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The loss of myelinating oligodendrocytes is a key characteristic of many neurological diseases, including Multiple Sclerosis (MS). In progressive MS, where effective treatment options are limited, peripheral immune cells can be found at the site of demyelination and are suggested to play a functional role during disease progression. In this study, we hypothesize that metabolic oligodendrocyte injury, caused by feeding the copper chelator cuprizone, is a potent trigger for peripheral immune cell recruitment into the central nervous system (CNS). We used immunohistochemistry and flow cytometry to evaluate the composition, density, and activation status of infiltrating T lymphocytes in cuprizone-intoxicated mice and post-mortem progressive MS tissues. Our results demonstrate a predominance of CD8 + T cells along with high proliferation rates and cytotoxic granule expression, indicating an antigenic and pro-inflammatory milieu in the CNS of cuprizone-intoxicated mice. Numbers of recruited T cells and the composition of lymphocytic infiltrates in cuprizone-intoxicated mice were found to be comparable to those found in progressive MS lesions. Finally, amelioration of the cuprizone-induced pathology by treating mice with laquinimod significantly reduces the number of recruited T cells. Overall, this study provides strong evidence that toxic demyelination is a sufficient trigger for T cells to infiltrate the demyelinated CNS. Further investigation of the mode of action and functional consequence of T cell recruitment might offer promising new therapeutic approaches for progressive MS., (© 2020 The Authors. GLIA published by Wiley Periodicals LLC.)

To unravel the failure of remyelination in multiple sclerosis (MS) and to test promising remyelinating treatments, suitable animal models like the well-established cuprizone model are required. However, this model is only standardized in young mice. This does not represent the typical age of MS patients. Furthermore, remyelination is very fast in young mice, hindering the examination of effects of remyelination-promoting agents. Thus, there is the need for a better animal model to study remyelination. We therefore aimed to establish the cuprizone model in aged mice. 6-month-old C57BL6 mice were fed with different concentrations of cuprizone (0.2-0.6%) for 5-6.5 weeks. De- and remyelination in the medial and lateral parts of the corpus callosum were analyzed by immunohistochemistry. Feeding aged mice 0.4% cuprizone for 6.5 weeks resulted in the best and most reliable administration scheme with virtually complete demyelination of the corpus callosum. This was accompanied by a strong accumulation of microglia and near absolute loss of mature oligodendrocytes. Subsequent remyelination was initially robust but remained incomplete. The remyelination process in mature adult mice better represents the age of MS patients and offers a better model for the examination of regenerative therapies.

Remyelination is the natural repair mechanism in demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis. Several animal models have been used to study demyelination and remyelination. Among toxic animal models, oral administration of the toxin cuprizone leads to white and gray matter demyelination. In contrast, focal demyelination models include the stereotactic application of a toxin such as lysolecithin or ethidium bromide. The injection procedure generates a local disruption of the blood-brain barrier (BBB) and might thus trigger a local inflammatory reaction and consequently may influence demyelination and remyelination. In order to study such consequences, we applied stereotactic injections in the cuprizone model where demyelination and remyelination are mediated independent of this procedure. Immunohistochemistry was performed to detect the presence of lymphocytes and activated glial cells in the injection area. Blood protein stainings were used to assess the integrity of the BBB and myelin staining to evaluate demyelination and remyelination processes. Stereotactic injection led to a local disruption of the BBB as shown by local extravasation of blood proteins. Along the injection canal, T and B lymphocytes could be detected and there was a tendency of a higher microgliosis and astrocytosis. However, these changes did not influence demyelination and remyelination processes at the site of injection, in the corpus callosum, or in the cerebral cortex. Our results suggest that a local stereotactic injection has no major impact on CNS demyelination and remyelination.

Oligodendrocytes are the myelinating cells of the central nervous system. Since many studies of demyelinating diseases focus their research on this cell type, there is growing interest for obtaining reliable markers that can specifically recognize oligodendroglia. Established markers are the myelin-associated neurite outgrowth inhibitor (NogoA), the transcription factor Olig2, and the antibody CC-1, the latter being directed against the protein adenomatous polyposis coli (APC). Unfortunately, it has been discussed whether APC and Olig2 could recognize astrocytes under pathological conditions as well. Hence, we performed immunohistochemical studies using the oligodendroglial markers NogoA, APC, and Olig2 in a murine model of cuprizone induced demyelination. We have found that APC co-localizes with NogoA and does not co-localize with the astrocytic marker GFAP. Olig2 shows co-localization with APC but there is also a small population of Olig2/GFAP double positive cells. Some Olig2/GFAP double positive cells are found in the corpus callosum in a narrow time window in which oligodendrocyte precursor cells proliferate in this model. In other brain regions including the cerebral cortex and hippocampus and in all regions in untreated control mice double positive Olig2/GFAP cells do not occur. In conclusion, our results underline that APC and NogoA are reliable markers for detection of mature oligodendrocytes. Olig2 is a suitable marker to stain cells of oligodendroglial origin but could be combined with GFAP to exclude the GFAP positive population of cells from the quantification of oligodendroglia.

Multiple sclerosis is the leading cause of neurological disability in young adults affecting more than two million people worldwide. Although multiple sclerosis is generally considered as white matter disease, distinct pathological alterations are also found in the grey matter. Involvement of basal ganglia seems to be related to a set of symptoms such as fatigue, impaired cognition, and movement disturbance. Since no appropriate animal model for studying cortical deep grey matter demyelination is established, we reassessed the cuprizone mouse model to investigate basal ganglia demyelination. Mice were fed cuprizone for different time intervals. The myelin status was analyzed by classical histological staining and immunohistochemistry for myelin proteins and glia markers. Expression of oligodendrocyte and astroglia were investigated by PCR. Cuprizone intoxication induced a severe demyelination of distinct cortical deep grey matter sub-regions. Striosmomes, located within the caudate-putamen and the ventral part of the caudate nucleus displayed intense demyelination, whereas those within the globus pallidus and the head of the caudate nucleus were not affected. The matrix region, however, was equally affected in the medial and lateral region. Besides demyelination, we observed hypertrophic and hyperplastic astrocytosis and microglia cell invasion/local proliferation in the demyelinated areas. Young adult and aged mice were similarly affected as well as mice with different genetic backgrounds. We conclude that cuprizone-induced demyelination provides an adequate animal model to investigate appropriate therapy strategies for the prevention of cortical deep grey matter demyelination. The heterogeneity in local demyelination points at beginning remyelination during ongoing demyelination.