Abstract
Astrocytes, a major class of glial cells, regulate neurotransmitter systems, synaptic processing, ion homeostasis, antioxidant defenses and energy metabolism. Astrocyte cultures derived from rodent brains have been extensively used to characterize astrocytes' biochemical, pharmacological and morphological properties. The aims of this study were to develop a protocol for routine preparation and to characterize a primary astrocyte culture from the brains of adult (90 days old) Wistar rats. For this we used enzymatic digestion (trypsin and papain) and mechanical dissociation. Medium exchange occurred from 24 h after obtaining a culture and after, twice a week up to reach the confluence (around the 4th to 5th week). Under basal conditions, adult astrocytes presented a polygonal to fusiform and flat morphology. Furthermore, approximately 95% the cells were positive for the main glial markers, including GFAP, glutamate transporters, glutamine synthetase and S100B. Moreover, the astrocytes were able to take up glucose and glutamate. Adult astrocytes were also able to respond to acute H2O2 exposure, which led to an increase in reactive oxygen species (ROS) levels and a decrease in glutamate uptake. The antioxidant compound resveratrol was able to protect adult astrocytes from oxidative damage. A response of adult astrocytes to an inflammatory stimulus with LPS was also observed. Changes in the actin cytoskeleton were induced in stimulated astrocytes, most likely by a mechanism dependent on MAPK and Rho A signaling pathways. Taken together, these findings indicate that the culture model described in this study exhibits the biochemical and physiological properties of astrocytes and may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, as well as investigating compounds involved in cytotoxicity and cytoprotection.
Highlights
Astrocytes are key cells in the central nervous system (CNS) involved in the maintenance of the extracellular environment and in the stabilization of cell-cell communications under physiological and pathological conditions. [1,2,3,4,5]
Polyclonal anti-GFAP was from Dako; monoclonal anti-NeuN was from Millipore; monoclonal anti-CD11 was from Invitrogen; polyclonal anti-GLAST and anti-GLT1 were from Alpha Diagnostic; monoclonal anti-Aldehyde dehydrogenase family 1 member L1 was from Neuromab; monoclonal antiGAPDH was from Chemicon
Monotypic CNS cell cultures have significantly contributed to the understanding of brain properties
Summary
Astrocytes are key cells in the central nervous system (CNS) involved in the maintenance of the extracellular environment and in the stabilization of cell-cell communications under physiological and pathological conditions. [1,2,3,4,5]. Astrocytes regulate neurotransmitter systems; ionic homeostasis; the metabolic support of neurons; energy metabolism; synaptic information processing, plasticity and neuronal excitability; detoxification; defense against oxidative stress; metal sequestration; maintenance of the blood-brain barrier; guidance of neuronal migration; and immune function [5,6,7]. These versatile cells are the most resilient cells in the CNS [7]. Once taken up by astrocytes, glutamate is converted into glutamine by glutamine synthetase (GS – EC 6.3.1.2) [15,16,17,18]. Glutamate uptake is important for maintaining the levels of glutathione (GSH), a major antioxidant molecule in the brain [15,16,19,20]
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