Rationale: The preterm lung is susceptible to injury during transition to air breathing at birth. It remains unclear whether rapid or gradual lung aeration at birth causes less lung injury. Objectives: To examine the effect of gradual and rapid aeration at birth on: 1) the spatiotemporal volume conditions of the lung; and 2) resultant regional lung injury. Methods: Preterm lambs (125 6 1 d gestation) were randomized at birth to receive: 1) tidal ventilation without an intentional recruitment (no-recruitment maneuver [No-RM]; n = 19); 2) sustained inflation (SI) until full aeration (n = 26); or 3) tidal ventilation with an initial escalating/de-escalating (dynamic) positive end-expiratory pressure (DynPEEP; n = 26). Ventilation thereafter continued for 90 minutes at standardized settings, including PEEP of 8 cm H 2 O. Lung mechanics and regional aeration and ventilation (electrical impedance tomography) were measured throughout and correlated with histological and gene markers of early lung injury. Measurements and Main Results: DynPEEP significantly improved dynamic compliance (P , 0.0001). An SI, but not DynPEEP or NoRM , resulted in preferential nondependent lung aeration that became less uniform with time (P = 0.0006). The nondependent lung was preferential ventilated by 5 minutes in all groups, with ventilation only becoming uniform with time in the NoRM and DynPEEP groups. All strategies generated similar nondependent lung injury patterns. Only an SI caused greater upregulation of dependent lung gene markers compared with unventilated fetal controls (P , 0.05). Conclusions: Rapidly aerating the preterm lung at birth creates heterogeneous volume states, producing distinct regional injury patterns that affect subsequent tidal ventilation. Gradual aeration with tidal ventilation and PEEP produced the least lung injury.

BackgroundCurrent sustained lung inflation (SI) approaches use uniform pressures and durations. We hypothesized that gestational-age-related mechanical and developmental differences would affect the time required to achieve optimal lung aeration, and resultant lung volumes, during SI delivery at birth in lambs.Methods49 lambs, in five cohorts between 118 and 139 days of gestation (term 142 d), received a standardized 40 cmHO SI, which was delivered until 10 s after lung volume stability (optimal aeration) was visualized on real-time electrical impedance tomography (EIT), or to a maximum duration of 180 s. Time to stable lung aeration (T) within the whole lung, gravity-dependent, and non-gravity-dependent regions, was determined from EIT recordings.ResultsT was inversely related to gestation (P<0.0001, Kruskal-Wallis test), with the median (range) being 229 (85,306) s and 72 (50,162) s in the 118-d and 139-d cohorts, respectively. Lung volume at T increased with gestation from a mean (SD) of 20 (17) ml/kg at 118 d to 56 (13) ml/kg at 139 d (P=0.002, one-way ANOVA). There were no gravity-dependent regional differences in T or aeration.ConclusionsThe trajectory of aeration during an SI at birth is influenced by gestational age in lambs. An understanding of this may assist in developing SI protocols that optimize lung aeration for all infants.

The development of regional lung injury in the preterm lung is not well understood. This study aimed to characterize time-dependent and regionally specific injury patterns associated with early ventilation of the preterm lung using a mass spectrometry-based proteomic approach. Preterm lambs delivered at 124-127 days gestation received 15 or 90 minutes of mechanical ventilation (positive end-expiratory pressure = 8 cm H 2 O, VT = 6-8 ml/kg) and were compared with unventilated control lambs. At study completion, lung tissue was taken from standardized nondependent and dependent regions, and assessed for lung injury via histology, quantitative PCR, and proteomic analysis using Orbitrap-mass spectrometry. Ingenuity pathway analysis software was used to identify temporal and region-specific enrichments in pathways and functions. Apoptotic cell numbers were ninefold higher in nondependent lung at 15 and 90 minutes compared with controls, whereas proliferative cells were increased fourfold in the dependent lung at 90 minutes. The relative gene expression of lung injury markers was increased at 90 minutes in nondependent lung and unchanged in gravity-dependent lung. Within the proteome, the number of differentially expressed proteins was fourfold higher in the nondependent lung than the dependent lung. The number of differential proteins increased over time in both lung regions. A total of 95% of enriched canonical pathways and 94% of enriched cellular and molecular functions were identified only in nondependent lung tissue from the 90-minute ventilation group. In conclusion, complex injury pathways are initiated within the preterm lung after 15 minutes of ventilation and amplified by continuing ventilation. Injury development is region specific, with greater alterations within the proteome of nondependent lung.

Preterm newborns often require invasive support, however even brief periods of supported ventilation applied inappropriately to the lung can cause injury. Real-time quantitative reverse transcriptase-PCR (qPCR) has been extensively employed in studies of ventilation-induced lung injury with the reference gene 18S ribosomal RNA (18S RNA) most commonly employed as the internal control reference gene. Whilst the results of these studies depend on the stability of the reference gene employed, the use of 18S RNA has not been validated. In this study the expression profile of five candidate reference genes (18S RNA, ACTB, GAPDH, TOP1 and RPS29) in two geographical locations, was evaluated by dedicated algorithms, including geNorm, Normfinder, Bestkeeper and ΔCt method and the overall stability of these candidate genes determined (RefFinder). Secondary studies examined the influence of reference gene choice on the relative expression of two well-validated lung injury markers; EGR1 and IL1B. In the setting of the preterm lamb model of lung injury, RPS29 reference gene expression was influenced by tissue location; however we determined that individual ventilation strategies influence reference gene stability. Whilst 18S RNA is the most commonly employed reference gene in preterm lamb lung studies, our results suggest that GAPDH is a more suitable candidate.

The preterm lung is particularly vulnerable to ventilator-induced lung injury (VILI) as a result of mechanical ventilation. However the developmental and pathological cellular mechanisms influencing the changing patterns of VILI have not been comprehensively delineated, preventing the advancement of targeted lung protective therapies. This study aimed to use SWATH-MS to comprehensively map the plasma proteome alterations associated with the initiation of VILI following 60 minutes of standardized mechanical ventilation from birth in three distinctly different developmental lung states; the extremely preterm, preterm and term lung using the ventilated lamb model. Across these gestations, 34 proteins were differentially altered in matched plasma samples taken at birth and 60 minutes. Multivariate analysis of the plasma proteomes confirmed a gestation-specific response to mechanical ventilation with 79% of differentially-expressed proteins altered in a single gestation group only. Six cellular and molecular functions and two physiological functions were uniquely enriched in either the extremely preterm or preterm group. Correlation analysis supported gestation-specific protein-function associations within each group. In identifying the gestation-specific proteome and functional responses to ventilation we provide the founding evidence required for the potential development of individualized respiratory support approaches tailored to both the developmental and pathological state of the lung.