Tubers are neither static nor discrete: Evidence from serial diffusion tensor imaging

Abstract

Objective: To assess the extent and evolution of tissue abnormality of tubers, perituber tissue, and normal-appearing white matter (NAWM) in patients with tuberous sclerosis complex using serial diffusion tensor imaging.

Methods: We applied automatic segmentation based on a combined global-local intensity mixture model of 3T structural and 35 direction diffusion tensor MRIs (diffusion tensor imaging) to define 3 regions: tuber tissue, an equal volume perituber rim, and the remaining NAWM. For each patient, scan, lobe, and tissue type, we analyzed the averages of mean diffusivity (MD) and fractional anisotropy (FA) in a generalized additive mixed model.

Results: Twenty-five patients (mean age 5.9 years; range 0.5-24.5 years) underwent 2 to 6 scans each, totaling 70 scans. Average time between scans was 1.2 years (range 0.4-2.9). Patient scans were compared with those of 73 healthy controls. FA values were lowest, and MD values were highest in tubers, next in perituber tissue, then in NAWM. Longitudinal analysis showed a positive (FA) and negative (MD) correlation with age in tubers, perituber tissue, and NAWM. All 3 tissue types followed a biexponential developmental trajectory, similar to the white matter of controls. An additional qualitative analysis showed a gradual transition of diffusion values across the tissue type boundaries.

Conclusions: Similar to NAWM, tuber and perituber tissues in tuberous sclerosis complex undergo microstructural evolution with age. The extent of diffusion abnormality decreases with distance to the tuber, in line with known extension of histologic, immunohistochemical, and molecular abnormalities beyond tuber pathology.

Figure 1. Segmentation algorithm and distribution histograms of FA values of tuber, perituber, and NAWM tissues

(A–D) Manual and automated tuber segmentation. (A) Axial fluid-attenuated inversion recovery image, with tubers appearing as bright areas of T2 prolongation. (B) Manual segmentation; areas indicating tuber tissue are marked red. (C) Intensity likelihood of outlier voxels with color spectrum from blue (least likely) to red (most likely), given the global-local intensity mixture model. (D) Automated tuber segmentation, tuber areas indicated in red and perituber tissue in salmon, after removal of false-positive areas (e.g., CSF pulsation artifacts). (E) Example of distribution histograms of 3 tissue types in 3 scans of one patient. On the right, 3 axial FA maps are aligned, each acquired at a different time point. The superimposed automated segmentation reveals 3 different tissue types of the right frontal lobe (dark blue cortex not included): red (tuber), salmon (perituber), light blue (NAWM). On the left, the corresponding distribution histograms of FA values are displayed for each tissue type. For clarity, only the histograms of the right frontal lobe of the first acquired image are shown. The mean (dotted line) and median (vertical line) reflect the skew in each histogram. FA = fractional anisotropy; NAWM = normal-appearing white matter.

Figure 2. Diffusion measures over time in tuberous sclerosis complex

FA (A) and MD (B) measures are fitted with the GAMM, and evolution with age in years is shown for every lobe. Three different tissue types are color-coded: red (tuber), salmon (perituber), and light blue (normal-appearing white matter). White matter of controls is represented in black. Note the early, steep, linear increase before age 5 to 6 years and the slower increase thereafter. FA = fractional anisotropy; GAMM = generalized additive mixed model; MD = mean diffusivity.

Figure 3. Gradual transition between tissue types from tuber to deep NAWM

(A) Changes in FA measured along trajectories from tuber to deep NAWM in each lobe of 5 patients reveal a gradual transition between tissue types (example trajectories in black above plots). The boundaries between the tuber (red), perituber (salmon), and NAWM (light blue) tissue types are not discrete, and thus segmentation is dependent on factors such as imaging contrast, resolution, and arbitrary thresholding. Note the deep “dips” in the curves of the NAWM, reflecting areas with more complex white matter, for example, the crossing of pathways. (B) The trajectories of 5 healthy controls, also with some increase of FA values in deeper white matter pathways. FA = fractional anisotropy; NAWM = normal-appearing white matter.

Figure 4. Histopathology and immunohistochemistry of tuber tissue (A–D), perituber white matter (E–H), a microtuber area (I–L), and NAWM (M–P)

The gross specimen (top left), low-magnification H&E/LFB (top middle), and pocket of tuber pathology (microtuber, top right) indicate the studied areas. Tuber tissue (A) H&E/LFB staining demonstrates absence of cortical lamination and presence of cells of “ambiguous phenotype” in tubers, variably positive for glial (B) and neuronal markers (C). SMI 31 (phosphorylated neurofilament) positivity in the perikarya is a feature of dysplastic neurons (D). In the white matter of perituber tissue (E), scattered balloon cells are also present, as well as occurring in small confluent clusters in the microtuber (I). In more remote white matter (M), apparently normal myelination is seen. Reactive astrocytes, indicating gliosis likely related to seizure activity, are seen in tubers (B), as well as remote from the tuber, in (F) and (J), and in the NAWM (N). Scattered heterotopic neurons are seen in perituber tissue (G), in the microtuber (K), and are easy to appreciate at lower magnification in the NAWM (O). NFP stain of perituber white matter marks some dysplastic neurons (H), as does the synaptophysin stain in the microtuber area (L). (P) A normal NFP stain shows white matter, with axons taking a radial turn into the cortex. GFAP = glial fibrillary acidic protein; H&E/LFB = hematoxylin & eosin/Luxol fast blue; NAWM = normal-appearing white matter; NeuN = neuronal nuclear antigen; NFP = neurofilament protein; SMI 31 = phosphorylated neurofilament.

Figure 5. The changing view of TSC pathology

(A) Extent of neuropathology is indicated by the density of black dots. With a segmentation based on imaging density values, an arbitrary cutoff is established to obtain the borders of 3 tissue types: tuber area in red, perituber in salmon, and the remainder of NAWM in light blue. Tubers (and other macroscopic abnormalities) represent the “tip of the iceberg,” and in the direct vicinity but also more remotely, areas of tuber-like pathology are found as well. With distance from the tuber, the extent of pathology diminishes. (B) Studies using depth electrodes (diagonal black lines) to elucidate whether seizures start in or next to a tuber, may be subject to measuring abnormal activity of microscopic collections of tuber-like pathology next to the tuber. (C) Reported pockets of normal white matter diffusion in patients with TSC compared with controls may in fact describe regions (green circles) in which the pathologic burden is below imaging resolution. (D) Studies suggesting tuber volume changes over time may be biased by different segmentation thresholds because the border of tuber pathology is not discrete. From top to bottom, thresholding may be subject to subtle differences in acquisition (1.5T, 3T, MRI from different vendor), in maturation (myelin), and in partial voluming effects or angulation. NAWM = normal-appearing white matter; TSC = tuberous sclerosis complex.