Neurogenesis in the chronic lesions of multiple sclerosis

Abstract

Subcortical white matter in the adult human brain contains a population of interneurons that helps regulate cerebral blood flow. We investigated the fate of these neurons following subcortical white matter demyelination. Immunohistochemistry was used to examine neurons in normal-appearing subcortical white matter and seven acute and 59 chronic demyelinated lesions in brains from nine patients with multiple sclerosis and four controls. Seven acute and 44 of 59 chronic multiple sclerosis lesions had marked neuronal loss. Compared to surrounding normal-appearing white matter, the remaining 15 chronic multiple sclerosis lesions contained a 72% increase in mature interneuron density, increased synaptic densities and cells with phenotypic characteristics of immature neurons. Lesion areas with increased neuron densities contained a morphologically distinct population of activated microglia. Subventricular zones contiguous with demyelinated lesions also contained an increase in cells with phenotypes of neuronal precursors. These results support neurogenesis in a subpopulation of demyelinated subcortical white matter lesions in multiple sclerosis brains.

Fig. 1

Morphology and immunophenotype of white matter neurons. MAP2-positive neurons in subcortical white matter have fusiform cell bodies and one to three primary dendrites with additional arborizations that are oriented parallel to myelinated axons (A and B). Images are from multiple sclerosis normal-appearing white matter; neuronal morphology and distribution are identical to controls. White matter neurons also express other neuronal markers, such as calretinin, a marker of interneurons (C, red), nNOS (D, red) and vasoactive molecules, NPY (E, red) and somatostatin (F, green). White matter neurons also express the neuronal nuclear antigen, NeuN (F, red). Green immunostain in C–E is MAP2. (C–E) Multiple sclerosis normal-appearing white matter. (F) Multiple sclerosis lesion (with increased neurons). Scale bars: (A and B) 50 μm; (C–F) 20 μm.

Fig. 2

Inflammatory demyelination is associated with decreased numbers of white matter neurons. Acute (A–D) and chronic (E–H) lesions are characterized using immunohistochemistry for PLP (A and E) and MHC Class II (B, C and F). Normal myelin protein stain is characterized by diffuse intense immunoreactivity as shown in the lower portion of panels A and E. Normal MHC Class II immunoreactivity is shown in lower portion of panel F. An active lesion of multiple sclerosis characterized by loss of myelin (A*), myelin debris within the demyelinated area and an even distribution of MHC Class II-positive macrophages throughout the lesion (B and C). All active lesions had decreased neurons. Residual MAP2-positive neurons had shrunken perikarya (D, arrow) and fragmented dendrites (D, arrowheads). A chronic lesion shows loss of myelin (E*) and increased MHC Class II-positive cells at the lesion border (F). White matter neurons are greatly reduced in most chronic multiple sclerosis lesions (G, MAP2). nNOS-positive neurons with shrunken perikarya (H) extend processes to blood vessels (H*). Scale bars: (A, B, E–G) 200 μm; (C, D and H) 20 μm.

Fig. 3

A subset of chronic multiple sclerosis lesions shows an increased density of white matter neurons. The area of demyelination is demonstrated by lack of PLP immunostaining (A). Leukocyte common antigen immunostaining demonstrates the lesion border as well as increased staining within the lesion (B). Increased neuronal density is demonstrated by MAP2 staining (C). Neurons in non-lesion white matter extend long processes that run along axons (D, MAP2). Neurons that are increased in white matter lesions extend shorter dendrites (E, MAP2). Double-label immunofluorescence and confocal microscopy show that MAP2 immunoreactivity is not present in microglial cells indicating that the increased MAP2 immunoreactivity in these lesions is not due to phagocytosis by microglia (F, MAP2, green, microglial marker, Iba1, red). Microglial cell morphology predicts whether lesions will contain increased neurons. In multiple sclerosis lesions containing increased neurons, microglia are abundant and have large cell bodies and multiple thick processes (F, Iba1, red, G, MHC Class II). In lesions where neurons are absent, microglia density is low, and these cells extend few thin processes (H, MHC Class II). Scale bars: (A–C) 200 μm; (D and E) 100 μm; (F–H) 20 μm.

Fig. 4

Evidence for neurogenesis in multiple sclerosis lesions. Confocal image of a section double-labelled for MAP2 (A, green) and NeuN (A, red). All MAP2-positive cells are NeuN-positive (A, arrows), but some NeuN-positive cells are MAP2-negative (A, arrowheads). Schematic diagrams illustrate the normal distribution of white matter neurons (B) and the appearance of multiple sclerosis lesions with increased neurons (lower circle in C). In 15 of 59 lesions, MAP2-positive cell density is increased 72% (D) compared to locations closer to the cortex (upper circle in C). NeuN-positive cell density is increased 130% when compared to normal white matter (D). Synaptophysin-positive (red) puncta closely appose MAP2-labelled (green) neuronal soma and dendrites in a multiple sclerosis lesion (E) and in control white matter (F). Neurons in the lesions radiate highly branched dendrites and receive an increased density of synaptophysin-positive puncta compared with control neurons. Electron microscopy of synaptophysin-stained multiple sclerosis lesion with increased neuronal density (G) demonstrates that synaptophysin immunoreactivity is present in membrane-bound structures apposed to neurons consistent with presynaptic endings (dark staining on right, N = neuronal nucleus). Neurons in multiple sclerosis lesions also express proteins specific for interneurons including calbindin (H, green), somatostatin (H, red) and nNOS (I, red, MAP2, green). Scale bars: (A, E, F, H and I) 20 μm; (G) 250 nm. Error bars: SEM.

Fig. 5

Immature neurons are increased in the SVZ bordering multiple sclerosis lesions. NeuN-positive cells were quantified in the SVZ bordering periventricular white matter lesions (A, red box) and adjacent non-lesion areas (A, blue box). NeuN-positive cells are rare in the non-lesion SVZ (B, arrows) but frequent in the adjacent lesion SVZ (C, arrows). There is a 3.5-fold increase of NeuN-positive cell density in the SVZ of the lesions compared to the non-lesion area (D). Some NeuN-positive cells (E, NovaRed, F, red) in the SVZ bordering a periventricular white matter lesion have PSA-NCAM-positive processes (E, brown cytoplasmic staining, DAB, arrows) and Dlx2-positive nuclei (F, green, NeuN, red). Scale bars: (A) 2 mm; (B and C) 200 μm; (E and F) 20 μm. Error bars: SEM.