To date, the mechanisms of tissue injury and neurodegeneration in multiple sclerosis are poorly understood. Demyelination and axonal injury occurs on a background of chronic inflammation in the relapsing as well as in the progressive stage of the disease (Frischer et al., 2009
) and close contacts between activated microglia or macrophages and degenerating axons, myelin sheaths and oligodendrocytes have been described (Ferguson et al., 1997
; Trapp et al., 1998
; Kornek et al., 2000
). Reactive oxygen species and nitric oxide intermediates are produced by activated macrophages and microglia and are, thus, likely candidates to be involved in tissue injury in multiple sclerosis (Van Hoorsen et al., 2010
). Indeed, biochemical studies on multiple sclerosis tissue provided clear evidence for oxidized lipids and DNA within active multiple sclerosis lesions (Vladimirova et al., 1998
; Smith et al., 1999
; Lu et al.
) and upregulation of antioxidative proteins has been reported mainly in astrocytes (Van Hoorssen et al., 2008
). However, attempts to directly localize oxidized molecules to degenerating oligodendrocytes, axons and neurons have so far been unsatisfactory. In the current study, immunocytochemistry for oxidized lipids revealed their presence within macrophages, located in structures that morphologically resemble lipofuscin. This is not surprising, since lipofuscin appears to contain non-digestible remnants of oxidized cellular components, accumulating in macrophages or resident cells of the CNS, for instance neurons (Keller et al., 2004
; Wang et al., 2008
). Similarly, nitrosylated epitopes, recognized by antibodies against nitrotyrosine, were mainly seen in macrophages (Cross et al., 1998
; Liu et al., 2001
). The only exception is immunoreactivity in oligodendrocytes for nitrotyrosine, previously described in some active multiple sclerosis lesions, which suggests a role of peroxynitrite in their destruction (Jack et al., 2007
; Zeis et al., 2009
). In contrast to these previous studies, we show here that DNA and lipid oxidation is associated with ongoing demyelination and neurodegeneration in active multiple sclerosis lesions. Furthermore, we show for the first time that acute cell injury and cell death of oligodendrocytes, axons and neurons in multiple sclerosis is linked to profound cytoplasmic and nuclear oxidative damage. The reason for the different results between our current and previous studies is not entirely clear. The most likely explanation comes from our observation that oxidized DNA and lipids were mainly present in a small zone of active multiple sclerosis lesions, which represents that previously described as the area of initial demyelination (Marik et al., 2007
) or the ‘prephagocytic’ lesion (Barnett and Prineas, 2004
; Henderson et al., 2009
). Such lesions or lesion areas may not have been included in earlier studies. It has been shown previously that oxidized phospholipids and MDA epitopes are present in apoptotic cells as well as in apoptotic bodies ingested by macrophages (Chang et al., 1999
). Apoptotic oligodendrocytes are predominantly seen in multiple sclerosis lesions in areas of initial (prephagocytic) demyelination (Barnett and Prineas, 2004
; Marik et al., 2007
). Furthermore, apoptotic cell death through oxidative mechanisms may exert pro-inflammatory and immunogenic actions (Chang et al., 2004
), which in part may explain the progressive increase in inflammation with lesion maturation in multiple sclerosis (Marik et al., 2007
; Henderson et al., 2009
Analysing DNA oxidation in active multiple sclerosis, we found that the cells most severely affected are those morphologically resembling oligodendrocytes and that these cells in part show condensed and fragmented nuclei, morphologically resembling apoptosis. These cells could in part be identified by their expression of the oligodendrocyte marker TPPP-p25 (Höftberger et al., 2010
). Identification of dying oligodendrocytes by double staining is difficult, since many cellular proteins, used as specific cell markers, are degraded in the course of apoptosis or necrosis. Nevertheless, the selective demyelination and oligodendrocyte apoptosis related to the expression of apoptosis-inducing factor (Veto et al., 2010
) in active multiple sclerosis lesions is in line with the profound DNA oxidation seen in this study. The prominent labelling of myelin and oligodendrocyte-like cells by immunocytochemistry for MDA and oxidized phospholipids, as shown in this study, further supports the view that oxidative damage plays a major role in demyelination and oligodendrocyte destruction in active multiple sclerosis lesions. It must, however, be emphasized that DNA oxidation in active multiple sclerosis lesions is not restricted to oligodendrocytes, but also affects astrocytes in low incidence.
Immunocytochemistry for oxidized phospholipids provided further insights into the mechanisms of tissue injury in multiple sclerosis lesions. Besides the presence of oxidized phospholipids in oligodendrocytes and myelin, we also found a highly selective and profound expression of these neo-epitopes within degenerating axons and neurons. Accumulation of oxidized phospholipids in dystrophic axons may accumulate at sites of disturbed axonal transport. Alternatively, they may indicate radical-mediated damage as an initial change in axonal demise. We think that the second possibility is more likely. The half-life of oxidized lipids in a cell is estimated by minutes or few hours only (Keller and Matson, 1998
). Furthermore, only a small fraction of amyloid precursor protein reactive dystrophic axons were also stained for oxidized phospholipids and they were concentrated at sites of initial lesions. In contrast, abundant amyloid precursor protein reactive axonal spheroids, which are present in the centre of the lesions, were devoid of immunoreactivity for oxidized phospholipids. This suggests that for the first time it is possible to directly visualize acute injury of axons, neuronal cell bodies or the fragmentation of their dendrites as a direct consequence of radical-mediated injury.
Oxidized phospholipids were also present in astrocytes in active multiple sclerosis lesions. In contrast to oligodendrocytes and neurons, these cells, however, do not degenerate but appear to be capable of sequestering damaged cytoplasmic components into (autophagic) vacuoles. However, it has been shown before that astrocytes, too, show signs of injury, mainly reflected by retraction of their cell processes and altered expression of molecules, related to the formation of the glia limitans (Parrat and Prineas, 2010
; Sharma et al., 2010
It was interesting to note that different cell types within the multiple sclerosis lesions differentially accumulated MDA and oxidized phospholipids. While both types of oxidized lipid epitopes were seen in myelin and oligodendrocytes, axons and neurons exclusively accumulated oxidized phospholipids. One possible explanation could be that the cellular content of phosphatidylcholine, rich in polyunsaturated fatty acids, is higher in the grey matter compared with the white matter (Svennerholm, 1968
), suggesting that such fatty acids are preferentially present in neurons and axons.
In summary, our study provides evidence for an important role of oxidative damage in the pathogenesis of demyelination and neurodegeneration in multiple sclerosis lesions, which may act in addition to, or in cooperation with nitric oxide radicals, as described previously (Bagasra et al., 1995
; Zeis et al., 2009
). It further shows—for the first time—that the analysis of oxidized lipid epitopes in multiple sclerosis lesions allows identification of acute damage of oligodendrocytes, axons and neurons at different stages of lesion formation. Our data also suggest that oxidative damage is in part related to inflammation, that it affects different cellular components of the CNS, but that myelin, oligodendrocytes, neurons and axons may be more sensitive to oxidative damage than astrocytes.