Redefining control O2 conditions for rat medullary slices via quantification of oxidative and nitrosative stress: experiments in the caudal solitary complex (cSC)

Abstract

We have postulated that the control oxygen level (95%) currently used in brain slices is too high, potentially modulating redox sensitive systems and inducing redox stress. This can lead to hyperexcitability, increased metabolism, and cell death. To test this hypothesis, we have decreased our control oxygen level from 95% to 40% while using electrophysiology, amperometry (WPI Apollo 4000), and real‐time fluorescence imaging to measure changes in the cell membrane and levels of reactive oxygen and nitrogen species (RO/NS) production. At 6mL/min, pO2 measurements in the aCSF superfusion bath were 283.9 ± 0.13mmHg (mean ± SEM) at 40% O2 and increased to 593.3 ± 0.38mmHg in 95% O2. pO2 measurements recorded at the surface of the tissue slice were 119.18 ± 0.5mmHg in 40% O2 and increased to 356.2 ± 1.2mmHg in 95% O2. The decrease in recorded pO2 when measurements were taken on the surface of the slice indicates active O2 consumption by the tissue. Nitric oxide levels also significantly increased from 56.43 ± 0.14nM in 40% O2 to 65.45 ± 0.16nM in 95% O2. Electrophysiology recordings indicate that 95% O2 stimulates firing rate in ~70% of cSC neurons tested. Overall, use of a 40% control level of oxygen has proven to be effective as slices are still viable, showing normal electrical activity in addition to active consumption of oxygen and subsequent increases in production of RO/NS. (ONR)