Cortical neuronal densities and cerebral white matter demyelination in multiple sclerosis: a retrospective study

Abstract

Background: Demyelination of cerebral white matter is thought to drive neuronal degeneration and permanent neurological disability in individuals with multiple sclerosis. Findings from brain MRI studies, however, support the possibility that demyelination and neuronal degeneration can occur independently. We aimed to establish whether post-mortem brains from patients with multiple sclerosis show pathological evidence of cortical neuronal loss that is independent of cerebral white-matter demyelination.

Methods: Brains and spinal cords were removed at autopsy from patients, who had died with multiple sclerosis, at the Cleveland Clinic in Cleveland, OH, USA. Visual examination of centimetre-thick slices of cerebral hemispheres was done to identify brains without areas of cerebral white-matter discoloration that were indicative of demyelinated lesions (referred to as myelocortical multiple sclerosis) and brains that had cerebral white-matter discolorations or demyelinated lesions (referred to as typical multiple sclerosis). These individuals with myelocortical multiple sclerosis were matched by age, sex, MRI protocol, multiple sclerosis disease subtype, disease duration, and Expanded Disability Status Scale, with individuals with typical multiple sclerosis. Demyelinated lesion area in tissue sections of cerebral white matter, spinal cord, and cerebral cortex from individuals classed as having myelocortical and typical multiple sclerosis were compared using myelin protein immunocytochemistry. Neuronal densities in cortical layers III, V, and VI from five cortical regions not directly connected to spinal cord (cingulate gyrus and inferior frontal cortex, superior temporal cortex, and superior insular cortex and inferior insular cortex) were also compared between the two groups and with aged-matched post-mortem brains from individuals without evidence of neurological disease.

Findings: Brains and spinal cords were collected from 100 deceased patients between May, 1998, and November, 2012, and this retrospective study was done between Sept 6, 2011, and Feb 2, 2018. 12 individuals were identified as having myelocortical multiple sclerosis and were compared with 12 individuals identified as having typical multiple sclerosis. Demyelinated lesions were detected in spinal cord and cerebral cortex, but not in cerebral white matter, of people with myelocortical multiple sclerosis. Cortical demyelinated lesion area was similar between myelocortical and typical multiple sclerosis (median 4·45% [IQR 2·54-10·81] in myelocortical vs 9·74% [1·35-19·50] in typical multiple sclerosis; p=0·5512). Spinal cord demyelinated area was significantly greater in typical than in myelocortical multiple sclerosis (median 3·81% [IQR 1·72-7·42] in myelocortical vs 13·81% [6·51-29·01] in typical multiple sclerosis; p=0·0083). Despite the lack of cerebral white-matter demyelination in myelocortical multiple sclerosis, mean cortical neuronal densities were significantly decreased compared with control brains (349·8 neurons per mm² [SD 51·9] in myelocortical multiple sclerosis vs 419·0 [43·6] in controls in layer III [p=0·0104]; 355·6 [46·5] vs 454·2 [48·3] in layer V [p=0·0006]; 366·6 [50·9] vs 458·3 [48·4] in layer VI [p=0·0049]). By contrast, mean cortical neuronal densities were decreased in typical multiple sclerosis brains compared with those from controls in layer V (392·5 [59·0] vs 454·2 [48·3]; p=0·0182) but not layers III and VI.

Interpretation: We propose that myelocortical multiple sclerosis is a subtype of multiple sclerosis that is characterised by demyelination of spinal cord and cerebral cortex but not of cerebral white matter. Cortical neuronal loss is not accompanied by cerebral white-matter demyelination and can be an independent pathological event in myelocortical multiple sclerosis. Compared with control brains, cortical neuronal loss was greater in myelocortical multiple sclerosis cortex than in typical multiple sclerosis cortex. The molecular mechanisms of primary neuronal degeneration and axonal pathology in myelocortical multiple sclerosis should be investigated in future studies.

Funding: US National Institutes of Health and National Multiple Sclerosis Society.

Figure 1:. Demyelination in Myelocortical Multiple Sclerosis (MCMS) and Typical Multiple Sclerosis (TMS)

Panel A shows a centimeter-thick slice from a TMS brain that contains a large white-matter lesion (arrow). Panel B shows a centimeter-thick slice from an MCMS brain without white-matter lesions. Panel C shows a normally-myelinated spinal cord section stained for PLP. Panels D and E show similar degrees of spinal cord demyelination in tissue sections from TMS (D) and MCMS (E). Panel F shows normally- myelinated cortex stained for PLP. Panels G and H show subpial cortical lesions in TMS (G) and MCMS (H) brains. Scale bars: C-H=2 mm. Black lines separate spinal cord gray and white matter (C-E) and cortical and subcortical white matter (F-H).

Figure 2:. Neuronal Loss in the Absence of Cerebral White-Matter Demyelination

Panel A shows a cresyl violet-stained hemispheric section from a TMS brain. Neuronal densities were compared in cortical layers III, V, and VI (B, yellow indicates neurons with an area greater than 60 μm) in the five cortical areas labeled in A. Panels C and D show staining for myelin proteolipid protein and the distribution of demyelinated lesions (blue=white-matter demyelination, pink=subpial demyelination) in hemispheric sections from TMS (C) and MCMS (D). A significant correlation between reduced cortical neuronal density and increased cerebral white-matter lesion volume was found in TMS, but not in MCMS (E; Pearson’s Correlation). Dashed lines in (E) indicate 95% CI. IFG=inferior frontal gyrus, STG=superior temporal gyrus, INi=inferior insular, INs superior insula, CG=cingulate gyrus. Scale bars: B=200 μm

Figure 3:. Magnetic Resonance Imaging in Myelocortical and Typical Multiple Sclerosis

Panels A and B show representative T2-weighted, fluid-attenuated inversion recovery images from postmortem TMS (A) and MCMS (B) brains.

Figure 4:. Pathological Correlates of Cerebral White-Matter Imaging Abnormalities in Myelocortical Multiple Sclerosis

An MCMS brain slice (A) is co-registered with T1-weighted (B), T2-weighted (C), and MTR (D) images. The red circle indicates a normal-appearing white-matter (NAWM) region as defined by no imaging abnormalities. The blue circle indicates a region of interest that is abnormal in T2-weighted, T1-weighted and MTR images (T2T1MTR). Panels E and F show axonal staining in tissue sections of the NAWM (E) and T2T1MTR (F) regions. Panel G compares measurements of myelin area, axonal area, axonal numbers, and axonal diameter in 46 NAWM and 31 T2T1MTR regions. Myelin area and axon count were significantly smaller in T2T1MTR regions (Student’s t-tests), whereas axonal diameter (Mann-Whitney U Test) was significantly higher. Furthermore, average axonal diameter negatively correlates with normalized T1-weighted intensity (H) and MTR (I). Individual dot colors represent individual patients (n=10), and dashed lines indicate 95% CIs in H and I. Data in H and I were analyzed by linear models where group, age, and sex were specified as fixed factors and subject was specified as a random factor. Scale bars: E and F=20 μm