Discussion

In accordance to the peculiarities of chronic progressive MS [18, 20, 22, 45], the present study described exacerbating locomotor system dysfunction and progressively increasing demyelination in chronic TME. Remyelination occurred with a delayed onset, about 4 months after first signs of demyelination were detected. The insufficiency of the remyelination process was further substantiated by morphometric analysis of the density of intralesional CNPase positive cells. These cells were significantly reduced between 56 and 196 dpi.

Similar findings have been reported previously for TME [46, 47]. In contrast to TME,, a fast and complete remyelination was reported for most other models of MS including anti-MOG antibody augmented experimental autoimmune encephalomyelitis (EAE) in Lewis rats [23], cuprizone toxicity in C57BL6 mice [26, 28], lysolecithin induced demyelination in the caudal cerebellar peduncle of Sprague Dawley rats [31], and murine hepatitis virus (MHV) A-59 strain infection of C57BL6 mice [29, 48]. Causes for insufficient remyelination in chronic progressive MS and TME are multiple and include (i) depletion of OPCs, (ii) inability of OPCs to proliferate and / or differentiate within the lesion due to a non-permissive environment; and (iii) axonal loss or the inability of chronically demyelinated axons to be remyelinated [49, 50].

Occurence of NG2-positive OPCs in TME

Some lesions in MS patients contain OPCs / pre-OGs, and still remain chronically demyelinated. More importantly, these cells do not differentiate into stable myelinating oligodendrocytes [21, 35, 36, 51]. Similarly, within the demyelinated lesions of TMEV-infected mice a rapid increase in the number of elongated bi- or oligopolar NG2-positive cells, interpreted as OPCs, was followed by a slow and less pronounced increase in stellate-shaped NG2-positive cells, resembling pre-OGs [33]. Accordingly, similar cellular morphologies have been described for NG2-positive cells within demyelinated lesions in MS [21], EAE [17, 23, 52], cuprizone-toxicity [26], MHV-induced demyelination [29], and traumatic brain injuries [53, 54]. In TMEV-infected mice, the number of intralesional NG2-positive cells was transiently elevated between 28 and 98 dpi, indicating that a proliferating cell population that possesses the intrinsic capacity to initiate remyelination can be found at least during the first months of lesion development. A similar assumption has been reported following infection of SJL/J mice with the DA-strain of TMEV [55]. A significant increase in

the number of intralesional OPCs has also been described in other models of demyelination including EAE [17, 23, 52], cuprizone toxicity [26, 50], ethidium bromide induced lesions [56], anti-GalC-antibodies / complement induced lesions [57], and MHV-induced demyelination [29]. The immunohistological demonstration of a temporarily increased population of NG2-positive cells was supported by the detection of a mild transient upregulation of NG2 and PDGFα-R mRNA in the spinal cords of TMEV-infected mice. The observed significant positive linear correlation between the amount of NG2-positive cells and NG2 mRNA suggests that NG2 mRNA expression represents a useful molecular marker for OPCs, as described by others [58].

Intralesional NG2-positive cells were detected throughout the observation period in TME, and they showed a transient upregulation despite a persisting TMEV-infection. Therefore, it seems unlikely that a virus-induced depletion of OPCs is the predominating pathogenetic mechanism causing insufficient remyelination. Still, a sustained loss of differentiated and NG2-negative regenerating OGs cannot be ruled out. A loss of differentiated OGs seemed to be responsible for the failure of remyelination in repeated adoptive transfer experiments using encephalitogenic T-cells / anti-MOG antibodies [59], or prolonged cuprizone feeding [50].

Consequently, further studies concerning the fate of oligodendroglial cells in TME are necessary to further elucidate the fate of OPCs during disease progression.

Differentiation of NG2-positive OPCs in TME

The histological and ultrastructural demonstration of remyelinated axons and the slight increase in patchy colocalization of NG2 and CNPase at 196 dpi suggested that at least some NG2-positive OPCs differentiated into OGs. Similarly, NG2/CNPase coexpressing cells were found within areas of demyelination in EAE [17, 23]. Similar to the present study, NG2/CNPase coexpression was restricted to cellular processes within remyelinating areas in EAE. [17]. In contrast to TME, the proliferation of OPCs was followed by a fast and complete remyelination in most other models of MS [23, 24, 26, 31, 57]. The marked difference in timing and extent of remyelination may be explained by an insufficient oligodendroglial differentiation of the OPCs in TME (Fig.12). The hypothesis of inhibited oligodendroglial differentiation was further supported by the either unchanged or mildly decreased gene expression of the myelin proteins CNPase and MBP, and a reduction of the exon 2 containing splice-variants of MBP compared to total MBP mRNA. Similarly to the presented results, SJL/J mice infected with the DA-strain of TMEV demonstrated a downregulation of PLP and

Figure 4-12: Developmental stages of cells of the oligodendrocyte lineage (black arrows) during Theiler`s murine encephalomyelitis (TME; grey bars and arrows). During their physiological development from multipotent neural progenitors, the cells of the oligodendrocyte lineage express stage specific markers like NG2, PDGFα-R, CNPase and MBP. In TME, a transiently increased number of intralesional NG2-positive oligodendrocyte progenitor cells (OPCs) / pre-oligodendrocytes was detected. An irreversible loss of CNPase-positive oligodendrocytes as well as a downregulation of MBP gene expression indicated an insufficient oligodendroglial differentiation of these OPCs. Furthermore, a possible alternative differentiation of the OPCs towards GFAP-positive astrocytes leads to the formation of an astroglial scar. Conclusively, it is assumed that a dysregulation of OPC-differentiation by unknown directly or indirectly TMEV-related factors represents the predominating pathogenetic mechanism causing delayed and limited remyelination in TME. CNPase = 2´,3´- cyclic nucleotide 3´-phosphodiesterase; GFAP = glial fibrillary acidic protein; MBP = myelin basic protein; NG2

= nerve/glial antigen 2; PDGFα-R = platelet derived growth factor α-receptor; TMEV = Theiler`s murine encephalomyelitis virus.

MBP mRNA by in situ hybridization [47]. In contrast, an increase of the exon 2 containing variants of MBP has been described in association with rapid and efficient remyelination in passive EAE in mice [60] and MHV-induced demyelination [61].

The slightly increased patchy intralesional NG2/GFAP colocalization at 196 dpi suggested a possible astrocytic differentiation of at least some NG2-positive OPCs. Similar findings,

though still discussed controversially, have been reported for MHV-induced demyelination [24, 29], and traumatic CNS injury [53, 54, 62]. However, a ~5-fold increased number of intralesional GFAP-positive astrocytes was detected at 98 and 196 dpi in the present investigation, confirming previous reports about astrocytic scaring in TME [55, 63]. Although astrogliosis is the name-giving pathological hallmark of MS, only little is known about the quantitative aspects of this alteration in other models of demyelination. In contrast to the present study, the response of astroglia is not very marked in EAE [23]. Similar to TMEV-induced demyelination, astrocytes responded to traumatic brain injury by altered gene-expression, hypertrophy and proliferation [53, 62]. In the present study, GFAP mRNA was upregulated between 28 and 196 dpi and there was a moderate significant positive linear correlation between the number of GFAP-positive astrocytes and GFAP mRNA. This is in agreement with the results of previous studies [63]. Interestingly, a low significant positive linear correlation was detected between NG2-positive cells and GFAP mRNA. This seems to further support the hypothesis of an astrocytic differentiation of at least some NG2-positive OPCs. In vitro about 80% of NG2-positive OPCs develop into GFAP-positive astrocytes after incubation with a serum-enriched medium [64, 65]. Whether a similar mechanism might operate after disruption of the blood-brain-barrier in TME needs to be investigated in future studies.

Axonal injury in TME

Interestingly, early-onset axonal injury was detected at 14 dpi. Similarly, early-onset axonal injury has been reported at 7 dpi for DA-infected SJL/J mice, and is thought to result from Wallerian degeneration induced by neuronal death due to TMEV-induced acute polioencephalitis [66, 67]. In the present investigation, early-onset axonal injury was followed by progressive axonal injury throughout the studied time period. This finding is in accordance with the demonstration of axonal loss in DA-infected SJL/J mice at ~200 dpi [6, 68]. Similar to previous studies in TME, ultrastructural examination revealed that the major part of the demyelinated axons displayed an unaltered morphology at least until 98 dpi [9-11]. Therefore a failure of remyelination solely due to a loss of axons seems to be unlikely. Moreover, similar to MS, the observed progressive axonal injury seems to result from secondary progressive axonal degeneration and atrophy due to primary demyelination [21, 22]. This secondary axonal loss might account for the reduced amount of non-pNF-positive axons detected within the lesion centers at 196 dpi. Ultrastructural examination confirmed that there were basically only few intact axons left within the astrogliotic lesion centers. However,

further studies are needed to quantitatively assess the ratio of early-onset (primary) axonal-injury to secondary axonal axonal-injury in TME.

Summary

In summary, the occurrence of chronic progressive inflammatory demyelination and exacerbating locomotor system dysfunction in persistently TMEV-infected SJL/J-mice further supported the usefulness of TME as a model for the chronic progressive form of MS [2, 4]. A prominent and transient increase in intralesional NG2-positive OPCs / pre-OGs was demonstrated briefly after the onset of demyelination. This was followed by a delayed and insufficient remyelination and marked astrogliosis. The presented findings indicated a dysregulation of OPC maturation as a key mechanism of remyelination failure in TME, resulting in a shift from oligodendroglial towards astrocytic differentiation. Possible pathways leading to a dysregulation of OPC oligodendroglial differentiation include serum-derived factors which are known to induce an astrocytic differentiation of OPCs in vitro [64, 65], inhibiting factors like myelin debris [56], activated LINGO-1 [69], and an activated jagged-1, notch-1 signaling pathway [70]. Furthermore, early-onset axonal injury should be considered as an additional and possibly TME-specific contributing factor.