title: Ascorbate protects neurons against oxidative stress: a Raman microspectroscopic study.
abstract: Oxidative stress due to excessive accumulation of reactive oxygen or nitrogen species in the brain as seen in certain neurodegenerative diseases can have deleterious effects on neurons. Hydrogen peroxide, endogenously generated in neurons under normal physiological conditions, can produce an excess of hydroxyl radical via a Fenton mediated mechanism. This may induce acute oxidative injury if not scavenged or removed effectively by antioxidants. There are several biochemical assay methods to estimate oxidative injury in cells; however, they do not provide information on the biochemical changes as the cells get damaged progressively under oxidative stress. Raman microspectroscopy offers the possibility of real time monitoring of the chemical composition of live cells undergoing oxidative stress under physiological conditions. In the present study, a hippocampal neuron coculture was used to observe the acute impact of hydroxyl radicals generated by hydrogen peroxide in the presence of Fe(2+) (Fenton reaction). Raman peaks related to nucleic acids (725, 782, 1092, 1320, 1340, 1420, and 1576 cm(-1)) showed time-dependent changes over the experimental period (60 min), indicating the breakdown of the phosphodiester backbone as well as nuclear bases. Interestingly, ascorbic acid (a potent antioxidant) when cotreated with Fenton reactants showed protection of cells as inferred from the Raman spectra, presumably by scavenging hydroxyl radicals. Little or no change in the Raman spectra was observed for untreated control cells and for cells exposed to Fe(2+) only, H2O2 only, and ascorbate only. A live-dead assay study also supported the current observations. Hence, Raman microspectroscopy has the potential to be an excellent noninvasive tool for early detection of oxidative stress that is seen in neurodegenerative diseases.
authors.forename: Abhaya
authors.initials: A
authors.lastname: Dutta
authors.forename: Rekha
authors.initials: R
authors.lastname: Gautam
authors.forename: Sreejata
authors.initials: S
authors.lastname: Chatterjee
authors.affiliation: LaserLaB, Faculty of Sciences, VU University Amsterdam , 1081 HV Amsterdam, The Netherlands.
authors.forename: Freek
authors.initials: F
authors.lastname: Ariese
authors.forename: Sujit Kumar
authors.initials: SK
authors.lastname: Sikdar
authors.forename: Siva
authors.initials: S
authors.lastname: Umapathy
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology*; D002412:Cations / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D018920:Coculture Techniques; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D017665:Hydroxyl Radical / Q000378:metabolism; D007501:Iron / Q000633:toxicity; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology*; D017208:Rats, Wistar; D013059:Spectrum Analysis, Raman / Q000379:methods; D013997:Time Factors
chemical_list: D000975:Antioxidants; D002412:Cations; D018696:Neuroprotective Agents; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D007501:Iron; D001205:Ascorbic Acid
keywords: Raman spectroscopy; hippocampal neuron; neurodegenerative disease; oxidative stress; reactive oxygen species