Differences between cultured astrocytes from neonatal and adult Wistar rats: focus on in vitro aging experimental models.

Astrocytes play key roles regulating brain homeostasis and accumulating evidence has suggested that glia are the first cells that undergo functional changes with aging, which can lead to a decline in brain function. In this context, in vitro models are relevant tools for studying aged astrocytes and, here, we investigated functional and molecular changes in cultured astrocytes obtained from neonatal or adult animals submitted to an in vitro model of aging by an additional period of cultivation of cells after confluence. In vitro aging induced different metabolic effects regarding glucose and glutamate uptake, as well as glutamine synthetase activity, in astrocytes obtained from adult animals compared to those obtained from neonatal animals. In vitro aging also modulated glutathione-related antioxidant defenses and increased reactive oxygen species and cytokine release especially in astrocytes from adult animals. Interestingly, in vitro aged astrocytes from adult animals exposed to pro-oxidant, inflammatory, and antioxidant stimuli showed enhanced oxidative and inflammatory responses. Moreover, these functional changes were correlated with the expression of the senescence marker p21, cytoskeleton markers, glutamate transporters, inflammatory mediators, and signaling pathways such as nuclear factor κB (NFκB)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1). Alterations in these genes are remarkably associated with a potential neurotoxic astrocyte phenotype. Therefore, considering the experimental limitations due to the need for long-term maintenance of the animals for studying aging, astrocyte cultures obtained from adult animals further aged in vitro can provide an improved experimental model for understanding the mechanisms associated with aging-related astrocyte dysfunction.

Lipopolysaccharide Induces Gliotoxicity in Hippocampal Astrocytes from Aged Rats: Insights About the Glioprotective Roles of Resveratrol.

Astrocytes may undergo a functional remodeling with aging, acquiring a pro-inflammatory state. In line with this, resveratrol represents an interesting strategy for a healthier brain aging since it can improve glial functions. In the present study, we investigated the glioprotective role of resveratrol against lipopolysaccharide (LPS)-induced gliotoxicity in hippocampal aged astrocytes. Astrocyte cultures were obtained from aged rats (365 days old) and challenged in vitro with LPS in the presence of resveratrol. Cultured astrocytes from newborn rats were used as an age comparative for evaluating LPS gliotoxicity. In addition, aged rats were submitted to an acute systemic inflammation with LPS. Hippocampal astrocyte cultures were also obtained from these LPS-stimulated aged animals to further investigate the glioprotective effects of resveratrol in vitro. Overall, our results show that LPS induced a higher inflammatory response in aged astrocytes, compared to newborn astrocytes. Several inflammatory and gene expression alterations promoted by LPS in aged astrocyte cultures were similar in hippocampal tissue from aged animals submitted to in vivo LPS injection, corroborating our in vitro findings. Resveratrol, in turn, presented anti-inflammatory effects in aged astrocyte cultures, which were associated with downregulation of p21 and pro-inflammatory cytokines, Toll-like receptors (TLRs), and nuclear factor κB (NFκB). Resveratrol also improved astroglial functions. Upregulation of sirtuin 1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) represent potential molecular mechanisms associated with resveratrol-mediated glioprotection. In summary, our data show that resveratrol can prime aged astrocytes against gliotoxic stimuli, contributing to a healthier brain aging.

Sulforaphane Induces Glioprotection After LPS Challenge.

Sulforaphane is a natural compound that presents anti-inflammatory and antioxidant properties, including in the central nervous system (CNS). Astroglial cells are involved in several functions to maintain brain homeostasis, actively participating in the inflammatory response and antioxidant defense systems. We, herein, investigated the potential mechanisms involved in the glioprotective effects of sulforaphane in the C6 astrocyte cell line, when challenged with the inflammogen, lipopolysaccharide (LPS). Sulforaphane prevented the LPS-induced increase in the expression and/or release of pro-inflammatory mediators, possibly due to nuclear factor κB and hypoxia-inducible factor-1α activation. Sulforaphane also modulated the expressions of the Toll-like and adenosine receptors, which often mediate inflammatory processes induced by LPS. Additionally, sulforaphane increased the mRNA levels of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO1), both of which mediate several cytoprotective responses. Sulforaphane also prevented the increase in NADPH oxidase activity and the elevations of superoxide and 3-nitrotyrosine that were stimulated by LPS. In addition, sulforaphane and LPS modulated superoxide dismutase activity and glutathione metabolism. Interestingly, the anti-inflammatory and antioxidant effects of sulforaphane were blocked by HO1 pharmacological inhibition, suggesting its dependence on HO1 activity. Finally, in support of a glioprotective role, sulforaphane prevented the LPS-induced decrease in glutamate uptake, glutamine synthetase activity, and glial-derived neurotrophic factor (GDNF) levels, as well as the augmentations in S100B release and Na+, K+ ATPase activity. To our knowledge, this is the first study that has comprehensively explored the glioprotective effects of sulforaphane on astroglial cells, reinforcing the beneficial effects of sulforaphane on astroglial functionality.

Gliotoxicity and Glioprotection: the Dual Role of Glial Cells.

Glial cells (astrocytes, oligodendrocytes and microglia) are critical for the central nervous system (CNS) in both physiological and pathological conditions. With this in mind, several studies have indicated that glial cells play key roles in the development and progression of CNS diseases. In this sense, gliotoxicity can be referred as the cellular, molecular, and neurochemical changes that can mediate toxic effects or ultimately lead to impairment of the ability of glial cells to protect neurons and/or other glial cells. On the other hand, glioprotection is associated with specific responses of glial cells, by which they can protect themselves as well as neurons, resulting in an overall improvement of the CNS functioning. In addition, gliotoxic events, including metabolic stresses, inflammation, excitotoxicity, and oxidative stress, as well as their related mechanisms, are strongly associated with the pathogenesis of neurological, psychiatric and infectious diseases. However, glioprotective molecules can prevent or improve these glial dysfunctions, representing glial cells-targeting therapies. Therefore, this review will provide a brief summary of types and functions of glial cells and point out cellular and molecular mechanisms associated with gliotoxicity and glioprotection, potential glioprotective molecules and their mechanisms, as well as gliotherapy. In summary, we expect to address the relevance of gliotoxicity and glioprotection in the CNS homeostasis and diseases.