Sex‐Dependent Differences in Inflammation One‐Year After Post‐Natal Hyperoxia Exposure in Rats
Authors: Wanek Sarah Catherine , Braun Rudolf , Goss Kara N. , Eldridge Marlowe 
Published: April 2020

Rationale

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth. It is induced by oxidative stress caused by hyperoxia exposure during the vulnerable saccular or early alveolar stage of lung development resulting in alveolar growth arrest and initiation of cellular senescence. Senescence is characterized by inhibition of the cell cycle through 2 major tumor suppressor pathways: the proteins p53/p21 and the p16Ink4a/retinoblastoma protein leading to stringent growth arrest. Previous results suggest that p16 is highly expressed in hyperoxia male rats and p21 is highly expressed in the hyperoxia female rats at one year. We hypothesized that hyperoxia‐induced senescence would lead to differences in inflammation in male or female rats as they age.

Methods

Newborn Sprague Dawley rats were exposed to hyperoxic (HYP, 85%, n=13) or normoxic (NORM, 21%, n=9) conditions for 14 days and then remained in normoxic conditions until they were 1‐year‐old. At 1 year, the left lung was inflated and fixed (4% formaldehyde) and the right lobes were flash frozen. Pulmonary tissue was preserved at −80°C until analyzed for mRNA expression of proteins p16, p21, and interleukin‐17 (IL‐17) via TaqMan qPCR. Differences in expression were calculated by the ddCt method using the reference gene GAPDH. Two‐way ANOVA was utilized for comparison of ddCt values between sexes in HYP and NORM rats for each target gene. Western blots were performed for quantification of p16 and p21 in the 1‐year‐old rats of both sexes and treatments. IL‐6 expression in the 1‐year‐old rats was assessed via an ELISA.

Results

Expression of p16 in the lung tissue mRNA was significantly higher in the 1‐year‐old HYP male rats compared to NORM male rats; whereas, the 1‐year‐old female rats of both treatments did not present a difference in p16 mRNA expression. At 1 year, p21 expression in the mRNA of HYP females and HYP males was higher compared to NORM females and NORM males with no difference between the sexes. The same results were observed in the level of protein at 1 year. At day 21, HYP males and HYP females had a significant upregulation of p16 in their mRNA compared to NORM rats (p<0.01), while p21 was slightly upregulated in the mRNA of HYP males and HYP females. IL‐17 was expressed in both NORM and HYP 1‐year‐old female mRNA. Contrarily, 1‐year‐old HYP male mRNA presented IL‐17 expression while there was no detectable expression in the mRNA of the 1‐year‐old NORM males. At day 21, there was no detectable IL‐17 expression in the mRNA of either sex of both treatment groups. The IL‐6 expression at 1 year was detected in the plasma of the HYP male rats, but was not detectable in the NORM male rats. Alternatively, the 1‐year‐old female rats of both treatments expressed IL‐6.

Conclusion

The significantly higher p16 expression in the 1‐year HYP male rats suggests that senescence is more evident in the HYP males compared to HYP female rats. Detectable IL‐6 as well as IL‐17 expression in the 1‐year‐old HYP male rats also may support this development of a severely high expression of p16 in this particular treatment and sex. The measurement of mRNA expression at intermediate time points would allow for further analysis of the development of senescence in this BPD model.

Support or Funding Information

NIH‐NHLBI R01‐HL115061