Superoxide (•O2-) production in CA1 neurons of rat hippocampal slices exposed to graded levels of oxygen
Superoxide (•O2-) production in CA1 neurons of rat hippocampal slices exposed to graded levels of oxygen
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DOI：
10.1152/jn.01003.2006
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发表时间：
2007-08-01
影响因子：
2.5
通讯作者：
Dean, Jay B.
中科院分区：
文献类型：
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作者：
D'Agostino, Dominic P.;Putnam, Robert W.;Dean, Jay B.
CA1 海马神经元的神经信号、可塑性和病理学都与氧化还原环境以及组织氧合作用密切相关。本研究检验了这样的假设：高氧超融合液 (95% O-2) 会导致切片中 CA1 神经元超氧阴离子（中心点 O-2(-)）产生随时间依赖性增加，并且随着氧浓度的降低而减少。将断奶大鼠的海马切片（400μm）与荧光探针二氢乙锭（DHE）一起孵育，该探针可检测细胞内中心点O-2（-）的产生。使用10μM DHE加载切片30分钟并使用一侧灌注维持或使用2.5μM DHE连续加载并使用两侧灌注维持（36℃）。 DHE 的连续加载和两侧灌注给出了中心点 O-2(-) 产生的最高时间分辨率测量，这是通过 4 小时内每分钟荧光强度单位 (FIU) (FIU/min +/- SE) 的增加来估计的。超氧化物的产生（2.5 μM DHE，两侧灌注）在 95% O-2 中最大（6.6 +/- 0.4 FIU/min），并且在与抗氧化剂（100 μM 褪黑激素、25 μM MnT-MPyP）共同暴露和较低水平的 O-2（60、40 和 20% O-2，5.3 +/- 0.3，分别为 3.3 +/- 0.1 和 1.6 +/- 0.2 FIU/分钟）。 4 小时后（乙锭同二聚体-1 染色）CA1 细胞死亡在 95% O-2 中最大，在 40 和 20% O-2 中最低。 CA1 神经元在 20% O-2 中产生诱发动作电位 > 4 小时，表明在较低氧合水平下的活力。我们得出结论，CA1 神经元中心点 O-2(-) 的产生和细胞死亡随着氧浓度乘积（= PO2 x 时间）的增加而增加。此外，应考虑较低水平的氧气（20-40%）和抗氧化剂，以尽量减少脑切片中超氧化物诱导的氧化应激。
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 (center dot O-2(-)) production in CA1 neurons in slices, which will decrease as oxygen concentration is decreased. Hippocampal slices (400 mu m) from weaned rats were incubated with the fluorescent probe dihydroethidium (DHE), which detects intracellular center dot O-2(-) production. Slices were loaded for 30 min using 10 mu M DHE and maintained using one-sided superfusion or continuously loaded using 2.5 mu M 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 center dot 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 mu M 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 mu M melatonin, 25 mu M MnT-MPyP) 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 center dot O-2(-) production and cell death in CA1 neurons increases in response to increasing oxygen concentration product (= PO2 x time). Additionally, lower levels of oxygen (20-40%) and antioxidants should be considered to minimize superoxide-induced oxidative stress in brain slices.