Cortical pathologies and astrocyte heterogeneity in rodent models of multiple sclerosis and Parkinson’s disease

Schmitz, Birte Katharina; Rink, Lothar (Thesis advisor); Kipp, Markus (Thesis advisor); Spehr, Marc (Thesis advisor)

Aachen (2020, 2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Many neuroinflammatory and neurodegenerative disorders are characterized by characteristic (histo-) pathological changes. In Parkinson’s disease (PD), for example, it is well known that dopaminergic neurons in the substantia nigra of the midbrain degenerate, which results in the dopaminergic denervation of the striatum. To take another example, multiple sclerosis (MS) is characterized by focal, inflammatory lesions of the white matter leading to oligodendrocyte degeneration, demyelination and axonal degeneration. Despite such "classical" pathological events, neuroinflammatory and neurodegenerative disorders are as well characterized by "non-classical" pathologies. In PD, we now know that besides the striatum other brain areas are subjected to the dopaminergic denervation. In MS, not just the white but as well the gray matter shows widespread and severe demyelination. In this thesis, "non-classical" pathologies in PD and MS animal models are investigated. Special emphasis is placed on astrocytes which are important regulators of brain homeostasis and pathologies. In the first study, pathological changes of cortical and subcortical brain structures were investigated applying the intrastriatal 6-hydroxydopamine (6-OHDA) mouse model, as they may be of particular interest for the understanding of motor and non-motor symptoms in PD. As expected, intrastriatal 6-OHDA injection induced retrograde degeneration of dopaminergic neurons within the substantia nigra pars compacta, but less so within the ventral tegmental area. Furthermore, a region-specific loss of tyrosine hydroxylase (TH)+ fiber density has been observed in distinct extrastriatal structures among the cingulate- and motor cortex. Interestingly, the loss of cortical TH+ fibers was not paralleled by changes in microglia or astrocyte densities or microglia morphology. Besides, in the globus pallidus and thalamus the loss of TH-immunoreactive fibers was paralleled by a strong increase in microglia densities. These results demonstrate that intrastriatal injections of 6-OHDA lead to pathological changes in both cortical and subcortical structures, which can be reproducibly and reliable examined in this model. The identification of putative disease-specific or regionally enriched reactive astrocyte subtypes could be particularly relevant to understand the selective susceptibility of neuronal populations in certain brain regions in PD. The objective of this second study was to reveal histological similarities and differences in astrocyte numbers and morphologies during 6-OHDA-induced striatal denervation depending on the marker protein used. Marked differences were already evident in the non-lesioned mouse striatum. While astrocytes in the striatum expressed vimentin (VIM) and glial fibrillary acidic protein (GFAP) in the primary thick processes, aldehyde dehydrogenase 1 family member L1 (ALDH1L1) and S100 calcium-binding protein β (S100β) were additionally expressed in fine branchlets and leaflets. After toxin-induced denervation GFAP- and VIM-immunoreactivity showed the strongest activation of astrocytes, while astrocyte densities marked by ALDH1L1 and brain lipid binding protein (BLBP) were comparatively lower. In contrast, S100β-immunoreactivity did not present astrocyte activation. This study clearly illustrates that pathological changes of astrocytes occur to a great extent in the MFB 6-OHDA mouse model of PD. Astrocytes are a highly heterogeneous cell population, and different antibodies could possibly visualize different aspects of this intricate heterogeneity. Once peripheral inflammatory cells invade the brain, such MS foci often evolve into large lesions in the white but not in the gray matter, which could be due to the increased amount of myelin debris in the white matter. To support the hypothesis that a lower myelin content within the cortical gray matter could possibly limit lesion expansion, lysophosphatidylcholine (LPC) will be stereotactically injected into either the cortex or the white matter corpus callosum (CC) and lesion size and neuroinflammatory response will be compared following immunohistochemical approaches. Seven days after LPC injection a substantial proportion of the CC was demyelinated, whereas the extent of demyelination was by far less intense in the cortical region. Besides, increased demyelination of the CC was paralleled by an extensive microgliosis and a moderate astrocytosis, whereas both cellular parameters were less severe in the cortical gray matter. This implicates that local neuroinflammatory cascades, triggered by myelin debris, are pivotal factors for promoting lesion expansion in MS. Reasons underlying lesion development in MS remain largely elusive, however astrocytes appear to play a pivotal role. Morphological characterization of astrocytes could contribute to our understanding of cell type function, since differences in morphologies are believed to relate to the functional status. This study analyzed astrocyte morphology in the experimental cuprizone model of MS after activation of astrocytes by the innate immune system. GFAP+ astrocytes of the medial CC were 3D reconstructed within the Neurolucida 360 software and analyzed in the Neurolucida Explorer. The one-week cuprizone diet significantly increased the mean branch volumes. Besides, the average branch diameter was enlarged in distinct Shell radii compared to controls. In summary, the applied approach can examine morphological alterations of GFAP+ astrocytes.