An in vitro model for analysis of oxidative death in primary mouse astrocytes.
Astrocytes provide a vital protective function in the brain. These cells are also vulnerable to oxidative stress, thus their loss of function could contribute to neurodegeneration. The goal of this study is to develop a cell culture model to study oxidative stress in astrocytes. Enriched astrocytic cultures were generated from neonatal mice. tertiary-butyl hydroperoxide (t-bOOH) was used as an exogenous peroxide and lactate dehydrogenase (LDH) release as a measure of loss of viability. Exposure to t-bOOH resulted in a linear increase in astrocytic death reaching 91.2% after 4 h exposure. That cell death was due to oxidative injury, was shown by the ability of the antioxidant N,N'-diphenyl-1,4-phenylenediamine (DPPD) to protect the t-bOOH treated cells. The involvement of iron in cell toxicity was demonstrated by the ability of the iron specific chelator desferal (DF) to completely prevent t-bOOH induced LDH release. Cells treated with a lipid soluble iron compound 3,5, 5-trimethyl (hexanoyl) ferrocene (TMH-Ferrocene), were more vulnerable to t-bOOH whereas neither ferrous ammonium sulfate (FAS) nor ferric ammonium citrate (FAC) had an effect. The increased sensitivity of the cells exposed to TMHF was reversible with the iron chelator desferal. Addition of recombinant human heavy chain ferritin or human apo-transferrin (Tf) did not alter LDH release. Electron microscopic analysis indicated astrocytes exposed to t-bOOH exhibited mitochondrial swelling prior to cell death (lactate dehydrogenase release). Additional increases in mitochondrial swelling were seen when the astrocytes were exposed to the lipophilic iron compound TMH-ferrocene and t-bOOH. These studies show that astrocytes are exquisitely sensitive to oxidative stress and that their vulnerability is related to and enhanced by iron. Decreased mitochondrial function in response to oxidative stress may precede cell death.
Robb
S J
SJ
George M. Leader Family Laboratory, Department of Neuroscience and Anatomy, Penn State University College of Medicine, M.S. Hershey Medical Center, Hershey, PA 17033, USA.
Connor
J R
JR
D000818:Animals; D001253:Astrocytes / Q000473:pathology*; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D007501:Iron / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000648:ultrastructure; D008954:Models, Biological; D018384:Oxidative Stress / Q000502:physiology*; D010545:Peroxides / Q000633:toxicity; D011485:Protein Binding; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide
D010545:Peroxides; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D007501:Iron