079 - Evaluating the Contribution of Chorioamnionitis to the Lung Phenotype of the Premature Rabbit Model of Bronchopulmonary Dysplasia.

Publication Number: 79.4766

Francesca Stretti, Chiesi Farmceutici, Parma, Emilia-Romagna, Italy; Giorgio Aquila, Chiesi Farmaceutici Spa, Parma, Emilia-Romagna, Italy; Emilie Goffinon, Katholieke Universiteit Leuven, Schaerbeek, Brussels Hoofdstedelijk Gewest, Belgium; Yannick Regin, KU LEUVEN, Leuven, Vlaams-Brabant, Belgium; Marnel Greyling, KU Leuven, Leuven, Vlaams-Brabant, Belgium; Gino Villetti, Chiesi Farmaceutici, Parma, Emilia-Romagna, Italy; Xabier Murgia Esteve, Consultant, Berango, Pais Vasco, Spain; Jaan Toelen, KU Leuven, Leuven, Vlaams-Brabant, Belgium; Francesca Ricci, chiesi farmaceutici S.p.A, PARMA, Emilia-Romagna, Italy

Background: Chorioamnionitis (CA) is an inflammation of fetal membranes caused by intrauterine bacterial infections. It underlies up to 70% of premature births and predisposes premature infants to bronchopulmonary dysplasia (BPD). CA, in combination with postnatal insults like mechanical ventilation and oxygen therapy, increases the risk of developing BPD significantly.

Objective: The study aimed to establish a novel preterm rabbit BPD multi-hit model, including prematurity, hyperoxia, and CA, to mimic this clinical scenario.

Design/Methods: Pregnant rabbits received an intra-amniotic injection of either lipopolysaccharide (LPS 1, 2.5, or 5 μg/sac) or saline (control) three days prior to delivery. All rabbits were delivered prematurely at 28 days of gestation (term 31 days) and exposed to 50% O2 until postnatal day (PND) 7. Body weight, survival, pulmonary function, histology, and biomolecular outcomes were assessed.

Results: The intra-amniotic dosing of LPS at 5 µg/sac resulted in a higher mortality of the fetuses at birth (Figure 1A) and a meager survival rate at PND7 compared to the 1 and 2.5 µg/sac LPS doses (Figure 1B). Preterm rabbits injected with both the 1 and 2.5 μg/sac LPS doses showed a partial reduction of body weight at birth (33g ± 4.6 and 33g ± 6, respectively), compared to controls (35.8g ± 4.7), which did not persist at PND7. Both the 1 and 2.5 μg/sac LPS doses slightly improved inspiratory capacity (Figure 2A) and reduced tissue damping (Figure 2B) in preterm rabbits compared to controls at PND7. However, both LPS doses worsened preterm rabbits’ lung morphology at PND7, as evidenced by the higher mean wall transection length in LPS groups, a measure of the interalveolar septal thickness (Figure 2C). LPS (2.5 μg/sac) significantly increased TGF-β and TNF-α mRNA levels but did not affect VEGFA mRNA expression levels (Figure 3).

Conclusion(s): The antenatal LPS dose of 2.5 µg/sac seems the most promising for establishing a model of CA in the preterm rabbit, as evidenced by the increased width of the alveolar septa and the persistent inflammation. However, preliminary results suggest a paradoxical effect with this dose, as indicated by ameliorated lung function parameters, which require confirmation in further experimental sessions.

Figure 1. Survival
Survival in premature rabbits at birth (A) and at PND7 (B) in saline-injected does (n=18) and LPS-injected does at 1 μg/sac (LPS 1 μg/sac, n=6), 2.5 μg/sac (LPS 2.5 μg/sac, n=13), or 5 μg/sac (LPS 5 μg/sac, n=1), following 7-days exposure to 50% O2.

Figure 2. Lung function and histomorphometry
(A) Total inspiratory capacity (IC/body weight), (B) tissue damping (G) and (C) mean wall transection length (Lmw) at PND7 in premature rabbits exposed to 50% O2 from saline-injected does (saline, n=18) or from LPS-injected does at 1 μg/sac (LPS 1 μg/sac, n=6), or 2.5 μg/sac (LPS 2.5 μg/sac, n=13). Results are presented as mean ± SEM and analyzed with one-way ANOVA, corrected for multiple comparisons. *p < 0.05, **p < 0.01.

Figure 3. qPCR analysis
qPCR analysis of TNF-α (A), TGF-β (B) and VEGFA (C) expression at PND7 in premature rabbits exposed to 50% O2 from saline-injected does (saline, n=18), or from LPS-injected does at 2.5 μg/sac (LPS 2.5 μg/sac, n=13). Results are presented as mean ± SEM and analyzed with t-test. **p < 0.01.