Late Passage Cultivation Induces Aged Astrocyte Phenotypes in Rat Primary Cultured Cells

Abstract

Astrocytes play various important roles such as maintaining brain homeostasis, supporting neurons, and secreting inflammatory mediators to protect the brain cells. In aged subjects, astrocytes show diversely changed phenotypes and dysfunctions. But, the study of aged astrocytes or astrocytes from aged subjects is not yet sufficient to provide a comprehensive understanding of their important processes in the regulation of brain function. In this study, we induced an in vitro aged astrocyte model through late passage cultivation of rat primary cultured astrocytes. Astrocytes were cultured until passage 7 (P7) as late passage astrocytes and compared with passage 1 (P1) astrocytes as early passage astrocytes to confirm the differences in phenotypes and the effects of serial passage. In this study, we confirmed the morphological, molecular, and functional changes of late passage astrocytes showing aging phenotypes through SA-β-gal staining and measurement of nuclear size. We also observed a reduced expression of inflammatory mediators including IL-1β, IL-6, TNFα, iNOS, and COX2, as well as dysregulation of wound-healing, phagocytosis, and mitochondrial functions such as mitochondrial membrane potential and mitochondrial oxygen consumption rate. Culture-conditioned media obtained from P1 astrocytes promoted neurite outgrowth in immature primary cultures of rat cortices, which is significantly reduced when we treated the immature neurons with the culture media obtained from P7 astrocytes. These results suggest that late passage astrocytes show senescent astrocyte phenotypes with functional defects, which makes it a suitable model for the study of the role of astrocyte senescence on the modulation of normal and pathological brain aging.

Senescent Phenotype of Astrocytes Leads to Microglia Activation and Neuronal Death

“…Senescent astrocytes in our study also found these similar changes of indicators. What's more is that the mitochondrial function of senescent astrocytes may decline with increased passaging [24], especially the level of proton leak and ATP, but we found that increased passaging did not affect the level of ATP, which is different from the research before, speculating that this discrepancy may be caused by the difference of length of culturing time and selfregulation of astrocyte itself, but ATP level of the model of oxidative stress of astrocyte indeed changed.…”

G-quadruplexes Stabilization Upregulates CCN1 and Accelerates Aging in Cultured Cerebral Endothelial Cells

“…CEC treated with PDS showed higher γH2AX fluorescence intensity compared with control cells (Figures 3A,B). We also observed that CEC treated with PDS showed a significant increase in their nuclear size, which is also a feature of cell senescence (Rey et al, 2017;Bang et al, 2021) (Figure 3C). Another cohort of cultured primary CEC was treated either with PDS, or with a vehicle, and stained for SA-β-galactosidase.…”

Blood-brain barrier dysfunction promotes astrocyte senescence through albumin-induced TGFβ signaling activation

“…Previous studies have demonstrated that senescent astrocytes have substantially reduced functional capacity compared to their normal counterparts. For example, senescent astrocytes exhibited impaired would healing ability, phagocytic uptake, metabolic function, and neuroprotective capacity (Bang et al, 2021; Pertusa et al, 2007). Among their numerous functions in the adult brain, astrocytes are required for BBB maintenance (Heithoff et al, 2021).…”