Radial diffusivity predicts demyelination in ex vivo multiple sclerosis spinal cords

Abstract

Objective: Correlation of diffusion tensor imaging (DTI) with histochemical staining for demyelination and axonal damage in multiple sclerosis (MS) ex vivo human cervical spinal cords.

Background: In MS, demyelination, axonal degeneration, and inflammation contribute to disease pathogenesis to variable degrees. Based upon in vivo animal studies with acute injury and histopathologic correlation, we hypothesized that DTI can differentiate between axonal and myelin pathologies within humans.

Methods: DTI was performed at 4.7 T on 9 MS and 5 normal control fixed cervical spinal cord blocks following autopsy. Sections were then stained for Luxol fast blue (LFB), Bielschowsky silver, and hematoxylin and eosin (H&E). Regions of interest (ROIs) were graded semi-quantitatively as normal myelination, mild (<50%) demyelination, or moderate-severe (>50%) demyelination. Corresponding axonal counts were manually determined on Bielschowsky silver. ROIs were mapped to co-registered DTI parameter slices. DTI parameters evaluated included standard quantitative assessments of apparent diffusion coefficient (ADC), relative anisotropy (RA), axial diffusivity and radial diffusivity. Statistical correlations were made between histochemical gradings and DTI parameters using linear mixed models.

Results: Within ROIs in MS subjects, increased radial diffusivity distinguished worsening severities of demyelination. Relative anisotropy was decreased in the setting of moderate-severe demyelination compared to normal areas and areas of mild demyelination. Radial diffusivity, ADC, and RA became increasingly altered within quartiles of worsening axonal counts. Axial diffusivity did not correlate with axonal density (p=0.091).

Conclusions: Increased radial diffusivity can serve as a surrogate for demyelination. However, radial diffusivity was also altered with axon injury, suggesting that this measure is not pathologically specific within chronic human MS tissue. We propose that radial diffusivity can serve as a marker of overall tissue integrity within chronic MS lesions. This study provides pathologic foundation for on-going in vivo DTI studies in MS.

Figure 1. White matter tract ROIs

Regions of interest are drawn to incorporate spinal cord white matter WMTs including dorsal white matter, left and right lateral white matter, and left and right ventral white matter WMTs.

Figure 2. Luxol fast blue myelin staining and scoring

An axial LFB staining of cervical spinal cord (panel D) exhibits variable degrees of demyelination in an MS subject. Panel A demonstrates relatively normal myelination. Panel B represents severe demyelination. Panel C represents mild demyelination.

Figure 3. Co-registration of histology to MRI images

A. A Luxol fast blue stained histologic slice is labeled with three ROIs (1–3). B. The histology image is warped with co-registered pixels to axial and radial diffusivity parameter maps (panels C and D).

Figure 4. DTI parameter means

Box plots of DTI parameters with boxes representing 25th–75^th percentiles. Whiskers represent two standard deviations from the mean. Outliers are represented by circles and stars. Axial, radial, and mean diffusivity were each different between MS subjects and controls (p<0.001 for each) but no difference was appreciated for RA (p=0.54)

Figure 5. Demyelination versus radial diffusivity

Radial diffusivity increases with increased degree of demyelination. Means are surrounded by 95% confidence intervals.

Figure 6. Degree of demyelination versus relative anisotropy

Areas of moderate to severe demyelination demonstrate decreased relative anisotropy compared to controls but other categories are not statistically distinguishable. Means are surrounded by 95% confidence intervals.

Figure 7. Axonal density versus axial diffusivity

Poor correlation is demonstrated between the axonal count, separated by quartile, and axial diffusivity. The lowest quartile represents ROIs with the lowest axonal counts. The highest quartile represents ROIs with the highest axonal counts. Means are surrounded by 95% confidence intervals.

Figure 8. Radial diffusivity by myelination and axonal density

Six groups of varying pathology demonstrate increase radial diffusivity with increase degree of demyelination and axonal loss. Means are surrounded by 95% confidence intervals.