Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models

Abstract

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4(+) T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8(+) T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4(+) T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.

Fig. 1

Oxidative burst, oxidative injury and iron accumulation in MS lesions. Expression of p22phox in the normal white matter (NWM) of a control brain (a), the normal-appearing white matter (NAWM; b), the zone of initial (pre-phagocytic) demyelination (c) and in the plaque centre of an active lesion (d) from an acute MS patient, defined by the presence of LFB and MOG reactive myelin degradation products in macrophages (inserts). Individual microglia in the control brain expressed p22phox. There was profound microglial activation in the NAWM with the formation of microglial nodules, a massive p22phox expression in initial areas of active demyelination and a low expression on macrophages containing myelin debris in the plaque centre. iNOS expression in acute MS in the NAWM (e) and in initial areas of demyelination (f) was weak and restricted to a few cells with microglial morphology. In the active plaque centre, iNOS was mainly expressed on macrophages (g). Immunoreactivity of E06 staining for oxidised phospholipids was low or absent in the NWM of controls (h) and very high in areas of initial demyelination in acute MS (i). Double staining for E06 (blue) and cell markers (brown) documented its presence in oligodendrocytes (stained for TPPPp25; j) and cytoplasmic granules in astrocytes (stained for GFAP; k). Intense E06 reactivity was also found in the cytoplasm of cortical neurons, showing beading and fragmentation of their cell processes (l). Iron staining of the MS brain revealed profound iron accumulation in the subcortical white matter (m; arrowheads) and at the edge of active lesions (m; arrows) from a SPMS patient. Established lesions showed a reduced iron staining (m; asterisks). Young control brains revealed a relatively low iron content mainly found in oligodendrocytes (n; age 30). The aged human control (o; age 84) and MS (m; age 57) brains displayed high amounts of non-haeme iron in oligodendrocytes and myelin. High iron content was present at the edge of an active lesion from a RRMS patient in microglia (p). Within the centre of an active lesion, the iron load was reduced and iron reactivity was mainly found in (perivascular) macrophages (q). Scale bar 50 μm except for m = 1 cm

Fig. 2

Expression patterns of genes related to oxidative stress, mitochondria and iron metabolism differ between passive transfer EAE and active white matter MS lesions. The heat maps show colour-coded relative quantities of genes with known functions in oxidative stress pathways (a) and iron metabolism (b). Additionally, mitochondrially encoded genes (c) as well as nuclear-encoded genes with important mitochondrial functions (d) are depicted. The gene expression in lower spinal cords of young (4 months) and old (8 months) Lewis rats suffering from MBP-specific T cell-induced EAE was determined at two time points (day 6, peak of disease; day 10, recovery phase) via RT-qPCR. Transcriptional changes in periplaque white matter, initial (pre-phagocytic) lesions and active demyelinating lesions, derived from 3 or 4 acute MS cases, were investigated by whole-genome microarrays. Relative quantities were calculated for each individual gene by comparing the different time points and lesions with untreated age-matched Lewis rats or normal white matter from control cases, respectively. A detailed description of genes and primers/probes is presented in the supplementary data (Supplementary Table 3 and 4). d6 day 6, d10 day 10, PP periplaque white matter, I initial (pre-phagocytic) lesion, A active demyelinating lesion, n.d. not determined

Fig. 3

Oxidative burst, oxidative injury and iron accumulation in acute and chronic models of CD4⁺ T cell-mediated EAE. a–i Acute EAE induced by passive transfer of MBP-reactive encephalitogenic CD4⁺ T cells in aged Lewis rats (14-month old) at the peak of the disease (6 days after T cell transfer). Inflammation was associated with profound microglial activation and macrophage infiltration at the lesions, immunoreactive for the pan-microglia/macrophage marker Iba-1 (a). In contrast, p22phox (b) and iNOS (c) expressions were more restricted and mainly found on cells with macrophage morphology. No E06 reactivity (oxidised phospholipids) was detected in inflammatory lesions (d) but was occasionally found in degenerating neurons (for instance in the cerebellar cortex of an aged control rat; e). Iron accumulated in the basal ganglia of a 14-month old EAE rat (f) while it was absent from the basal ganglia of 2-month old rats (g). In old rats, iron accumulated in oligodendrocytes and myelin in the basal ganglia (h). Within inflammatory EAE lesions, few macrophages and microglia showed iron accumulation (i). j–o Chronic relapsing/progressive EAE in C57BL/6 mice immunised with MOG_35–55. This model is characterised by large inflammatory demyelinating lesions with extensive axonal injury and loss in the spinal cord. The lesions contained abundant CD3⁺ T cells (j) and Iba-1⁺ macrophages (k). In addition, we found massive Iba-1 expression in cells with microglial morphology in the adjacent white and grey matter (k). The macrophages in the lesions also expressed p22phox (l), while iNOS expression was restricted to a limited number of perivascular macrophages (m). E06 reactivity (oxidised phospholipids) was sparse or absent in the lesions (n), although there were few axonal spheroids with weak E06 staining (insert). Single lesion-associated meningeal macrophages accumulated iron (o). p–x Chronic relapsing EAE in DA rats immunised with MOG_1–125. Initial lesions presenting with a perivenous pattern of demyelination (p) showed scattered p22phox (q) and iNOS (r) expressions. p22phox in these lesions was mainly seen in granulocytes (q, insert). In contrast, in the chronic phase of the disease, there was an extensively active demyelination in the spinal cord grey and white matter (t) indicated by the presence of macrophages with myelin degradation products (t, insert). The lesions contained high numbers of ED1⁺ macrophages (v) but their majority did not express p22phox (u) or iNOS (s). Only few macrophages in the lesions expressed p22phox (u, insert). Oxidised phospholipids did not accumulate in the active chronic lesions (w), but individual neurons with morphological evidence for retrograde degeneration were strongly labelled (w, insert). Within lesions, few macrophages and microglia showed iron accumulation (x). Scale bar 50 μm except for f = 0.25 cm and j, k, n, o, p = 0.5 mm

Fig. 4

Oxidative burst, oxidative injury and iron accumulation in CD8⁺ T cell-mediated EAE, in innate immunity-driven inflammation and in toxic cuprizone-mediated demyelination. a–f Brain inflammation induced by CD8⁺ T cells. Perivascular inflammatory infiltrates were associated with massive microglial activation in the adjacent tissue (a; Iba-1). Perivascular inflammatory cells and to lower degree also the surrounding microglia highly expressed p22phox (b), while iNOS expression was minor and restricted to a few perivascular cells (c). The immunohistochemistry for oxidised phospholipids (E06; d) was negative. Iron deposition was lacking in most lesions (e) but occurred in few lesions within perivascular macrophages (f). g–m Inflammatory demyelinating lesions induced by local injection of LPS into the spinal cord dorsal column. In the early inflammatory stage (1 day after LPS injection), p22phox⁺ granulocytes (g) and iNOS⁺ macrophages (h) were present in the lesions. At the peak of active demyelination (9 days after LPS injection), lesions were densely infiltrated by macrophages and showed microglial activation at the lesion edge (i; Iba-1, asterisk). Macrophages in these lesions and, to a lower degree, microglia at the lesion edges showed expression of p22phox (j). Despite a very strong expression of p22phox in active lesions (j), the expression of iNOS was very low (k). There was a weak reactivity for oxidised phospholipids in the lesions (l; E06). Individual perivascular macrophages were iron positive (m). n–s Actively demyelinating lesion in the corpus callosum induced by cuprizone diet. Extensive loss of myelin was seen in the corpus callosum (n), which was densely packed with macrophages and microglia (o), although their majority did not express p22phox (p) and iNOS only very weakly (q). Additionally, there was no evidence for oxidised phospholipids (r; E06) or iron deposition (s) in the lesions. Scale bar 50 μm

Fig. 5

Oxidative burst, oxidative injury and iron accumulation in inflammatory demyelinating lesions in MHV-JHM coronavirus-induced encephalomyelitis. a–i Basic characterisation of inflammatory demyelinating lesions in the spinal cord of an infected Lewis rat with demyelinating encephalomyelitis after infection with MHV-JHM coronavirus. There was extensive plaque-like demyelination (a; MBP) with relative preservation of axons (b and f; e NAWM; Bodian silver impregnation). Virus antigen was present in the periplaque white and grey matter (c; immunohistochemistry for nucleocapsid protein N) in neurons (g) as well as in glia cells in the white matter (h). Active demyelination was associated with profound T cell-mediated inflammation (d), the majority of inflammatory cells being CD8⁺ T cells (blue; i) and ED1⁺ macrophages (brown; i). j–n Determination of microglial activation in MHV-JHM coronavirus-induced lesions. Activated microglia formed microglial nodules in the NAWM reactive for Iba-1 (j) and p22phox (l). The edge of an active lesion is shown with immunocytochemistry for macrophages/microglia (Iba-1; k), for myelin (MBP; m) and for expression of NADPH oxidase (p22phox; n). Active demyelination was associated with profound microglial activation (k; Iba-1). Their numbers increased in the periplaque white matter (PPWM) towards the lesion edge (k; left side) showing the highest density at the zone of initial demyelination (k, middle) and decreasing towards the centre of the lesion, where many cells exhibited a macrophage phenotype (k; right side). Immunohistochemistry for p22phox in serial-cut adjacent sections from the lesion (n) showed a staining pattern similar to that of Iba-1. iNOS expression was sparse in the lesions (o). p–u Accumulation of oxidative damage and iron in MHV-JHM coronavirus-induced lesions. Lesions showed abundant reactivity for oxidised phospholipids (p, q, s E06) in myelin (p), in cells with apoptotic nuclei (q), in macrophage granules (q) and in axonal spheroids (s). In addition, nuclei within the lesions contained oxidised DNA (8OHdH reactivity; r). Most lesions did not show iron deposition (t), although iron-positive perivascular macrophages accumulated in individual lesions (u). Scale bar 50 μm except for a–d = 0.5 mm