In this study hippocampal demyelination in the cuprizone model was described and the role of Polysialic acid in de- and remyelination was further investigated. The fact that cuprizone treatment causes demyelination of the hippocampus has been previously reported (Hoffmann et al. 2008). In the first part of our study loss of myelin in the hippocampus as well as the cellular response to cuprizone administration in C57BL/6 mice were described in detail. It was shown that demyelination of the hippocampus after 6 weeks of treatment with 0.2% cuprizone was prominent. Distinct formations within the hippocampus had a variable temporal pattern of demyelination, reaching complete demyelination at different time points. A zonal distribution of demyelination in the cuprizone model has also been described for the corpus callosum (Stidworthy et al.
2003). Moreover, astroglia accumulation and microglia infiltration were observed within the various hippocampal structures. This is in accordance with microglia and astroglia accumulation reported in cortical, cerebellar, and corpus callosum cuprizone-induced demyelination (Skripuletz et al. 2008; Lindner et al. 2008b; Gudi et al. 2009; Skripuletz et al. 2009b; Norkute et al. 2009; Hiremath et al. 1998; Remington et al. 2007).
Accumulation of nestin-positive cells was also described in the cuprizone-treated hippocampus and it has been linked to reactive astrogliosis. Changes in the expression of PSA during demyelination of the hippocampus were also recorded. PSA is a marker of neuronally-committed precursors (Kempermann et al. 2004). Distinct hippocampal formations within the hippocampus reacted differentially to cuprizone administration. PSA staining on the hippocampal commissure as well as on the young neurons of the subgranular layer of the dentate gyrus was completely abolished after 6 weeks of
treatment. On the other hand it was only faintly reduced on the fibers of the mossy fiber tract.
Our results, where comparable, were similar to the ones reported in another study on cuprizone-induced hippocampal demyelination (Norkute et al. 2009). Only one significant difference existed between the two studies. While we reported hippocampal microgliosis during cuprizone treatment, Norkute et al. did not. As the mouse strain in both cases was C57BL/6, differences cannot be attributed to strain dependency. Cuprizone feeding conditions and animal ages were also similar. Microglia detection though was performed using different markers. Norkute et al. used Iba-1 and F4/80, macrophage-microglia markers that are expressed in resting microglia, while their expression is upregulated in activated microglia (Imai et al. 1996; Ohsawa et al. 2004; Chen et al. 2005; Austyn and Gordon 1981). We studied microglia activation through immunolabeling for Mac-3, a lysosomal antigen, which is also used for detecting inflammatory macrophages and activated microglia. It is possible that the difference pinpointed between the two studies is a result of the above markers recognizing different subpopulations within the activated microglia pool. In multiple sclerosis tissue, when lesions were classified according to location, microgliosis was rare in intrahippocampal lesions, while moderate microglia infiltration was observed in mixed intrahippocampal-perihippocampal lesions (Geurts et al.
2007). In another study microglia infiltration extent was used in order to classify hippocampal lesions according to their activation stage. No microglia accumulation was considered to characterize inactive lesions, while reduced amount of activated microglia in the centre and high cellular density in the lesion edge was considered to characterize chronic active lesions. Finally, strong microglia infiltration was found in one lesion that was considered acute active (Papadopoulos et al. 2009). It is obvious that microglia
accumulation in multiple sclerosis lesions within the hippocampus is among others affected by the stage of activity and location, and trying to make parallels between multiple sclerosis and experimentally-induced lesions in this case is difficult.
In general, our study offered a better insight on the events defining toxin-induced demyelination. Demyelinated lesions described in the hippocampus of multiple sclerosis patients (Papadopoulos et al. 2009; Geurts et al. 2007; Sicotte et al. 2008) have been thought to contribute to cognitive function impairments known to occur in many multiple sclerosis patients (Rao et al. 1991). Subsequently, better understanding of their pathogenesis is of great importance. Here we outlined cellular events during cuprizone-induced hippocampal demyelination and showed the usefulness of the murine cuprizone model in studying hippocampal demyelinated lesions. These results can be used as reference for investigations of hippocampal demyelination in future studies.
The second part of the study attempted to further explore the role of PSA expression in remyelination after a demyelinating event. As PSA is re-expressed on the denuded axons of demyelinated lesions in multiple sclerosis, while the adjacent intact white matter has no PSA-expressing axons, PSA is considered to be an inhibitor of remyelination (Charles et al. 2002). Further elucidation of its function in the lesion is required. Mice deficient for St8siaIV, one of the two enzymes responsible for PSA synthesis on NCAM were submitted to cuprizone treatment and their de- and remyelination were compared to wildtype mice. It was shown that reduced PSA synthesis is associated with a faster onset of remyelination in the corpus callosum and higher myelin content in the cortex as soon as cuprizone is withdrawn from the rodent chow. This adds to the data supporting an inhibitory role for PSA in remyelination in vivo. As quantification of NG2-positive OPC in the demyelinated lesions does not differ between wildtype and deficient mice, it is
suggested that reduced PSA expression does not affect OPC recruitment, but rather their maturation to myelin-producing oligodendrocytes. This is also supported by the fact that in the cortex, 4 days after withdrawal of cuprizone, Nogo-A-positive mature oligodendrocytes were more abundant in the St8siaIV-/- than in the St8siaIV+/+ mice. PSA expression changes exhibit no differences between the two strains. In the St8siaIV-/- animals, PSA has not been found to be expressed in the cortex, dendrites and axons of the stratum pyramidalis neurons of the hippocampus, or the hippocampal mossy fiber tract, as shown before (Eckhardt et al. 2000).Knock-out animals though express PSA on the hippocampal commissure fibers, newborn neurons of the subgranular zone in the dentate gyrus, and diffusely on the stratum lacunosum moleculare as wildtype animals do. These formations exhibit complete or slight loss of PSA staining during cuprizone treatment, which is similar to both strains. Hippocampal demyelination is also similar in extent and temporal pattern between the wildtype and St8siaIV deficient mice. Finally, the investigation of developmental myelination of the St8siaIV-/- mice showed no significant alterations to the wildtype strain.
This study provided further data supporting the assumption that PSA expression interferes with remyelination in vivo. It is shown that modulation of PSA may improve regeneration processes and that PSA could act as a target in future regeneration treatments.
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