Introduction: Astrocytopathy is known to be an early feature of different neuroinflammatory diseases. However, the impact of astrocyte loss and repopulation on the development and progression of demyelinating lesions in complex etiologies, such as multiple sclerosis, is difficult to determine. To more easily analyse astrocyte- oligodendrocyte-interactions during lesion formation and progression in the human brain, diseases like Central pontine myelinolysis (CPM) can be used as a less complex model of demyelinating disorders. CPM is a rare neurological condition characterized by damage to the myelin sheath of pontine nerves after osmotic shifts in serum. Astrocytopathy is regarded to be the first event in the pathogenesis of CPM lesions. Methods: Histological investigation of autopsy tissue from human CPM patients was performed. Lesions were staged considering the myelination and the appearance of different astrocyte subtypes, which was used to judge behaviour of the astrocytic compartment. Further, dynamics of oligodendrocyte loss and repopulation were analysed and compared to the astrocytic repopulation. Results: Early-staged lesions were largely demyelinated and showed an overall reduction of astrocyte densities. The few astrocytes present showed a bipolar morphology and were APQ4-negative, indicating an immature state. Intermediate- stage lesions were still largely demyelinated, but had increased overall densities of astrocytes, which did not yet reflect densities observed in the perilesion. Astrocytes appeared mostly ramified and AQP4-positive, indicating maturity. Nevertheless, bipolar astrocytes were still observable, indicating that repopulation was not yet finalized. Late-stage CPM-lesions were at least partially remyelinated. Astrocytes were detectable in overall densities comparable to the perilesion and showed a ramified (or even reactive morphology), as well as regular expression of AQP4. Investigating the oligodendrocytes, intralesional densities were reduced in early- and intermediate-stage lesions when compared to the perilesion. Re-increase in oligodendrocyte densities was first observable in late-stage lesions, but did not reach perilesional levels. Conclusion: The study at hand indicates that the recovery of demyelinated osmolyte- induced pontine lesions follows a distinct time-course. Repopulation of the lesion with oligodendrocytes is not carried out until lesions are completely repopulated with functional resident astrocytes, as indicated by the ramified morphology and the expression of AQP4. Further studies will be needed to determine, whether the appearance of immature astrocytes, indicating an ongoing repopulation of lesions with astrocytes, correlates with an inefficient repair of demyelinated lesions.:List of Abbreviations.................................................................................................................6 1 Introduction................................................................................................................7 1.1 Osmotic Demyelinating Syndrome......................................................................... 7 1.2 Clinical manifestation............................................................................................. 9 1.3 Diagnosis and Management of CPM.....................................................................11 1.4 Aetiology of Central Pontine Myelinolysis.................................. ......................... 14 1.5 The brain, its adaptation to hyponatraemia and response to correction – pathophysiology of CPM............................................................................................16 1.6 Pathology of myelin............................................................................................. 19 1.6.1 Astrocytopathy and oligodendrocytopathy.................................................................................................20 1.7 Aims of the study................................................................................................. 23 2 Material und Methods............................................................................................. 24 2.1 Patient tissue........................................................................................................ 24 2.2 Histology and immunohistochemistry................................................................................................24 2.2.1 Basic concepts........................................................................................... ......24 2.2.2 Hematoxylin and Eosin (HE)............................................................................. 26 2.2.3 Luxol Fast Blue/ Periodic Acid Schiff stain........................................................27 2.2.4 Immunohistochemistry. Application and Protocol.............................................28 2.3 Implementation.................................................................................................... 31 2.4 Estimation of demyelination................................................................................. 32 2.5 Analysis of cell density and proliferation.............................................................. 32 2.6 Data plotting and statistical analysis.................................................................... 32 3 Results..................................................................................................................... 33 3.1 Patient cohort....................................................................................................... 33 3.2 Characteristics of demyelination.......................................................................... 35 3.3 CPM lesion and disease staging.......................................................................... 37 3.4 Astrocytes within human CPM lesions................................................................. 42 3.4.1 Astrocyte densities are decreased in early CPM lesions....................................42 3.4.2 Astrocytes in CPM– morphological distinctions.................................................45 3.5 Oligodendrocyte densities within human CPM lesions.........................................48 3.6 Macrophages and activated microglia.................................................................. 54 3.6.1 KiM1P – a marker for infiltrating macrophages and activated microglia............54 3.6.2 Proliferating Iba1+ cells are observed in all lesion stages..................................58 4 Discussion................................................................................................................ 61 4.1 Lesion Staging...................................................................................................... 61 4.2 Astrocytes in the pathogenesis of CPM............................................................... 65 4.3 Oligodendrocyte pathology in CPM..................................................................... 69 4.4 Mechanisms of regeneration in human CPM lesions............................................72 4.5 Summary, interpretation and limitations of our study............................................78 5 Conclusion and Outlook.......................................................................................... 80 6 Bibliography............................................................................................................. 82 7 List of Tables.............................................................................................................91 8 List of Figures.......................................................................................................... 92 9 Appendix.................................................................................................................. 94 9.1 Declaration of Authenticity.....................................................................................94 9.2 Acknowledgements...............................................................................................95

Adaptation to osmotic imbalance is crucial for cell survival and many organisms have developed complex mechanisms to counteract the changes induced by aniosmolarity. In mammals, the central nervous system is one of the most vulnerable organs after sudden changes in osmolarity. This is best exemplified by two common clinical disorders, brain edema resulting from acute hyponatremia and brain dehydration after hypernatremia. In clinical practice, hyponatremia is the most common electrolyte disorder and carries a significant mortality and morbidity. However, correction of hyponatremia should be undertaken with great caution as failure to adapt to rapid changes in chronic hyponatremia will cause rapid and fatal demyelination of the central nervous system. This syndrome is called osmotic demyelination syndrome (ODS) or central pontine myelinolysis. In this work, we investigated the pathophysiology and new diagnostic and treatments tools for osmotic demyelination syndrome. Using a rat model, we demonstrated the efficacy of the neuroprotective agent minocycline in osmotic demyelination syndrome. We also compared treatment with dexamethasone and re-lowering of serum sodium after rapid correction of hyponatremia and we showed that re-lowering of serum sodium is better than administration of dexamethasone. We explored the mechanisms underlying brain demyelination in ODS and demonstrated that the rupture of the blood brain barrier is not necessary for demyelination and that activated microglia does not play a key role in brain demyelination but can potentiate the lesions induced by rapid correction of hyponatremia. We also investigated the role of astrocytes during the development of osmotic demyelination and demonstrated that astrocytes are a major component of the physiopathology of osmotic demyelination. We showed that early and massive astrocyte apoptosis delineates the regions of future myelin damage and found that astrocyte death induces severe upregulation of myelinolytic cytokines and destruction of astrocyte oligodendrocyte junctions with subsequent disruption of panglial syncitium. <p>Finally, as there are currently no markers of demyelination, we investigated the astroglial protein S100B in ODS and found a significant release of S100B during development of ODS, which correlated with astrocyte damage. We also showed that the increase in S100B is prevented by protective treatment of hyponatremia with urea and demonstrated that serum levels of S100B could be used as a prognosis factor in ODS .<p>All together, our work has revealed a central role of astrocytes in the pathophysiology of ODS and clarified the importance of blood barrier dysfunction and microglial activation. This work also proposes new diagnostic and treatment tools for ODS. <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished