Thalamic atrophy in multiple sclerosis is associated with tract disconnection and altered microglia

Abstract

Thalamic atrophy already occurs in the early stages of multiple sclerosis (MS) and continues progressively throughout the disease. Demyelination is one of the main pathological hallmarks of MS and yet, thalamic demyelination does not correlate well with thalamic atrophy. By combining post-mortem magnetic resonance imaging with immunohistochemistry of thalami from 13 control and 13 MS donors, we investigated the underlying pathological contributors of thalamic atrophy and pathology. We first assessed the volumes of four thalamic nuclei groups (anterior, lateral, medial and posterior). Then, diffusion weighted imaging was used to assess the microstructural integrity of white matter tracts connecting each thalamic nuclei group. In addition, we studied myelination, inflammation, neurodegeneration and microglial activation by immunohistochemistry. We uncovered that medial and posterior thalamic nuclei were more atrophic compared to the anterior and lateral nuclei. Bilateral posterior nuclei and the right medial and anterior nuclei showed reduced fractional anisotropy in connected white matter tracks. We further show that microglial cells in the mediodorsal nuclei have an increased density and morphological complexity in MS compared to control donors. Microglia show signs of phagocytosis of pre-synapses, although we did not observe an overall synaptic loss in the thalamus of MS donors. These microglial changes within mediodorsal nuclei correlated with lower medial thalamic volume. Taken together, this study provides evidence that thalamic (subnuclear) atrophy relates tostructural thalamic network disconnection and within-thalamic microglial changes, but not thalamic demyelination. These findings could impact future treatment strategies aimed at thalamic neuroprotection.

Keywords: Atrophy; Microglia; Multiple sclerosis; Post-mortem; Thalamus.

Fig. 1

Increased atrophy in the thalamus of progressive MS donors. a Example of brain segmentation on 3D-T1 and 3D-FLAIR scans. b Normalized total brain volume of control and MS donors. c Percentage of different brain volume fractions of control and MS donors. d Percentage of white matter volume lesion of control and MS donors. e Example of thalamic segmentation on 3D-T1 and 3D-FLAIR scans. f Percentage of each thalamic nuclei volume of control and MS donors. Individual datapoints indicate data from an individual donor, columns and error bars show mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. MS multiple sclerosis

Fig. 2

Increased disconnection in the thalamus of progressive MS donors. a Example of brain streamlines on 3D-T1 and 3D-FLAIR scans. b Mean FA per streamline for each left thalamic nuclei. c Mean FA per streamline for each right thalamic nuclei. d Heatmap indicating partial correlation values between mean FA and thalamic volume. Colour key represents high (red) and low (blue) positive correlations. Individual datapoints indicate data from an individual donor, columns and error bars show mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. FA fractional anisotropy, MS multiple sclerosis

Fig. 3

Limited peripheral infiltration and lesions in the MDn of progressive MS donors. a Schematic overview for MDn identification based on autopsy and post-mortem MRI, Nissl and Synaptophysin stainings. b Representative images displaying PLP and HLA-DR expression of the thalamus of control and MS donors. Zoom-in shows the MDn. c Percentage of total demyelination in MDn. d Representative images showing HuC/HuD⁺ neurons in the MDn of control and MS subjects. e Quantification of HuC/HuD⁺ neurons in the MDn of control and MS donors. f Representative staining of CD3⁺ T cells and CD19⁺ B cells in a MS donor. g Quantification of CD3⁺ T cells and CD19⁺ B cells in the MDn of control and MS donors. Individual datapoints indicate averaged data from an individual donor, columns and error bars show mean ± SEM; ** P < 0.01. Filled datapoints show MS donors with lesions in the MDn. Scale bars = 200 µm (b), 100 µm (d), 50 µm (f). AD anterodorsal nucleus, AV anteroventral nucleus, CeME central medial nucleus, iml internal medullary lamina, MDn mediodorsal nuclei, MDMC mediodorsal nucleus, magnocellular part, PRF prerubral field (ruber nucleus), PT paratenial nucleus, PV paraventricular nucleus, Re reuniens nucleus, STh subthalamic nucleus, VLPE ventral posterolateral nucleus, external part, VLPI ventral posterolateral nucleus, internal part, ZI zona incerta, MS multiple sclerosis

Fig. 4

Microglial morphology complexity is increased in progressive MS. a Representative stainings showing P2Y12⁺ and IBA1⁺ microglia in MDn of control and progressive MS donors. b Quantification of the microglial density and mean fluorescence intensity of P2Y12 in IBA1⁺ microglia. c Representative images showing HLA-DR + and IBA1 + microglia in MDn of control and progressive MS donors. d Quantification of the mean fluorescence intensity of HLA-DR in IBA1⁺ cells. e Images of individual P2Y12⁺ IBA1⁺ microglia in MDn nuclei (top panels) and the corresponding tracing outlines (bottom panels). f Sholl-derived measurements. Non-linear curve fit of the average number of microglial branch intersections per 0.3 µm step from the cell soma. Area under the curve (AUC) and maximal number of intersections of microglial cell morphology averaged per donor. Individual datapoints indicate averaged data from an individual donor, columns and error bars show mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001. Filled datapoints show MS donors with lesions in the MDn. Scale bars = 20 µm (a, c, e). MFI mean fluorescence intensity, MDn mediodorsal nucleus, MS multiple sclerosis

Fig. 5

Microglial pre-synapses engulfment is enhanced in progressive MS. a Representative images of control and MS sections stained for IBA1 (microglia), LAMP1 (lysosomes) and Synaptophysin (pre-synapses) in the MDn. b Quantification of Synaptophysin⁺ spots in IBA1⁺ microglia (pre-synapses engulfment) and in IBA1⁺ LAMP1⁺ microglial lysosomes (pre-synapses phagocytosis). c Representatives images of synaptophysin in the MDn. d Percentage of pre-synaptic volume in control and MS donors. Individual datapoints indicate averaged data from an individual donor, columns and error bars show mean ± SEM; * P < 0.05. Filled datapoints show MS donors with lesions in the MDn. Scale bars = 5 µm (a), 10 µm (c). MDn mediodorsal nucleus; MS: multiple sclerosis

Fig. 6

Thalamic atrophy and disconnection correlates with microglial changes. a Heatmap indicating partial correlation values between MRI variables (medial thalamic volume and medial thalamic mean FA) and immunohistochemistry variables. Colour key represents positive (red) and negative (blue) correlations. * P < 0.05, ** P < 0.01. b Scatter plot of correlation between microglial morphology and medial thalamic volume. c Scatter plots of specific correlation between microglial characteristics and medial mean FA. Individual datapoints indicate averaged data from an individual donor. Filled datapoints show MS donors with lesions in the MDn. FA fractional anisotropy, GM grey matter, MDn mediodorsal nucleus, MS multiple sclerosis