ (λ₂ + λ₃)/2, describes the mean ADC magnitude of water molecules diffusing perpendicular to the tract (Song et al., 2002).

We have hypothesized that the underlying pathology might be discerned by evaluating directional diffusivities. We previously showed that in dysmyelinated shiverer mouse CNS white matter, where there is little or no myelin sheath surrounding intact axons, λ_‖ was not affected (Song et al., 2002). In contrast, the magnitude of λ_⊥ increased, reflecting the increased freedom of motion perpendicular to axons due to the lack of myelin (Song et al., 2002). It is also our hypothesis that axonal degeneration without (or prior to) demyelination will result in a decrease in both λ_‖ and λ_⊥. The degree of decrease in λ_⊥ is expected to be relatively much smaller than that in λ_‖ and is less likely to be detectable experimentally since λ_⊥ is ∼10–30% of λ_‖ in magnitude. Thus, we propose that axonal degeneration in the absence of demyelination will result in a detectable decrease of λ_‖.

To test our hypothesis, we used a mouse model of retinal ischemia that causes an acute inner retinal degeneration Kawai et al., 2001, Rosenbaum et al., 2001 with axonal degeneration in optic nerve. Subsequent to this retinal degeneration, secondary myelin fragmentation occurs (“Wallerian degeneration”) (Adachi et al., 1996). A longitudinal DTI examination of the optic nerve in this mouse model was conducted and correlated with neurofilament and myelin basic protein (MBP) immunostaining (Kim et al., 2001) at 3 and 7 days after the injury.