The Effects of Hypergravity on Key Characteristics and Reactivity of Primary Murine Astrocytes for Potential CNS Injury Regeneration

Kurzfassung

Many challenges are standing in the way of finding possible regeneration approaches to central nervous system (CNS) injuries, due to the multiple and complex intrinsic and extrinsic factors which play a role in the formation of the glial scar. Although the glial scar does prevent the widespread of damaged tissue, it inhibits neuronal regeneration as well as axonal regrowth, which is the reason CNS injuries are mostly deemed irreversible. Over the years, there have been many attempts at trying to find methods that promote neuronal regeneration. However, to this day, researchers have not been able to detect a simple and accessible way to do so, which is mostly due to the complexity of the whole process. It is also important to mention that an exact definition of reactive astrocytes, which play a major role in the formation of the glial scar, and how to identify them has not been made thus far. In this thesis, the focus lies specifically on this point. Possible regeneration approaches could be easier to attain if the exact mechanisms of the formation of the glial scar are better understood. In previous studies, it has been shown that astrocytes decrease their cell migration rate under 2g hypergravity conditions in contrast to 1g. Noteworthy is that under 2g conditions, neurons – in contrast to astrocytes – increase their neurite numbers as well as their neurite projection lengths. These results demonstrate that gravity might play a crucial role in obtaining viable regeneration approaches, where neuronal regeneration mechanisms and astrocytic migration could selectively be influenced. To further investigate these seemingly promising results, protocols for the examination of key astrocytic parameters were optimized within the framework of the thesis. These key parameters include proliferation and apoptosis rates, spreading and adherence rates, astrocytic reactivity markers and astrocytic cytoskeletal dynamics, which was made possible by studying actin dynamics in LifeAct cells. The goal was not only to better understand these mechanisms but to also see how the exposure to hypergravity influences them. The live-staining protocol with Annexin-V and Propidium iodide (PI) was optimized to quantify the apoptosis rates of the samples. Additionally, a double stain protocol with DAPI and Ki67 was also optimized for the identification of proliferating cells, which should simplify the quantification process of proliferation rates. Regarding the spreading and adherence assay, the results of the three tested cultures showed a significant decrease in cell spreading area in the cultures exposed to 2g conditions achieved on the Multi Sample Incubator Centrifuge (MuSIC). Control measurements of potential vibrations were conducted during the operating centrifuge to exclude that no additional forces besides the heightened g-load are affecting the samples. Furthermore, several reactive astrocyte markers other than GFAP were tested out, which should build the basis for a scoring method that should make reactive astrocyte identification more concrete, as it will not only include several reactivity markers but also the other above-mentioned key parameters. Lastly, the LifeAct experiments were conducted several times and the protocol for live imaging was optimized accordingly so that phototoxicity caused by the LEDs of the microscope can be completely avoided in future experiments. Overall, this thesis contributes to identifying and understanding key astrocytic parameters which play a fundamental role in the process of the formation of a glial scar. This should then help in finding possible approaches to reverse or hinder this prosses and make regeneration post CNSinjuries more attainable.