White matter changes in the perforant path area in patients with amyotrophic lateral sclerosis

Abstract

Objective: The aim of this study was to test the hypothesis that white matter degeneration of the perforant path - as part of the Papez circuit - is a key feature of amyotrophic lateral sclerosis (ALS), even in the absence of frontotemporal dementia (FTD) or deposition of pTDP-43 inclusions in hippocampal granule cells.

Methods: We used diffusion Magnetic Resonance Imaging (dMRI), polarized light imaging (PLI) and immunohistochemical analysis of post mortem hippocampus specimens from controls (n = 5) and ALS patients (n = 14) to study white matter degeneration in the perforant path.

Results: diffusion Magnetic Resonance Imaging demonstrated a decrease in fractional anisotropy (P = 0.01) and an increase in mean diffusivity (P = 0.01) in the perforant path in ALS compared to controls. PLI-myelin density was lower in ALS (P = 0.05) and correlated with fractional anisotropy (r = 0.52, P = 0.03). These results were confirmed by immunohistochemistry; both myelin (proteolipid protein, P = 0.03) and neurofilaments (SMI-312, P = 0.02) were lower in ALS. Two out of the fourteen ALS cases showed pTDP-43 pathology in the dentate gyrus, but with comparable myelination levels in the perforant path to other ALS cases.

Conclusion: We conclude that degeneration of the perforant path occurs in ALS patients and that this may occur before, or independent of, pTDP-43 aggregation in the dentate gyrus of the hippocampus. Future research should focus on correlating the degree of cognitive decline to the amount of white matter atrophy in the perforant path.

Keywords: amyotrophic lateral sclerosis; diffusion MRI; frontotemporal dementia; hippocampus; perforant path; polarized light imaging.

Figure 1

The circuit of Papez with a schematic representation of the hippocampal formation. The perforant path (blue) starts in the entorhinal cortex and projects through the subiculum via the molecular layer of the hippocampus to the dentate gyrus and the CA3 region of the hippocampus (circled‐cross indicates out of screen – i.e. anterior‐posterior – orientation). The alvear path (yellow) projects via the CA1 region into the hippocampus. CA, cornu ammonis; DG, dentate gyrus; EC, entorhinal cortex; PaS, parasubiculum; PreS, presubiculum; Sub, subiculum.

Figure 2

Perforant path microstructure estimated from diffusion Magnetic Resonance Imaging (dMRI) data. (a) Tractography in the perforant pathway running from the subiculum to the dentate gyrus for two controls and two amyotrophic lateral sclerosis (ALS) cases. These tracts were used for extracting the mean diffusivity (MD), radial diffusivity (RD), axial diffusivity (AD) and fractional anisotropy (FA) values within the perforant path. Tracts are superimposed on the T1‐weighted anatomical scan. (b) The FA value in the part of the perforant path that runs from the subiculum to the dentate gyrus is lower in ALS cases compared to controls (P = 0.013). The MD value is significantly higher in the perforant path in the ALS specimen compared to controls (P = 0.015) as well as the AD (P = 0.026) and the RD (P = 0.026). All P‐values given for a one‐tailed t‐test. The boxplot centre lines depict the median for each metric; box limits, the 25th and 75th percentiles of the metrics; the whiskers extend to the most extreme data points excluding outliers; raw data points are given by the black dots.

Figure 3

Polarized light imaging data from a control case. The transmittance map reflects the amount of light passing through the tissue. The retardance map shows the degree to which the light is shifted due to interaction with the myelin. The fibre orientation map colour‐codes the directionality of the myelinated fibres indicated by the colour wheel. This map is formed by combining the retardance and in‐plane map. The anatomical regions within the hippocampus are delineated and the approximate direction of the perforant path is indicated with the yellow arrows. Circled‐cross represents anterior‐posterior direction of the perforant path. The maps are a composite of multiple field‐of‐views. CA, cornu ammonis; DG, dentate gyrus; EC, entorhinal cortex; PreSub, presubiculum and Sub, subiculum. Scale‐bar = 3 mm.

Figure 4

Polarized light imaging retardance – reflecting myelin density – in the perforant path. The upper part of the figure depicts two exemplar retardance maps from a control – and an amyotrophic lateral sclerosis (ALS) case. Here, the red arrows indicate the approximate location of the perforant path. Retardance was significantly lower in ALS patients compared to controls. Furthermore, the changes in fractional anisotropy (FA) were possibly driven by myelin density, supported by significant correlation between FA and retardance were significantly correlated across all subjects. A correlation between mean diffusivity (MD) and retardance was not found. See caption Figure 2 for boxplot description. Scale‐bar = 3 mm.

Figure 5

pTDP‐43 pathology in the granule cell layer (gcl) of the dentate gyrus. Cases were stratified by absence (a) or presence (b) of pTDP‐43 inclusions (coloured brown) in the granule cells of the dentate gyrus. Blue nuclei. Scale‐bar = 50 μm.

Figure 6

Histological data processing. The left column shows stained sections with a snapshot for myelin (PLP), neurofilaments (SMI‐312), and activated microglia (CD68). The middle column depicts the segmentation of the images in the left column. A colour‐based segmentation was applied to separate DAB‐stain‐positive pixels (red) from the background tissue (black) and nontissue pixels (white). After processing, the stained area fraction was calculated per 100 × 100 pixels resulting in a down‐sampled image reflecting the stained area fraction over the entire slice (right column). Scale‐bar = 30 μm.

Figure 7

Immunohistochemistry in amyotrophic lateral sclerosis (ALS) and controls. The stained area fraction of myelin (PLP) and neurofilaments (SMI‐312) was significantly lower in the perforant paths of ALS patients compared to controls. The area fractions of activated microglia (visualized with CD68; marker for inflammation) were similar in both groups. Two out of the fourteen ALS cases exhibited pTDP‐43 pathology within the dentate gyrus, used for neuropathological characterization of FTD. Furthermore, myelin area fractions from the immunohistochemistry stains correlated with myelin density estimated from the polarized light imaging retardance maps and also with FA derived from diffusion Magnetic Resonance Imaging (dMRI). The two ALS‐FTD cases had lowered myelin density estimates, but no difference was observed compared to pure ALS cases. See caption Figure 2 for boxplot description.