Superoxide (*O2- ) production in CA1 neurons of rat hippocampal slices exposed to graded levels of oxygen.
Neuronal signaling, plasticity, and pathologies in CA1 hippocampal neurons are all intimately related to the redox environment and, thus tissue oxygenation. This study tests the hypothesis that hyperoxic superfusate (95% O(2)) causes a time-dependent increase in superoxide anion (*O(2)(-)) production in CA1 neurons in slices, which will decrease as oxygen concentration is decreased. Hippocampal slices (400 microm) from weaned rats were incubated with the fluorescent probe dihydroethidium (DHE), which detects intracellular *O(2)(-) production. Slices were loaded for 30 min using 10 microM DHE and maintained using one-sided superfusion or continuously loaded using 2.5 microM DHE and maintained using two-sided superfusion (36 degrees C). Continuous loading of DHE and two-sided superfusion gave the highest temporal resolution measurements of *O(2)(-) production, which was estimated by the increase in fluorescence intensity units (FIUs) per minute (FIU/min +/- SE) over 4 h. Superoxide production (2.5 microM DHE, 2-sided superfusion) was greatest in 95% O(2) (6.6 +/- 0.4 FIU/min) and decreased significantly during co-exposure with antioxidants (100 microM melatonin, 25 microM MnTMPyP) and lower levels of O(2) (60, 40, and 20% O(2) at 5.3 +/- 0.3, 3.3 +/- 0.1, and 1.6 +/- 0.2 FIU/min, respectively). CA1 cell death after 4 h (ethidium homodimer-1 staining) was greatest in 95% O(2) and lowest in 40 and 20% O(2). CA1 neurons generated evoked action potentials in 20% O(2) for >4 h, indicating viability at lower levels of oxygenation. We conclude that .O(2)(-) production and cell death in CA1 neurons increases in response to increasing oxygen concentration product (= PO(2) x time). Additionally, lower levels of oxygen (20-40%) and antioxidants should be considered to minimize superoxide-induced oxidative stress in brain slices.
D'Agostino
Dominic P
DP
Department of Molecular Pharmacology and Physiology, College of Medicine, University of South Florida, Tampa, Florida 33612, USA.
Putnam
Robert W
RW
Dean
Jay B
JB
D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004996:Ethidium / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D006624:Hippocampus / Q000166:cytology*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D009474:Neurons / Q000187:drug effects*; D010100:Oxygen / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D014475:Uncoupling Agents / Q000494:pharmacology
D000975:Antioxidants; D014475:Uncoupling Agents; C067883:dihydroethidium; D013481:Superoxides; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D004996:Ethidium; D010100:Oxygen