Antenatal Endotoxin Impairs Lung Mechanics and Increases Sensitivity to Ventilator-Induced Lung Injury in Newborn Rat Pups

Perinatal inflammation due to chorioamnionitis and ventilator-induced lung injury (VILI) at birth is independent risk factors for the development of bronchopulmonary dysplasia (BPD). We have previously shown that antenatal endotoxin (ETX) causes abnormal lung structure and function in 2-week-old rats, but whether ETX impairs lung mechanics at birth and increases risk for VILI is unknown. Fetal rats were exposed to 10 μg endotoxin or saline via intra-amniotic injection. At birth (D0) or 7 days (D7), rats received 90 min of lung protective ventilation [PROTECT group; tidal volume (Vt) = 6 ml/kg with positive end expiratory pressure (PEEP) = 2 cmH2O]; P20 ventilation [plateau pressure (Pplat) = 20 cmH2O, PEEP = 0]; or P24 ventilation (Pplat = 24 cmH2O, PEEP = 0, only applied to D7). Prior to prolonged ventilation at D0, endotoxin-exposed rats had decreased compliance and inspiratory capacity (IC) compared to controls. At D7, endotoxin was associated with reduced compliance. High-pressure ventilation (P20 and P24) tended to increase IC and compliance in all saline-treated groups. Ventilation at D0 with P20 increased IC and compliance when applied to saline-treated but not endotoxin-exposed pups. At D7, P24 ventilation of endotoxin-exposed pups increased elastance, bronchoalveolar lavage protein content, and IL-1b and TEN-C mRNA expression in comparison to the saline group. In summary, antenatal endotoxin exposure alters lung mechanics at birth and 1 week of life and increases susceptibility to VILI as observed in lung mechanics, alveolocapillary barrier injury, and inflammatory mRNA expression. We speculate that antenatal inflammation primes the lung for a more marked VILI response, suggesting an adverse synergistic effect of antenatal and postnatal exposures.

Supplementary Figure S1. Pulmonary system elastance (H) measured prior to prolonged ventilation at D0 was significantly elevated (A, n=18-25). This difference did not persist at D7 (C. n=23-25). The ETX-P24 group demonstrated increased elastance (demarcated by shaded boxes and red arrows) that was significantly greater than CTL-P24 and the other ETX ventilation groups (n=6-9). Figure S2. Lung tissue damping (G) was significantly elevated by ETX at D0 prior to prolonged ventilation (A, n=18-25). Ventilation at D0 tended to increase G (indicated by shaded boxes and upward pointing red arrows) and significant differences were present between CTL and ETX for . At D7, the CTL animals showed decreased G with P20 and P24 ventilation. Figure S3. Central airway resistance (RN) was significantly elevated by ETX at D7 prior to ventilation (n=23-25). D0 did not show significant ETX effects (n=18-24). PROTECT ventilation at D7 caused a modest but significant decrease in RN as indicated by shaded boxes and downward pointing triangles (n=8). Ventilation of CTL-P20 (D0) and CTL-P24 (D7) rats caused a modest increase in RN that is indicated by upward pointing triangles.

Quasi-static Compliance:
The quasi-static compliance calculated at an airway pressure of 3 cmH2O (Cst3) followed the same trends as Cst6 ( Figure S4). Prior to ventilation, D0 ETX animals demonstrated a marked decrease in lung compliance that did not persist at D7. Ventilation of the D0 pups caused increased compliance in all but the CTL-PROTECT group, and the CTL-P20 animals showed higher Cst3 than ETX-P20. At D7, increasing ventilation pressure tended to increase Cst3 with the exception of ETX-P24. Figure S4. Pulmonary system compliance at a pressure of 3 cmH2O (Cst3) is significantly decreased by ETX at D0 prior to prolonged ventilation (A, n=23-27). Ventilation tended to increase Cst3 (indicated by shaded boxes and red triangles). P24 ventilation at D7 increased Cst3 in CTL but not in ETX.

Tidal Volume:
The delivered tidal volume (scaled by body weight) was calculated from ventilator waveform data that was corrected for cannula resistance and gas compressibility by the ventilator software. During PROTECT ventilation ( Figure S5A) Vt was elevated slightly above the prescribed 6 mL/kg and the D7-CTL animals showed significantly higher PROTECT Vt than D0-CTL. During P20 ventilation ( Figure S5B, blue) the D0 animals showed significantly higher Vt than the D7 animals. However, D0-P20 and D7-P24 tidal volumes were similar, suggesting that those two groups may be compared directly. Figure S5. Delivered tidal volume for D0 (grey shaded) and D7 pups during PROTECT (green, n = 11-17), P20 (blue, n = 8-14), and P24 ventilation (red, n = 8). Shaded boxes indicate endotoxin treatment (ETX).

Lung Neutrophil Content:
Myeloperoxidase (MPO) activity in lung homogenate was measured to assess lung neutrophil content ( Figure S6) and no significant differences were observed. At D0, there was a trend towards increased MPO activity in the lungs of animals exposed to antenatal ETX prior to postnatal ventilation. At D7, there was trend towards increased MPO activity with increasing ventilation pressure from PROTECT to P20 to P24. Figure S6. Measurement of Myeloperoxidase (MPO) activity in whole lung tissue of animals at birth (D0) and day 7 of postnatal life (D7). There was a not a significant change in MPO activity in animals exposed to antenatal endotoxin (ETX) and postnatal mechanical ventilation as compared to animals who received postnatal mechanical ventilation alone at D0 or D7 (n=4-5).

Supplementary
Lung Structure: At D7 prior to ventilation, ETX increased the mean linear intercept (MLI, Figure S7) and there was a trend towards decreased radial alveolar counts (RAC, Figure S8). Both of these findings point towards alveolar simplification with ETX treatment. Mechanical ventilation of D7-ETX caused a decrease in the MLI which, given that total lung volumes tended to increase, was an unexpected finding. It is possible that this observation stems from differential structural changes in the alveoli and alveoli ducts. Figure 8 shows substantial enlargement of the alveolar ducts due to the retraction of the alveolar septa. Our prior studies in mouse ventilator induced lung injury demonstrate a reduction in alveolar size while volume is shifted to the ducts (Smith et al., 2020). In that case, the MLI could conceivably decrease as there are many more alveoli than ducts in the numeric average. Technical aspects of the tissue preparation, such as variable lung inflation levels, could also explain contribute to this somewhat unexpected result. At D0, no significant changes were observed in the MLIs. Figure S7. Mean linear intercept (MLI) prior to mechanical ventilation (NV) was increased by antenatal endotoxin (ETX) at day 7 of life (D7). In the D7-ETX pups, mechanical ventilation (PROTECT and P24) decreased the MLI. At D0, no significant alterations were observed. (n=3-6). Figure S8. Radial alveolar counts (RACs) at day 7 of life (D7) are a measure of lung complexity. Prior to mechanical ventilation, antenatal endotoxin treatment (ETX) tended to decrease the RAC. Mechanical ventilation tended to decrease RAC in both ETX and control (CTL) pups. n=3.

Supplementary Tables:
The supplementary tables show the number of animals (n), the mean, and the standard error (SE). Table S1 shows lung mechanics derived from the quasi-static pressure volume loops (IC,Cst3,Cst6,and Cst15). Table S2 summarizes the results of the FOT measurements. Table S3 details the BAL protein  measurements in the D7 rats, and Tables S4 and S5 show gene expression for both D0 and D7.