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Regenerative capacity of reactive astrocytes in vitro and in vivo
Regenerative capacity of reactive astrocytes in vitro and in vivo
Reactive astrogliosis is a reaction of the central nervous system (CNS) common to diverse types of injury, but only upon invasive injury a subset of reactive astrocytes acquires proliferative capacity in vivo and exhibits stem cell potential through self-renewal and multipotency in vitro. Given that in the adult mammalian brain only adult neural stem cells (aNSCs) located in specific niches are able to self-renew and give rise to neurons, it is important to test to which extent reactive astrocytes can enact their NSC potential also in vivo when exposed to different environmental conditions. For this purpose, experimental mouse models were used to investigate (i) whether and to which extent astrocytes in the injured cerebral cortex grey matter exhibit self-renewal in vivo when exposed to sequential pathological stimuli; (ii) whether reactive astrocytes can give rise to different cell types in vivo when placed in neurogenesis-supportive environments. In order to analyze the proliferative behavior of reactive astrocytes in the adult murine cerebral cortex in response to repetitive pathological stimuli, I established a double labeling paradigm based on sequential delivery of two thymidine analogues, BrdU and EdU. Furthermore, in order to verify the results obtained with this paradigm I performed clonal analysis of reactive astrocytes using GlastCreERT2-mediated recombination in the R26-Confetti reporter line. Results from both experimental paradigms demonstrate that a distinct subset of reactive astrocytes within the cortical parenchyma is able to re-enter the cell cycle and give rise to 3-cell clones upon repetitive injuries, which had so far not been observed. Furthermore, astrocyte cell-cycle reentry is modulated by monocyte infiltration, as it was increased in their absence in transgenic CCR2-/- mice. Moreover, we used BrdU and EdU double labeling to investigate whether proliferation was a property confined to a specific subset of astrocytes, or if different sets of reactive astrocytes could be activated to enter cell cycle. Our analysis showed that the astrocyte proliferative pool is not fixed, and new astrocytes can be recruited into proliferation upon a second pathological event. Intriguingly, our results suggest a strong drive towards astroglial population homeostasis, which has so far not been described in these cells. To analyze the differentiation capacity of RAs in vivo, cortical reactive astrocytes and aNSCs from the subependymal zone (SEZ) of adult actin-GFP mice cultured as neurospheres were transplanted heterotypically into the adult dentate gyrus (DG) and the E13 embryonic brain. Our analysis showed that the progeny of reactive astrocytes remained restricted within the glial lineage in both environments, whereas aNSCs gave rise to immature neurons in the DG and to mature neurons in a few regions of the embryonic brain. Taken together although reactive astrocytes show multipotency and can give rise to neurons in vitro, they are largely unable to generate neurons in vivo. Furthermore, in light of a recently reopened debate that questions reactive astrocyte stem cell potential, our results from transplantation experiments provoked further investigation on this matter. There is new evidence supporting that all neurospheres obtained from injured cortical tissue are actually originated from SEZ aNSCs. As our transplantation results showed a distinct differentiation profile of reactive astrocytes when compared to aNSCs in both host environments, we performed experiments to add evidence to this debate. For this purpose, we developed two independent experimental paradigms in which cortical cells (but not SEZ cells) were labeled prior to injury through double transgenic Emx1-GFP mice and through delivery of AAV-iCre into the cortex of floxed GFP-Reporter mice. Our results obtained with both experimental paradigms show that cells of cortical origin can give rise to neurospheres in vitro following stab wound lesion. Altogether, through this study we have achieved many novel insights into astrocyte reaction to injury, regulation of astrocytic population through different proliferation strategies, and into the stem cell potential of reactive astrocytes in vivo. Our findings advance the general understanding of astrocyte biology in the context of CNS pathology and open the way to many new questions in this field.
reactive astrocytes, adult neural stem cells, traumatic brain injury
Canhos, Luisa Lange
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Canhos, Luisa Lange (2018): Regenerative capacity of reactive astrocytes in vitro and in vivo. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)
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Abstract

Reactive astrogliosis is a reaction of the central nervous system (CNS) common to diverse types of injury, but only upon invasive injury a subset of reactive astrocytes acquires proliferative capacity in vivo and exhibits stem cell potential through self-renewal and multipotency in vitro. Given that in the adult mammalian brain only adult neural stem cells (aNSCs) located in specific niches are able to self-renew and give rise to neurons, it is important to test to which extent reactive astrocytes can enact their NSC potential also in vivo when exposed to different environmental conditions. For this purpose, experimental mouse models were used to investigate (i) whether and to which extent astrocytes in the injured cerebral cortex grey matter exhibit self-renewal in vivo when exposed to sequential pathological stimuli; (ii) whether reactive astrocytes can give rise to different cell types in vivo when placed in neurogenesis-supportive environments. In order to analyze the proliferative behavior of reactive astrocytes in the adult murine cerebral cortex in response to repetitive pathological stimuli, I established a double labeling paradigm based on sequential delivery of two thymidine analogues, BrdU and EdU. Furthermore, in order to verify the results obtained with this paradigm I performed clonal analysis of reactive astrocytes using GlastCreERT2-mediated recombination in the R26-Confetti reporter line. Results from both experimental paradigms demonstrate that a distinct subset of reactive astrocytes within the cortical parenchyma is able to re-enter the cell cycle and give rise to 3-cell clones upon repetitive injuries, which had so far not been observed. Furthermore, astrocyte cell-cycle reentry is modulated by monocyte infiltration, as it was increased in their absence in transgenic CCR2-/- mice. Moreover, we used BrdU and EdU double labeling to investigate whether proliferation was a property confined to a specific subset of astrocytes, or if different sets of reactive astrocytes could be activated to enter cell cycle. Our analysis showed that the astrocyte proliferative pool is not fixed, and new astrocytes can be recruited into proliferation upon a second pathological event. Intriguingly, our results suggest a strong drive towards astroglial population homeostasis, which has so far not been described in these cells. To analyze the differentiation capacity of RAs in vivo, cortical reactive astrocytes and aNSCs from the subependymal zone (SEZ) of adult actin-GFP mice cultured as neurospheres were transplanted heterotypically into the adult dentate gyrus (DG) and the E13 embryonic brain. Our analysis showed that the progeny of reactive astrocytes remained restricted within the glial lineage in both environments, whereas aNSCs gave rise to immature neurons in the DG and to mature neurons in a few regions of the embryonic brain. Taken together although reactive astrocytes show multipotency and can give rise to neurons in vitro, they are largely unable to generate neurons in vivo. Furthermore, in light of a recently reopened debate that questions reactive astrocyte stem cell potential, our results from transplantation experiments provoked further investigation on this matter. There is new evidence supporting that all neurospheres obtained from injured cortical tissue are actually originated from SEZ aNSCs. As our transplantation results showed a distinct differentiation profile of reactive astrocytes when compared to aNSCs in both host environments, we performed experiments to add evidence to this debate. For this purpose, we developed two independent experimental paradigms in which cortical cells (but not SEZ cells) were labeled prior to injury through double transgenic Emx1-GFP mice and through delivery of AAV-iCre into the cortex of floxed GFP-Reporter mice. Our results obtained with both experimental paradigms show that cells of cortical origin can give rise to neurospheres in vitro following stab wound lesion. Altogether, through this study we have achieved many novel insights into astrocyte reaction to injury, regulation of astrocytic population through different proliferation strategies, and into the stem cell potential of reactive astrocytes in vivo. Our findings advance the general understanding of astrocyte biology in the context of CNS pathology and open the way to many new questions in this field.