The same surgical procedures were performed as described above and rats were assigned into 2 groups at 10 min after CPR (Fig. 1 C): (1) The post-resuscitation normoxic therapy group (n = 6) included those that were successfully resuscitated from 10-min asphyxia CA and treated with inhaled 30% oxygen (CA-Normo) following the initial 10 min of 100% oxygen, and (2) The post-resuscitation hyperoxia group (n = 6) included those treated with inhaled 100% oxygen (CA-Hypero) during the entire observational period. For all rats, the brain and heart tissues were collected at 2 h after CPR for mRNA extraction, followed by complementary DNA (cDNA) synthesis and real-time PCR. Additionally, tissues of control (naive) rats were collected to create a reference for mRNA gene expression.
RNA isolation, reverse transcription, and real-time PCR analysis for the brain, heart, and lung samples extracted at 2 h after CA were performed according to manufacturer instructions. Briefly, total RNAs were extracted and reverse transcribed using TRIzol Reagent® (Invitrogen, Carlsbad, CA) and SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (Invitrogen, Carlsbad, CA), respectively. Real-time PCR was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems™, Waltham, MA) on the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany). All primers were purchased from Thermofisher: Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, TaqMan Assay ID: Rn01775763_g1), Interleukin-1 beta (Il1b, Rn00580432_m1), Interleukin-6 (Il6, Rn01410330_m1), Transforming growth factor-beta 1 (Tgfb1, Rn00572010_m1), Intercellular adhesion molecule-1 (Icam1, Rn00564227_m1), Nuclear factor-kappa beta 1 (Nfkb1, Rn01399583_m1), Tumor necrosis factor (Tnf) receptor-associated factor-6 (Traf6, Rn01512911_m1), Caspase-3 (Casp3, Rn00563902_m1), Caspase-9 (Casp9, Rn00581212_m1), Epidermal growth factor (Egf, Rn00563336_m1), and B-cell leukemia/lymphoma-2 (Bcl2) associated X protein (Bax, Rn02532082_g1).
RNA isolation, reverse transcription, and real-time PCR analysis for the brain, heart, and lung samples extracted at 2 h after CA were performed according to manufacturer instructions. Briefly, total RNAs were extracted and reverse transcribed using TRIzol Reagent® (Invitrogen, Carlsbad, CA) and SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme (Invitrogen, Carlsbad, CA), respectively. Real-time PCR was performed using TaqMan™ Fast Advanced Master Mix (Applied Biosystems™, Waltham, MA) on the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany). All primers were purchased from Thermofisher: Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, TaqMan Assay ID: Rn01775763_g1), Interleukin-1 beta (Il1b, Rn00580432_m1), Interleukin-6 (Il6, Rn01410330_m1), Transforming growth factor-beta 1 (Tgfb1, Rn00572010_m1), Intercellular adhesion molecule-1 (Icam1, Rn00564227_m1), Nuclear factor-kappa beta 1 (Nfkb1, Rn01399583_m1), Tumor necrosis factor (Tnf) receptor-associated factor-6 (Traf6, Rn01512911_m1), Caspase-3 (Casp3, Rn00563902_m1), Caspase-9 (Casp9, Rn00581212_m1), Epidermal growth factor (Egf, Rn00563336_m1), and B-cell leukemia/lymphoma-2 (Bcl2) associated X protein (Bax, Rn02532082_g1).
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