335 - Changes in Signaling molecular Pathways and development of lung fibrosis and alveolar simplification in Neonatal Mouse Model after Prolonged hyperoxia exposure

Publication Number: 1319.335

Suraj Karanje, Michigan State University, Lansing, MI, United States; Ranga Prasanth Thiruvenkataramani, Michigan State University College of Human Medicine, Lansing, MI, United States; Amal Abdul-Hafez, Michigan State University, East Lansing, MI, United States; Burra V. Madhukar, Michigan State University College of Human Medicine, E. Lansing, MI, United States; Said Omar, Michigan State University College of Human Medicine, Lansing, MI, United States; Hady Omar, Michigan State University College of Human Medicine, East Lansing, MI, United States; James Wagner, Michigan State University, East Lansing, MI, United States

Background: Prolonged hyperoxia in premature neonates is associated with lung injury and bronchopulmonary dysplasia (BPD). Several studies have suggested peroxisome proliferator activated receptor gamma (PPARg) and aryl hydrocarbon receptor (AhR) signaling pathways to play a role in protection against lung injury and development of BPD

Objective: To evaluate the temporal progression of lung fibrosis, alveolar development and protein & mRNA expression of protective signaling pathways in neonatal mice exposed to hyperoxia

Design/Methods: Neonatal mice were exposed to N:normoxia 21% or H:hyperoxia (85% O₂ continuously from postnatal day 1 (P1) to P28. Lungs were collected at P7, P10, P14, and P28 (n = 5/ group). Collagen density was quantified on Masson's Trichrome-stained lung sections to determine the % of lung fibrosis. Alveolarization was assessed using the radial alveolar count (RAC). Lung tissues were homogenized for total RNA & protein extraction. qRT-PCR and Western blotting were performed to detect mRNA and protein levels of PPARγ, NFATc1,Nrf2,AhR,Cyp1a1,and RelB. Data were analyzed using ANOVA and post hoc using Tukey test

Results: Hyperoxia caused progressive septal thickening, fibrosis and alveolar simplification. The collagen density in the lung increased in H group from 1.5 to 6.6% (p <.001) at P7 & P28 V in N group .6 to 2.5%. The difference was more significant after P14 (fig-1). RAC was lower in H group from P7 to P28 (5.8 vs 3.3;p <.01) and N group showed relatively preserved alveolarization (8.1 vs 6.5; p<.01). H compared with N group had higher protein and mRNA levels of PPARγ on P7 & 28 and lower on P10 & 14 in both groups whereas NFATc1 mRNA levels peaked on P10 in both groups. The mRNA levels were lower in the H group. NFATc1 protein levels increased on P10,14 and peaked on P28 in H compared with N group. Nrf2 protein levels increased compared with N at all time-points, but mRNA did not change in response to hyperoxia. AhR mRNA and protein levels decreased after hyperoxia exposure. The AhR target gene, Cyp1a1 mRNA level decreased in hyperoxia in P10,14 and 28 and protein levels showed a slight increase. The inflammatory mediator RelB gene mRNA increased in H group P7 preceding the increase of RelB protein on P10

Conclusion(s): Our data demonstrates that prolonged hyperoxia exposure leads to interstitial lung fibrosis, impaired alveolar formation, and increased alveolar simplification. With prolonged hyperoxia, defense mechanisms involving activation or crosstalk between AhR and Nrf2 and between Nrf2 and PPARγ signaling pathways, but this is insufficient to prevent lung parenchymal damage

Figure-1: Fibrosis in lung parenchyma using Masson staining
Increasing fibrosis noted from D7 to D28 in hyperoxia exposed neonatal mice and is statistically significant

Table-1: Transcription factor changes in Neonatal mouse exposed to hyperoxia


Figure-2: Degree of fibrosis in neonatal mice lung parenchyma
There is a postnatal increase in fibrosis in both normoxia and hyperoxia groups overtime with p<0.001 using ANOVA with repeated measures. There is a significant increase in fibrosis between normoxia and hyperoxia on Day 7,10,14,28 with p<0.001.