New oligodendrocytes exhibit more abundant and accurate myelin regeneration than those that survive demyelination

Abstract

Oligodendrocytes that survive demyelination can remyelinate, including in multiple sclerosis (MS), but how they do so is unclear. In this study, using zebrafish, we found that surviving oligodendrocytes make few new sheaths and frequently mistarget new myelin to neuronal cell bodies, a pathology we also found in MS. In contrast, oligodendrocytes generated after demyelination make abundant and correctly targeted sheaths, indicating that they likely also have a better regenerative potential in MS.

Extended Data Figure 1. Characterisation of the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A) Schematic illustrating the Tg(mbp:TRPV1-tagRFPt) demyelination model made using Biorender. The rat ortholog of the TRPV1 channel is expressed in myelinating oligodendrocytes and is activated by addition of csn which drives cation influx. Zebrafish TRPV1 channels are insensitive to csn, therefore csn treatment specifically results in damage to myelinating glia which express the rat ortholog of the TRPV1 channel. (B) Confocal images of myelinating oligodendrocytes in the Tg(mbp:EGFP; mbp:TRPV1- tagRFPt) zebrafish line at 4dpf showing oligodendrocytes co-expressing EGFP and tagRFPt in the merged image. Scale bar, 20 µm. (C) Brightfield images of a zebrafish containing the Tg(mbp:TRPV1-tagRFPt) transgene (TRPV1+ve), or wildtype siblings without the Tg(mbp:TRPV1-tagRFPt) transgene (TRPV1-ve) which show no developmental differences at 4dpf. Scale bars, 500µm. (D and E) Confocal images of the (D) Tg(mbp:EGFP-CAAX) line and (E) the Tg(mbp:EGFP) line pre-treatment (indicated here as pre-t) at 4dpf, and 3hpt. Zebrafish not containing the Tg(mbp:TRPV1-tagRFPt) transgene show no disruption to myelin or oligodendrocytes following a 2 hour treatment of 10µM csn. Scale bars, 20µm. (F) Confocal images of myelin visualised in Tg(mbp:EGFP-CAAX; mbp:TRPV1-tagRFPt) zebrafish, with control (DMSO) and csn treated animals in top and bottom panels respectively pre-treatment (pre-t) at 4dpf, 3hpt, 1dpt and 3dpt. Scale bar, 20µm.

Extended Data Figure 2. Csn treatment induces severe demyelination in the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A) Transmission electron microscopy images of DMSO and csn treated Tg(mbp:TRPV1-tagRFPt) zebrafish at 1dpt show numerous large calibre myelinated axons (blue) in DMSO treated animals and numerous large calibre unmyelinated axons (yellow) in csn treated animals. Scale bar, 1µm. (B) Quantification of the number of myelinated axons>0.4µm diameter in the dorsal spinal cord at 1dpt in DMSO (median=20.00, IQR=16.50-22.00) versus csn (median = 0.00, IQR=0.00-1.50) treated zebrafish, p<0.0001. Unpaired two-tailed t-test with Welch’s correction. N=5 zebrafish per condition. Data are presented as median with IQR. (C) Quantification of the number of unmyelinated axons>0.4µm diameter in the dorsal spinal cord at 1dpt in DMSO (mean=10.00±5.24SD) versus csn (mean=27.40±9.61SD) treated zebrafish, p=0.0075. Unpaired two-tailed t-test. N=5 zebrafish per condition. Data are presented as mean±SEM. (D) Quantification of the number of axons>0.4µm diameter in the dorsal spinal cord at 1dpt in DMSO (mean=29.40±8.26SD) versus csn (mean=28.00±9.95SD) treated zebrafish, p=0.8148. Unpaired two-tailed t-test. N=5 zebrafish per condition. Data are presented as mean±SEM. (E) Quantification of the number of axons with>3 myelin wraps in the dorsal spinal cord at 1dpt in DMSO (median=20.00, IQR=15.00-20.00) versus csn (median=0.00,IQR=0.00-1.50) treated zebrafish, p=0.0079. Two-tailed Mann-Whitney t-test. N=5 zebrafish per condition. Data are presented as median with IQR (F) Quantification of the number of axons with≤3 myelin wraps in the dorsal spinal cord at 1dpt in DMSO (median=1.00, IQR=0.00-3.00) versus csn (median=0.00, IQR=0.00-0.00) treated zebrafish, p=0.1667. Two-tailed Kolmogorov-Smirnov test. N=5 zebrafish per condition. Data is presented as median with IQR.(G) Transmission electron microscopy images of DMSO and csn treated Tg(mbp:TRPV1-tagRFPt) zebrafish at 3dpt show numerous large calibre myelinated axons (>3 myelin wraps highlighted in blue, ≤3 myelin wraps highlighted in orange) and unmyelinated axons (highlighted in yellow) in DMSO and csn treated animals. Scale bar, 1µm. (H) Quantification of the number of myelinated axons >0.4µm diameter in the dorsal spinal cord at 3dpt in DMSO (mean=24.20±9.83SD) versus csn (mean=20.83±5.78SD) treated zebrafish, p=0.4963. Unpaired two-tailed t-test. N=5 DMSO treated zebrafish, N=6 csn treated zebrafish. Data are presented as mean±SEM.(I) Quantification of the number of unmyelinated axons>0.4µm diameter in the dorsal spinal cord at 3dpt in DMSO (mean=23.20±6.76SD) versus csn (mean=30.40±4.39SD) treated zebrafish, p=0.0809. Unpaired two-tailed t-test. N=5 DMSO treated zebrafish, N=6 csn treated zebrafish. Data are presented as mean±SEM.(J) Quantification of the number of axons>0.4µm diameter in the dorsal spinal cord at 3dpt in DMSO (mean=47.60±10.92SD) versus csn (mean=51.00±6.45SD) treated zebrafish, p=0.5359. Unpaired two-tailed t-test. N=5 DMSO treated zebrafish, N=6 csn treated zebrafish. (K) Quantification of the number of axons with>3 myelin wraps in the dorsal spinal cord at 3dpt in DMSO (median=16.00, IQR=15.50-27.00) versus csn (median=8.50, IQR=3.75-11.50) treated zebrafish, p=0.0152. Two-tailed Mann-Whitney t-test. N=5 DMSO treated zebrafish, N=6 csn treated zebrafish. (L) Quantification of the number of axons with≤3 myelin wraps in the dorsal spinal cord at 3dpt in DMSO (mean=4.00±3.24SD) versus csn (mean=12.50±5.01SD) treated zebrafish, p=0.0099. Unpaired two-tailed t-test. N=5 DMSO treated zebrafish, N=6 csn treated zebrafish.

Extended Data Figure 3. Csn treatment induces minimal oligodendrocyte loss in the Tg(mbp:TRPV1-tagRFPt) model.

(A) Confocal images of myelinating oligodendrocytes visualised in Tg(mbp:EGFP; mbp:TRPV1-tagRFPt) zebrafish, with control (DMSO) and csn treated animals in top and bottom panels respectively pre-treatment (pre-t) at 4dpf, 3hpt, 1dpt and 3dpt. Scale bar, 20 µm. (B-F) Quantification of myelinating oligodendrocyte number in DMSO and csn treated Tg(mbp:EGFP; mbp:TRPV1-tagRFPt) zebrafish over time. (B) Pre-treatment DMSO (mean=58.60±13.23SD) versus csn (mean=57.20±7.64SD), p=0.7753. Data are presented as mean±SEM. (C) 3hpt DMSO (mean=65.00±14.93SD) versus csn (mean=44.11±10.36SD), p=0.0041. Data are presented as mean±SEM. (D) 1dpt DMSO (mean=76.90±14.14SD) versus csn (mean=61.70±7.39SD), p=0.0075. Data are presented as mean±SEM. (E) 3dpt DMSO (mean=99.00±15.93SD) versus csn (mean=85.70±11.83SD), p=0.0444. Data are presented as mean±SEM. (F) 5dpt DMSO (mean=104.5±12.95SD) versus csn (mean=108.5±15.10SD), p=0.5328. (B-F) Unpaired two-tailed t-tests. Pre-treatment N=10 zebrafish per treatment group, 3hpt N=8 (DMSO) and 9 (csn) treated zebrafish, 1dpt N=10zebrafish per treatment group, 3dpt N=11 zebrafish (DMSO) and 10 zebrafish (DMSO), 5 dpt N=10 zebrafish per treatment group. Data are presented as mean±SEM. Each data point represents total (dorsal + ventral) oligodendrocyte number analysed per imaged area per zebrafish.

Extended Data Figure 4. Characterisation of single oligodendrocyte loss and myelin debris phagocytosis following demyelination in the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A) Confocal images of a single oligodendrocyte labelled with mbp:EGFP-CAAX in the Tg(mbp:TRPV1-tagRFPt) line pre-treatment (pre-t) at 4dpf, and 3hpt. An example of oligodendrocyte cell death is demonstrated here by the disappearance of a tagRFPt+ve cell body following csn treatment in the same zebrafish before and after demyelination, whilst 2 tagRFPt+ve oligodendrocytes which survive demyelination are seen neighbouring it at 3hpt. Arrows indicate the location of the oligodendrocyte cell body which undergoes cell death, or where it was prior to demyelination. Scale bar, 20µm. (B) Confocal images of microglia / macrophage engulfment of myelin debris following demyelination at 1dpt. Arrowheads highlight the location of myelin debris engulfment. Scale bar, 20µm. (C) Quantification of the number of mpeg+ve cells (macrophages / microglia) in a 4-somite section of the spinal cord at pre-treatment (mean=1.38±0.74SD), 1dpt (mean=2.14±1.07SD), 2dpt (mean=3.14±0.38SD), 3dpt (mean=2.57±1.90SD) and 4dpt (mean=3.00±1.29SD) (where treatment was a DMSO control). Pre-t vs 1dpt p=0.7196, pre-t vs 2dpt p=0.0505, pre-t vs 3dpt p=0.3106, pre-t vs 4dpt p=0.0844, 1dpt vs 2dpt p=0.5190, 1dpt vs 3dpt p=0.9597, 1dpt vs 4dpt p=0.6588, 2dpt vs 3dpt p=0.8930, 2dpt vs 4dpt p=0.9994, 3dpt vs 4dpt p=0.9597. Ordinary one-way ANOVA with Tukey’s multiple comparison test. Pre-treatment N=8 zebrafish , 1–4dpt N=7 zebrafish. Data are presented as mean±SEM. (D) Quantification of the number of mpeg+ve cells (macrophages / microglia) in a 4-somite section of the spinal cord at pre-treatment (mean=1.56±1.01SD), 1dpt (mean=7.88±2.75SD), 2dpt (mean=10.38±3.42SD), 3dpt (mean=9.12±2.48SD) and 4dpt (mean=6.63±1.92SD) (where treatment was a demyelinating csn treatment). Pre-t vs 1dpt p<0.0001, pre-t vs 2dpt p<0.0001, pre-t vs 3dpt p<0.0001, pre-t vs 4dpt p=0.0011, 1dpt vs 2dpt p=0.2586, 1dpt vs 3dpt p=0.8394, 1dpt vs 4dpt p=0.8394, 2dpt vs 3dpt p=0.8394, 2dpt vs 4dpt p=0.0294, 3dpt vs 4dpt p=0.2586. Ordinary one-way ANOVA with Tukey’s multiple comparison test. Pre-treatment N=9 zebrafish, 1–4dpt N=8 zebrafish. Data are presented as mean±SEM.

Extended Data Figure 5. ROCK inhibitor treatment further increases myelin mistargeting by surviving oligodendrocytes in the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A) Confocal images of single oligodendrocytes which have not undergone demyelination treated with DMSO (control) or Y27632 ROCK inhibitor and imaged at 4dpf. Scale bar, 20µm. (B) Confocal images of single surviving oligodendrocytes followed over time from prior to demyelination (csn treatment) at 4dpf through to 3dpt. Following demyelination oligodendrocytes were treated with either DMSO (control) or Y27632 ROCK inhibitor. Scale bars, 20µm. (C) Quantification of the number of sheaths produced in oligodendrocytes which have not been demyelinated in control (mean=16.08±5.73SD) and Y27632 ROCK inhibitor (mean=19.35±4.08SD) treated zebrafish, p=0.0386. Unpaired two-tailed t-test. N=24 oligodendrocytes from 24 zebrafish (control), N=20 oligodendrocytes from 20 zebrafish (Y27632). Data are presented as mean±SEM. (D) Quantification of the average sheath length (µm) produced in oligodendrocytes which have not been demyelinated in control (mean=28.62±7.18SD) and Y27632 ROCK inhibitor (mean=27.26±6.91SD) treated zebrafish, p=0.5308. Unpaired two-tailed t-test. N=24 oligodendrocytes from 24 zebrafish (control), N=20 oligodendrocytes from 20 zebrafish (Y27632). Data are presented as mean±SEM.(E) Quantification of the number of mistargeted myelin profiles produced in oligodendrocytes which have not been demyelinated in control (median=0.00, IQR=0.00-0.00) and Y27632 ROCK inhibitor (median=0.00, IQR=0.00-0.00) treated zebrafish, p>0.9999. Two-tailed Mann-Whitney test. N=24 oligodendrocytes from 24 zebrafish (control), N=20 oligodendrocytes from 20 zebrafish (Y27632). Data are presented as median with IQR.(F) Quantification of the number of sheaths produced per oligodendrocyte following demyelination in control (median = 3.00, 25th percentile 1.50, 75th percentile 3.00) and Y27632 ROCK inhibitor (median=4.00, IQR=1.00-4.50) treated zebrafish, p=0.5964. Two-tailed Mann-Whitney t-test. N=5 oligodendrocytes from 5 zebrafish (control), N=9 oligodendrocytes from 9 zebrafish (Y27632). Data are presented as median with IQR. (G) Quantification of the number of mistargeted myelin profiles produced per oligodendrocyte following demyelination in control (median=1.00, IQR=1.00-2.00) and Y27632 ROCK inhibitor (median=3.00, IQR=1.50-4.75) treated zebrafish, p=0.0414. Unpaired two tailed t-test with Welch’s correction . N=5 oligodendrocytes from 5 zebrafish (control), N=9 oligodendrocytes from 9 zebrafish (Y27632). Data are presented as mean±SEM.

Extended Data Figure 6. Mistargeted myelin profiles are present in remyelinating lesions in motor cortex tissue from people with MS.

(A) Low magnification image of chromogenic immunohistochemistry for proteolipid protein (PLP - brown) and NeuN (blue) in human MS motor cortex. Outline of quantified areas shown with lesion area highlighted in red, perilesion area highlighted in green, normal appearing grey matter (NAGM) in purple and white matter indicated by ‘WM’ in white. Images 1 and 2 show examples of quantified areas in 2 different human MS motor cortex samples. Scale bars, 2000µm. (B and C) High magnification images of chromogenic immunohistochemistry for proteolipid protein (PLP - brown) and NeuN (blue) in human MS motor cortex. (B) Images 1-9 show example images of PLP+ve wrapped NeuN+ve cells (myelinated neuronal cell bodies). Images 1 and 3-9 scale bars, 20µm. Image 2 scale bar, 10µm. (C) Images 1-5 show example images of PLP+ve wrapped NeuN-ve cells (oligodendrocytes). Scale bars, 20µm. (D) Fluorescent immunohistochemistry for NeuN (white), PLP (red) and Hoechst (nuclei-blue) in human MS motor cortex. Arrows indicate the location of PLP+ve wrapped NeuN+veHoechst+ve cells (myelinated neuronal cell body). Scale bar, 20µm. (E) Images 1-5 show example images of CNPase+ve wrapped NeuN+ve cells (myelinated neuronal cell bodies). Scale bars, 20µm. (F) Fluorescent immunohistochemistry for NeuN (green), CNPase (magenta) and Hoechst (nuclei-blue) in human MS motor cortex. Arrows indicate the location of CNPase+ve wrapped NeuN+veHoechst+ve cells. Scale bar, 20µ

Extended Data Figure 7. Extensive remyelination by newly generated oligodendrocytes in the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A) Confocal images of csn treated zebrafish with oligodendrocytes newly generated after demyelination. Arrows show position of oligodendrocyte cell bodies. Scale bars, 20µm. (B) Quantification of the total myelin produced per oligodendrocyte (calculated by multiplying number of sheaths per oligodendrocyte by the average sheath length per oligodendrocyte) (mean=521.5±138.30SD), versus the same oligodendrocytes 3dpt (mean=47.18±26.57SD) and by newly differentiated oligodendrocytes at 3dpt (mean=491.80±199.10SD). Pre-treatment versus surviving p<0.0001, pre-treatment versus newly differentiated p=0.8271, surviving versus newly differentiated p<0.0001. Ordinary one-way ANOVA with Tukey’s multiple comparison test. N=15 oligodendrocytes from 15 zebrafish (pre-treatment and surviving). N=20 oligodendrocytes from 11 zebrafish (newly differentiated). Data are presented as mean±SEM.

Extended Data Figure 8. Summary Schematic.

Summary schematic outlining the responses of oligodendrocytes which survive demyelination and those newly generated after demyelination made using Biorender.

Figure 1. Remyelination by oligodendrocytes that survive demyelination in the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A) Confocal images of single surviving oligodendrocytes followed over time from pre-treatment (Pre-t at 4dpf) to 3dpt. Scale bars, 20μm. (B) The percentage of different oligodendrocyte fates of the 23 oligodendrocytes that survived demyelination. (C) Quantification of the number of myelin sheaths produced by the same oligodendrocytes pre-treatment (median=18.00, IQR=13.00-20.00) and 3dpt (median=2.00, IQR=1.00-3.00), p<0.0001. Two-tailed Wilcoxon matched-pairs signed rank test. N=23 zebrafish. (D) Quantification of the average length of myelin sheaths produced by the same oligodendrocytes pre-treatment (mean=30.93±5.76SD) and 3dpt (mean=25.47±9.16SD), p=0.0655. Paired two-tailed t-test. N=15 zebrafish. (E) Quantification of the number of mistargeted myelin profiles produced by the same oligodendrocytes pre-treatment (median=0.00, IQR=0.00-0.00) and 3dpt (median=1.00, IQR=0.00-1.00), p=0.0012. Two-tailed Wilcoxon matched-pairs signed rank test. N=23 zebrafish. (F) Quantification of the average distance of myelin structures (sheaths and mistargeted myelin) to the oligodendrocyte cell body for the same oligodendrocytes pre-treatment (median=16.64, IQR=14.47-18.69) and 3dpt (median=8.24, IQR=4.73-16.33), p=0.0008. Two-tailed Wilcoxon matched-pairs signed rank test. N=22 zebrafish. (G) Quantification of the average distance of myelin structures to the oligodendrocyte cell body (μm) for myelin sheaths (median=11.97, IQR=7.36-16.24) and mistargeted myelin profiles (median=6.62, IQR=4.53-14.86) in surviving myelinating oligodendrocytes at 3dpt, p=0.3627. Two-tailed Mann-Whitney t-test. N=15 zebrafish (sheaths), N=13 zebrafish (mistargeted myelin). (H) Confocal images of single surviving oligodendrocytes at 3dpt which mistarget their myelin around neuronal cell bodies (CB). Scale bars, 10μm. (I-J’) Transmission electron microscopy images of a myelinated neuronal cell body surrounded by cell bodies which are not myelinated. (I) Scale bar=1μm. (J). Scale bar=0.2μm. In all figures, 1 oligodendrocyte was analysed per zebrafish. Data are presented as median with IQR in figures (C and E - G) and mean±SD in figure (D). In figure (A) and (H) Arrows show the position of oligodendrocyte cell bodies, and arrowheads show positions of mistargeted myelin.

Figure 2. Myelin sheath dynamics of surviving oligodendrocytes following demyelination in the Tg(mbp:TRPV1-tagRFPt) zebrafish model.

(A-C) Confocal images of zoomed regions of oligodendrocytes that survive demyelination and form new sheaths from (A and B) pre-existing processes and (C) de novo sheath formation imaged over time at 1dpt, 3dpt, 5dpt and 7dpt. Arrows indicate locations of processes and arrow heads highlight the locations of newly formed myelin sheaths. Scale bars=10 μm. (D) Quantification of the number of sheaths produced per oligodendrocyte by the same cells following demyelination over time at 1dpt (median=2.00, IQR=1.00-2.00), 3dpt (median=1.00, IQR=1.00-2.00), 5dpt (median= 1.00, IQR = 1.00 - 2.00), 7dpt (median = 1.00, IQR = 1.00 - 2.00). 1dpt vs 3dpt p>0.9999, 1dpt vs 5dpt p>0.9999, 1dpt vs 7dpt p>0.9999, 3dpt vs 5dpt p>0.9999, 3dpt vs 7dpt p>0.9999, 5dpt vs 7dpt p>0.9999. Friedman test with Dunn’s multiple comparisons test. Data are presented as median with IQR. N=23 oligodendrocytes from 23 zebrafish. (E) Quantification of the average sheath length produced per oligodendrocyte by the same cells following demyelination over time at 1dpt (mean=14.68±9.12 SD), 3dpt (mean=28.75±16.62 SD), 5dpt (mean=28.98±14.98 SD), 7dpt (mean=28.32±14.12SD). 1dpt vs 3dpt p=0.0009, 1dpt vs 5dpt p=0.0006, 1dpt vs 7dpt p=0.0006, 3dpt vs 5dpt p=0.9296, 3dpt vs 7dpt p=0.9296, 5dpt vs 7dpt p=0.3654. Mixed-effects analysis with the Geisser-Greenhouse correction and Holm-Sidak’s multiple comparisons test. Data are presented as mean±SD. At 1dpt N=18 oligodendrocytes from 18 zebrafish, at 3dpt N=21 oligodendrocytes from 21 zebrafish, at 5 and 7dpt N=20 oligodendrocytes from 20 zebrafish. (F) Quantification of the number of mistargeted myelin profiles produced per oligodendrocyte following demyelination over time at 1dpt (median=0.00, IQR=0.00-1.00), 3dpt (median=0.00, IQR=0.00-1.00), 5dpt (median=1.00, IQR=0.00-1.00), 7dpt (median=0.00, IQR=0.00-1.00). 1dpt vs 3dpt p>0.9999, 1dpt vs 5dpt p>0.9999, 1dpt vs 7dpt p>0.9999, 3dpt vs 5dpt p>0.9999, 3dpt vs 7dpt p>0.9999, 5dpt vs 7dpt p>0.9999. Friedman test with Dunn’s multiple comparisons test. Data are presented as median with IQR. N=23 oligodendrocytes from 23 zebrafish. (G) Quantification of the average distance of myelin (sheaths and mistargeted myelin profiles) to the oligodendrocyte cell body (μm) per oligodendrocyte following demyelination over time at 1dpt (mean=7.52±4.53SD), 3dpt (mean= 9.89±5.58SD), 5dpt (mean=10.74±7.33SD), 7dpt (mean=9.69±6.16SD). 1dpt vs 3dpt p=0.1270, 1dpt vs 5dpt p=0.1643, 1dpt vs 7dpt p=0.2562, 3dpt vs 5dpt p=0.9067, 3dpt vs 7dpt p=0.9965, 5dpt vs 7dpt p=0.2543. Mixed-effects analysis, with Geisser-Greenhouse correction and Tukey’s multiple comparisons test. Data are presented as mean±SD. At 1dpt N=20 oligodendrocytes from 20 zebrafish, at 3, 5 and 7dpt N=23 oligodendrocytes from 23 zebrafish. (H) Quantification of the sheath dynamics of oligodendrocytes that survive demyelination and maintain their sheath number from 1 - 7dpt. N=10 oligodendrocytes from 10 zebrafish. (I) Quantification of the sheath dynamics of oligodendrocytes that survive demyelination and increase sheath number from 1 - 7dpt. N=7 oligodendrocytes from 7 zebrafish. (J) Quantification of the sheath dynamics of oligodendrocytes that survive demyelination and decrease sheath number from 1 - 7dpt. N=6 oligodendrocytes from 6 zebrafish.

Figure 3. Mistargeted myelin profiles are present in remyelinating lesions in motor cortex tissue from people with MS.

(A and B) Low magnification images of chromogenic immunohistochemistry for proteolipid protein (PLP - brown) and NeuN (blue) in (A) human control (people without MS) motor cortex and (B) human MS motor cortex. Asterisks indicate lesion areas. Scale bars, 100μm. (C and D) High magnification images of myelin-wrapped (PLP - brown) neuronal cell bodies (NeuN - blue) in human MS motor cortex, at positions indicated by black arrows. (C) shows a high magnification view of the region highlighted in the black box in (B). Scale bars, 10μm. (E) Low magnification image of fluorescent immunohistochemistry for NeuN (white), PLP (red) and Hoechst (nuclei-blue) in human MS motor cortex. White arrows indicate locations of myelin wrapped (PLP+ve) neuronal (NeuN+ve) cell bodies (Hoechst+ve). Merged channel image scale bar, 20μm. Split channel scale bars, 10μm. (F) shows a high magnification view of the region highlighted in the white box in (E). (G) High magnification images of fluorescent immunohistochemistry for CNPase (magenta), NeuN (green) and Hoechst (nuclei-blue). White arrows indicate the location of myelin wrapped (CNPase+ve) neuronal (NeuN+ve) cell bodies (Hoechst+ve) close to the oligodendrocyte cell body. Scale bar, 20μm. H) Quantification of the number of myelinated neuronal cell bodies per grey matter area (cm²) in control grey matter (median=0.00, 25th percentile 0.00, 75th percentile 0.82), MS lesion area (median 11.65, IQR=0.00-21.06), MS perilesion area (median=81.63, IQR=38.65-134.30) and MS normal appearing grey matter (NAGM) (median = 0.61, IQR=0.00-2.32). Control versus MS lesion p>0.9999, control versus MS perilesion p=0.0076, control versus MS NAGM p>0.9999, MS lesion versus MS perilesion p=0.1735, MS lesion versus MS NAGM p>0.9999, MS perilesion versus MS NAGM p=0.0321. Kruskal-Wallis test with Dunn’s multiple comparisons test. Data are presented as median with IQR. All samples are from human motor cortex tissue: N=5 human tissue samples (from 5 different individuals) per condition.