Detection of CO by gas chromatography/mass spectrometry.

For in vivo detection of CO levels in the cerebral circulation in newborn pigs, samples of pCSF (0.4 ml) were collected from the brain surface underneath the cranial window at 10-min intervals during the 2.5-h experimentation period that included basal normoxia conditions, asphyxia, and reventilation. To detect in vivo astrocytic CO production during normoxic, asphyxic, and reventilation periods, cortical astrocytes were freshly isolated from the brains of normoxic and 1-h asphyxic animals. Samples of pCSF, freshly isolated cortical astrocytes, or astrocytes cultured on Cytodex beads were placed in Thermo Scientific 9-mm leak-proof vessels, sealed with tight rubber seals, and incubated with Krebs solution for 1 h at 37°C. The ¹³C¹⁸O standard was added to all samples for the purpose of CO quantification. The concentration of CO in the headspace gas was detected based on the peak areas corresponding to ¹²C¹⁶O and ¹³C¹⁸O using an Agilent 5975 GC/MSD ChemStation (Agilent Technologies, Santa Clara, CA) and normalized to the protein amount in the samples, as previously described in detail elsewhere (26, 48).