The pathologic substrates of subtle MTR and DTI abnormalities in NAWM of MS brains have not been specifically addressed in previous MRI-pathology correlation reports. The present study used image-guided sampling of MS brain tissue acquired through rapid autopsy to investigate the pathologic correlates of these MRI characteristics in non-lesional WM. The following observations were made: (i) the pathologic substrates for subtle MTR abnormality in MS NAWM vary depending on proximity to WM lesions; (ii) reduced MTR is associated with axonal swelling in NAWM regions close to WM lesions but not in regions far from WM lesions; (iii) NAWM regions with reduced MTR (either far from or close to lesions) have increased numbers of enlarged microglia / macrophages, and the density of enlarged microglia correlates with DTI measures; (iv) regions of NAWM with reduced MTR often neighbor demyelinating cortical lesions. These findings suggest that a portion of the MTR abnormality in MS NAWM can be accounted for by axonal degeneration and microglial activation. Chronic cortical plaques might also contribute indirectly to reducing MTR in subcortical NAWM by activating nearby microglia. We did not observe significant differences in myelin density, blood vessel number, or plasma IgG deposition (“leaky” vessels) in NAWM regions with slightly abnormal MTR as compared to NAWM regions with normal MTR. Numbers of astrocytic glial cells did not differ in the studied NAWM ROIs.
Consistent with prior reports,(32
) we confirmed that there are fewer axons in T2T1MTR lesions as compared to NAWM. We did not find significant differences in axonal counts between the three types of non-lesional WM ROIs (sa-WM Close, sa-WM Far and NAWM ROIs). However, there were more swollen axons in both T2T1MTR lesions and sa-WM Close ROIs as compared with sa-WM Far and NAWM ROIs (). In earlier morphological studies of MS brains and spinal cord, reduced axonal density and swollen degenerating axons were found around demyelinated plaques and within defined fiber tracts emerging from plaques.(2
) Our current observations are generally in line with previous reports and suggest that MTR abnormality in NAWM around MS lesions might partly be explained by axonal destruction in the lesions followed by secondary axonal degeneration and increase in axonal water in peri-plaque WM.
In the present study of MRI correlates of pathology, we did not find differences in myelin, astrogliosis or vascular changes in sa-WM Close or sa-WM Far ROIs as compared with NAWM ROIs. On the other hand, sa-WM ROIs were associated with microglial activation, as indicated by significantly increased density of activated MHCII(+) microglia and macrophages and formation of microglial nodules. Low or moderate density perivascular cuffs of mononuclear leukocytes were also noted in sa-WM Close and sa-WM Far regions. We did not observe tissue infiltration by T-lymphocytes in the studied ROIs except in T2T1MTR chronic lesions.
It is important to note that sa-WM is different both radiologically and pathologically from “dirty appearing white matter” (DAWM).(17
) Radiologically, DAWM is defined as WM with diffusely abnormal signal intensity on T2-weighted weighted images, whereas sa-WM has normal T2 signal intensity. Like sa-WM regions, DAWM is located outside of focally demyelinated plaques and has lower MTR than NAWM.(17
) However, unlike sa-WM, DAWM is associated with prominent loss of myelin, chronic fibrillary gliosis, presence of inflammatory infiltrates and blood-brain barrier alterations.(38
) Therefore, it is expected that there would be differences in the pathologic characteristics that determine MRI abnormalities in sa-WM and DAWM, and the findings in these two distinct types of non-lesional MS brain tissue cannot be directly compared.
In this study, subtypes of NAWM regions were defined based on MTR and distance to lesions. Demyelination has been proposed to be the major pathologic substrate for decreased MTR within MS lesions,(29
) and our findings of relatively strong correlations between MTR and myelin density across all ROIs are consistent with previous reports. However, additional pathologic features may contribute to the MTR signal.(27
) When we excluded lesions from the analysis, MTR was no longer correlated with myelin density, suggesting that other pathologic processes were responsible for MTR differences in NAWM. In non-lesional WM where myelin is preserved, we mainly observed differences in axonal pathology and activated microglia in regions with slightly abnormal MTR as compared to NAWM with normal MTR. To our knowledge, there have not been prior studies on the pathologic correlates of abnormal MRI measures in NAWM as there have been for lesions.
We also investigated pathologic correlates of DTI measurements because DTI is another method used to quantify tissue damage in NAWM in vivo. Pathologic correlates of DTI changes have also been described in lesions, (42
) but not in non-lesional WM of MS brains. The current study used in situ
imaging, short post-mortem intervals, and contrast ratios to minimize the potential impact of post-mortem changes in DTI measures.(45
) In our analysis of DTI parameters across all ROIs (non-lesional WM and focally demyelinated lesions), FA, MD, λ
were all moderately correlated with markers of myelin and axonal damage, but not with enlarged microglia/macrophages. Interestingly, in the non-lesional regions, decreases in FA and λ
were associated with activated microglia and the presence of microglial nodules. There was also a trend for a correlation between MD and axonal count in the non-lesional regions. Overall, these results suggest that in non-lesional WM, subtle increases in MD may be indicative of axonal loss, and decreases in FA and λ
may signal microglial activation.
Importantly, we also found that many sa-WM far ROIs were located near cortical lesions. These ROIs were associated with microglial activation. We also noted variable numbers of dystrophic swollen axons in sa-WM far ROIs without significant differences as compared with NAWM. Previous MRI and histopathologic studies suggested that grey matter and white matter abnormalities might occur largely independently from each other, or evolve in different time frames.(3
) It is plausible, however, that activated microglia and secondary axonal degeneration in sa-WM Far regions might be causally related to nearby cortical pathology.
In summary, this study addresses a previously unexplored question: What are the pathologic substrates of subtle MRI changes consistently observed in MS NAWM in vivo
, including the slight downward shift in the MTR histogram and changes in DTI measurements? Due to the strong correlations between MTR and myelin in lesions, shifts in the MTR histogram are often assumed to be due to changes in myelin. However, in non-lesional WM we did not observe differences in MBP immunoreactivity between NAWM regions with different MTRs. Our data suggest that subtle MTR abnormality in NAWM in close proximity to WM lesions can be attributed to axonal degeneration and microglial activation. In contrast, subtle MTR abnormalities in NAWM far from lesions are associated with marked microglial activation but not with axonal pathology. Previous studies show that MTR and DTI abnormalities in MS NAWM correlate with disability, but are not altered by currently available treatments.(6
) A better understanding of the substrate for these changes may lead to more targeted imaging outcome measures for new therapies that target secondary axonal damage and microglial activation in NAWM.