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Neuroimmunol Neuroinflammation 2020;7:51-67.10.20517/2347-8659.2019.21© The Author(s) 2020.
Open AccessOriginal Article

Substantial subpial cortical demyelination in progressive multiple sclerosis: have we underestimated the extent of cortical pathology?

1Institute of Life Sciences, Swansea University Medical School, Swansea University, Swansea SA2 8PP, UK.

2Department of Brain Sciences, Faculty of Medicine, Imperial College London, London W5 3EH, UK.

3Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales NSW 2006, Australia.

Correspondence Address: Dr. Owain W. Howell, Institute of Life Sciences, Swansea University Medical School, Swansea University, Singleton Park, Swansea, SA2 8PP, UK. E-mail: o.w.howell@swansea.ac.uk

    This article belongs to the Special Issue Role of Neuroinflammation in Multiple Sclerosis
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    Aim: Multiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease. Much of the complex symptomatology relates to pathology outside the classic white matter plaque, whereby lesions of the cortical grey matter, which are difficult to resolve by conventional clinical imaging, are in part predictive of outcome. We investigated the extent of grey matter pathology in whole coronal macrosections to reassess the contribution of cortical pathology to total demyelinating lesion area in progressive MS.

    Methods: Twenty-two cases of progressive MS were prepared as whole bi-hemispheric macrosections for histology, immunostaining and quantitative analysis of lesion number and relative area, leptomeningeal inflammation and microglial/macrophage activation.

    Results: Cortical grey matter demyelination was seen in all cases, which was more extensive than in white and deep grey matter (hippocampus, thalamus and basal ganglia) and accounted for 0.8%-60.2% of the entire measurable cortical ribbon. The pattern of cortical grey matter demyelination was predominantly subpial (mean 90.9%, range 60%-100%, of total cortical grey matter lesion area) and cases with the largest areas of subpial cortical lesions had more and larger deep grey matter lesions, greater numbers of activated microglia/macrophages, both in lesions as well as in normal cortical grey matter, together with elevated leptomeningeal inflammation and lymphoid-like structures. White matter lesion area was unchanged when compared with the progressive MS cases with little subpial cortical demyelination.

    Conclusion: Analysis of whole coronal macrosections reveals cortical demyelination is more extensive than reported by conventional histological methods. Cases of progressive MS with substantial subpial cortical demyelination that is independent of underlying white matter lesion area support the implications that these lesions may in-part arise through different pathogenetic mechanisms. Biomarkers and/or imaging correlates of this subpial pathology are required if we are to fully comprehend the clinical disease process.


    Neocortical grey matter demyelination, microglial activation and neurodegeneration, which may in-part be driven by inflammation of the overlying leptomeninges, are important pathological processes influencing the clinical severity and outcome of multiple sclerosis (MS)[1]. No biomarker or widely available imaging technology can fully report the extent of neocortical pathology, which is essential for optimal disease management.

    Neocortical and cerebellar cortical pathology is evident from the earliest stages of MS[2-5]. It is a pathological hallmark of progressive MS[1,2] and closely associates with clinical severity at all stages[6]. For example, the extent of cortical pathology is better at predicting disease outcome than white matter pathology[7]. Radiological imaging of grey matter and cortical atrophy are predictive of the conversion to clinically definite MS[8-10] or the risk of transitioning to the progressive phase and disability[7,11]. Nevertheless, even high fidelity, non-routine, imaging technologies fail to identify lesions of the most superficial cortical layers, whilst neuropathological assessment of standard size tissue blocks often underestimates the extent of cortical lesions, which can occupy the cortical ribbon over multiple contiguous gyri and sulci[2,12].

    Lesions of the superficial layers of the neocortex (subpial cortical grey matter lesions) are more numerous and sometimes associate topographically with leptomeningeal foci of immune cell aggregates that resemble lymphoid-like structures (alternatively termed ectopic B cell follicle structures) seen in other autoimmune, chronic inflammatory and infectious diseases[13,14]. Progressive MS cases with leptomeningeal lymphoid-like structures exhibit a gradient of cortical tissue damage extending away from the pial surface, suggestive of factor(s) in the CSF that promote underlying cortical inflammation and injury[15-17], possibly through the activation of microglia. The degree of leptomeningeal inflammation correlates with the extent of cortical demyelination, neurodegeneration and microglial activation in acute and progressive stages[4,13,18,19], whilst white matter lesion area is not changed. These findings suggest that subpial cortical lesions may arise through partly independent mechanisms compared with lesions located further from the CSF-filled spaces of the pia or ventricular lumen[17].

    The study of whole bi-hemispheric coronal macrosections, although technically challenging, can reveal hitherto undisclosed aspects of MS pathology and better report the extent of global pathology. We analysed the distribution and histological characteristics of cortical grey matter, white matter and deep grey matter lesions, together with inflammation of the brain and overlying leptomeninges, using whole brain coronal sections from 22 cases of progressive MS. We demonstrate the sometimes surprising extent of subpial cortical grey matter demyelinating pathology that can be seen and highlight the association between subpial and periventricular grey matter lesions, the dissociation between subpial lesion load and white matter lesion area, and confirm the close relationship between leptomeningeal inflammation and subpial cortical lesion pathology. This data support efforts to develop brain imaging and biomarker technologies for the identification of subpial grey matter lesions to improve disease prediction and monitoring.


    Post-mortem cohort

    Formalin fixed, whole brains [n = 22; median age 57 years (range 30-71 years), median disease duration 21.5 years (2-40 years), female = 17] were available from clinically and neuropathologically validated cases of secondary progressive MS (see Table 1 for details). All cases were provided by the UK Multiple Sclerosis Society Tissue Bank, Imperial College London, with appropriate research ethics approval (08/MRE09/31+5). Case selection was based on availability of whole brains with well-preserved leptomeninges and accompanying detailed clinical and neuropathology summaries, collected between February 2004 and December 2008. Some of these cases have previously been reported[18], but all analysis and data presented here are unique to this manuscript.

    Table 1

    Cases and sections used in this study

    CaseSections analysedSexAge at death (years)Disease duration (years)MS typeCause of death
    MS202    A2, P1 F58                  23SPMSPulmonary embolism
    MS204    A3, P1, P3 M58                  19SPMSLeukaemia and MS
    MS212    A3, P1, P3 F47                  29SPMSMultiple sclerosis/bronchopneumonia
    MS214    P1 F51                  31SPMSMultiple sclerosis
    MS217    A3, P1, P3 F57                  15SPMSSuicide
    MS223    A2 F45                  2SPMSMultiple sclerosis/bronchopneumonia
    MS224    A2, P1, P3 F59                  33SPMSMultiple sclerosis/bronchopneumonia
    MS226    A2, P3 M64                  27SPMSMultiple sclerosis/pneumonia
    MS253    A3, P1, P3 F37                  16SPMSMultiple sclerosis/pulmonary embolism
    MS257    A2, P1, P3 F49                  22SPMSAspiration pneumonia
    MS258    A2, P1, P3 M46                  20SPMSMultiple sclerosis
    MS278    A3, P1, P3 M30                  21SPMSPneumonia
    MS293    P1, P3 F53                  18SPMSMultiple sclerosis
    MS295    P1, P3 F71                  15SPMSBronchopneumonia
    MS323    A4, P1 F62                  31SPMSMultiple sclerosis/sepsis
    MS336    A2, P1, P3 F57                  27SPMSMultiple sclerosis/resp failure
    MS344    P1 F57                  15SPMSMultiple sclerosis/septicaemia
    MS360    A2, P1, P3 M55                  40SPMSMultiple sclerosis
    MS361    P1 F60                  34SPMSMultiple sclerosis
    MS366    P1 F61                  19SPMSMultiple sclerosis/bronchopneumonia
    MS387    P3 F43                  11SPMSMultiple sclerosis
    MS395    A3, P3 M63                  26SPMSMultiple sclerosis/chest infection

    Individual progressive MS brains were dissected into 1-cm-thick coronal sections, cut in an anterior direction from the mammillary bodies as coronal bi-hemispheric sections (A1, A2 and A3), or posteriorly from the mammillary bodies towards the occipital lobe (sections P1, P2 and P3) such that each coronal section contained several different cyto-architectonic areas [Figure 1]. For example, coronal section A3 includes frontal cortex and poles of the temporal gyrus; coronal section P1 includes motor and somatosensory cortex, thalamus and anterior hippocampus; and coronal section P3 includes parietal and occipital lobe and occipital horn of the lateral ventricle. Areas of interest for comparison were subdivided into: (1) cortical (neocortex); (2) white matter; and (3) hippocampus and deep grey matter [comprising caudate, pallidum (interna and externa), putamen, thalamus, hypothalamus and hippocampus and dentate gyrus].

    Figure 1. Study setup and sampling protocol. Whole coronal tissue slabs sectioned along coronal planes A3, P1 and P3 were prepared at three levels from immersion fixed brains (A) (created with Biorender.com). Histology and immunohistochemistry (anti-PLP immunostaining) revealed tissue anatomy and areas of frank demyelination (B) (MS278 section A3, MS202 section P1 and MS360 section P3). Stained and mounted tissue sections were digitised and colour masks depicting demyelinating lesions involving the neocortical grey matter (red colour mask), deep grey matter and hippocampus (green colour mask) and white matter (blue colour mask) were annotated for lesion quantification (C). (B, C) Scale bar = 2 cm. PLP: proteolipid protein

    Tissue preparation, staining and characterising demyelinated lesions

    Whole coronal tissue sections were processed under vacuum and paraffin wax embedded prior to sectioning at 10 µm onto 3-Aminopropyltriethoxysilane-silane (Sigma, UK) coated glass slides using a Reicheart-Jung tetrander microtome. Following dewaxing and rehydration, coronal sections from each case were stained with luxol fast blue (LFB) and cresyl violet to identify anatomical landmarks (grey and white matter) and to facilitate the assessment of total cortical, hippocampal and deep grey matter and white matter areas. Subsequent sections were immunostained with anti-proteolipid protein (PLP) and anti-CD68 primary antisera [Table 2] and revealed by sequential anti-species specific biotinylated secondary and avidin-biotin complex (Vector Labs. Ltd.) horse-radish peroxidase conjugated tertiary detection reagent with ImmPACT DAB as the reporter chromogen (Vector). All slides were counterstained, dehydrated, cleared in xylene and DePeX mounted under glass coverslip (Ted Pella Inc. USA).

    Table 2

    List of primary antibodies used

    AntibodyDilutionTarget antigenCompany and product details
    Anti-CD681:400CD68 (macrosialin)          Dako; clone kp1
    Anti-CD201:125CD20          Dako; clone l26
    Anti-PLP1:1500Myelin proteolipid protein          BioRad; clone plpc1

    Cortical grey matter lesions (GMLs), revealed by anti-PLP immunohistochemistry, were grouped as Type I (leukocortical), Type II (intracortical and not contacting the pia or grey/white matter boundary), or Type III (extending from the pia, sometimes involving the entire breadth of the cortex and stopping at the grey/white matter boundary (Bo Type IV cortical GML[20]). Areas of sparse and patchy anti-PLP immunostaining, which reflect incomplete demyelination and/or remyelination, but are notably different to the most “normal” grey matter, were labelled as partially de/re-myelinated grey matter[4].

    White matter lesions (WMLs) were separately characterised as active inflammatory (confluent in CD68+ microglia/macrophages), chronic active (activated microglia/macrophages restricted to the lesion edge containing PLP+ and/or LFB+ myelin breakdown products) and chronic inactive lesions (few activated microglia/macrophages at the lesion edge without evidence of recent myelin phagocytosis). Additional areas of putative remyelinating/shadow plaque (by PLP+ myelin internodes and a classic LFB+ shadow appearance) were identified but not quantified. Demyelinating lesions of the hippocampus and deep grey matter were identified based on a clear loss of LFB and/or PLP+ immunostain and were characterised as active, chronic active or chronic inactive inflammatory demyelinating lesions[21].

    Slide digitisation, lesion masks and quantitative analysis

    Individual coronal sections were reviewed using an Olympus SZ60 stereo microscope 0.1-10 × magnification) and a Zeiss AxioImager Z1 (40-400 × magnification) to identify normal and pathological regions of interest in each slide, which were marked on A4-sized print-outs of the same slide captured using a conventional document scanner (HP Scanjet 300) at 1200 dpi.

    Quantifying the number and area of cortical, white matter and hippocampus and deep grey matter lesions

    The scanned images were used to guide our tracing of white and grey matter areas (from the LFB stained section), and areas of cortical, white matter and hippocampus and deep grey matter lesions (PLP+ slide) as colour-mask overlays using GNU image manipulation software (GIMP 2.10; see Figure 1). The modified high-resolution TIFF images were analysed in ImageJ (https://imagej.net/Fuiji/Downloads) to record the total number and area (mm2) of cortical grey matter, white matter and hippocampus and deep grey GML area per section, per case. We defined the maximum extent of a Type III lesion for lesion counting as an area of complete demyelination that extended over a maximum of two entire sulci and gyri, as some Type III lesions extended across three or more gyri or involved the entire superficial cortical grey matter in a single hemisphere. Therefore, our Type III lesion count represents individual, small, Type III lesions, as well as large subpial lesions, subdivided and quantified as two or more separate lesions. In addition to measuring the area of cortical GMLs, the area and relative extent of cortex identified as de/re-myelinated cortical grey matter was recorded per section, per case.

    To produce illustrative “heat maps” that depict the burden of cortical GMLs in cases defined by lymphoid-like structure status (absence/presence), we first identified all cortical GMLs in section P1 per case and superimposed these lesions as “layer” images on a line-drawn representative whole coronal brain section (adapted from plate 34[22]) in GIMP. The final overlaid schema, comprising the “layer” masks of each case sampled at the P1 coronal level, revealed the absolute number of lesions by the relative depth of colour at that site.

    Determining leptomeningeal inflammation

    A measure of relative leptomeningeal immune cell infiltration per case was reported. Briefly, meningeal infiltrates were graded by assessing the extent of Nissl+ counter-stained cellular infiltrates of the intact cerebral leptomeninges, with the most notable infiltrate per case, rather than the average extent of infiltration, being reported. None to mild leptomeningeal inflammation was rated 0+ (0-5 cells per 100 × microscopic field of view; equivalent to 440-µm length of leptomeningeal tissue); diffuse and modest rated ++ (equivalent to an infiltrate of 5-50 loosely packed cells); or substantial infiltration rated +++ (based on > 50 cells in a tightly packed infiltrate[18]). Bona fide leptomeningeal lymphoid-like structures characterised by the presence of an anti-CD35+ reticular network, proliferating B cells (Ki67 antigen+) and immunoglobulin+ plasma cells[23] were previously reported in a subset of these cases[18] and the lymphoid-like structure status (presence or absence of detectable structures) is detailed in is detailed in the results section.

    Quantifying activated microglia/macrophages

    The density of CD68+ microglia/macrophages in cortical GML centres, normal appearing cortical grey matter or WML centre or normal appearing white matter was quantified from four 40 × images (Zeiss AxioImager Z1 and Axiocam Hrc camera) per region of interest captured from cingulate, pre-central gyrus (including pre- and post-central superior gyrus), insula cortex in parietal lobe and temporal lobe (medial) of each available P1 coronal macrosection, per case. In the absence of a demyelinating lesion, only data from the normal appearing tissue were reported for that region, per case. All data were averaged for lesion or normal appearing status across the sampled regions and reported as density of CD68+ cells/mm2, for comparison between groups.

    Statistical analysis

    Group means or medians were compared and plotted using GraphPad Prism 7. All data were assumed to be non-normally distributed given the small sample size[24]. The unpaired Kruskal-Wallis with Dunn’s multiple comparison post-test was used for three-group comparisons [i.e., when comparing the average area (mm2) of cortical and subcortical lesions per section, per case], whilst the Mann-Whitney U test was used when comparing two groups (i.e., per cent hippocampus and deep GML in cortical High vs. Low MS). Fischer’s exact test compared the actual vs. anticipated proportion of males/females and other clinical variables, such as seizures (yes/no) or lymphoid-like structure status (absent/present), between groups. Correlative analysis used Spearman’s method and Spearman r and P values were reported. A two-tailed P value of > 0.05 was considered significant.


    We conducted a study of whole coronal sections to better understand the burden of demyelination and inflammation in a cohort of 22 cases of secondary progressive MS (SPMS).

    Cortical grey matter is disproportionality demyelinated in progressive MS

    Cortical grey matter (GM), hippocampus and deep GM and WMLs were seen in all 22 cases analysed, with lesions noticeable across the coronal planes sampled [Figures 1 and 2A]. The area of demyelination, determined from the analysis of LFB and anti-PLP immuno-stained sections, varied considerably amongst the MS cohort [Figure 2B]. Quantification of the total area occupied by cortical GMLs, hippocampus and deep GML and WMLs revealed the total area of cortical GML (mean 589 mm2, range 25-2563 mm2) to be significantly greater than the total hippocampus and deep GML area (mean 81 mm2, range 0-382 mm2) and subcortical WML area (mean 203 mm2, range 2-617 mm2; Figure 2B). The relative area of cortical GML (per cent GML of total cortical GM) was almost twice that of the relative area of WML of total section WM per case (17.9% ± 2.6% compared with 8.9% ± 1.3% for cortical GML and subcortical WML, respectively, P = 0.023, non-parametric Mann-Whitney U test).

    Figure 2. Demyelinating lesions of the cortical grey matter, deep grey matter and hippocampus and white matter. Anti-PLP immunostaining of sections through the rostral-caudal extent of MS212, at coronal levels A3, P1 and P3 (A). Note the widespread demyelination of the cortical grey matter (red colour mask). Quantification of the area of demyelinated tissues revealed the total area of demyelinated cortical grey matter to exceed that of the total measured area of deep grey matter and white matter (B). Data points represent the sum of all demyelinated lesions, per region of interest per case with mean and SD depicted. Groups compared by Kruskal-Wallis and Dunn’s multiple comparison post-test. Cx: cortical; GML: grey matter lesion; WML: white matter lesion. Scale bar = 1 cm

    Type III cortical grey matter lesions are the most frequent and can affect the entire cortical ribbon in a whole coronal section

    We noted cases with little and others with expansive areas of cortical GM demyelination in our progressive MS cohort [Figure 3A]. We counted the number of separate lesions of the neocortex and white matter and showed that the subpial cortical GMLs (Type III and IV lesions; Figure 3B) were more numerous than any other cortical grey matter lesion type or white matter lesion (Figure 3C, P < 0.0001). In total, 1884 lesions were identified, of which 1363 (72.3%) were subpial (Type III or IV) cortical GMLs.

    Figure 3. Subpial cortical grey matter lesions are the most frequent and occupy the greatest area. Anti-PLP immunostaining of section A3 of MS253, MS224 and MS257 revealed subpial (Type III/IV cortical lesions, red mask), intracortical (Type II cortical lesion, orange mask) and leukocortical (Type I cortical lesion, yellow mask) lesions. Lesions of the deep grey matter (green) and lesions of white matter (blue mask) are also shown (A). Subpial (Type III/IV) lesions were predominant and often spanned multiple gyri (B). Of 1884 separate lesions, 72.3% were subpial cortical grey matter lesions (C). By plotting the average area of each lesion type (calculated per section) and represented per case, we show the heterogeneity of lesion size across our cohort (D), and how subpial cortical lesions contribute a large component of the lesion burden, especially in those cases most affected (from MS366 to MS257, for example). Ave: average; Cx: cortical; GML: grey matter lesion; PLP: proteolipid protein immunohistochemistry; WML: white matter lesion. (A) Scale bar = 1 cm; (B) Scale bar = 1 mm

    Subpial cortical GMLs accounted for the largest area of all lesion types measured. We plotted the measured area of each lesion type, with the bar height representing the total average area (mm2) of demyelination per section, per case, to show the substantial contribution subpial GMLs made to the total lesion area [Figure 3D]. Arranging cases according to the total measured area of demyelination [from left (highest), to right (lowest)] highlighted that subpial GMLs overwhelmingly accounted for the different total lesion areas between cases with relatively high or low total demyelinated lesion area. In cases with relatively lower lesion area (cases MS361-MS295), the area of subpial GML as a per cent of total measured lesion area was far less than in cases with the higher total lesion area (34.5%, range 8.0%-60.1% vs. 74.3%, range 54.3%-94.9%; P < 0.0001, non-parametric Mann-Whitney U test). We compared the relative extent of demyelination in cortical and subcortical tissues and showed that the relative area of total cortical GML was not statistically associated with the relative area of WML (r = 0.135, P = 0.549; Table 3). The area of subpial cortical GML was significantly associated with the extent of hippocampus and deep GML area (Spearman r = 0.458, P = 0.036), whilst leukocortical/intracortical GML area was significantly associated with WML area (Spearman r = 0.771, P < 0.0001; Table 3).

    Table 3

    Correlating cortical, white matter and hippocampus and deep grey matter lesions

    % Cx GML III/IV% Hippo and deep GML% WML% Total Cx GML
    % Cx GML I/II  r = -0.005
      P = 0.982
            r = 0.224
            P = 0.328
    r = 0.771
    P < 0.0001
          r = 0.051
          P = 0.827
    % Cx GML III/IV        r = 0.458
            P = 0.037
    r = 0.092
    P = 0.684
          r = 0.988
          P < 0.0001
    % Hippo and deep GMLr = 0.169
    P = 0.464
          r = 0.495
          P = 0.023
    % WML      r = 0.135
          P = 0.549

    Pathological correlates of a high subpial cortical grey matter lesion load

    To understand the clinical and pathological associations that might co-exist with subpial cortical GM demyelination, we subdivided the cohort based on relative extent of cortical GML area per section, per case [Table 4 and Figure 4]. Cases with 15% or greater relative cortical GML of total cortical GM (n = 9 cases in total; Figure 4A, which represent cases MS366-MS257 in graph Figure 3D) were designated cortical GML High MS (mean cortical GML area 35.6%; range 15.6%-60.2%), whilst the others (n = 13 cases, cortical GML less than 12% total cortical GM area) were designated as cortical GML Low MS (mean cortical GML area 5.5%; range 0.9%-12.0%).

    Table 4

    Progressive MS cases defined by relative cortical grey matter lesion load

    CaseSex2yr RROnsetProgW’chairDiedDurationPm delayBrain weight (g)Seizures (Y/N)LLSMenin inflam (0-3)
    Cortical GM High MS
      MS202F     2  35  46    47  58      23    39        1200      NY3
      MS212F     1  18  33    35  47      29    66        1314      NY3
      MS214F     2  20  31    38  51      31    20        1140      n/dN2
      MS217F     1  42  49    50  57      15    29        1200      NY3
      MS257F     6  27  31    39  49      22    28        1168      YY3
      MS278M     1  9  23    23  30      21    60        1402      YY3
      MS323F     2  31  38    44  62      31    13        1207      YN1
      MS360M     1  15  40    51  55      40    18        1063      YY3
      MS366F     3  42  45    53  61      19    14        1090      NY3
      n = 97F:2M     2  27  38    44  55      23    31.9        1198.2      4Y:4N7/93
    Cortical GM Low MS
      MS204M     3  39  44    48  58      19    35        1180      NN1
      MS223F     4  43  43    45  45      2    72        1004      YN1
      MS224F     1  26  35    36  59      33    27        1100      NN1
      MS226F     5  37  51    58  64      27    27        1300      NN0
      MS253F     3  21  23    33  37      16    24        1259      NN2
      MS258M     2  26  35    38  46      20    44        1460      NN1
      MS293F     2  35  42    43  53      18    44        1250      YY3
      MS295F     5  56  60    62  71      15    42        1166      NN0
      MS336F     1  30  47    51  57      27    24        1226      NY3
      MS344F     6  42  43    47  57      15    14        1062      NN1
      MS361F     2  26  38    42  60      34    10        956      YN1
      MS387F     6  32  35    36  43      11    13        1115      NN2
      MS395M     1  37  45    52  63      26    4        958      NN1
      n = 1310F:3M     3  35  43    45  57      19    24.3        1161.3      3Y:10N2/131

    Figure 4. Defining a cohort of cortical High grey matter lesion MS. Nine progressive MS cases with the greatest relative area of subpial cortical demyelination were labelled as Cortical High GML MS and the remaining cases as Cortical Low GML MS (A). Cortical High GML MS had a greater area of demyelinated deep grey matter and hippocampus (B), but unchanged white matter lesion areas (C). The density of CD68+ microglia/macrophages was increased in both normal appearing GM (NGM) and GML of cortical High GML MS (D) but was only different in the underlying normal appearing WM (NWM) (E). Semi-quantitative evaluation of meningeal inflammation revealed cortical High GML MS to have increased inflammation of the leptomeninges (F). Examples of CD68+ microglia of a subpial GM lesion (G) and CD68+ microglia/macrophages in a white matter lesion (H). Examples of 1-3 rated inflammatory infiltrates (“+” to “+++”) of the forebrain meninges [I-N with (L-N) being higher power images of (I-K), respectively]. Please note that infiltrates (I, L, K, N) are near partially de/re-myelinated cortex of cortical High GML Case MS257. Cx: cortical; GML: grey matter lesion; NGM: normal appearing grey matter; NWM: normal appearing white matter; PLP: proteolipid protein immunohistochemistry; WML: white matter lesion. (G-K) Scale bar = 100µm; (L-N) Scale bar = 25 µm

    The relative area of hippocampus and deep GML was greater in cortical GML High MS [Figure 4B], whilst the relative area of WML was not different [Figure 4C]. Cortical GML High MS cases had a greater density of CD68+ microglia/macrophages in both GML centre and normal appearing GM regions, in comparison to cortical GML Low MS [Figure 4D and G], whilst the density of CD68+ microglia/macrophages was only increased in normal appearing WM of cortical GML High cases [Figure 4E and H]. Inflammation of the forebrain leptomeninges is associated topographically with subpial cortical demyelination and cases with semi-organised lymphoid-like structures are characterised by a more extensive cortical GML load[13]. Our designated cortical GML High MS cohort presented with a substantially increased median rating of leptomeningeal immune cell infiltrates (P = 0.003, Figure 4F), with seven of nine cases containing at least one bona fide lymphoid-like structure [Table 4]. Examples of leptomeningeal infiltrates rated as mild [+; Figure 4I and L], modest [++, Figure 4J and M] and substantial [+++, Figure 4K and N] are provided in Figure 4. Immune cell aggregates were associated both with areas of partially de/re-myelinated subpial cortical GM as well as areas of subpial GM demyelination [Figure 4].

    Areas of cortical GM defined as partially de/re-myelinated GM ranged from < 1% (0.9%, MS223) to 13.5% (MS257) of total cortical GM area per case but were not different between the cortical GML High and Low groups (area of partially de/re-myelinated GM = 8.01% vs. 6.13%, for cortical High vs. Low GML cases, respectively, P = 0.387). Rudimentary measures of gross brain pathology, such as brain weight at death and the measured area of grey and white matter per P1 section (as an indication of regional grey or white matter tissue atrophy; data not shown), did not reveal a difference between the cortical GML High and Low MS cases [Table 4].

    Clinical correlations of a high subpial cortical grey matter lesion load

    There was no difference in the proportion of males to females or of any reported clinical measure (such as the number of relapses in the first two years, the report of seizures or age of death) between cortical GML High and Low groups [Table 4]. There was no difference between groups with regards age of disease onset (P = 0.11), confirmed age at onset of progressive phase (P = 0.27) or disease duration (P = 0.15). Post-mortem delay did not differ between the groups (P = 0.75; Table 4).

    Leptomeningeal inflammation, lymphoid-like structures and cortical demyelination

    We identified four separate “+++” rated foci of substantial leptomeningeal infiltrates in a single P1 section of Case MS217, which displayed massive cortical, and exclusively subpial, GM demyelination (59.6% of cortical GM defined as GML; Figure 5A and B). Progressive MS cases with moderate-to-high leptomeningeal inflammation (rated ++/+++ or +++/+++ for immune cell infiltrates) had an increased relative cortical GML area compared with those cases with little-to-mild leptomeningeal infiltrates 7.8% (range 0.8%-27.7%) vs. 26.2% (range 0.8%-60.2%), P = 0.012, Mann-Whitney U test). The relative area of hippocampus and deep GML area (7.4%, range 0%-19.1% vs. 16.4% range 1.4%-69.2%, P = 0.159) and WML lesion area (10.1%, range 0.6%-42.1% vs. 10.0%, range 2.3%-26.5%, P = 0.981) was unchanged between leptomeningeal rated moderate-high cases vs. those rated with little-to-mild infiltrates, respectively. The relationship between the extent of leptomeningeal inflammation and cortical GML area is presented schematically as a heat map of all cortical GMLs observed (in olive green colour) in section P1 of cases with mild (rated 0 to +, n = 9) vs. modest/substantial infiltrates (++ to +++, n = 9; Figure 5C). Please note that four cases did not have P1 sections available for analysis [Table 1].

    Figure 5. Meningeal inflammation and subpial cortical demyelination. Example infiltrates of the leptomeninges overlying the neocortex in a single coronal macrosection. Aggregates visualised by LFB/haematoxylin histology were rich in CD20+ B cells (A). The location of the infiltrates in (A) are marked as asterisks (*) on the serial anti-PLP stained section (B) (Case MS217). By grouping the cases based on their relative extent of leptomeningeal inflammation (0/+ vs. ++/+++), we constructed schema depicting all neocortical grey matter lesions noted per P1 section, per case (n = 18). Depth of colour (see heatmap representing percent lesion occurrence with 100% indicating tissue was demyelinated in every P1 section in that group) illustrates how frequently an area of neocortex was demyelinated in meningeal inflammation rated 0/+ and meningeal inflammation rated ++/+++ MS (C). PLP: proteolipid protein. (A) Scale bar = 200 µm; (B) Scale bar = 1 cm


    Our quantitative histological analysis of whole coronal macrosections demonstrates cortical grey matter demyelination can be the preeminent pathology in some cases of progressive MS and can be used to identify a post-mortem cohort with a more severe pathological disease. Subpial cortical grey matter lesions associated with the extent of demyelinated deep grey matter and with infiltrates of the leptomeninges but importantly did not associate with white matter lesion load. Our study reinforces the concept of a CSF-derived (outside-in) driver of tissue pathology in SPMS.

    Substantial subpial cortical grey matter demyelination defines a subset of progressive MS cases

    Quantitative image analysis of whole coronal macrosections revealed the proportion of demyelinated cortical grey matter to be almost twice that of the proportion of demyelinated white matter. In those cases, with the greatest cortical pathology, labelled as cortical GML High MS, there was a near four-fold increase in relative area of cortical GML compared to WML. These data are in accordance with the work of Carassiti et al.[25] and are supportive of an MS post-mortem cohort characterised by a dominant neocortical demyelinating pathology[26]. Amongst our samples, we observed instances of hugely divergent grey over white matter pathology (for example, Case MS214 WML accounted for 3.2% of total WM, whilst cortical GML area was 43.4% of total cortex; Case MS217 WML area was 3.1% and cortical GML was 59.6%). Cortical GML pathology was overwhelmingly subpial in location, with an average of 90.9% of total cortical GML being characterised as subpial (Type III/IV) in distribution. Such extensive and disproportionate demyelination exceeds that previously reported by ourselves and others and highlights the benefit of working with whole coronal macrosection preparations[2,12,18] to better understand MS pathology.

    We have previously noted associations between lymphoid-like structure status and clinical disease measures, such as age of onset, age at progression and age at death[13,18]. Our cortical GML High and Low MS cases had a similar age of onset to that seen in our earlier studies (median age at onset of 27 and 35 years, respectively) but this did not represent a significant difference in our relatively small study. No other clinical-pathological correlation was noted, which in part reflects the complex and highly variable pathology of this disease, whereby synaptic, neuritic and neuronal loss, alongside demyelination and gliosis, variable impact on disease outcome.

    Demyelinating pathology at subpial and subependymal territories

    The correlation between the extent of demyelination of the hippocampus and deep grey (including the caudate, putamen, pallidum and thalamus) with that of subpial cortical GML suggests, at least in part, a shared pathological mechanism of lesion formation and/or expansion between these regions lining superficial surfaces of the pia and ventricles. Subpial demyelinating lesions are a pathological hallmark of MS[27] and clinical imaging shows that the thalamus of children with MS, a structure which is severely affected in progressive MS[28], displays a specific imaging abnormality as a surface-in gradient pattern from the ventricular margin[29]. Adult MS patients also display a gradient of magnetisation transfer ratio signal change from the superficial surfaces of the brain: a gradient of signal change, declining with distance across the white matter[30,31] when measured from the ventricular surface, and across the neocortex from the pial surface, which are most altered in progressive MS[32,33]. A magnetic resonance imaging (MRI) signature of gadolinium leptomeningeal enhancement, which may partly reflect inflammation of meninges[34], relates to the number and volume of cortical and thalamic lesions[35]. These imaging studies support the concepts of meningeal inflammation and outside-in CSF factor(s) in lesion genesis. Numerous post-mortem studies have demonstrated a topographical association between the extent of immune cell infiltration of the leptomeninges with cortical GM demyelination[13,23,36-40], which is also true for subpial tissue of the cerebellar cortex and spinal cord[41-44]. This subpial demyelinating pathology of the neocortex is associated with microglia activation, a gradient of tissue injury and disruption of the pial glial limiting membrane[16,45]. Therefore, the pattern of neocortical lesion location and deep GM pathology is suggestive, at least in part, of an effect of soluble cytotoxic mediators from the overlying CSF-filled spaces that contributes to the underlying disease process.

    Soluble mediators of subpial lesions

    Recently, we have demonstrated the overexpression of RNA transcripts associated with pleiotropic chemokines and cytokines in the isolated meningeal tissue from cases characterised by extensive leptomeningeal inflammation and subpial demyelination[46,47]. The profile of elevated mediators, many 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 signature of substantial cortical pathology[47]. 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 subclinical autoimmune encephalitis[46]. 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 making[48].

    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, with a gradient of lessening neuronal damage with distance from the pia[16]. 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 are enriched in MS patient CSF and can mediate neuroaxonal pathology and mitochondrial dysfunction[65], whilst simvastatin, a cholesterol-reducing therapy that enters the CNS, is associated with a slowing of brain atrophy in SPMS[67]. 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[53], and 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 field MRI detects only a fraction of all subpial lesions[68]. 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 (β = 0.8) predicted a cohort of at least 40 cases would be required (α = 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 interest, meaning that we could not, for example, report lesion measures for separate deep GM structures. Nevertheless, the use of whole coronal sections reduces observer variation between individual cases, increases the accuracy of observations, and ensures comparisons between different forebrain areas, which are invaluable for the study of this heterogeneous disease.

    In conclusion, our quantitative histological analysis revealed global grey matter demyelination and meningeal inflammation to be substantial in a subset of progressive MS brains. Cases defined by a substantial cortical GML load displayed greater microglia/macrophage activation, larger areas of deep grey matter pathology but little change in white matter lesion area, which highlights the partially separate pathogenetic mechanisms of lesion evolution (or susceptibility to damage) in these compartments. The distribution of subcortical and deep GML in MS is consistent with the presence of soluble proinflammatory factors in the subarachnoid space and ventricular CSF and furthers the need to identify companion biomarkers of cortical pathology to aid patient monitoring and therapeutic choice.


    Authors’ contributions

    Made substantial contributions to conception and design of the study: Griffiths L, Reynolds R, Howell OW

    Performed data analysis, interpretation and contributed to writing the manuscript and approved the final submitted document: Griffiths L, Reynolds R, Evans R, Bevan RJ, Rees MI, Gveric D, Neal JW, Howell OW

    Availability of data and materials

    Data generated during the current study is available on reasonable request.

    Financial support and sponsorship

    The UK MS Society Tissue Bank (www.ukmstissuebank.imperial.ac.uk) supplied the post-mortem samples used in this study and is supported by the Multiple Sclerosis Society of Great Britain and Northern Ireland. The authors would like to thank members of the UK MS Tissue Bank for their invaluable assistance. This work was supported by the St. Davids Medical Foundation, the British Neuropathological Society and the Medical Research Council (G0700356). Reynolds R has received speaking honoraria from Roche, Novartis and ECTRIMS and grant funding from MedImmune plc. Howell OW has received travel reimbursement or speaking honoria (paid to Swansea University) from Roche, the Neurology Academy and ECTRIMS. All other authors have no relevant disclosures.

    Conflicts of interest

    All authors declared that there are no conflicts of interest.

    Ethical approval and consent to participate

    The use of human post mortem tissue is covered by UK Research Ethics committee approval (study approval number 08/MRE09/31+5).

    Consent for publication

    Not applicable.


    © The Author(s) 2020.


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    Cite This Article

    Griffiths L, Reynolds R, Evans R, Bevan RJ, Rees MI, Gveric D, Neal JW, Howell OW. Substantial subpial cortical demyelination in progressive multiple sclerosis: have we underestimated the extent of cortical pathology?. Neuroimmunol Neuroinflammation 2020;7:51-67 . http://dx.doi.org/10.20517/2347-8659.2019.21



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      15 Nov 2021 08:14


    • Alarna Ullah   
      Herpes simplex virus is said to have no cure, just as i was told by my family doctor  After i got Infected with the virus March 4th, 2019, that herpes can not be cured but can be controlled, Although i was a very stubborn person i never believed in what my doctor told me about herpes having no cure. Am a very honest person who believe in nature and i also believed that there could be cure somewhere in the world, I kept my faith so strong and kept on doing my research till i saw an article shared by a testifier on How a herbal Doctor Razor from west Africa, I Decided to use his herbal medicine, so i Reached out to him and explained my situation to him, he promised to guide me, after 4 days of reaching out to him i received my medication sent to me by Doctor Razor and he instructed me to use it for 18 days, After Completion of his herbal medicine dosage, i went for a medical checkup as i was told by Doctor Razor. I am so happy and extremely excited that I got cured of this Virus. Massive gratitude to doctor Razor. Reach out to him via his clinic email : herbalistrazorherbalhome@gmail.com  On whatsapp/call his cell phone +2349065420442. Together we stand a chance to fight herpes. Let's continue to share this till The whole world Gets to know that there is a herbal cure for herpes.God bless you all. Doctor Razor's Website : https://herbalistrazorherb.wixsite.com/drrazorherbalhome
      Facebook Page : @HerbalistrazorMedicinalcure

      11 Nov 2021 21:13


    • patrice Arnaud   
      Many people with MULTIPLE SCLEROSIS, myself included, have found success with WORLDHERBSCLINIC MS HERBAL FORMULA. And when you need great HERBAL FORMULA side effect free recipes, WORLDHERBSCLINIC has the best!

      29 Sep 2021 08:36


    • Patiala legitimate   
      Do you need a quick loan?
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      21 Sep 2021 17:56


    • Nicole Liam   
      I NEVER BELIEVED IN HERBS BUT DOCTOR RAZOR MADE ME A BELIEVER. Massive Gratitude to doctor Razor who got my Genital Herpes cured a month ago. I got diagnosed with herpes on Feb 13, 2017.  I have been reading so many comments of some people who were cured from various diseases by Doctor Razor, but I never believed them. I was hurt and depressed, I was too curious and wanted to try Doctor Razor, then I contacted him through his email (drrazorherbalhome@gmail.com) when I contacted him, he assured me 100% that he will heal me, I pleaded with him to help me out. I also got His Website : https://herbalistrazorherb.wixsite.com/drrazorherbalhome.  My treatment was a great success, he healed me just as he promised. He sent me his medication and asked me to go for check up after 18 days of taking his herbal medicine. I agreed with him and I took his medication and went for check up, to my greatest surprise my result came out negative after the treatment, I'm really delighted that i'm completely cured and healthy again. I waited for a month to be very sure I was completely healed before writing this testimony. I did another blood test one week ago and it was still Herpes negative. so i guess it's time i recommend anyone going through Herpes HSV-1 or HSV-2, HIV, HPV, Hepatitis B, Diabetes, Cancer should reach him. EMAIL: drrazorherbalhome@gmail.com. WHATSAPP: +2349065420442  My greatest joy is having back my happiness  

      10 Sep 2021 10:00


    • mathias gibbs   
      I don't know where to start from because i am overwhelmed. I never thought I will be healed of HIV virus and Genital warts. These Diseases have affected my life in many way and I have lost everything, including my partner and my job in the course of finding solutions to these illnesses. I wish to share to those living with HIV virus and genital warts (HPV), and those suffering from various diseases not to give up, because i never knew i will one day be healed and fine. My faith got back after reading from those who got healed Dr O.Water with his herbal medicines. I hurriedly contacted this great Doctor on drwaterhivcurecentre@gmail.com. He gave me hope that i will be totally healed after the treatment, so i did all he requested and he sent me his herbal medicines with the instructions on how to use it. To my surprise, in the third week, I was noticing great changes in my body system and the warts totally disappeared, though i still did not believe until i was confirmed both HIV and Genital warts absolutely Negative. Please if you are living with Herpes,Cancer, Diabetes, Hepatitis or any disease it is my advise you write Dr O.Water  for his medicines. He is actually a wonderful man and Godsent, he is kind and very straight forward. His Email is  DRWATERHIVCURECENTRE@GMAIL.COM. His Whatsapp is +2349050205019 

      30 Aug 2021 08:19


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