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Griffiths et al. Neuroimmunol Neuroinflammation 2020;7:51-67 I http://dx.doi.org/10.20517/2347-8659.2019.21 Page 63

of which are associated with processes of lymphoid neogenesis, were mirrored by the finding of elevated
protein levels of many of the same factors in the matched post-mortem CSF, which were also differentially
expressed in an independent cohort of newly diagnosed MS patients with a clinical and radiological
[47]
signature of substantial cortical pathology . TNF and IFNγ are amongst those differentially expressed in
patients with a cortical phenotype and these cytokines mediate a rapid pattern of cortical demyelination
and microglia/macrophage activation when injected into the subarachnoid space of animals with
[46]
subclinical autoimmune encephalitis . These findings imply that lymphoid follicle-like structures, and
infiltrates outside of these semi-organised structures, are a source of damaging factors that drive subpial
pathology. Sampling CSF may reveal disease-relevant biomarkers of activity to aid therapeutic decision
[48]
making .
Subpial cortical GMLs are associated with the loss of neurons of the superficial cortical layers, an elevated
number of complement activated neurons and glia, together with substantial neuritic and synaptic
loss [13,16,45,49-53] . The most striking pathological changes are found in the superficial cortical laminae,
[16]
with a gradient of lessening neuronal damage with distance from the pia . Neurons and glia of the
MS cortex are exposed to elevated excitotoxins and reactive free radicals [54,55] , are energy depleted and
display mitochondrial pathology, which may contribute to further neurodegeneration [56-58] . There is an
imbalance between TNF receptor 1/2 anti-apoptotic pathways vs. pro-death signals in the progressive MS
cortex and oligodendrocytes are vulnerable to degenerate by a TNF receptor type 1/Receptor-interacting
protein kinase type 1 necroptotic cascade [59,60] . Immunoglobulins derived from meningeal plasma cells
are enriched in the MS neocortex, and products of central CD20+ B effector cells are directly toxic to
cultured neurons and oligodendrocytes [61-63] . Alongside myriad immune mediators, MS CSF is enriched in
bio-active lipids, whose levels associate with disease severity [64-66] . Lipid sterols, including key products of
cholesterol metabolism and ceramide, can be directly neurotoxic. For example, C16:0 and C24:0 ceramides
[65]
are enriched in MS patient CSF and can mediate neuroaxonal pathology and mitochondrial dysfunction ,
whilst simvastatin, a cholesterol-reducing therapy that enters the CNS, is associated with a slowing of brain
[67]
atrophy in SPMS . We currently have identified neither the combination of damaging factors that are
causative of injury nor the relationship between these mediators with clinical progression on an individual
level.

Lesions of the deeper cortical laminae (leukocortical or Type I cortical GMLs) are centred on cortical
veins, typically contain greater numbers of activated microglia/macrophages than subpial lesions , and
[53]
their relative area correlated with the extent of white matter lesion area in the same case. The statistical
association between cortical and subcortical lesions, both characterised by an inflamed central vein,
suggests they both share similar a mechanism of formation, which is in part different to lesions of the
superficial grey matter structures. MRI is adept at resolving leukocortical GMLs, whilst even ultra-high
[68]
field MRI detects only a fraction of all subpial lesions . Our finding of an association between the relative
area of leukocortical GML and WML area may explain the correlation between white matter and cortical
grey matter lesions reported by MRI.

Methodological considerations and study limitations
The analysis of whole coronal macrosections allows the study of cortical lesions in continuity, improves the
accuracy of their interpretation and the relationship with other pathological features, such as meningeal
inflammation or lesions in different anatomical sites [19,69,70] . The handling of such large tissue samples is
not trivial and this restricted the n number for our work, which may have meant our clinical-pathological
comparisons were statistically underpowered [for instance, to observe a significant difference in age
of onset between our GML High and Low MS cases, post-hoc power calculations (b = 0.8) predicted
a cohort of at least 40 cases would be required (a = 0.05)]. Whole brain immersion fixation often led
to some deformation of tissues, such that it was not always possible to align anatomical structures of