Extended exposure to low pO2
A custom designed pO2 modulation chamber (7 star systems, Delhi, India) was used. A constant temperature (25±5 ˚C) and relative humidity (50±5%) were maintained in the simulation chamber during experiments. Airflow of 2 L/min was provided to Sprague-Dawley (SD) rats undergoing hypobaric hypoxia exposure. The animals were provisioned with food and water ad libitum during the entire duration of low pO2 exposure. Age and gender matched male SD rats were exposed to either normoxia (n=5) or low pO2 of 8.19 kPa (equivalent altitude: 7620 m; simulated). Exposure lasted for 3 days (HD3), 7 days (HD7) and 14 days (HD14) (n=5 per group) of stay in the pO2 chamber. The rate of decrease of PB was ~4.1 kPa/min. Exposure to PB 40 kPa (pO2 8.19 kPa) began after 12 h each at PB 71 kPa and PB 59 kPa. The purpose of such a slow and graded low pO2 exposure was to minimize mortality of animals and mimic real-world conditions of altitude ascent. After the completion of normal and low pO2 exposures, the animals were sacrificed and their lungs and plasma collected. Further, quantitative proteomics investigations, biochemical assays, assessment of redox specific transcripts and validations using immuno-chemical methods were performed on these. All animals were sacrificed using approved procedures to minimize pain and suffering immediately after the exposure. Our study design was initially approved by the Institute’s Animal Ethics Committee (Fig. 1).